Eugene Butcher

All Publications

• Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss. Circulation research Andaloussi Mae, M., He, L., Nordling, S., Vazquez-Liebanas, E., Nahar, K., Jung, B., Li, X., Tan, B. C., Foo, J. C., Cazenave Gassiot, A., Wenk, M., Zarb, Y., Lavina, B., Quaggin, S. E., Jeansson, M., Gu, C., Silver, D., Michael Vanlandewijck, M., Butcher, E. C., Keller, A., Betsholtz, C. 2020

  Abstract

  Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level, and to correlate them with regional heterogeneities in BBB function and vascular phenotype. Methods and Results: We reveal transcriptional, morphological and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 is paradoxical given its wider role as TIE2 receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.

  View details for DOI 10.1161/CIRCRESAHA.120.317473

  View details for PubMedID 33375813

• Retinoic Acid Promotes Endothelial Cell Cycle Early G1 State to Enable Human Hemogenic Endothelial Cell Specification. Cell reports Qiu, J., Nordling, S., Vasavada, H. H., Butcher, E. C., Hirschi, K. K. 2020; 33 (9): 108465

  Abstract

  Development of blood-forming (hemogenic) endothelial cells that give rise to hematopoietic stem and progenitor cells (HSPCs) is critical during embryogenesis to generate the embryonic and postnatal hematopoietic system. We previously demonstrated that the specification of murine hemogenic endothelial cells is promoted by retinoic acid (RA) signaling and requires downstream endothelial cell cycle control. Whether this mechanism is conserved in human hemogenic endothelial cell specification is unknown. Here, we present a protocol to derive primordial endothelial cells from human embryonic stem cells and promote their specification toward hemogenic endothelial cells. Furthermore, we demonstrate that RA treatment significantly increases human hemogenic endothelial cell specification. That is, RA promotes endothelial cell cycle arrest to enable RA-induced instructive signals to upregulate the genes needed for hematopoietic transition. These insights provide guidance for the exvivo generation of autologous human hemogenic endothelial cells that are needed to produce human HSPCs for regenerative medicine applications.

  View details for DOI 10.1016/j.celrep.2020.108465

  View details for PubMedID 33264627

• Endothelial HIF-2alpha as a Key Endogenous Mediator Preventing Emphysema. American journal of respiratory and critical care medicine Pasupneti, S., Tian, W., Tu, A. B., Dahms, P., Granucci, E., Gandjeva, A., Xiang, M., Butcher, E., Semenza, G. L., Tuder, R., Jiang, X., Nicolls, M. R. 2020

  Abstract

  RATIONALE: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysema lungs exhibit decreased expression of hypoxia inducible factor-2alpha (HIF-2alpha)-regulated genes, and tobacco smoke decreases pulmonary HIF-2alpha levels. These findings suggest that decreased HIF-2alpha expression is important in the development of emphysema.OBJECTIVES: The objective of this study was to evaluate the roles of endothelial cell (EC) HIF-2alpha in the pathogenesis of emphysema in mice.METHODS: Mouse lungs were examined for emphysema following either the loss or overexpression of EC Hif-2alpha. Additionally, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for hepatocyte growth factor (HGF), a protein involved in alveolar development and homeostasis. Patient emphysema lungs were measured for endothelial HIF-2alpha expression.MEASUREMENTS AND MAIN RESULTS: EC Hif-2alpha deletion resulted in emphysema, in association with fewer ECs and pericytes. Following SU5416 exposure, EC Hif-2alpha knockout mice developed more severe emphysema, whereas EC Hif-2alpha-overexpressing mice were protected. EC Hif-2alpha knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2alpha expression.CONCLUSIONS: Here, we demonstrate a unique, protective role for pulmonary endothelial HIF-2alpha and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2alpha may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2alpha may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

  View details for DOI 10.1164/rccm.202001-0078OC

  View details for PubMedID 32515984

• Neutrophils Recirculate through Lymph Nodes to Survey Tissues for Pathogens. Journal of immunology (Baltimore, Md. : 1950) Bogoslowski, A., Wijeyesinghe, S., Lee, W., Chen, C., Alanani, S., Jenne, C., Steeber, D. A., Scheiermann, C., Butcher, E. C., Masopust, D., Kubes, P. 2020

  Abstract

  The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics.

  View details for DOI 10.4049/jimmunol.2000022

  View details for PubMedID 32205425

• A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature. Frontiers in cardiovascular medicine Xiang, M., Grosso, R. A., Takeda, A., Pan, J., Bekkhus, T., Brulois, K., Dermadi, D., Nordling, S., Vanlandewijck, M., Jalkanen, S., Ulvmar, M. H., Butcher, E. C. 2020; 7: 52

  Abstract

  Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN.

  View details for DOI 10.3389/fcvm.2020.00052

  View details for PubMedID 32426372

• Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells. Gastroenterology Ding, S. n., Song, Y. n., Brulois, K. F., Pan, J. n., Co, J. Y., Ren, L. n., Feng, N. n., Yasukawa, L. L., Sánchez-Tacuba, L. n., Wosen, J. E., Mellins, E. D., Monack, D. M., Amieva, M. R., Kuo, C. J., Butcher, E. C., Greenberg, H. B. 2020

  Abstract

  Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids.We analyzed transcriptome data from mouse Peyer's Patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells.A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids.We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

  View details for DOI 10.1053/j.gastro.2020.03.053

  View details for PubMedID 32247021

• Exploration of Cell Development Pathways through High-Dimensional Single Cell Analysis in Trajectory Space. iScience Dermadi, D. n., Bscheider, M. n., Bjegovic, K. n., Lazarus, N. H., Szade, A. n., Hadeiba, H. n., Butcher, E. C. 2020; 23 (2): 100842

  Abstract

  High-dimensional single cell profiling coupled with computational modeling is emerging as a powerful tool to elucidate developmental programs directing cell lineages. We introduce tSpace, an algorithm based on the concept of "trajectory space", in which cells are defined by their distance along nearest neighbor pathways to every other cell in a population. Graphical mapping of cells in trajectory space allows unsupervised reconstruction and exploration of complex developmental sequences. Applied to flow and mass cytometry data, the method faithfully reconstructs thymic T cell development and reveals development and trafficking regulation of tonsillar B cells. Applied to the single cell transcriptome of mouse intestine and C. elegans, the method recapitulates development from intestinal stem cells to specialized epithelial phenotypes more faithfully than existing algorithms and orders C. elegans cells concordantly to the associated embryonic time. tSpace profiling of complex populations is well suited for hypothesis generation in developing cell systems.

  View details for DOI 10.1016/j.isci.2020.100842

  View details for PubMedID 32058956

  View details for PubMedCentralID PMC6997593

• A molecular map of murine lymph node blood vascular endothelium at single cell resolution. Nature communications Brulois, K. n., Rajaraman, A. n., Szade, A. n., Nordling, S. n., Bogoslowski, A. n., Dermadi, D. n., Rahman, M. n., Kiefel, H. n., O'Hara, E. n., Koning, J. J., Kawashima, H. n., Zhou, B. n., Vestweber, D. n., Red-Horse, K. n., Mebius, R. E., Adams, R. H., Kubes, P. n., Pan, J. n., Butcher, E. C. 2020; 11 (1): 3798

  Abstract

  Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis.

  View details for DOI 10.1038/s41467-020-17291-5

  View details for PubMedID 32732867

• Single-Cell Survey of Human Lymphatics Unveils Marked Endothelial Cell Heterogeneity and Mechanisms of Homing for Neutrophils. Immunity Takeda, A., Hollmen, M., Dermadi, D., Pan, J., Brulois, K. F., Kaukonen, R., Lonnberg, T., Bostrom, P., Koskivuo, I., Irjala, H., Miyasaka, M., Salmi, M., Butcher, E. C., Jalkanen, S. 2019

  Abstract

  Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsularsinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.

  View details for DOI 10.1016/j.immuni.2019.06.027

  View details for PubMedID 31402260

• Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1 NATURE MEDICINE Yousef, H., Czupalla, C. J., Lee, D., Chen, M. B., Burke, A. N., Zera, K. A., Zandstra, J., Berber, E., Lehallier, B., Mathur, V., Nair, R. V., Bonanno, L. N., Yang, A. C., Peterson, T., Hadeiba, H., Merkel, T., Koerbelin, J., Schwaninger, M., Buckwalter, M. S., Quake, S. R., Butcher, E. C., Wyss-Coray, T. 2019; 25 (6): 988-+

  View details for DOI 10.1038/s41591-019-0440-4

  View details for Web of Science ID 000470844400031

• Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment FRONTIERS IN IMMUNOLOGY Pachynski, R. K., Wang, P., Salazar, N., Zheng, Y., Nease, L., Rosalez, J., Leong, W., Virdi, G., Rennier, K., Shin, W., Viet Nguyen, Butcher, E. C., Zabel, B. A. 2019; 10

  View details for DOI 10.3389/fimmu.2019.00983

  View details for Web of Science ID 000467341300001

• Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment. Frontiers in immunology Pachynski, R. K., Wang, P., Salazar, N., Zheng, Y., Nease, L., Rosalez, J., Leong, W. I., Virdi, G., Rennier, K., Shin, W. J., Nguyen, V., Butcher, E. C., Zabel, B. A. 2019; 10: 983

  Abstract

  Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy.

  View details for DOI 10.3389/fimmu.2019.00983

  View details for PubMedID 31139180

  View details for PubMedCentralID PMC6518384

• DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis. Proceedings of the National Academy of Sciences of the United States of America Graham, K. L., Werner, B. J., Moyer, K. M., Patton, A. K., Krois, C. R., Yoo, H. S., Tverskoy, M., LaJevic, M., Napoli, J. L., Sobel, R. A., Zabel, B. A., Butcher, E. C. 2019

  Abstract

  The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

  View details for PubMedID 30718413

• Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1. Nature medicine Yousef, H. n., Czupalla, C. J., Lee, D. n., Chen, M. B., Burke, A. N., Zera, K. A., Zandstra, J. n., Berber, E. n., Lehallier, B. n., Mathur, V. n., Nair, R. V., Bonanno, L. N., Yang, A. C., Peterson, T. n., Hadeiba, H. n., Merkel, T. n., Körbelin, J. n., Schwaninger, M. n., Buckwalter, M. S., Quake, S. R., Butcher, E. C., Wyss-Coray, T. n. 2019

  Abstract

  An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.

  View details for PubMedID 31086348

• Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio-protocol Czupalla, C. J., Yousef, H., Wyss-Coray, T., Butcher, E. C. 2018; 8 (22)

  Abstract

  The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. In the past, approaches to isolate endothelial cells have either involved the use of transgenic reporter mice or suffered from insufficiently pure cell populations and poor yield. This protocol here is based on well-established protocols that were modified and combined to allow single cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after careful removal of the meninges and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested in two steps resulting in a single cell suspension. Cells are stained with a cocktail of fluorochrome-conjugated antibodies identifying not only brain endothelial cells, but also potentially contaminating cell types such as pericytes, astrocytes, and lineage cells. Using flow cytometry, cell populations are separated and sorted directly into either RNA lysis buffer for bulk RNA analyses (e.g., RNA microarray and RNA-Seq) or in pure fetal bovine serum to preserve viability for other downstream applications such as single cell RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq). The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 3 * 105 cells from a pool of four adult mice.

  View details for PubMedID 30637296

• Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio-protocol Yousef, H., Czupalla, C. J., Lee, D., Butcher, E. C., Wyss-Coray, T. 2018; 8 (22)

  Abstract

  Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. In the past, approaches to isolate endothelial cells were dependent on transgenic mice and often resulted in insufficiently pure cell populations and poor yield. This protocol describes a technique that allows single-cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after perfusion and careful removal of the meninges, and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested resulting in a single cell suspension. Cells are stained with fluorochrome-conjugated antibodies identifying CD31+ brain endothelial cells, as well as CD45+CD11b+ myeloid cells for exclusion. Using flow cytometry, cell populations are separated and CD31+BECs are sorted in bulk into RNA later or as single cells directly into either RNA lysis buffer for single or bulk RNA-Seq analyses. The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 1 × 105 cells isolated from an adult mouse cortex/hippocampus.

  View details for DOI 10.21769/BioProtoc.3091

  View details for PubMedID 31032379

  View details for PubMedCentralID PMC6482965

• Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry BIO-PROTOCOL Yousef, H., Czupalla, C. J., Lee, D., Butcher, E. C., Wyss-Coray, T. 2018; 8 (22)

  View details for DOI 10.21769/BioProtoc.3091

  View details for Web of Science ID 000458027400011

• Endothelial HIF-2alpha is Required for the Maintenance of Airway Microvasculature. Circulation Jiang, X., Tian, W., Tu, A. B., Pasupneti, S., Shuffle, E., Dahms, P., Zhang, P., Cai, H., Dinh, T. T., Liu, B., Cain, C., Giaccia, A. J., Butcher, E. C., Simon, C., Semenza, G. L., Nicolls, M. R. 2018

  Abstract

  BACKGROUND: Hypoxia-inducible factors (HIFs), especially HIF-1alpha and HIF-2alpha, are key mediators of the adaptive response to hypoxic stress and play essential roles in maintaining lung homeostasis. Human and animal genetics studies confirm that abnormal HIF correlates with pulmonary vascular pathology and chronic lung diseases, but it remains unclear whether endothelial cell (EC) HIF production is essential for microvascular health. The large airway has an ideal circulatory bed for evaluating histologic changes and physiology in genetically-modified rodents.METHODS: The tracheal microvasculature of mice, with conditionally-deleted or overexpressed HIF-1alpha or HIF-2alpha, was evaluated for anatomy, perfusion, and permeability. Angiogenic signaling studies assessed vascular changes attributable to dysregulated HIF expression. An orthotopic tracheal transplantation model further evaluated the contribution of individual HIF isoforms in airway ECs.RESULTS: The genetic deletion of Hif-2alpha, but not Hif-1alpha, caused tracheal EC apoptosis, diminished pericyte coverage, reduced vascular perfusion, defective barrier function, overlying epithelial abnormalities and subepithelial fibrotic remodeling. HIF-2alpha promoted microvascular integrity in airways through endothelial angiopoietin-1/TIE2 signaling and Notch activity. In functional tracheal transplants, HIF-2alpha deficiency in airway donors accelerated graft microvascular loss, whereas HIF-2alpha or angiopoietin-1 overexpression prolonged transplant microvascular perfusion. Augmented endothelial HIF-2alpha in transplant donors promoted airway microvascular integrity and diminished alloimmune inflammation.CONCLUSIONS: Our findings reveal that the constitutive expression of endothelial HIF-2alpha is required for airway microvascular health.

  View details for PubMedID 30586708

• Gut-Selective Integrin-Targeted Therapies for Inflammatory Bowel Disease JOURNAL OF CROHNS & COLITIS Lamb, C. A., O'Byrne, S., Keir, M. E., Butcher, E. C. 2018; 12: S653–S668

  Abstract

  Integrins are cell surface receptors with bidirectional signalling capabilities that can bind to adhesion molecules in order to mediate homing of leukocytes to peripheral tissues. Gut-selective leukocyte homing is facilitated by interactions between α4β7 and its ligand, mucosal addressin cellular adhesion molecule-1 [MAdCAM-1], while retention of lymphocytes in mucosal tissues is mediated by αEβ7 binding to its ligand E-cadherin. Therapies targeting gut-selective trafficking have shown efficacy in inflammatory bowel disease [IBD], confirming the importance of leukocyte trafficking in disease pathobiology. This review will provide an overview of integrin structure, function and signalling, and highlight the role that these molecules play in leukocyte homing and retention. Anti-integrin therapeutics, including gut-selective antibodies against the β7 integrin subunit [etrolizumab] and the α4β7 integrin heterodimer [vedolizumab and abrilumab], and the non-gut selective anti-α4 integrin [natalizumab], will be discussed, as well as novel targeting approaches using small molecules.

  View details for PubMedID 29767705

• A Chimeric Antibody against ACKR3/CXCR7 in Combination with TMZ Activates Immune Responses and Extends Survival in Mouse GBM Models MOLECULAR THERAPY Salazar, N., Carlson, J. C., Huang, K., Zheng, Y., Oderup, C., Gross, J., Jang, A. D., Burke, T. M., Lewen, S., Scholz, A., Huang, S., Nease, L., Kosek, J., Mittelbronn, M., Butcher, E. C., Tu, H., Zabel, B. A. 2018; 26 (5): 1354–65

  Abstract

  Glioblastoma (GBM) is the least treatable type of brain tumor, afflicting over 15,000 people per year in the United States. Patients have a median survival of 16 months, and over 95% die within 5 years. The chemokine receptor ACKR3 is selectively expressed on both GBM cells and tumor-associated blood vessels. High tumor expression of ACKR3 correlates with poor prognosis and potential treatment resistance, making it an attractive therapeutic target. We engineered a single chain FV-human FC-immunoglobulin G1 (IgG1) antibody, X7Ab, to target ACKR3 in human and mouse GBM cells. We used hydrodynamic gene transfer to overexpress the antibody, with efficacy in vivo. X7Ab kills GBM tumor cells and ACKR3-expressing vascular endothelial cells by engaging the cytotoxic activity of natural killer (NK) cells and complement and the phagocytic activity of macrophages. Combining X7Ab with TMZ allows the TMZ dosage to be lowered, without compromising therapeutic efficacy. Mice treated with X7Ab and in combination with TMZ showed significant tumor reduction by MRI and longer survival overall. Brain-tumor-infiltrating leukocyte analysis revealed that X7Ab enhances the activation of M1 macrophages to support anti-tumor immune response in vivo. Targeting ACKR3 with immunotherapeutic monoclonal antibodies (mAbs) in combination with standard of care therapies may prove effective in treating GBM.

  View details for PubMedID 29606504

• Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bogoslowski, A., Butcher, E. C., Kubes, P. 2018; 115 (10): 2449–54

  Abstract

  Staphylococcus aureus is a skin- and respiratory tract-colonizing bacterium and is the leading cause of community-acquired skin infections. Dissemination of these bacteria into systemic circulation causes bacteremia, which has a high mortality rate. Therefore, understanding the immunologic barriers that prevent dissemination is critical to developing novel treatments. In this study, we demonstrate that an S. aureus breach across skin leads to some migration of the pathogen to the draining lymph node, but no further. While subcapsular sinus (SCS) macrophage in lymph nodes were important in detaining S. aureus, a rapid complement-dependent neutrophil recruitment (independent of the SCS macrophage) via high endothelial venules (HEVs) resulted in high numbers of neutrophils that intercepted the bacteria in the lymph nodes. Peripheral Node Addressin together with its two ligands, L-selectin and platelet P-selectin, are critical for recruiting neutrophils via the HEVs. Almost no neutrophils entered the lymph nodes via lymphatics. Neutrophils actively phagocytosed S. aureus and helped sterilize the lymph nodes and prevent dissemination to blood and other organs.

  View details for PubMedID 29378967

• Single-cell analysis of early progenitor cells that build coronary arteries. Nature Su, T. n., Stanley, G. n., Sinha, R. n., D'Amato, G. n., Das, S. n., Rhee, S. n., Chang, A. H., Poduri, A. n., Raftrey, B. n., Dinh, T. T., Roper, W. A., Li, G. n., Quinn, K. E., Caron, K. M., Wu, S. n., Miquerol, L. n., Butcher, E. C., Weissman, I. n., Quake, S. n., Red-Horse, K. n. 2018

  Abstract

  Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

  View details for PubMedID 29973725

• Development of a New Human CD22 transgenic Mouse Macauley, M. S., Bednar, K. J., Shanina, E., Ballet, R., Connors, E. P., Duan, S., Juan, J., Arlian, B. M., Kulis, M. D., Butcher, E. C., Fung-Leung, W., Rao, T. S., Paulson, J. C. OXFORD UNIV PRESS INC. 2017: 1249

  View details for Web of Science ID 000423267000191

• Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model. Journal of immunology (Baltimore, Md. : 1950) Bednar, K. J., Shanina, E., Ballet, R., Connors, E. P., Duan, S., Juan, J., Arlian, B. M., Kulis, M. D., Butcher, E. C., Fung-Leung, W. P., Rao, T. S., Paulson, J. C., Macauley, M. S. 2017; 199 (9): 3116-3128

  Abstract

  CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca2+) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22-/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22-/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22-/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22.

  View details for DOI 10.4049/jimmunol.1700898

  View details for PubMedID 28972089

  View details for PubMedCentralID PMC5679471

• A Mucosal and Cutaneous Chemokine Ligand for the Lymphocyte Chemoattractant Receptor GPR15 FRONTIERS IN IMMUNOLOGY Ocon, B., Pan, J., Dinh, T., Chen, W., Ballet, R., Bscheider, M., Habtezion, A., Tu, H., Zabel, B. A., Butcher, E. C. 2017; 8: 1111

  Abstract

  Chemoattractants control lymphocyte recruitment from the blood, contributing to the systemic organization of the immune system. The G protein-linked receptor GPR15 mediates lymphocyte homing to the large intestines and skin. Here we show that the 9 kDa CC-motif containing cationic polypeptide AP57/colon-derived sushi containing domain-2 binding factor (CSBF), encoded by C10orf99 in the human and 2610528A11Rik in the mouse, functions as a chemokine ligand for GPR15 (GPR15L). GPR15L binds GPR15 and attracts GPR15-expressing T cells including lymphocytes in colon-draining lymph nodes and Vγ3+ thymic precursors of dermal epithelial T cells. Patterns of GPR15L expression by epithelial cells in adult mice and humans suggest a homeostatic role for the chemokine in lymphocyte localization to the large intestines, as well as a role in homing to the epidermis during wound healing or inflammation. GPR15L is also significantly expressed in squamous mucosa of the oral cavity and esophagus with still poorly defined regulation. Identification of the chemotactic activity of GPR15L adds to its reported antibacterial and tumor cell growth regulatory functions and suggests the potential of targeting GPR15L-GPR15 interactions for modulation of mucosal and cutaneous inflammation.

  View details for PubMedID 28936214

• Trafficking receptor signatures define blood plasmablasts responding to tissue-specific immune challenge. JCI insight Seong, Y., Lazarus, N. H., Sutherland, L., Habtezion, A., Abramson, T., He, X., Greenberg, H. B., Butcher, E. C. 2017; 2 (6)

  Abstract

  Antibody-secreting cells are generated in regional lymphoid tissues and traffic as plasmablasts (PBs) via lymph and blood to target sites for local immunity. We used multiparameter flow cytometry to define PB trafficking programs (TPs, combinations of adhesion molecules and chemoattractant receptors) and their imprinting in patients in response to localized infection or immune insults. TPs enriched after infection or autoimmune inflammation of mucosae correlate with sites of immune response or symptoms, with different TPs imprinted during small intestinal, colon, throat, and upper respiratory immune challenge. PBs induced after intramuscular or intradermal influenza vaccination, including flu-specific antibody-secreting cells, display TPs characterized by the lack of mucosal homing receptors. PBs of healthy donors display diverse mucosa-associated TPs, consistent with homeostatic immune activity. Identification of TP signatures of PBs may facilitate noninvasive monitoring of organ-specific immune responses.

  View details for DOI 10.1172/jci.insight.90233

  View details for PubMedID 28352656

• Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo BLOOD Pan, J., Dinh, T. T., Rajaraman, A., Lee, M., Scholz, A., Czupalla, C. J., Kiefel, H., Zhu, L., Xia, L., Morser, J., Jiang, H., Santambrogio, L., Butcher, E. C. 2016; 128 (1): 104-109

  Abstract

  Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantialC activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

  View details for DOI 10.1182/blood-2015-12-684688

  View details for Web of Science ID 000379249600018

  View details for PubMedID 27207787

  View details for PubMedCentralID PMC4937354

• Vitamin D immunoregulation through dendritic cells. Immunology Bscheider, M., Butcher, E. C. 2016; 148 (3): 227-236

  Abstract

  Vitamin D (VD3) has been linked to immunological processes, and its supplementation may have a role in treatment or prevention of diseases with underlying autoimmune or pro-inflammatory states. As initiators of the immune responses, dendritic cells (DC) are a potential target of VD3 to dampen autoimmunity and inflammation, but the role of DC in VD3-mediated immunomodulation in vivo is not understood. In addition to being targets of VD3, DC can provide a local source of bioactive VD3 for regulation of T-cell responses. Here we review existing studies that describe the tolerogenic potential of VD3 on DC, and discuss them in the context of current understanding of DC development and function. We speculate on mechanisms that might account for the potent but poorly understood tolerogenic activities of VD3 and the role of DC as both targets and sources of this hormone.

  View details for DOI 10.1111/imm.12610

  View details for PubMedID 27040466

  View details for PubMedCentralID PMC4913286

• Leukocyte Trafficking to the Small Intestine and Colon. Gastroenterology Habtezion, A., Nguyen, L. P., Hadeiba, H., Butcher, E. C. 2016; 150 (2): 340-354

  Abstract

  Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.

  View details for DOI 10.1053/j.gastro.2015.10.046

  View details for PubMedID 26551552

• Generation and transcriptional programming of intestinal dendritic cells: essential role of retinoic acid MUCOSAL IMMUNOLOGY Zeng, R., Bscheider, M., Lahl, K., Lee, M., Butcher, E. C. 2016; 9 (1): 183-193

  Abstract

  The vitamin A metabolite retinoic acid (RA) regulates adaptive immunity in the intestines, with well-characterized effects on IgA responses, Treg induction, and gut trafficking of T- and B-effector cells. It also controls the generation of conventional dendritic cell (cDC) precursors in the bone marrow and regulates cDC subset representation, but its roles in the specialization of intestinal cDC subsets are understudied. Here we show that RA acts cell intrinsically in developing gut-tropic pre-mucosal dendritic cell (pre-μDC) to effect the differentiation and drive the specialization of intestinal CD103(+)CD11b(-) (cDC1) and of CD103(+)CD11b(+) (cDC2). Systemic deficiency or DC-restricted antagonism of RA signaling resulted in altered phenotypes of intestinal cDC1 and cDC2, and reduced numbers of cDC2. Effects of dietary deficiency were most apparent in the proximal small intestine and were rapidly reversed by reintroducing vitamin A. In cultures of pre-μDC with Flt3L and granulocyte-macrophage colony-stimulating factor (GM-CSF), RA induced cDC with characteristic phenotypes of intestinal cDC1 and cDC2 by controlling subset-defining cell surface receptors, regulating subset-specific transcriptional programs, and suppressing proinflammatory nuclear factor-κB-dependent gene expression. Thus, RA is required for transcriptional programming and maturation of intestinal cDC, and with GM-CSF and Flt3L provides a minimal environment for in vitro generation of intestinal cDC1- and cDC2-like cDC from specialized precursors.

  View details for DOI 10.1038/mi.2015.50

  View details for Web of Science ID 000367653800015

  View details for PubMedCentralID PMC4698111

• Generation and transcriptional programming of intestinal dendritic cells: essential role of retinoic acid. Mucosal immunology Zeng, R., Bscheider, M., Lahl, K., Lee, M., Butcher, E. C. 2016; 9 (1): 183-93

  Abstract

  The vitamin A metabolite retinoic acid (RA) regulates adaptive immunity in the intestines, with well-characterized effects on IgA responses, Treg induction, and gut trafficking of T- and B-effector cells. It also controls the generation of conventional dendritic cell (cDC) precursors in the bone marrow and regulates cDC subset representation, but its roles in the specialization of intestinal cDC subsets are understudied. Here we show that RA acts cell intrinsically in developing gut-tropic pre-mucosal dendritic cell (pre-μDC) to effect the differentiation and drive the specialization of intestinal CD103(+)CD11b(-) (cDC1) and of CD103(+)CD11b(+) (cDC2). Systemic deficiency or DC-restricted antagonism of RA signaling resulted in altered phenotypes of intestinal cDC1 and cDC2, and reduced numbers of cDC2. Effects of dietary deficiency were most apparent in the proximal small intestine and were rapidly reversed by reintroducing vitamin A. In cultures of pre-μDC with Flt3L and granulocyte-macrophage colony-stimulating factor (GM-CSF), RA induced cDC with characteristic phenotypes of intestinal cDC1 and cDC2 by controlling subset-defining cell surface receptors, regulating subset-specific transcriptional programs, and suppressing proinflammatory nuclear factor-κB-dependent gene expression. Thus, RA is required for transcriptional programming and maturation of intestinal cDC, and with GM-CSF and Flt3L provides a minimal environment for in vitro generation of intestinal cDC1- and cDC2-like cDC from specialized precursors.

  View details for DOI 10.1038/mi.2015.50

  View details for PubMedID 26129652

  View details for PubMedCentralID PMC4698111

• Role and species-specific expression of colon T cell homing receptor GPR15 in colitis. Nature immunology Nguyen, L. P., Pan, J., Dinh, T. T., Hadeiba, H., O'Hara, E., Ebtikar, A., Hertweck, A., Gökmen, M. R., Lord, G. M., Jenner, R. G., Butcher, E. C., Habtezion, A. 2015; 16 (2): 207-213

  Abstract

  Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

  View details for DOI 10.1038/ni.3079

  View details for PubMedID 25531831

• Role and species-specific expression of colon T cell homing receptor GPR15 in colitis NATURE IMMUNOLOGY Nguyen, L. P., Pan, J., Dinh, T. T., Hadeiba, H., O'Hara, E., Ebtikar, A., Hertweck, A., Goekmen, M. R., Lord, G. M., Jenner, R. G., Butcher, E. C., Habtezion, A. 2015; 16 (2): 207-?

  Abstract

  Lymphocyte recruitment maintains intestinal immune homeostasis but also contributes to inflammation. The orphan chemoattractant receptor GPR15 mediates regulatory T cell homing and immunosuppression in the mouse colon. We show that GPR15 is also expressed by mouse TH17 and TH1 effector cells and is required for colitis in a model that depends on the trafficking of these cells to the colon. In humans GPR15 is expressed by effector cells, including pathogenic TH2 cells in ulcerative colitis, but is expressed poorly or not at all by colon regulatory T (Treg) cells. The TH2 transcriptional activator GATA-3 and the Treg-associated transcriptional repressor FOXP3 robustly bind human, but not mouse, GPR15 enhancer sequences, correlating with receptor expression. Our results highlight species differences in GPR15 regulation and suggest it as a potential therapeutic target for colitis.

  View details for DOI 10.1038/ni.3079

  View details for Web of Science ID 000348143100013

• Leukocyte Chemoattractant Receptors in Human Disease Pathogenesis ANNUAL REVIEW OF PATHOLOGY: MECHANISMS OF DISEASE, VOL 10 Zabel, B. A., Rott, A., Butcher, E. C. 2015; 10: 51-81

  Abstract

  Combinations of leukocyte attractant ligands and cognate heptahelical receptors specify the systemic recruitment of circulating cells by triggering integrin-dependent adhesion to endothelial cells, supporting extravasation, and directing specific intratissue localization via gradient-driven chemotaxis. Chemoattractant receptors also control leukocyte egress from lymphoid organs and peripheral tissues. In this article, we summarize the fundamental mechanics of leukocyte trafficking, from the evolution of multistep models of leukocyte recruitment and navigation to the regulation of chemoattractant availability and function by atypical heptahelical receptors. To provide a more complete picture of the migratory circuits involved in leukocyte trafficking, we integrate a number of nonchemokine chemoattractant receptors into our discussion. Leukocyte chemoattractant receptors play key roles in the pathogenesis of autoimmune diseases, allergy, inflammatory disorders, and cancer. We review recent advances in our understanding of chemoattractant receptors in disease pathogenesis, with a focus on genome-wide association studies in humans and the translational implications of mechanistic studies in animal disease models.

  View details for DOI 10.1146/annurev-pathol-012513-104640

  View details for Web of Science ID 000348560600003

  View details for PubMedID 25387059

• Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model. Journal of visualized experiments : JoVE Pachynski, R. K., Scholz, A., Monnier, J., Butcher, E. C., Zabel, B. A. 2015

  Abstract

  Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.  Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune "escape," including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

  View details for DOI 10.3791/52657

  View details for PubMedID 25938949

• A Novel CMKLR1 Small Molecule Antagonist Suppresses CNS Autoimmune Inflammatory Disease PLOS ONE Graham, K. L., Zhang, J. V., Lewen, S., Burke, T. M., Dang, T., Zoudilova, M., Sobel, R. A., Butcher, E. C., Zabel, B. A. 2014; 9 (12)

  Abstract

  Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.

  View details for DOI 10.1371/journal.pone.0112925

  View details for Web of Science ID 000347114900022

  View details for PubMedID 25437209

  View details for PubMedCentralID PMC4249827

• Absence of Nkx2-3 Homeodomain Transcription Factor Reprograms the Endothelial Addressin Preference for Lymphocyte Homing in Peyer's Patches JOURNAL OF IMMUNOLOGY Kellermayer, Z., Mihalj, M., Labadi, A., Czoempoely, T., Lee, M., O'Hara, E., Butcher, E. C., Berta, G., Balogh, A., Arnold, H., Balogh, P. 2014; 193 (10): 5284-5293

  Abstract

  Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin β receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

  View details for DOI 10.4049/jimmunol.1402016

  View details for Web of Science ID 000345023400056

• Absence of Nkx2-3 homeodomain transcription factor reprograms the endothelial addressin preference for lymphocyte homing in Peyer's patches. Journal of immunology Kellermayer, Z., Mihalj, M., Lábadi, Á., Czömpöly, T., Lee, M., O'Hara, E., Butcher, E. C., Berta, G., Balogh, A., Arnold, H., Balogh, P. 2014; 193 (10): 5284-5293

  Abstract

  Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin β receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

  View details for DOI 10.4049/jimmunol.1402016

  View details for PubMedID 25320278

• Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing NATURE IMMUNOLOGY Lee, M., Kiefel, H., LaJevic, M. D., Macauley, M. S., O'Hara, E., Pan, J., Paulson, J. C., Butcher, E. C. 2014; 15 (10): 982-U129

  Abstract

  Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

  View details for DOI 10.1038/ni.2983

  View details for Web of Science ID 000342564800014

  View details for PubMedCentralID PMC4222088

• Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing. Nature immunology Lee, M., Kiefel, H., LaJevic, M. D., Macauley, M. S., Kawashima, H., O'Hara, E., Pan, J., Paulson, J. C., Butcher, E. C. 2014; 15 (10): 982-995

  Abstract

  Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

  View details for DOI 10.1038/ni.2983

  View details for PubMedID 25173345

• Orphan chemoattractant receptor GPR15 mediates dendritic epidermal T-cell recruitment to the skin EUROPEAN JOURNAL OF IMMUNOLOGY Lahl, K., Sweere, J., Pan, J., Butcher, E. 2014; 44 (9): 2577-2581

  Abstract

  Homing of murine dendritic epidermal T cells (DETCs) from the thymus to the skin is regulated by specific trafficking receptors during late embryogenesis. Once in the epidermis, Vγ3δ1 TCR DETCs are maintained through self-renewal and participate in wound healing. GPR15 is an orphan G protein-linked chemoattractant receptor involved in the recruitment of regulatory T cells to the colon. Here we show that GPR15 is highly expressed on fetal thymic DETC precursors and on recently recruited DETCs, and mediates the earliest seeding of the epidermis, which occurs at the time of establishment of skin barrier function. DETCs in GPR15(-/-) mice remain low at birth, but later participation of CCR10 and CCR4 in DETC homing allows DETCs to reach near normal levels in adult skin. Our findings establish a role for GPR15 in skin lymphocyte homing and suggest that it may contribute to lymphocyte subset targeting to diverse epithelial sites.

  View details for DOI 10.1002/eji.201444628

  View details for Web of Science ID 000342818900006

  View details for PubMedCentralID PMC4165750

• Orphan chemoattractant receptor GPR15 mediates dendritic epidermal T-cell recruitment to the skin. European journal of immunology Lahl, K., Sweere, J., Pan, J., Butcher, E. 2014; 44 (9): 2577-2581

  Abstract

  Homing of murine dendritic epidermal T cells (DETCs) from the thymus to the skin is regulated by specific trafficking receptors during late embryogenesis. Once in the epidermis, Vγ3δ1 TCR DETCs are maintained through self-renewal and participate in wound healing. GPR15 is an orphan G protein-linked chemoattractant receptor involved in the recruitment of regulatory T cells to the colon. Here we show that GPR15 is highly expressed on fetal thymic DETC precursors and on recently recruited DETCs, and mediates the earliest seeding of the epidermis, which occurs at the time of establishment of skin barrier function. DETCs in GPR15(-/-) mice remain low at birth, but later participation of CCR10 and CCR4 in DETC homing allows DETCs to reach near normal levels in adult skin. Our findings establish a role for GPR15 in skin lymphocyte homing and suggest that it may contribute to lymphocyte subset targeting to diverse epithelial sites.

  View details for DOI 10.1002/eji.201444628

  View details for PubMedID 24838826

  View details for PubMedCentralID PMC4165750

• Circulating human rotavirus specific CD4 T cells identified with a class II tetramer express the intestinal homing receptors a4ß7 and CCR9. Virology Parra, M., Herrera, D., Calvo-Calle, J. M., Stern, L. J., Parra-López, C. A., Butcher, E., Franco, M., Angel, J. 2014; 452-453: 191-201

  Abstract

  Using a consensus epitope prediction approach, three rotavirus (RV) peptides that induce cytokine secretion by CD4 T cells from healthy volunteers were identified. The peptides were shown to bind HLA-DRB1(⁎)0101 and then used to generate MHC II tetramers. RV specific T cell lines specific for one of the three peptides studied were restricted by MHC class II molecules and contained T cells that bound the tetramer and secreted cytokines upon activation with the peptide. The majority of RV and Flu tetramer(+) CD4 T cells in healthy volunteers expressed markers of antigen experienced T cells, but only RV specific CD4 T cells expressed intestinal homing receptors. CD4 T cells from children that received a RV vaccine, but not placebo recipients, were stained with the RV-VP6 tetramer and also expressed intestinal homing receptors. Circulating RV-specific CD4 T cells represent a unique subset that expresses intestinal homing receptors.

  View details for DOI 10.1016/j.virol.2014.01.014

  View details for PubMedID 24606696

• Circulating human rotavirus specific CD4 T cells identified with a class II tetramer express the intestinal homing receptors alpha 4 beta 7 and CCR9 VIROLOGY Parra, M., Herrera, D., Calvo-Calle, J. M., Stern, L. J., Parra-Lopez, C. A., Butcher, E., Franco, M., Angel, J. 2014; 452: 191-201

  Abstract

  Using a consensus epitope prediction approach, three rotavirus (RV) peptides that induce cytokine secretion by CD4 T cells from healthy volunteers were identified. The peptides were shown to bind HLA-DRB1(⁎)0101 and then used to generate MHC II tetramers. RV specific T cell lines specific for one of the three peptides studied were restricted by MHC class II molecules and contained T cells that bound the tetramer and secreted cytokines upon activation with the peptide. The majority of RV and Flu tetramer(+) CD4 T cells in healthy volunteers expressed markers of antigen experienced T cells, but only RV specific CD4 T cells expressed intestinal homing receptors. CD4 T cells from children that received a RV vaccine, but not placebo recipients, were stained with the RV-VP6 tetramer and also expressed intestinal homing receptors. Circulating RV-specific CD4 T cells represent a unique subset that expresses intestinal homing receptors.

  View details for DOI 10.1016/j.virol.2014.01.014

  View details for Web of Science ID 000333000400021

• Comparative transcriptional and functional profiling defines conserved programs of intestinal DC differentiation in humans and mice NATURE IMMUNOLOGY Watchmaker, P. B., Lahl, K., Lee, M., Baumjohann, D., Morton, J., Kim, S. J., Zeng, R., Dent, A., Anse, K. M., Diamond, B., Hadeiba, H., Butcher, E. C. 2014; 15 (1): 98-108

  Abstract

  Dendritic cells (DCs) that orchestrate mucosal immunity have been studied in mice. Here we characterized human gut DC populations and defined their relationship to previously studied human and mouse DCs. CD103(+)Sirpα(-) DCs were related to human blood CD141(+) DCs and to mouse intestinal CD103(+)CD11b(-) DCs and expressed markers of cross-presenting DCs. CD103(+)Sirpα(+) DCs aligned with human blood CD1c(+) DCs and mouse intestinal CD103(+)CD11b(+) DCs and supported the induction of regulatory T cells. Both CD103(+) DC subsets induced the TH17 subset of helper T cells, while CD103(-)Sirpα(+) DCs induced the TH1 subset of helper T cells. Comparative analysis of transcriptomes revealed conserved transcriptional programs among CD103(+) DC subsets and identified a selective role for the transcriptional repressors Bcl-6 and Blimp-1 in the specification of CD103(+)CD11b(-) DCs and intestinal CD103(+)CD11b(+) DCs, respectively. Our results highlight evolutionarily conserved and divergent programming of intestinal DCs.

  View details for DOI 10.1038/ni.2768

  View details for Web of Science ID 000328800500017

  View details for PubMedID 24292363

• A novel CMKLR1 small molecule antagonist suppresses CNS autoimmune inflammatory disease. PloS one Graham, K. L., Zhang, J. V., Lewén, S., Burke, T. M., Dang, T., Zoudilova, M., Sobel, R. A., Butcher, E. C., Zabel, B. A. 2014; 9 (12)

  Abstract

  Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.

  View details for DOI 10.1371/journal.pone.0112925

  View details for PubMedID 25437209

• Retinoic acid regulates the development of a gut-homing precursor for intestinal dendritic cells. Mucosal immunology Zeng, R., Oderup, C., Yuan, R., Lee, M., Habtezion, A., Hadeiba, H., BUTCHER, E. C. 2013; 6 (4): 847-856

  Abstract

  The vitamin A metabolite retinoic acid (RA) regulates intestinal immune responses through immunomodulatory actions on intestinal dendritic cells (DCs) and lymphocytes. Here, we show that RA also controls the generation of gut-tropic migratory DC precursors, referred to as pre-mucosal DCs (pre-μDCs). Pre-μDCs express the gut trafficking receptor α4β7 and home preferentially to the intestines. They develop in the bone marrow (BM), can differentiate into CCR9⁺ plasmacytoid DCs as well as conventional DCs (cDCs), but preferentially give rise to CD103⁺ intestinal cDCs. Generation of pre-μDCs in vivo in the BM or in vitro is regulated by RA and RA receptor α (RARα) signaling. The frequency of pre-μDCs is reduced in vitamin A-deficient animals and in animals treated with RAR inhibitors. The results define a novel vitamin A-dependent, RA-regulated developmental sequence for DCs and identify a targeted precursor for CD103⁺ cDCs in the gut.

  View details for DOI 10.1038/mi.2012.123

  View details for PubMedID 23235743

  View details for PubMedCentralID PMC3612556

• Canonical and noncanonical wnt proteins program dendritic cell responses for tolerance. Journal of immunology Oderup, C., Lajevic, M., Butcher, E. C. 2013; 190 (12): 6126-6134

  Abstract

  Ag-presenting dendritic cells (DCs) interpret environmental signals to orchestrate local and systemic immune responses. They govern the balance between tolerance and inflammation at epithelial surfaces, where the immune system must provide robust pathogen responses while maintaining tolerance to commensal flora and food Ags. The Wnt family of secreted proteins, which control epithelial and hematopoietic development and homeostasis, is emerging as an important regulator of inflammation. In this study, we show that canonical and noncanonical Wnts directly stimulate murine DC production of anti-inflammatory cytokines. Wnt3A triggers canonical β-catenin signaling and preferentially induces DC TGF-β and VEGF production, whereas Wnt5A induces IL-10 through alternative pathways. The Wnts also alter DC responses to microbe- or pathogen-associated molecular patterns, inhibiting proinflammatory cytokine induction in response to TLR ligands and promoting DC generation of Foxp3(+) regulatory T cells. Moreover, although both Wnts suppress proinflammatory responses to bacterial endotoxin and to TLR1/2, TLR7, and TLR9 ligands, Wnt5A, but not Wnt3A, inhibits IL-6 production in response to the viral mimic, polyinosinic:polycytidylic acid. Thus, Wnt family members directly and differentially regulate DC functions, an ability that may contribute to the balance between tolerance and inflammation at epithelial sites of exposure to microbes and environmental Ags.

  View details for DOI 10.4049/jimmunol.1203002

  View details for PubMedID 23677472

• Plasmacytoid dendritic cells promote rotavirus-induced human and murine B cell responses. journal of clinical investigation Deal, E. M., Lahl, K., Narváez, C. F., Butcher, E. C., Greenberg, H. B. 2013; 123 (6): 2464-2474

  Abstract

  B cell-dependent immunity to rotavirus, an important intestinal pathogen, plays a significant role in viral clearance and protects against reinfection. Human in vitro and murine in vivo models of rotavirus infection were used to delineate the role of primary plasmacytoid DCs (pDCs) in initiating B cell responses. Human pDCs were necessary and sufficient for B cell activation induced by rotavirus. Type I IFN recognition by B cells was essential for rotavirus-mediated B cell activation in vitro and murine pDCs and IFN-α/β-mediated B cell activation after in vivo intestinal rotavirus infection. Furthermore, rotavirus-specific serum and mucosal antibody responses were defective in mice lacking functional pDCs at the time of infection. These data demonstrate that optimal B cell activation and virus-specific antibody secretion following mucosal infection were a direct result of pDC-derived type I IFN. Importantly, viral shedding significantly increased in pDC-deficient mice, suggesting that pDC-dependent antibody production influences viral clearance. Thus, mucosal pDCs critically influence the course of rotavirus infection through rotavirus recognition and subsequent IFN production and display powerful adjuvant properties to initiate and enhance humoral immunity.

  View details for DOI 10.1172/JCI60945

  View details for PubMedID 23635775

  View details for PubMedCentralID PMC3668833

• Thymus-homing dendritic cells in central tolerance. European journal of immunology Hadeiba, H., Butcher, E. C. 2013; 43 (6): 1425-1429

  Abstract

  Central tolerance is critical in establishing a peripheral T-cell repertoire purged of functional autoreactive T cells. One of the major requirements for effective central tolerance is the presentation of self and other innocuous antigens (Ags), including food, gut flora, or airway allergens, to developing T cells in the thymus. This seemingly challenging task can be mediated in some cases by ectopic expression of tissue-specific Ags by thymic epithelial cells or by entry of systemic blood-borne Ags into the thymus. More recently, thymic homing peripheral dendritic cells (DCs) have been proposed as cellular transporters of peripheral tissue-specific Ags or foreign innocuous Ags. The aim of this viewpoint is to discuss the three principal thymic DC populations and their trafficking properties in the context of central tolerance. We will first discuss the importance of peripheral DC trafficking to the thymus and then compare and contrast the three DC subsets. We will describe how they were characterized, describe their trafficking to and their microenvironmental positioning in the thymus, and discuss the functional consequence of thymic trafficking and localization on thymic selection events.

  View details for DOI 10.1002/eji.201243192

  View details for PubMedID 23616226

• Chemerin Is an Antimicrobial Agent in Human Epidermis PLOS ONE Banas, M., Zabieglo, K., Kasetty, G., Kapinska-Mrowiecka, M., Borowczyk, J., Drukala, J., Murzyn, K., Zabel, B. A., Butcher, E. C., Schroeder, J. M., Schmidtchen, A., Cichy, J. 2013; 8 (3)

  Abstract

  Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val(66)-Pro(85), which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.

  View details for DOI 10.1371/journal.pone.0058709

  View details for Web of Science ID 000317562600033

  View details for PubMedID 23527010

  View details for PubMedCentralID PMC3604073

• Plasmablast frequency and trafficking receptor expression are altered in pediatric ulcerative colitis INFLAMMATORY BOWEL DISEASES Tarlton, N. J., Green, C. M., Lazarus, N. H., Rott, L., Wong, A. P., Abramson, O. N., Bremer, M., Butcher, E. C., Abramson, T. 2012; 18 (12): 2381-2391

  Abstract

  The incidence of pediatric ulcerative colitis (UC), a chronic autoinflammatory disease of the colon, is on the rise. Although an increased infiltration of B cells from the peripheral blood into the colon occurs in UC, B-cell trafficking is understudied. We hypothesized that the frequency of circulating plasmablasts (PBs) and their trafficking receptor (TR) expression may be indicative of the location and degree of pathology in pediatric UC.We conducted multicolor flow cytometry analyses of circulating IgA(+/-) PBs and IgA(+) memory B cells (MBCs) in pediatric UC patients with remission, mild, moderate, and severe state of disease (n = 12), and healthy pediatric (n = 2) and adult donors (n = 11).Compared to healthy donors the average frequency of PBs among total peripheral blood lymphocytes is increased 30-fold during severe UC activity, and positively correlates with Pediatric Ulcerative Colitis Activity Index score, C-reactive protein level, and erythrocyte sedimentation rate. A greater percent of PBs in severe patients express the gut-homing receptors α4β7 and CCR10, and the inflammatory homing molecule P-selectin ligand (P-sel lig). The percent of IgA(+) MBCs expressing α4β7, however, is reduced. Furthermore, expression of the small intestine TR CCR9 is decreased on α4β7(high) PBs, and on α4β7(high) /CCR10(high) PBs and MBCs in these patients, consistent with preferential cell targeting to the colon.Peripheral blood PBs with a colon-homing phenotype (α4β7/CCR10/P-sel lig) are elevated in children with severe UC. Screening this B-cell subset may provide a complementary approach in monitoring disease activity or therapeutic efficacy in pediatric UC.

  View details for DOI 10.1002/ibd.22962

  View details for Web of Science ID 000311242400022

  View details for PubMedID 22488927

  View details for PubMedCentralID PMC3404263

• Plasmacytoid Dendritic Cells and Anti-Inflammatory Intestinal IgA Production Crohn's-and-Colitis-Foundation's-National-Clinical-and-Research Conference on Advances in Inflammatory Bowel Diseases Lahl, K., Deal, E., Greenberg, H., Butcher, E. WILEY-BLACKWELL. 2012: S108–S108

  View details for Web of Science ID 000311172600275

• The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses JOURNAL OF EXPERIMENTAL MEDICINE Pachynski, R. K., Zabel, B. A., Kohrt, H. E., Tejeda, N. M., Monnier, J., Swanson, C. D., Holzer, A. K., Gentles, A. J., Sperinde, G. V., Edalati, A., Hadeiba, H. A., Alizadeh, A. A., Butcher, E. C. 2012; 209 (8): 1427-1435

  Abstract

  Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.

  View details for DOI 10.1084/jem.20112124

  View details for Web of Science ID 000307016500006

  View details for PubMedID 22753924

  View details for PubMedCentralID PMC3409495

• Expression, Regulation, and Function of Atypical Chemerin Receptor CCRL2 on Endothelial Cells JOURNAL OF IMMUNOLOGY Monnier, J., Lewen, S., O'Hara, E., Huang, K., Tu, H., Butcher, E. C., Zabel, B. A. 2012; 189 (2): 956-967

  Abstract

  Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)β(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.

  View details for DOI 10.4049/jimmunol.1102871

  View details for Web of Science ID 000306119800054

  View details for PubMedID 22696441

  View details for PubMedCentralID PMC3428203

• Janus-like opposing roles of CD47 in autoimmune brain inflammation in humans and mice JOURNAL OF EXPERIMENTAL MEDICINE Han, M. H., Lundgren, D. H., Jaiswal, S., Chao, M., Graham, K. L., Garris, C. S., Axtell, R. C., Ho, P. P., Lock, C. B., Woodard, J. I., Brownell, S. E., Zoudilova, M., Hunt, J. F., Baranzini, S. E., Butcher, E. C., Raine, C. S., Sobel, R. A., Han, D. K., Weissman, I., Steinman, L. 2012; 209 (7): 1325-1334

  Abstract

  Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

  View details for DOI 10.1084/jem.20101974

  View details for Web of Science ID 000306174300008

  View details for PubMedID 22734047

  View details for PubMedCentralID PMC3405500

• Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Issa, M. E., Muruganandan, S., Ernst, M. C., Parlee, S. D., Zabel, B. A., Butcher, E. C., Sinal, C. J., Goralski, K. B. 2012; 302 (11): C1621-C1631

  Abstract

  The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.

  View details for DOI 10.1152/ajpcell.00187.2011

  View details for Web of Science ID 000305396500006

  View details for PubMedID 22460713

  View details for PubMedCentralID PMC3378017

• The chemoattractant chemerin as a natural tumor suppressive cytokine. 48th Annual Meeting of the American-Society-of-Clinical-Oncology (ASCO) Pachynski, R. K., Zabel, B., Tejeda, N., Monnier, J., Holzer, A. K., Gentles, A., Kohrt, H. E., Hadeiba, H., Alizadeh, A. A., Butcher, E. AMER SOC CLINICAL ONCOLOGY. 2012

  View details for Web of Science ID 000318009803142

• Plasmacytoid Dendritic Cells Transport Peripheral Antigens to the Thymus to Promote Central Tolerance IMMUNITY Hadeiba, H., Lahl, K., Edalati, A., Oderup, C., Habtezion, A., Pachynski, R., Nguyen, L., Ghodsi, A., Adler, S., Butcher, E. C. 2012; 36 (3): 438-450

  Abstract

  Central tolerance can be mediated by peripheral dendritic cells (DCs) that transport innocuous antigens (Ags) to the thymus for presentation to developing T cells, but the responsible DC subsets remained poorly defined. Immature plasmacytoid DCs (pDCs) express CCR9, a chemokine receptor involved in migration of T cell precursors to the thymus. We show here that CCR9 mediated efficient thymic entry of endogenous or i.v. transfused pDCs. pDCs activated by Toll-like receptor (TLR) ligands downregulated CCR9 and lost their ability to home to the thymus. Moreover, endogenous pDCs took up subcutaneously injected fluorescent Ag and, in the absence of TLR signals, transported Ag to the thymus in a CCR9-dependent fashion. Injected, Ag-loaded pDCs effectively deleted Ag-specific thymocytes, and this thymic clonal deletion required CCR9-mediated homing and was prevented by infectious signals. Thus, peripheral pDCs can contribute to immune tolerance through CCR9-dependent transport of peripheral Ags and subsequent deletion of Ag-reactive thymocytes.

  View details for DOI 10.1016/j.immuni.2012.01.017

  View details for Web of Science ID 000302048400015

  View details for PubMedID 22444632

  View details for PubMedCentralID PMC3315699

• Disruption of the Chemokine-Like Receptor-1 (CMKLR1) Gene Is Associated with Reduced Adiposity and Glucose Intolerance ENDOCRINOLOGY Ernst, M. C., Haidl, I. D., Zuniga, L. A., Dranse, H. J., Rourke, J. L., Zabel, B. A., Butcher, E. C., Sinal, C. J. 2012; 153 (2): 672-682

  Abstract

  Adipose tissue secretes a variety of bioactive signaling molecules, termed adipokines, which regulate numerous biological functions including appetite, energy balance, glucose homeostasis, and inflammation. Chemerin is a novel adipokine that regulates adipocyte differentiation and metabolism by binding to and activating the G protein-coupled receptor, chemokine like receptor-1 (CMKLR1). In the present study, we investigated the impact of CMKLR1 deficiency on adipose development, glucose homeostasis, and inflammation in vivo. Herein we report that regardless of diet (low or high fat), CMKLR1(-/-) mice had lower food consumption, total body mass, and percent body fat compared with wild-type controls. CMKLR1(-/-) mice also exhibited decreased hepatic and white adipose tissue TNFα and IL-6 mRNA levels coincident with decreased hepatic dendritic cell infiltration, decreased adipose CD3+ T cells, and increased adipose natural killer cells. CMKLR1(-/-) mice were glucose intolerant compared with wild-type mice, and this was associated with decreased glucose stimulated insulin secretion as well as decreased skeletal muscle and white adipose tissue glucose uptake. Collectively these data provide compelling evidence that CMKLR1 influences adipose tissue development, inflammation, and glucose homeostasis and may contribute to the metabolic derangement characteristic of obesity and obesity-related diseases.

  View details for DOI 10.1210/en.2011-1490

  View details for Web of Science ID 000299928200016

  View details for PubMedID 22186410

  View details for PubMedCentralID PMC3275396

• A Disintegrin and Metalloproteinase 10 Regulates Antibody Production and Maintenance of Lymphoid Architecture JOURNAL OF IMMUNOLOGY Chaimowitz, N. S., Martin, R. K., Cichy, J., Gibb, D. R., Patil, P., Kang, D., Farnsworth, J., Butcher, E. C., McCright, B., Conrad, D. H. 2011; 187 (10): 5114-5122

  Abstract

  A disintegrin and metalloproteinase 10 (ADAM10) is a zinc-dependent proteinase related to matrix metalloproteinases. ADAM10 has emerged as a key regulator of cellular processes by cleaving and shedding extracellular domains of multiple transmembrane receptors and ligands. We have developed B cell-specific ADAM10-deficient mice (ADAM10(B-/-)). In this study, we show that ADAM10 levels are significantly enhanced on germinal center B cells. Moreover, ADAM10(B-/-) mice had severely diminished primary and secondary responses after T-dependent immunization. ADAM10(B-/-) displayed impaired germinal center formation, had fewer follicular Th cells, decreased follicular dendritic cell networks, and altered chemokine expression in draining lymph nodes (LNs). Interestingly, when spleen and LN structures from immunized mice were analyzed for B and T cell localization, tissues structure was aberrant in ADAM10(B-/-) mice. Importantly, when ADAM10-deficient B cells were stimulated in vitro, they produced comparable Ab as wild type B cells. This result demonstrates that the defects in humoral responses in vivo result from inadequate B cell activation, likely because of the decrease in follicular Th cells and the changes in structure. Thus, ADAM10 is essential for the maintenance of lymphoid structure after Ag challenge.

  View details for DOI 10.4049/jimmunol.1102172

  View details for Web of Science ID 000296767300020

  View details for PubMedID 21998451

  View details for PubMedCentralID PMC4006936

• Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine Cathepsins JOURNAL OF IMMUNOLOGY Kulig, P., Kantyka, T., Zabel, B. A., Banas, M., Chyra, A., Stefanska, A., Tu, H., Allen, S. J., Handel, T. M., Kozik, A., Potempa, J., Butcher, E. C., Cichy, J. 2011; 187 (3): 1403-1410

  Abstract

  Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

  View details for DOI 10.4049/jimmunol.1002352

  View details for Web of Science ID 000292874200041

  View details for PubMedID 21715684

  View details for PubMedCentralID PMC3140563

• Panhematin provides a therapeutic benefit in experimental pancreatitis GUT Habtezion, A., Kwan, R., Akhtar, E., Wanaski, S. P., Collins, S. D., Wong, R. J., Stevenson, D. K., Butcher, E. C., Omary, M. B. 2011; 60 (5): 671-679

  Abstract

  Acute pancreatitis (AP) can result in pancreatic necrosis and inflammation, with subsequent multi-organ failure. AP is associated with increased neutrophil recruitment and a rise in pro-inflammatory cytokines such as TNFα. Pretreatment with haemin, results in recruitment of haem-oxygenase-1 (HO-1)(+) macrophages and protects against experimental pancreatitis. It is not clear whether modulation of HO-1 after onset of disease has a protective role. In this study, we tested the utility of Panhematin, a water-soluble haemin formulation, in activating and inducing pancreatic HO-1, and as a therapeutic agent in treating mouse acute pancreatitis.We defined the distribution of radiolabelled haemin, then used in vivo HO-1-luciferase bioluminescence imaging and the CO-release assay to test Panhematin-induced upregulation of HO-1 transcription and activity, respectively. Using two well-defined AP murine models, we tested the therapeutic benefit of Panhematin, and quantified cytokine release using a luminex assay.Intravenously administered Panhematin induces rapid recruitment of HO-1(+) cells to the pancreas within 2 h and de novo splenic HO-1 transcription by 12 h. Despite high baseline spleen HO-1 activity, the pancreas is particularly responsive to Panhematin-mediated HO-1 induction. Panhematin-treated mice, at various time points after AP induction had significant reduction in mortality, pancreatic injury, together with upregulation of HO-1 and downregulation of pro-inflammatory cytokines and CXCL1, a potent neutrophil chemoattractant.Despite AP-associated mortality and morbidity, no effective treatment other than supportive care exists. We demonstrate that Panhematin leads to: (i) rapid induction and activation of pancreatic HO-1 with recruitment of HO-1(+) cells to the pancreas, (ii) amelioration of AP even when given late during the course of disease, and (iii) a decrease in leucocyte infiltration and pro-inflammatory cytokines including CXCL1. The utility of Panhematin at modest doses as a therapeutic in experimental pancreatitis, coupled with its current use and safety in humans, raises the potential of its applicability to human pancreatitis.

  View details for DOI 10.1136/gut.2010.217208

  View details for Web of Science ID 000289076700015

  View details for PubMedID 21159893

  View details for PubMedCentralID PMC3580958

• Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Habtezion, A., Toivola, D. M., Asghar, M. N., Kronmal, G. S., Brooks, J. D., Butcher, E. C., Omary, M. B. 2011; 108 (4): 1445-1450

  Abstract

  Keratin 8 (K8) is a major intermediate filament protein present in enterocytes and serves an antiapoptotic function in hepatocytes. K8-null mice develop colonic hyperplasia and colitis that are reversed after antibiotic treatment. To investigate the pathways that underlie the mechanism of colonocyte hyperplasia and the normalization of the colonic phenotype in response to antibiotics, we performed genome-wide microarray analysis. Functional annotation of genes that are differentially regulated in K8(-/-) and K8(+/+) isolated colon crypts (colonocytes) identified apoptosis as a major altered pathway. Exposure of K8(-/-) colonocytes or colon organ ("organoid") cultures, but not K8(-/-) small intestine organoid cultures, to apoptotic stimuli showed, surprisingly, that they are resistant to apoptosis compared with their wild-type counterparts. This resistance is not related to inflammation per se because T-cell receptor α-null (TCR-α(-/-)) and wild-type colon cultures respond similarly upon induction of apoptosis. Following antibiotic treatment, K8(-/-) colonocytes and organ cultures become less resistant to apoptosis and respond similarly to the wild-type colonocytes. Antibiotics also normalize most differentially up-regulated genes, including survivin and β4-integrin. Treatment of K8(-/-) mice with anti-β4-integrin antibody up-regulated survivin, and induced phosphorylation of focal adhesion kinase with decreased activation of caspases. Therefore, unlike the proapoptotic effect of K8 mutation or absence in hepatocytes, lack of K8 confers resistance to colonocyte apoptosis in a microflora-dependent manner.

  View details for DOI 10.1073/pnas.1010833108

  View details for Web of Science ID 000286594800046

  View details for PubMedID 21220329

  View details for PubMedCentralID PMC3029736

• Heme oxygenase-1 is induced in peripheral blood mononuclear cells of patients with acute pancreatitis: a potential therapeutic target AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY Habtezion, A., Kwan, R., Yang, A. L., Morgan, M. E., Akhtar, E., Wanaski, S. P., Collins, S. D., Butcher, E. C., Kamal, A., Omary, M. B. 2011; 300 (1): G12-G20

  Abstract

  Heme oxygenase-1 (HO-1) induction by hemin or Panhematin protects against experimental pancreatitis. As a preclinical first step toward determining whether HO-1 upregulation is a viable target in acute pancreatitis (AP) patients, we tested the hypothesis that HO-1 expression in peripheral blood mononuclear cell (PBMC) subsets of hospitalized patients with mild AP is upregulated then normalizes upon recovery and that cells from AP patients have the potential to upregulate their HO-1 ex vivo if exposed to Panhematin. PBMCs were isolated on days 1 and 3 of hospitalization from the blood of 18 AP patients, and PMBC HO-1 levels were compared with PMBCs of 15 hospitalized controls (HC) and 7 volunteer healthy controls (VC). On day 1 of hospitalization, AP patients compared with VCs had higher HO-1 expression in monocytes and neutrophils. Notably, AP monocyte HO-1 levels decreased significantly upon recovery. Panhematin induced HO-1 in ex vivo cultured AP PBMCs more readily than in HC or VC PBMCs. Furthermore, PBMCs from acutely ill AP patients on day 1 were more responsive to HO-1 induction compared with day 3 upon recovery. Similarly, mouse splenocytes had enhanced HO-1 inducibility as their pancreatitis progressed from mild to severe. In conclusion, AP leads to reversible PBMC HO-1 upregulation that is associated with clinical improvement and involves primarily monocytes. Leukocytes from AP patients or mice with AP are primed for HO-1 induction by Panhematin, which suggests that Panhematin could offer a therapeutic benefit.

  View details for DOI 10.1152/ajpgi.00231.2010

  View details for Web of Science ID 000285744300002

  View details for PubMedID 20966033

  View details for PubMedCentralID PMC3025514

• Synovial Tissue-Infiltrating Natural Killer Cells in Osteoarthritis and Periprosthetic Inflammation ARTHRITIS AND RHEUMATISM Huss, R. S., Huddleston, J. I., Goodman, S. B., Butcher, E. C., Zabel, B. A. 2010; 62 (12): 3799-3805

  Abstract

  Infiltrating immune cells play a central role in degenerative joint disease associated with osteoarthritis (OA) and particle-mediated periprosthetic osteolysis. The goal of this study was to characterize a newly identified population of synovial tissue-infiltrating natural killer (NK) cells obtained from patients with OA or patients with periprosthetic joint inflammation.Synovial and interfacial tissue samples were collected from patients with OA who were undergoing primary or revision total joint replacement (TJR) surgery. The histologic features of OA synovium obtained from patients undergoing primary surgery and interfacial tissue obtained from patients undergoing revision surgery were determined by immunohistochemistry and immunofluorescence. Synovial tissue-infiltrating NK cells were evaluated for the expression of surface receptors, using flow cytometry. Chemoattractant and cytokine protein and RNA levels in synovial and interfacial tissue and fluid were assessed by Luminex assay and real-time quantitative polymerase chain reaction. Cytokine production and degranulation by stimulated synovial tissue versus normal blood NK cells were evaluated by intracellular cytokine staining.NK cells comprised nearly 30% of the CD45+ mononuclear cell infiltrate in synovial tissue obtained from patients undergoing primary TJR and from patients undergoing revision TJR. NK cells from both groups expressed CXCR3, CCR5, L-selectin, α4 integrins, and cutaneous lymphocyte antigen. Synovial fluid from patients undergoing revision surgery contained elevated concentrations of the NK cell attractants CCL4, CCL5, CXCL9, and CXCL10; all levels in synovial fluid obtained from patients undergoing revision surgery were higher than those in synovial fluid from patients undergoing primary surgery. Cytokine-stimulated interferon-γ production was significantly impaired in NK cells derived from primary and revision TJRs compared with blood NK cells.NK cells are a principal tissue-infiltrating lymphocyte subset in patients with OA and patients with periprosthetic inflammation and display a quiescent phenotype that is consistent with postactivation exhaustion.

  View details for DOI 10.1002/art.27751

  View details for PubMedID 20848566

• IL-17 Regulates Adipogenesis, Glucose Homeostasis, and Obesity JOURNAL OF IMMUNOLOGY Zuniga, L. A., Shen, W., Joyce-Shaikh, B., Pyatnova, E. A., Richards, A. G., Thom, C., Andrade, S. M., Cua, D. J., Kraemer, F. B., Butcher, E. C. 2010; 185 (11): 6947-6959

  Abstract

  Inflammatory mediators have the potential to impact a surprising range of diseases, including obesity and its associated metabolic syndrome. In this paper, we show that the proinflammatory cytokine IL-17 inhibits adipogenesis, moderates adipose tissue (AT) accumulation, and regulates glucose metabolism in mice. IL-17 deficiency enhances diet-induced obesity in mice and accelerates AT accumulation even in mice fed a low-fat diet. In addition to potential systemic effects, IL-17 is expressed locally in AT by leukocytes, predominantly by γδ T cells. IL-17 suppresses adipocyte differentiation from mouse-derived 3T3-L1 preadipocytes in vitro, and inhibits expression of genes encoding proadipogenic transcription factors, adipokines, and molecules involved in lipid and glucose metabolism. IL-17 also acts on differentiated adipocytes, impairing glucose uptake, and young IL-17-deficient mice show enhanced glucose tolerance and insulin sensitivity. Our findings implicate IL-17 as a negative regulator of adipogenesis and glucose metabolism in mice, and show that it delays the development of obesity.

  View details for DOI 10.4049/jimmunol.1001269

  View details for Web of Science ID 000284311500062

  View details for PubMedID 21037091

  View details for PubMedCentralID PMC3001125

• Chemoattractant Receptors and Lymphocyte Egress from Extralymphoid Tissue: Changing Requirements during the Course of Inflammation JOURNAL OF IMMUNOLOGY Brown, M. N., Fintushel, S. R., Lee, M. H., Jennrich, S., Geherin, S. A., Hay, J. B., Butcher, E. C., Debes, G. F. 2010; 185 (8): 4873-4882

  Abstract

  Memory/effector T cells traffic efficiently through extralymphoid tissues, entering from the blood and leaving via the afferent lymph. During inflammation, T cell traffic into the affected tissue dramatically increases; however, the dynamics and mechanisms of T cell exit from inflamed tissues are poorly characterized. In this study, we show, using both a mouse and a sheep model, that large numbers of lymphocytes leave the chronically inflamed skin. Many T cells capable of producing IFN-γ and IL-17 also entered the draining afferent lymph, demonstrating that memory/effector T cells egress from sites of inflammation. Whereas efficient egress from acutely inflamed skin required lymphocyte-expressed CCR7, chronic inflammation promoted significant CCR7-independent exit as well. Lymphocyte exit at late time points of inflammation was sensitive to pertussis toxin but was only partially affected by the drug FTY720, implying the contribution of alternative chemoattractant receptors other than spingosine 1-phosphate receptor 1. Our data show that CCR7 is an important receptor for lymphocyte egress from both resting and inflamed extralymphoid tissues, but that alternative exit receptors come into play during chronic inflammation.

  View details for DOI 10.4049/jimmunol.1000676

  View details for Web of Science ID 000282525900041

  View details for PubMedID 20833836

  View details for PubMedCentralID PMC3327166

• Regulation of the Atypical Chemerin Receptor, CCRL2, on Activated Brain Endothelial Cells 10th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Monnier, J., Zabel, B., Butcher, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S63–S63

  View details for DOI 10.1016/j.clim.2010.03.193

  View details for Web of Science ID 000277953700177

• Chemokine-Like Receptor-1 Expression by Central Nervous System-Infiltrating Leukocytes and Involvement in a Model of Autoimmune Demyelinating Disease JOURNAL OF IMMUNOLOGY Graham, K. L., Zabel, B. A., Loghavi, S., Zuniga, L. A., Ho, P. P., Sobel, R. A., Butcher, E. C. 2009; 183 (10): 6717-6723

  Abstract

  We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35-55 (MOG(35-55)), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG(35-55)-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

  View details for DOI 10.4049/jimmunol.0803435

  View details for Web of Science ID 000271765700076

  View details for PubMedID 19864606

  View details for PubMedCentralID PMC2904075

• Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Skrzeczynska-Moncznik, J., Wawro, K., Stefanska, A., Oleszycka, E., Kulig, P., Zabel, B. A., Sulkowski, M., Kapinska-Mrowiecka, M., Czubak-Macugowska, M., BUTCHER, E. C., Cichy, J. 2009; 380 (2): 323-327

  Abstract

  Interferon alpha-producing plasmacytoid dendritic cells (pDC) are crucial contributors to pro-inflammatory or tolerogenic immune responses and are important in autoimmune diseases such as psoriasis. pDC accumulate in the lesional skin of psoriasis patients, but are rarely found in the affected skin of patients with atopic dermatitis (AD). While homeostatic chemokine CXCL12 and inducible pro-inflammatory CXCR3 chemokine ligands may regulate pDC influx to psoriatic skin, the mechanism responsible for selective pDC recruitment in psoriasis vs. AD remains unknown. Circulating pDC from normal donors express a limited number of chemoattractant receptors, including CXCR3 and CMKLR1 (chemokine-like receptor 1). In this work, we demonstrate that circulating pDC from normal donors as well as psoriasis and AD patients express similar levels of CXCR3 and responded similarly in functional migration assays to CXCL10. We next found that blood pDC from normal, AD, and psoriasis patients express functional CMKLR1. In contrast to normal skin, however, lesional skin from psoriasis patients contains the active form of the CMKLR1 ligand chemerin. Furthermore, in affected skin from psoriatic patients the level of active chemerin was generally higher than in AD skin. Taken together, these results indicate that local generation of active chemerin may contribute to pDC recruitment to psoriatic skin.

  View details for DOI 10.1016/j.bbrc.2009.01.071

  View details for Web of Science ID 000263742200022

  View details for PubMedID 19168032

• Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets JOURNAL OF BIOLOGICAL CHEMISTRY Du, X., Zabel, B. A., Myles, T., Allen, S. J., Handel, T. M., Lee, P. P., Butcher, E. C., Leung, L. L. 2009; 284 (2): 751-758

  Abstract

  Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH(2)-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH(2)-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

  View details for DOI 10.1074/jbc.M805000200

  View details for Web of Science ID 000262122900008

  View details for PubMedID 19010784

  View details for PubMedCentralID PMC2613638

• Chemerin and the recruitment of NK cells to diseased skin 36th Winter School on Molecule Interactions in Health and Disease Skrzeczynska-Moncznik, J., Stefanska, A., Zabel, B. A., Kapinska-Mrowiecka, M., Butcher, E. C., Cichy, J. ACTA BIOCHIMICA POLONICA. 2009: 355–60

  Abstract

  Natural killer (NK) cells play a major role in the initial control of many viral pathogens and in the rejection of tumors. Consistent with their roles as immune sentinels, NK cells are found in inflamed skin, including lichen planus, psoriasis and atopic dermatitis (AD) lesions. In oral lichen planus lesions, the recruitment as well as intradermal colocalization of NK cells and pDC (plasmacytoid dendritic cells) appear to be mediated by chemerin, a recently identified protein ligand for chemokine-like receptor 1 (CMKLR1), a chemoattractant receptor expressed by both cell types. Dendritic cells can regulate NK cell activity, and NK cells can regulate DC-mediated responses. Since chemerin was recently implicated in recruitment of pDC to psoriatic skin, in this work we determined whether chemerin facilitates interactions between NK and pDC in psoriatic plaques through controlling influx of NK cells to diseased skin. We demonstrate that circulating NK cells from normal donors as well as psoriasis and AD patients respond similarly in functional migration assays to chemerin. However, differences in the distribution of NK cells and pDC in skin lesions suggest that recruitment of both NK cells and pDC is unlikely to be controlled solely by chemerin.

  View details for Web of Science ID 000267607200018

  View details for PubMedID 19543554

• Modeling the Role of Homologous Receptor Desensitization in Cell Gradient Sensing JOURNAL OF IMMUNOLOGY Lin, F., Butcher, E. C. 2008; 181 (12): 8335-8343

  Abstract

  G-protein-coupled chemoattractant receptors signal transiently upon ligand binding to effect cell orientation and motility but then are rapidly desensitized. The importance of desensitization has been unclear, because mutated nondesensitizable receptors mediate efficient chemotaxis. We hypothesized that homologous receptor desensitization is required for cellular navigation in fields of competing attractants. Modeling of receptor-mediated orientation shows that desensitization allows integration of attractant signals. Cells expressing normal receptors are predicted to 1) orient preferentially to distant gradients; 2) seek an intermediate position between balanced agonist sources; 3) and can be repositioned between chemoattractant-defined microenvironmental domains by modest changes in receptor number. In contrast, in the absence of desensitization, orientation is dominated by local agonist sources, precluding continued navigation. Furthermore, cell orientation in competing ligand gradients depends on the relative kinetic rates of receptor desensitization and recycling. We propose that homologous receptor desensitization is critical for cellular navigation in complex chemoattractant fields.

  View details for Web of Science ID 000261583000020

  View details for PubMedID 19050250

• A role for leukocyte-endothelial adhesion mechanisms in epilepsy NATURE MEDICINE Fabene, P. F., Mora, G. N., Martinello, M., Rossi, B., Merigo, F., Ottoboni, L., Bach, S., Angiari, S., Benati, D., Chakir, A., Zanetti, L., Schio, F., Osculati, A., Marzola, P., Nicolato, E., Homeister, J. W., Xia, L., Lowe, J. B., McEver, R. P., Osculati, F., Sbarbati, A., Butcher, E. C., Constantin, G. 2008; 14 (12): 1377-1383

  Abstract

  The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately one percent of the world population, are not well understood. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1, encoded by Selplg) and leukocyte integrins alpha(4)beta(1) and alpha(L)beta(2). Inhibition of leukocyte-vascular interactions, either with blocking antibodies or by genetically interfering with PSGL-1 function in mice, markedly reduced seizures. Treatment with blocking antibodies after acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with the potential leukocyte involvement in epilepsy in humans, leukocytes were more abundant in brains of individuals with epilepsy than in controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.

  View details for DOI 10.1038/nm.1878

  View details for Web of Science ID 000261393600024

  View details for PubMedID 19029985

  View details for PubMedCentralID PMC2710311

• CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease NATURE IMMUNOLOGY Hadeiba, H., Sato, T., Habtezion, A., Oderup, C., Pan, J., Butcher, E. C. 2008; 9 (11): 1253-1260

  Abstract

  Dendritic cells (DCs) are 'professional' antigen-presenting cells that are key in the regulation of immune responses. Here we characterize a unique subset of tolerogenic DCs that expressed the chemokine receptor CCR9 and migrated to the CCR9 ligand CCL25, a chemokine linked to the homing of T cells and DCs to the gut. CCR9(+) DCs were of the plasmacytoid DC (pDC) lineage, had an immature phenotype and rapidly downregulated CCR9 in response to maturation-inducing pDC-restricted Toll-like receptor ligands. CCR9(+) pDCs were potent inducers of regulatory T cell function and suppressed antigen-specific immune responses both in vitro and in vivo, including inhibiting acute graft-versus-host disease induced by allogeneic CD4(+) donor T cells in irradiated recipients. Our results identify a highly immunosuppressive population of pDCs present in lymphoid tissues.

  View details for DOI 10.1038/ni.1658

  View details for Web of Science ID 000260248600012

  View details for PubMedID 18836452

  View details for PubMedCentralID PMC2901237

• An Indispensable Role for the Chemokine Receptor CCR10 in IgA Antibody-Secreting Cell Accumulation JOURNAL OF IMMUNOLOGY Morteau, O., Gerard, C., Lu, B., Ghiran, S., Rits, M., Fujiwara, Y., Law, Y., Distelhorst, K., Nielsen, E. M., Hill, E. A., Kwan, R., Lazarus, N. H., Butcher, E. C., Wilson, E. 2008; 181 (9): 6309-6315

  Abstract

  The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.

  View details for Web of Science ID 000260659000057

  View details for PubMedID 18941222

  View details for PubMedCentralID PMC2748758

• Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis JOURNAL OF EXPERIMENTAL MEDICINE Zabel, B. A., Nakae, S., Zuniga, L., Kim, J., Ohyama, T., Alt, C., Pan, J., Suto, H., Soler, D., Allen, S. J., Handel, T. M., Song, C. H., Galli, S. J., Butcher, E. C. 2008; 205 (10): 2207-2220

  Abstract

  Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell-expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell-dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other "silent" or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

  View details for DOI 10.1084/jem.20080300

  View details for Web of Science ID 000259656700005

  View details for PubMedID 18794339

  View details for PubMedCentralID PMC2556791

• Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking NATURE IMMUNOLOGY Sigmundsdottir, H., Butcher, E. C. 2008; 9 (9): 981-987

  Abstract

  Lymphocytes are imprinted during activation with trafficking programs (combinations of adhesion and chemoattractant receptors) that target their migration to specific tissues and microenvironments. Cytokines contribute, but, for gut and skin, evolution has cleverly adapted external cues from food (vitamin A) and sunlight (ultraviolet-induced vitamin D3) to imprint lymphocyte homing to the small intestines and T cell migration into the epidermis. Dendritic cells are essential: they process the vitamins to their active metabolites (retinoic acid and 1,25(OH)(2)D3) for presentation with antigen to lymphocytes, and they help export environmental cues through lymphatics to draining lymph nodes, to program the trafficking and effector functions of naive T and B cells.

  View details for DOI 10.1038/ni.f.208

  View details for Web of Science ID 000258559600006

  View details for PubMedID 18711435

  View details for PubMedCentralID PMC3171274

• Lymphocyte electrotaxis in vitro and in vivo JOURNAL OF IMMUNOLOGY Lin, F., Baldessari, F., Gyenge, C. C., Sato, T., Chambers, R. D., Santiago, J. G., Butcher, E. C. 2008; 181 (4): 2465-2471

  Abstract

  Electric fields are generated in vivo in a variety of physiologic and pathologic settings, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration (i.e., electrotaxis) of epithelial cells, endothelial cells, fibroblasts, and neutrophils suggesting a potential role in cell positioning during wound healing. In the present study, we investigated the ability of lymphocytes to respond to applied direct current (DC) electric fields. Using a modified Transwell assay and a simple microfluidic device, we show that human PBLs migrate toward the cathode in physiologically relevant DC electric fields. Additionally, electrical stimulation activates intracellular kinase signaling pathways shared with chemotactic stimuli. Finally, video microscopic tracing of GFP-tagged immunocytes in the skin of mouse ears reveals that motile cutaneous T cells actively migrate toward the cathode of an applied DC electric field. Lymphocyte positioning within tissues can thus be manipulated by externally applied electric fields, and may be influenced by endogenous electrical potential gradients as well.

  View details for Web of Science ID 000258345300026

  View details for PubMedID 18684937

• Critical role for the chemokine receptor CXCR6 in homeostasis and activation of CD1d-restricted NKT cells JOURNAL OF IMMUNOLOGY Germanov, E., Veinotte, L., Cullen, R., Chamberlain, E., Butcher, E. C., Johnston, B. 2008; 181 (1): 81-91

  Abstract

  NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6+/- and CXCR6-/- mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6-/- animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1+, but not immature NK1.1- NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1- cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1+ NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6-/- mice following in vivo stimulation with alpha-galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN-gamma production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-gamma, were reduced in CXCR6-/- liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis.

  View details for Web of Science ID 000257404900012

  View details for PubMedID 18566372

• Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs BLOOD Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Baker, E. M., Landan, G., Herman, E. I., Butcher, E. C., Contag, C. H., Negrin, R. S. 2008; 111 (5): 2919-2928

  Abstract

  In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.

  View details for DOI 10.1182/blood-2007-09-112789

  View details for PubMedID 17989315

• NMR assignment of human chemerin, a novel chemoattractant BIOMOLECULAR NMR ASSIGNMENTS Allen, S. J., Zabel, B. A., Kirkpatrick, J., Butcher, E. C., Nietlispach, D., Handel, T. M. 2007; 1 (2): 171-173

  Abstract

  Chemerin is a potent chemoattractant for cells expressing the GPCR CMKLR1, and is thought to play important roles in cell migration and recruitment to sites of tissue damage and inflammation. Here we report the NMR assignments of the 15.6 kDa active form of uniformly (15)N, (13)C labeled chemerin.

  View details for DOI 10.1007/s12104-007-9047-7

  View details for Web of Science ID 000258721700006

  View details for PubMedID 19636857

• Regulation of chemerin bioactivity by plasma carboxypeptidase N, carboxypeptidase B (TAFIa) and platelets 49th Annual Meeting of the American-Society-of-Hematology Du, X., Zabel, B., Allen, S. J., Handel, T. M., Lee, P. P., Butcher, E. C., Leung, L. L. AMER SOC HEMATOLOGY. 2007: 126A–127A

  View details for Web of Science ID 000251100800409

• INKT cells require CCR4 to localize to the airways and to induce airway hyperreactivity JOURNAL OF IMMUNOLOGY Meyer, E. H., Wurbel, M., Staton, T. L., Pichavant, M., Kan, M. J., Savage, P. B., DeKruyff, R. H., Butcher, E. C., Campbell, J. J., Umetsu, D. T. 2007; 179 (7): 4661-4671

  Abstract

  iNKT cells are required for the induction of airway hyperreactivity (AHR), a cardinal feature of asthma, but how iNKT cells traffic to the lungs to induce AHR has not been previously studied. Using several models of asthma, we demonstrated that iNKT cells required the chemokine receptor CCR4 for pulmonary localization and for the induction of AHR. In both allergen-induced and glycolipid-induced models of AHR, wild-type but not CCR4-/- mice developed AHR. Furthermore, adoptive transfer of wild-type but not CCR4-/- iNKT cells reconstituted AHR in iNKT cell-deficient mice. Moreover, we specifically tracked CCR4-/- vs wild-type iNKT cells in CCR4-/-:wild-type mixed BM chimeric mice in the resting state, and when AHR was induced by protein allergen or glycolipid. Using this unique model, we showed that both iNKT cells and conventional T cells required CCR4 for competitive localization into the bronchoalveolar lavage/airways compartment. These results establish for the first time that the pulmonary localization of iNKT cells critical for the induction of AHR requires CCR4 expression by iNKT cells.

  View details for Web of Science ID 000249752100043

  View details for PubMedID 17878364

  View details for PubMedCentralID PMC2564604

• Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism JOURNAL OF BIOLOGICAL CHEMISTRY Goralski, K. B., McCarthy, T. C., Hanniman, E. A., Zabel, B. A., Butcher, E. C., Parlee, S. D., Muruganandan, S., Sinal, C. J. 2007; 282 (38): 28175-28188

  Abstract

  Obesity is an alarming primary health problem and is an independent risk factor for type II diabetes, cardiovascular diseases, and hypertension. Although the pathologic mechanisms linking obesity with these co-morbidities are most likely multifactorial, increasing evidence indicates that altered secretion of adipose-derived signaling molecules (adipokines; e.g. adiponectin, leptin, and tumor necrosis factor alpha) and local inflammatory responses are contributing factors. Chemerin (RARRES2 or TIG2) is a recently discovered chemoattractant protein that serves as a ligand for the G protein-coupled receptor CMKLR1 (ChemR23 or DEZ) and has a role in adaptive and innate immunity. Here we show an unexpected, high level expression of chemerin and its cognate receptor CMKLR1 in mouse and human adipocytes. Cultured 3T3-L1 adipocytes secrete chemerin protein, which triggers CMKLR1 signaling in adipocytes and other cell types and stimulates chemotaxis of CMKLR1-expressing cells. Adenoviral small hairpin RNA targeted knockdown of chemerin or CMKLR1 expression impairs differentiation of 3T3-L1 cells into adipocytes, reduces the expression of adipocyte genes involved in glucose and lipid homeostasis, and alters metabolic functions in mature adipocytes. We conclude that chemerin is a novel adipose-derived signaling molecule that regulates adipogenesis and adipocyte metabolism.

  View details for DOI 10.1074/jbc.M700793200

  View details for Web of Science ID 000249455600067

  View details for PubMedID 17635925

• Lack of lymphoid chemokines CCL19 and CCL21 enhances allergic airway inflammation in mice INTERNATIONAL IMMUNOLOGY Xu, B., Aoyama, K., Kusumoto, M., Matsuzawa, A., Butcher, E. C., Michie, S. A., Matsuyama, T., Takeuchi, T. 2007; 19 (6): 775-784

  Abstract

  Lymphoid chemokines CCL19 and CCL21 are crucial for the recruitment of circulating naive T cells into lymph nodes. However, it is not completely known how they contribute to the development of allergic diseases. To determine whether the lack of CCL19 and CCL21 affects allergic airway inflammation, CCL19- and CCL21-deficient [paucity of lymph node T cells (plt/plt)] and wild-type (WT) mice were immunized intra-peritoneally and then challenged intra-nasally with chicken ovalbumin (OVA). Plt/plt mice developed more severe allergic airway inflammation characterized by increased eosinophils and lymphocytes in bronchoalveolar lavage (BAL) and profound inflammation in peribronchiolar and perivascular regions than did WT mice. CD4+ alpha4 integrin+ and CD4+ beta7 integrin+ T cells were significantly increased in the BAL of OVA-immunized and OVA-challenged (OVA/OVA) plt/plt mice compared with OVA/OVA WT mice. Moreover, there were higher levels of IL-4 and IL-13 mRNAs and lower levels of IL-2 and IFN-gamma mRNAs in inflamed lungs of OVA/OVA plt/plt mice compared with OVA/OVA WT mice. Plt/plt mice produced higher levels of total and OVA-specific IgE antibody. Thus, our results suggest that lack of lymphoid chemokines CCL19 and CCL21 enhances allergic airway inflammation by modulating the recruitment of CD4+ T cells into the lung, the balance between Th1 and Th2 cytokines and the IgE production.

  View details for DOI 10.1093/intimm/dxm046

  View details for Web of Science ID 000248177800010

  View details for PubMedID 17513879

• Selectin, platelet plays a critical role in granulocyte access to the pregnant mouse uterus under physiological and pathological conditions BIOLOGY OF REPRODUCTION Fernekorn, U., Butcher, E. C., Behrends, J., Karsten, C. M., Roebke, A., Schulze, T. J., Kirchner, H., Kruse, A. 2007; 76 (4): 645-653

  Abstract

  Leukocyte recruitment to the pregnant mouse uterus is associated with highly regulated patterns of expression of vascular adhesion receptors. One striking observation is the localized expression of mucosal vascular addressin cell adhesion molecule (MADCAM1) and selectin, platelet (SELP, formerly P-selectin) by maternal vessels in the vascular zone (VZ) during the first half of pregnancy. From midgestation onwards, endothelial cells lining the maternal vessels of the VZ in addition express vascular cell adhesion molecule-1 (VCAM1). The predominant cell population within these vessels is monocyte-like cells. Granulocytes and low numbers of lymphocytes are also present. Murine fetal trophoblast cells are almost devoid of adhesion molecules, including SELP. In contrast, spontaneous abortions of allogeneic pregnancies are characterized by dramatic upregulation of SELP on maternal VZ vessels and on fetal trophoblast cells. Upregulation of SELP is associated with a dramatic influx of highly activated granulocytes, which infiltrate the vessels and tissue of the VZ and the trophoblast. The majority of the activated granulocytes within the trophoblast undergo nuclear fragmentation, which can be detected by TUNEL staining. To demonstrate that SELP is involved in the recruitment of granulocytes to the pregnant uterus, we undertook long-term in vivo inhibition studies using a monoclonal antibody to inhibit the contribution of SELP to leukocyte trafficking to the decidua. In addition, the pregnant uteri of syngeneic Selp(-/-) x Selp(-/-) mice were investigated and compared to the controls. Our results clearly demonstrate the importance of SELP for granulocyte access to the pregnant mouse uterus under physiological and pathological conditions.

  View details for DOI 10.1095/biolreprod.106.056192

  View details for Web of Science ID 000245092900013

  View details for PubMedID 17151351

• Staphylococcus aureus-derived staphopain B, a potent cysteine protease activator of plasma chemerin JOURNAL OF IMMUNOLOGY Kulig, P., Zabel, B. A., Dubin, G., Alllen, S. J., Ohyama, T., Potempa, J., Handel, T. M., Butcher, E. C., Cichy, J. 2007; 178 (6): 3713-3720

  Abstract

  Chemerin is an attractant for cells that express the serpentine receptor CMKLR1, which include immature plasmacytoid dendritic cells (pDC) and macrophages. Chemerin circulates in the blood where it exhibits low biological activity, but upon proteolytic cleavage of its C terminus, it is converted to a potent chemoattractant. Enzymes that contribute to this conversion include host serine proteases of the coagulation, fibrinolytic, and inflammatory cascades, and it has been postulated that recruitment of pDC and macrophages by chemerin may serve to balance local tissue immune and inflammatory responses. In this work, we describe a potent, pathogen-derived proteolytic activity capable of chemerin activation. This activity is mediated by staphopain B (SspB), a cysteine protease secreted by Staphylococcus aureus. Chemerin activation is triggered by growth medium of clinical isolates of SspB-positive S. aureus, but not by that of a SspB(null) mutant. C-terminal processing by SspB generates a chemerin isoform identical with the active endogenous attractant isolated from human ascites fluid. Interestingly, SspB is a potent trigger of chemerin even in the presence of plasma inhibitors. SspB may help direct the recruitment of specialized host cells, including immunoregulatory pDC and/or macrophages, contributing to the ability of S. aureus to elicit and maintain a chronic inflammatory state.

  View details for Web of Science ID 000244942400044

  View details for PubMedID 17339469

• DCs metabolize sunlight-induced vitamin D3 to 'program' T cell attraction to the epidermal chemokine CCL27 NATURE IMMUNOLOGY Sigmundsdottir, H., Pan, J., Debes, G. F., Alt, C., Habtezion, A., Soler, D., Butcher, E. C. 2007; 8 (3): 285-293

  Abstract

  During adaptive immune responses, dendritic cells activate T cells and endow them with specific homing properties. Mechanisms that 'imprint' specific tropisms, however, are not well defined. We show here that 1,25(OH)(2)D(3), the active form of vitamin D3, signaled T cells to express CC chemokine receptor 10, which enabled them to migrate to the skin-specific chemokine CCL27 secreted by keratinocytes of the epidermis. In contrast, 1,25(OH)(2)D(3) suppressed the gut-homing receptors alpha4beta7 and CCR9. Vitamin D3, the inactive prohormone naturally generated in the skin by exposure to the sun, was processed by dendritic cells and T cells to the active metabolite, providing a mechanism for the local regulation of T cell 'epidermotropism'. Our findings support a model in which dendritic cells process and 'interpret' locally produced metabolites to 'program' T cell homing and microenvironmental positioning.

  View details for DOI 10.1038/ni1433

  View details for Web of Science ID 000244275100014

  View details for PubMedID 17259988

• Prevention of acute graft-versus-host disease by blocking T cell entry to secondary lymphoid organs 96th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Schulz, S., Beilhack, A., Baker, J., Nishimura, R., Landan, G., Baker, E., Herman, E., BUTCHER, E. C., Contag, C. H., Negrin, R. S. NATURE PUBLISHING GROUP. 2007: 259A–259A

  View details for Web of Science ID 000244922402023

• Prevention of acute-versus-host disease by blocking t cell entry to secondary lymphoid organs 96th Annual Meeting of the United-States-and-Canadian-Academy-of-Pathology Schultz, S., Beilhack, A., Baker, J., Nishimura, R., Landan, G., Baker, E., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. NATURE PUBLISHING GROUP. 2007: 259A–259A

  View details for Web of Science ID 000244935302023

• The germinal center response is impaired in the absence of T cell-expressed CXCR5 EUROPEAN JOURNAL OF IMMUNOLOGY Arnold, C. N., Campbell, D. J., Lipp, M., Butcher, E. C. 2007; 37 (1): 100-109

  Abstract

  Germinal centers support the differentiation of memory B cells and long-lived antibody-secreting cells during infection or upon vaccination. Here, we constructed mice with T cells that selectively lack the chemokine receptor CXCR5 to determine if expression of this receptor by T cells is mandatory for germinal center formation and function. In these animals, germinal centers that are properly localized in B cell follicles and contain T cells do form after immunization with a thymus-dependent antigen. However, fewer and smaller germinal centers form, resulting in a significant reduction in the frequency of germinal center B cells. The defect in germinal center formation is paralleled by decreased frequencies of isotype-switched antibody-secreting cells in the spleen and bone marrow and reduced serum concentrations of total and high-affinity hapten-specific IgG1. The results demonstrate that although CXCR5-dependent T cell positioning is important for maximal induction and expansion of germinal centers, stimulation of isotype class switching, and development of antibody-secreting cells that seed the spleen and bone marrow, it is not absolutely required for the formation and function of follicular germinal centers.

  View details for DOI 10.1002/eji.200636486

  View details for Web of Science ID 000243600400012

  View details for PubMedID 17171760

• Chemokine-like receptor 1 expression by macrophages in vivo: Regulation by TGF-beta and TLR ligands EXPERIMENTAL HEMATOLOGY Zabel, B. A., Ohyama, T., Zuniga, L., Kim, J., Johnston, B., Allen, S. J., Guido, D. G., Handel, T. M., Butcher, E. C. 2006; 34 (8): 1106-1114

  Abstract

  Chemokine-like receptor 1 (CMKLR1) is expressed by human antigen presenting cells and binds to chemerin, a proteolytically activatable chemoattractant. Here we assessed the expression of mCMKLR1 on mouse leukocytes, focusing on ex vivo dendritic cells (DC) and macrophages. mCMKLR1-expressing cells were evaluated for functional responses to chemerin. We examined the regulation of mCMKLR1 expression by exposure to toll-like receptor (TLR) ligands and cytokines. Finally, we evaluated ex vivo human ascites macrophages for huCMKLR1 expression and chemerin responsiveness.A novel anti-mCMKLR1 monoclonal antibody was generated to assess mCMKLR1 expression by mouse leukocytes using flow cytometry. Mouse bone marrow-derived DC precursors, mouse peritoneal macrophages, and human ascites leukocytes were examined in functional assays (in vitro chemotaxis and intracellular calcium mobilization).During DC differentiation from bone marrow, mCMKLR1 is upregulated early and then diminishes with time in culture. Most DC in vivo do not detectably express the receptor. In contrast, freshly isolated F4/80+CD11b+ mouse serosal macrophages express mCMKLR1, bind a fluorescently labeled chemerin peptide, and display calcium signaling and migration to the active ligand. Interestingly, macrophage mCMKLR1 is suppressed by proinflammatory cytokines and TLR ligands, whereas treatment with TGF-beta upregulates the receptor. A small population of blood-borne F4/80+CD11b+ macrophages also expresses mCMKLR1. Freshly isolated macrophages from human ascites fluid express CMKLR1 and are chemerin responsive, as well.The conserved expression of CMKLR1 by macrophages in mouse and man, coupled with the stimuli-specific regulation of CMKLR1, may reflect a critical role for CMKLR1:chemerin in shaping the nature (either proinflammatory or suppressive) in macrophage-mediated immune responses.

  View details for DOI 10.1016/j.exphem.2006.03.011

  View details for Web of Science ID 000239751900017

  View details for PubMedID 16863918

• Chemoattractants, extracellular proteases, and the integrated host defense response EXPERIMENTAL HEMATOLOGY Zabel, B. A., Zuniga, L., Ohyama, T., Allen, S. J., Cichy, J., Handel, T. M., Butcher, E. C. 2006; 34 (8): 1021-1032

  Abstract

  The host response to tissue injury and/or infection is dependent on the action of numerous extracellular proteases. Proteolytic cascades trigger blood clotting, fibrinolysis, and complement activation, while proteases released upon leukocyte degranulation are integral to the processes of inflammation and immunity. Modulation of effector protein activity by proteases provides a critical layer of posttranslational control that enables rapid enzymatic regulation of target proteins. This report reviews the emerging literature describing a novel class of proteolytic targets, leukocyte chemoattractants, and, in particular, chemerin, a dendritic cell and macrophage chemoattractant activated by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades. As chemoattractants are critical for both systemic leukocyte positioning by triggering integrin activation and subsequent recruitment from circulation, and local intratissue leukocyte positioning via chemotaxis, modulation of attractant activities by proteases may have profound effects on the immune response.

  View details for DOI 10.1016/j.exphem.2006.05.003

  View details for Web of Science ID 000239751900007

  View details for PubMedID 16863908

• A cell-surface molecule involved in organ-specific homing of lymphocytes JOURNAL OF IMMUNOLOGY Gallatin, W. M., Weissman, I. L., Butcher, E. C. 2006; 177 (1): 5-9

  View details for Web of Science ID 000238471400002

  View details for PubMedID 16785489

• Short-term homing assay reveals a critical role for lymphocyte function-associated antigen-1 in the hepatic recruitment of lymphocytes in graft-versus-host disease JOURNAL OF HEPATOLOGY Sato, T., Habtezion, A., Beilhack, A., Schulz, S., Butcher, E., Thorlacius, H. 2006; 44 (6): 1132-1140

  Abstract

  The liver is a major target organ of graft versus host disease (GvHD) with massive infiltration of alloreactive lymphocytes resulting in hepatitis and hepatocyte injury. Although adhesive mechanisms have been implicated in the biology of GvHD hepatitis, the identity of homing receptors involved in the initial recruitment of cells from the blood is not known.We have developed a short-term homing assay in a model of murine GvHD. Splenocytes from donors at an active stage of GvHD were injected intravenously into adoptive recipients also undergoing GvHD. The recruitment of cells to the liver was assessed 6h after cell transfer.Activated donor CD8 and CD4 lymphocytes expressed lymphocyte function antigen-1 (LFA-1), alpha4-integrins, and P-selectin binding ligands, and localized more efficiently than naïve T cells. Immunoneutralization of LFA-1 reduced the recruitment of CD8 and CD4 lymphocytes to the liver by more than 60%. Anti-LFA-1 antibody also markedly reduced infiltration of lymphocytes in periportal areas and protected against hepatocellular damage.We demonstrate a critical role of LFA-1 in the recruitment of activated lymphocytes to the liver and in immune-cell mediated hepatitis. LFA-1 may be an effective therapeutic target for protecting the liver following bone marrow transplantation.

  View details for DOI 10.1016/j.jhep.2005.11.042

  View details for Web of Science ID 000237984400016

  View details for PubMedID 16466827

• Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA(+) plasmablast recruitment to the intestinal lamina propria after rotavirus infection JOURNAL OF IMMUNOLOGY Feng, N., Jaimes, M. C., Lazarus, N. H., Monak, D., Zhang, C., Butcher, E. C., Greenberg, H. B. 2006; 176 (10): 5749-5759

  Abstract

  Rotaviruses (RV) are the most important cause of severe childhood diarrheal disease. In suckling mice, infection with RV results in an increase in total and virus-specific IgA(+) plasmablasts in the small intestinal lamina propria (LP) soon after infection, providing a unique opportunity to study the mechanism of IgA(+) cell recruitment into the small intestine. In this study, we show that the increase in total and RV-specific IgA(+) plasmablasts in the LP after RV infection can be blocked by the combined administration of Abs against chemokines CCL25 and CCL28, but not by the administration of either Ab alone. RV infection in CCR9 knockout mice still induced a significant accumulation of IgA(+) plasmablasts in the LP, which was blocked by the addition of anti-CCL28 Ab, confirming the synergistic role of CCL25 and CCL28. The absence of IgA(+) plasmablast accumulation in LP following combined anti-chemokine treatment was not due to changes in proliferation or apoptosis in these cells. We also found that coadministration of anti-CCL25 and anti-CCL28 Abs with the addition of anti-alpha(4) Ab did not further inhibit IgA(+) cell accumulation in the LP and that the CCL25 receptor, CCR9, was coexpressed with the intestinal homing receptor alpha(4)beta(7) on IgA(+) plasmablasts. Finally, we showed that RV infection was associated with an increase in both CCL25 and CCL28 in the small intestine. Hence, our findings indicate that alpha(4)beta(7) along with either CCR9 or CCR10 are sufficient for mediating the intestinal migration of IgA(+) plasmablasts during RV infection.

  View details for Web of Science ID 000237705200010

  View details for PubMedID 16670280

• CD8(+) recent thymic emigrants home to and efficiently repopulate the small intestine epithelium NATURE IMMUNOLOGY Staton, T. L., Habtezion, A., Winslow, M. M., Sato, T., Love, P. E., Butcher, E. C. 2006; 7 (5): 482-488

  Abstract

  Prevailing knowledge dictates that naive alphabeta T cells require activation in lymphoid tissues before differentiating into effector or memory T cells capable of trafficking to nonlymphoid tissues. Here we demonstrate that CD8(+) recent thymic emigrants (RTEs) migrated directly into the small intestine. CCR9, CCL25 and alpha(4)beta(7) integrin were required for gut entry of CD8(+) RTEs. After T cell receptor stimulation, intestinal CD8(+) RTEs proliferated and acquired a surface phenotype resembling that of intraepithelial lymphocytes. CD8(+) RTEs efficiently populated the gut of lymphotoxin-alpha-deficient mice, which lack lymphoid organs. These studies challenge the present understanding of naive alphabeta T cell trafficking and suggest that RTEs may be involved in maintaining a diverse immune repertoire at mucosal surfaces.

  View details for DOI 10.1038/ni1319

  View details for Web of Science ID 000237008800013

  View details for PubMedID 16582913

• RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kalesnikoff, J., Rios, E. J., Chen, C. C., Nakae, S., Zabel, B. A., BUTCHER, E. C., Tsai, M., Tam, S. Y., Galli, S. J. 2006; 103 (8): 2659-2664

  Abstract

  We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.

  View details for Web of Science ID 000235554900034

  View details for PubMedID 16533754

• Prevention of acute graft-versus-host disease despite compensatory function of lymphoid organs in vivo 32nd Annual Meeting of the American-Society-for-Blood-and-Marrow-Transplantation Beilback, A., Schulz, S., Baker, J., Beilback, G. F., Nishimura, R., Landan, G., Baker, E. M., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. ELSEVIER SCIENCE INC. 2006: 11–11

  View details for Web of Science ID 000235344100024

• T cell chemotaxis in a simple microfluidic device LAB ON A CHIP Lin, F., Butcher, E. C. 2006; 6 (11): 1462-1469

  Abstract

  This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.

  View details for DOI 10.1039/b607071j

  View details for Web of Science ID 000241526500012

  View details for PubMedID 17066171

• iNKT-Cells require CCR4 binding of CCL17 to localize to the airways where they are necessary and sufficient for inducing airway hyperreactivity. 6th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Meyer, E., Wurbel, M., Staton, T., Savage, P., DeKruyff, R., Campbell, J., Butcher, E., Umetsu, D. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S22–S22

  View details for DOI 10.1016/j.clim.2006.04.205

  View details for Web of Science ID 000237924300050

• Prevention of acute graft-versus-host disease despite compensatory function of lymphoid organs in vivo. 47th Annual Meeting of the American-Society-of-Hematology Beilhack, A., Schulz, S., Baker, J., Beilhack, G. F., Nishimura, R., Landan, G., Baker, E. M., Herman, E. I., BUTCHER, E. C., Contag, C. H., Negrin, R. S. AMER SOC HEMATOLOGY. 2005: 173A–173A

  View details for Web of Science ID 000233426001051

• Hemin-activated macrophages home to the pancreas and protect from acute pancreatitis via heme oxygenase-1 induction JOURNAL OF CLINICAL INVESTIGATION Nakamichi, I., Habtezion, A., Zhong, B. H., Contag, C. H., BUTCHER, E. C., Omary, M. B. 2005; 115 (11): 3007-3014

  Abstract

  Hemin upregulates heme oxygenase-1 (HO-1), a stress-induced enzyme implicated in protection from a variety of injuries while its related isoform HO-2 is constitutively expressed. The role of hemin or HO-1 in the pancreas and their potential modulation of pancreatic injury are unknown. We show that HO-1 is induced in pancreatitis caused by caerulein and more prominently in severe pancreatitis caused by feeding a choline-deficient diet (CDD). Intraperitoneal hemin administration dramatically increases peritoneal and pancreas macrophages that overexpress HO-1 in association with pancreatic induction of the chemoattractants monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha but not RANTES or macrophage inflammatory protein-2. Hemin administration before CDD feeding protected 8 of 8 mice from lethality while 7 of 16 controls died. Protection is mediated by HO-1-overexpressing macrophages since hemin-primed macrophages home to the pancreas after transfer to naive mice and protect from CDD-induced pancreatitis. Suppression of hemin-primed peritoneal cell HO-1 using HO-1-specific small interfering RNA prior to cell transfer abolishes protection from CDD-induced pancreatitis. Similarly, hemin pretreatment in caerulein-induced pancreatitis reduces serum amylase and lipase, decreases pancreatic trypsin generation, and protects from lung injury. Therefore, hemin-like compounds or hemin-activated macrophages may offer novel therapeutic approaches for preventing acute pancreatitis and its pulmonary complication via upregulation of HO-1.

  View details for DOI 10.1172/JCI24912

  View details for Web of Science ID 000233022100011

  View details for PubMedID 16239966

  View details for PubMedCentralID PMC1257535

• Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades JOURNAL OF BIOLOGICAL CHEMISTRY Zabel, B. A., Allen, S. J., Kulig, P., Allen, J. A., Cichy, J., Handel, T. M., BUTCHER, E. C. 2005; 280 (41): 34661-34666

  Abstract

  Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the "rapid responder" leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum- and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation.

  View details for DOI 10.1074/jbc.M504868200

  View details for Web of Science ID 000232403900035

  View details for PubMedID 16096270

• Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues NATURE IMMUNOLOGY Debes, G. F., Arnold, C. N., Young, A. J., Krautwald, S., Lipp, M., Hay, J. B., BUTCHER, E. C. 2005; 6 (9): 889-894

  Abstract

  Lymphocytes travel throughout the body to carry out immune surveillance and participate in inflammatory reactions. Their path takes them from blood through tissues into lymph and back to blood. Molecules that control lymphocyte recruitment into extralymphoid tissues are well characterized, but exit is assumed to be random. Here, we showed that lymphocyte emigration from the skin was regulated and was sensitive to pertussis toxin. CD4(+) lymphocytes emigrated more efficiently than CD8(+) or B lymphocytes. T lymphocytes in the afferent lymph expressed functional chemokine receptor CCR7, and CCR7 was required for T lymphocyte exit from the skin. The regulated expression of CCR7 by tissue T lymphocytes may control their exit, acting with recruitment mechanisms to regulate lymphocyte transit and accumulation during immune surveillance and inflammation.

  View details for DOI 10.1038/ni1238

  View details for Web of Science ID 000231369800018

  View details for PubMedID 16116468

  View details for PubMedCentralID PMC2144916

• Chemoattractant induces LFA-1 associated PI 3K activity and cell migration that are dependent on Fyn signaling. FASEB journal Bernardini, G., Kim, J. Y., Gismondi, A., Butcher, E. C., Santoni, A. 2005; 19 (10): 1305-1307

  Abstract

  The activation state of integrins on leukocytes is tightly controlled by the association of intracellular molecules to the cytoplasmic domain of the integrin. To identify signaling intermediates involved in chemoattractant receptor-induced integrin activation, we analyzed the ability of LFA-1 integrin to associate with members of Src tyrosine kinase and phosphatidylinositol 3Kinase (PI 3K) families following fMLP stimulation of a lymphoid T cell line stably expressing the fMLP receptor (fPR). fMLP-induced cell activation resulted in a rapid increase of integrin-associated PI 3Kinase activity in G protein and Src kinase dependent manner. We show, upon fMLP-stimulation, a rapid and transient association of the Src tyrosine kinase Fyn with LFA-1. Also, by transiently expressing mutant forms of this kinase, we demonstrated that Fyn is required for the integrin associated PI 3K activity. Furthermore, overexpression of the mutant form of Fyn resulted in a significant impairment of fMLP-induced cell migration through ICAM-1-coated filters, while ICAM-1 independent migration was not affected. Our observations indicate that chemoattractant receptor engagement induces Fyn-dependent PI 3K activation in association with LFA-1 and suggests that Fyn is necessary to initiate and/or to regulate chemoattractant-mediated LFA-1 activation to promote directional migration.

  View details for PubMedID 15955842

• Surface phenotype of Peyer's patch germinal center cells: Implications for the role of germinal centers in B cell differentiation (Reprinted) JOURNAL OF IMMUNOLOGY BUTCHER, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R., Weissman, I. L. 2005; 175 (3): 1363-1372

  View details for Web of Science ID 000233648000002

  View details for PubMedID 16034071

• Can cell systems biology rescue drug discovery? NATURE REVIEWS DRUG DISCOVERY Butcher, E. C. 2005; 4 (6): 461-467

  Abstract

  The focus of innovation in current drug discovery is on new targets, yet compound efficacy and safety in biological models of disease - not target selection - are the criteria that determine which drug candidates enter the clinic. We consider a biology-driven approach to drug discovery that involves screening compounds by automated response profiling in disease models based on complex human-cell systems. Drug discovery through cell systems biology could significantly reduce the time and cost of new drug development.

  View details for DOI 10.1038/nrd1754

  View details for Web of Science ID 000229578100016

  View details for PubMedID 15915152

• Chemoattractant induces LFA-1 associated PI3K activity and cell migration that are dependent on Fyn signaling FASEB JOURNAL Bernardini, G., Kim, J. Y., Gismondi, A., BUTCHER, E. C., Santoni, A. 2005; 19 (8): 1305-?

  Abstract

  The activation state of integrins on leukocytes is tightly controlled by the association of intracellular molecules to the cytoplasmic domain of the integrin. To identify signaling intermediates involved in chemoattractant receptor-induced integrin activation, we analyzed the ability of LFA-1 integrin to associate with members of Src tyrosine kinase and phosphatidylinositol 3Kinase (PI 3K) families following fMLP stimulation of a lymphoid T cell line stably expressing the fMLP receptor (fPR). fMLP-induced cell activation resulted in a rapid increase of integrin-associated PI 3Kinase activity in G protein and Src kinase dependent manner. We show, upon fMLP-stimulation, a rapid and transient association of the Src tyrosine kinase Fyn with LFA-1. Also, by transiently expressing mutant forms of this kinase, we demonstrated that Fyn is required for the integrin associated PI 3K activity. Furthermore, overexpression of the mutant form of Fyn resulted in a significant impairment of fMLP-induced cell migration through ICAM-1-coated filters, while ICAM-1 independent migration was not affected. Our observations indicate that chemoattractant receptor engagement induces Fyn-dependent PI 3K activation in association with LFA-1 and suggests that Fyn is necessary to initiate and/or to regulate chemoattractant-mediated LFA-1 activation to promote directional migration.

  View details for DOI 10.1096/fj.04-3352fje

  View details for Web of Science ID 000230207800015

• Keratin-8-deficient mice develop chronic spontaneous Th2 colitis amenable to antibiotic treatment JOURNAL OF CELL SCIENCE Habtezion, A., Toivola, D. M., BUTCHER, E. C., Omary, M. B. 2005; 118 (9): 1971-1980

  Abstract

  Keratin 8 (K8) is the major intermediate filament protein present in intestinal epithelia. Depending on the mouse genetic background, absence of K8 causes embryonic lethality or colonic hyperplasia and colitis. We studied disease progression, the inflammatory responses, and role of luminal bacteria in K8-null mice in order to characterize the intestinal pathology of K8-associated colitis. Colon lymphocytes were isolated for analysis of their phenotype and cytokine production, and vascular and lymphocyte adhesion molecule expression in K8-/- mice of varying ages. K8-/- mice had a marked increase in TCR(beta)-positive/CD4-positive T cells infiltrating the colon lamina propria, in association with enhanced Th2 cytokine (IL-4, IL-5 and IL-13) production. K8-/- mice show early signs of inflammation even prior to weaning, that increases with age, and their epithelial cells overexpress MHC class II antigens. The chronic colitis is related to increased CD4-positive infiltrating T cells displaying memory and naive phenotypes, and an altered vascular endothelium with aberrant expression of peripheral node addressin. Analysis of normal gut-specific homing molecules, reveals an increased number of alpha(4)beta(7)-positive cells and vascular mucosal addressin cell adhesion molecule-1 in K8-null colons. Antibiotic treatment markedly decreased colon inflammation and ion transporter AE1/2 mistargeting, indicating that luminal bacteria play an important role in the observed phenotype. Therefore, K8-null mice develop chronic spontaneous Th2-type colitis due to a primary epithelial rather than immune cell defect, which is amenable to antibiotic therapy. These mice provide a model to investigate epithelial-leukocyte and epithelial-microbial cross-talk.

  View details for DOI 10.1242/jcs.02316

  View details for Web of Science ID 000229458700020

  View details for PubMedID 15840656

• Modified representational difference analysis: isolation of differentially expressed mRNAs from rare cell populations ANALYTICAL BIOCHEMISTRY O'Hara, E. F., Williams, M. B., Rott, L., Abola, P., Hansen, N., Jones, T., Gurjal, M. R., Federspiel, N., BUTCHER, E. C. 2005; 336 (2): 221-230

  Abstract

  Representational difference analysis of cDNAs (cDNA-RDA) is a sensitive subtractive hybridization technique capable of isolating rare mRNAs differentially expressed in two cell populations. cDNA-RDA can detect sequences represented at 0.0001% in the starting mRNA. By using reverse transcriptase polymerase chain reaction (PCR), cDNA-RDA also lends itself to studies in which samples are derived from limited numbers of cells. Standard cDNA-RDA protocols depend upon the presence of specific restriction enzyme sites in each cDNA, typically enzymes with four base recognition sequences. These sites are used to reduce the cDNA size range and provide primer sites for subsequent PCR amplification. Consequently, transcripts containing fewer than two of the chosen restriction sites are undetectable by cDNA-RDA. We have developed a restriction enzyme site-independent cDNA-RDA protocol called modified RDA (MRDA). We constructed MRDA test sequences from random hexamer-primed cDNA, thereby increasing the representation of mRNAs which are excluded by cDNA-RDA protocols. MRDA is also more efficient than cDNA-RDA at removing highly expressed housekeeping genes during the subtractive hybridization process, thereby allowing more efficient isolation of preferentially expressed mRNAs. Using MRDA, we isolated cDNAs differentially expressed between limited numbers of human CD4(+) naive and memory T lymphocyte subsets and skin- and gut-homing memory T cell subsets.

  View details for DOI 10.1016/j.ab.2004.10.014

  View details for Web of Science ID 000226289000010

  View details for PubMedID 15620887

• Chemokine-like receptor 1 expression and Chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood JOURNAL OF IMMUNOLOGY Zabel, B. A., Silverio, A. M., BUTCHER, E. C. 2005; 174 (1): 244-251

  Abstract

  Plasmacytoid dendritic cells (pDCs) are versatile cells of the immune response, secreting type I IFNs and differentiating into potent immunogenic or tolerogenic APCs. pDCs can express adhesion and chemokine receptors for lymphoid tissues, but are also recruited by unknown mechanisms during tissue inflammation. We use a novel mAb specific for serpentine chemokine-like receptor 1 (CMKLR1) to evaluate its expression by circulating leukocytes in humans. We show that CMKLR1 is expressed by circulating pDCs in human blood, whereas myeloid DCs (mDCs) as well as lymphocytes, monocytes, neutrophils, and eosinophils are negative. We identify a major serum agonist activity for CMKLR1 as chemerin, a proteolytically activated attractant and the sole known ligand for CMKLR1, and we show that chemerin is activated during blood coagulation and attracts pDC but not mDC in ex vivo chemotaxis assays. We conclude that CMKLR1 expression and chemerin-mediated chemotaxis distinguish circulating pDCs from mDCs, providing a potential mechanism for their differential contribution to or regulation of immune responses at sites of bleeding or inflammatory protease activity.

  View details for Web of Science ID 000225852900028

  View details for PubMedID 15611246

• Role for CXCR6 in recruitment of activated CD8(+) lymphocytes to inflamed liver JOURNAL OF IMMUNOLOGY Sato, T., Thorlacius, H., Johnston, B., Staton, T. L., Xiang, W. K., Littman, D. R., BUTCHER, E. C. 2005; 174 (1): 277-283

  Abstract

  Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6(-/-), H-2D(b) lymphocytes into BDF(1), H-2D(bxd) recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6(-/-) or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6(-/-) CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6(-/-) and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.

  View details for Web of Science ID 000225852900032

  View details for PubMedID 15611250

• Functional involvement of P-selectin and MAdCAM-1 in the recruitment of alpha 4 beta 7-integrin-expressing monocyte-like cells to the pregnant mouse uterus EUROPEAN JOURNAL OF IMMUNOLOGY Fernekorn, U., BUTCHER, E. C., Behrends, J., Hartz, S., Kruse, A. 2004; 34 (12): 3423-3433

  Abstract

  Leukocyte recruitment to the pregnant mouse uterus has been suggested to be associated with highly regulated expression of distinct patterns of vascular adhesion receptors. One of the most striking observations is the combined expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and P-selectin by maternal vessels of the vascular zone during the critical period of initial placenta development. The predominant cell population within these vessels is of the monocyte/macrophage lineage and expresses the mucosal integrin alpha4beta7, which represents the ligand for MAdCAM-1; neutrophils and lymphocytes are rare. To directly assess the importance of identified adhesion receptors, we undertook long-term in vivo inhibition studies using monoclonal antibodies to inhibit the contribution of MAdCAM-1 in leukocyte trafficking to the decidua or to deplete alpha4beta7(+) leukocytes. In addition, implantation sites of mouse strains genetically deficient in specific adhesion receptors were investigated. Our results underline the importance of predicted adhesion pathways in the recruitment of monocyte-like cells, especially those expressing alpha4beta7. Interestingly, maternal/fetal units with inhibited recruitment of alpha4beta7(+) leukocytes or the absence of these cells are characterized by reduced size and frequency of uterine NK cells.

  View details for Web of Science ID 000225677200014

  View details for PubMedID 15484189

• Systems biology in drug discovery NATURE BIOTECHNOLOGY Butcher, E. C., BERG, E. L., Kunkel, E. J. 2004; 22 (10): 1253-1259

  Abstract

  The hope of the rapid translation of 'genes to drugs' has foundered on the reality that disease biology is complex, and that drug development must be driven by insights into biological responses. Systems biology aims to describe and to understand the operation of complex biological systems and ultimately to develop predictive models of human disease. Although meaningful molecular level models of human cell and tissue function are a distant goal, systems biology efforts are already influencing drug discovery. Large-scale gene, protein and metabolite measurements ('omics') dramatically accelerate hypothesis generation and testing in disease models. Computer simulations integrating knowledge of organ and system-level responses help prioritize targets and design clinical trials. Automation of complex primary human cell-based assay systems designed to capture emergent properties can now integrate a broad range of disease-relevant human biology into the drug discovery process, informing target and compound validation, lead optimization, and clinical indication selection. These systems biology approaches promise to improve decision making in pharmaceutical development.

  View details for DOI 10.1038/nbt1017

  View details for Web of Science ID 000224326100026

  View details for PubMedID 15470465

• Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children JOURNAL OF VIROLOGY Jaimes, M. C., Rojas, O. L., Kunkel, E. J., Lazarus, N. H., Soler, D., BUTCHER, E. C., Bass, D., Angel, J., Franco, M. A., Greenberg, H. B. 2004; 78 (20): 10967-10976

  Abstract

  We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.

  View details for DOI 10.1028/JVI.78.20.10967-10976.2004

  View details for Web of Science ID 000224229000014

  View details for PubMedID 15452217

  View details for PubMedCentralID PMC521846

• Unique gene expression program of human germinal center T helper cells BLOOD Kim, C. H., Lim, H. W., Kim, J. R., Rott, L., Hillsamer, P., BUTCHER, E. C. 2004; 104 (7): 1952-1960

  Abstract

  Gene expression profiling was used to compare the gene expression patterns of human germinal center (GC) T helper (Th) cells with other CD4+ T-cell subsets (naive, central, and effector memory T cells). GC-Th cells, specifically localized in germinal centers to help B cells, are distantly related to central and effector memory T cells in global gene expression profiles. GC-Th cells displayed substantial differences in mRNA for adhesion molecules, chemoattractant receptors, and cytokines compared with other populations. Distinct expression of transcriptional factors by GC-Th cells is consistent with the hypothesis that they may be different from other T cells in cell lineage. Interestingly, CXCL13, a critical chemokine for B-cell entry to lymphoid follicles, is one of the most highly up-regulated genes in GC-Th cells. GC-Th cells (but not other T cells) produce and secrete large amounts of functional CXCL13 upon T-cell receptor activation, a process that is dependent on costimulation, requires translation and transcription, and is dramatically enhanced by activation in the presence of GC-B cells. This study revealed for the first time the unique gene expression program of GC-Th cells.

  View details for DOI 10.1182/blood-2004-03-1206

  View details for Web of Science ID 000224112800015

  View details for PubMedID 15213097

• CCL28 controls immunoglobulin (Ig)A plasma cell accumulation in the lactating mammary gland and IgA antibody transfer to the neonate JOURNAL OF EXPERIMENTAL MEDICINE Wilson, E., BUTCHER, E. C. 2004; 200 (6): 805-809

  Abstract

  The accumulation of immunoglobulin (Ig)A antibody-secreting cells (ASCs) in the lactating mammary gland leads to secretion of antibodies into milk and their passive transfer to the suckling newborn. This transfer of IgA from mother to infant provides transient immune protection against a variety of gastrointestinal pathogens. Here we show that the mucosal epithelial chemokine CCL28 is up-regulated in the mammary gland during lactation and that IgA ASCs from this tissue express CCR10 and migrate to CCL28. In vivo treatment with anti-CCL28 antibody blocks IgA ASC accumulation in the mammary gland, inhibiting IgA antibody secretion into milk and the subsequent appearance of antibody in the gastrointestinal tract of nursing neonates. We propose that CCL28 is a key regulator of IgA ASC accumulation in the mammary gland and thus controls the passive transfer of IgA antibodies from mother to infant.

  View details for DOI 10.1084/jem.20041069

  View details for Web of Science ID 000224105500011

  View details for PubMedID 15381732

  View details for PubMedCentralID PMC2211970

• Rapid structure-activity and selectivity analysis of kinase inhibitors by BioMAP analysis in complex human primary cell-based models ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES Kunkel, E. J., Plavec, I., Nguyen, D., Melrose, J., Rosler, E. S., Kao, L. T., Wang, Y. K., Hytopoulos, E., Bishop, A. C., Bateman, R., Shokat, K. M., BUTCHER, E. C., Berg, E. L. 2004; 2 (4): 431-441

  Abstract

  Rapid, quantitative methods for characterizing the biological activities of kinase inhibitors in complex human cell systems could allow the biological consequences of differential target selectivity to be monitored early in development, improving the selection of drug candidates. We have previously shown that Biologically Multiplexed Activity Profiling (BioMAP) permits rapid characterization of drug function based on statistical analysis of protein expression data sets from complex primary human cellbased models of disease biology. Here, using four such model systems containing primary human endothelial cells and peripheral blood mononuclear cells in which multiple signaling pathways relevant to inflammation and immune responses are simultaneously activated, we demonstrate that BioMAP analysis can detect and distinguish a wide range of inhibitors directed against different kinase targets. Using a panel of p38 mitogen-activated protein kinase antagonists as a test set, we show further that related compounds can be distinguished by unique features of the biological responses they induce in complex systems, and can be classified according to their induction of shared (on-target) and secondary activities. Statistical comparisons of quantitative BioMAP profiles and analysis of profile features allow correlation of induced biological effects with chemical structure and mapping of biological responses to chemical series or substituents on a common scaffold. Integration of automated BioMAP analysis for prioritization of hits and for structure-activity relationship studies may improve and accelerate the design and selection of optimal therapeutic candidates.

  View details for Web of Science ID 000223849000011

  View details for PubMedID 15357924

• An integrative biology approach for analysis of drug action in models of human vascular inflammation. FASEB journal Kunkel, E. J., Dea, M., Ebens, A., Hytopoulos, E., Melrose, J., Nguyen, D., Ota, K. S., Plavec, I., Wang, Y., Watson, S. R., Butcher, E. C., Berg, E. L. 2004; 18 (11): 1279-1281

  Abstract

  Unexpected drug activities discovered during clinical testing establish the need for better characterization of compounds in human disease-relevant conditions early in the discovery process. Here, we describe an approach to characterize drug function based on statistical analysis of protein expression datasets from multiple primary human cell-based models of inflammatory disease. This approach, termed Biologically Multiplexed Activity Profiling (BioMAP), provides rapid characterization of drug function, including mechanism of action, secondary or off-target activities, and insights into clinical phenomena. Using three model systems containing primary human endothelial cells and peripheral blood mononuclear cells in different environments relevant to vascular inflammation and immune activation, we show that BioMAP profiles detect and discriminate multiple functional drug classes, including glucocorticoids; TNF-alpha antagonists; and inhibitors of HMG-CoA reductase, calcineurin, IMPDH, PDE4, PI-3 kinase, hsp90, and p38 MAPK, among others. The ability of cholesterol lowering HMG-CoA reductase inhibitors (statins) to improve outcomes in rheumatic disease patients correlates with the activities of these compounds in our BioMAP assays. In addition, the activity profiles identified for the immunosuppressants mycophenolic acid, cyclosporin A, and FK-506 provide a potential explanation for a reduced incidence of posttransplant cardiovascular disease in patients receiving mycophenolic acid. BioMAP profiling can allow integration of meaningful human biology into drug development programs.

  View details for PubMedID 15208272

• Antigen-specific lymphocyte sequestration in lymphoid organs: Lack of essential roles for alpha(L) and alpha(4) integrin-dependent adhesion or G alpha(i) protein-coupled receptor signaling JOURNAL OF IMMUNOLOGY Arnold, C. N., BUTCHER, E. C., Campbell, D. J. 2004; 173 (2): 866-873

  Abstract

  Selective lymphocyte sequestration was described over 30 years ago as the transient withdrawal of Ag-specific lymphocytes from the circulation as a result of their activation in secondary lymphoid organs. We used a TCR-transgenic adoptive transfer system to further characterize the Ag and adjuvant dependence of this process in mice. In addition, we examined the contribution of the alpha(L) and alpha(4) integrin chains as well as Galpha(i) protein-coupled receptor signaling to the retention of Ag-specific T cells in peripheral lymph nodes. Our results demonstrate that selective lymphocyte sequestration is T cell autonomous and adjuvant independent, and that the duration of sequestration is not controlled by the continued presence of Ag in secondary lymphoid organs. This process is not critically dependent on the alpha(L) and alpha(4) integrin chains or Galpha(i) protein-coupled receptor signaling. Selective lymphocyte sequestration may be mediated by redundant mechanisms and/or controlled by novel or nonclassical adhesion or trafficking molecules.

  View details for Web of Science ID 000222621000019

  View details for PubMedID 15240673

• Murine CD8(+) recent thymic emigrants are (alpha)(E) integrin-positive and CC chemokine ligand 25 responsive JOURNAL OF IMMUNOLOGY Staton, T. L., Johnston, B., BUTCHER, E. C., Campbell, D. J. 2004; 172 (12): 7282-7288

  Abstract

  Recent thymic emigrants (RTE) are an important subpopulation of naive CD8+ T cells because of their ability to reconstitute a diverse immune system after periods of T cell depletion. In neonatal mice, the majority of peripheral T lymphocytes are RTE, cells that have recently left the thymus to populate the periphery. Postulating that these cells could have unique trafficking mechanisms, we compared adhesion molecule and chemokine receptor expression of neonatal RTE with mature adult lymphocytes. Neonatal CD8+ splenocytes uniformly express alpha(E) integrin and exhibit a high responsiveness to CC chemokine ligand (CCL25) (as compared with adult CD8+ splenocytes). Mature CD8+ thymocytes have a similar alpha(E) integrin(+) CCL25 responsive phenotype, as do adult CD8+ RTE identified by intrathymic FITC injection. With increasing age, the frequency of CD8+ alpha(E) integrin(+) splenocytes decreases, roughly correlating with thymic involution. Moreover, halting thymic output by thymectomy accelerates the age-dependent decline in peripheral CD8+ alpha(E) integrin(+) RTE phenotype cells. Low expression of CD44 distinguishes these CD8+ RTE from a population of memory phenotype alpha(E) integrin(+) CD8+ cells that are CD44(high). We conclude that CD8+ RTE have unique adhesive and chemotactic properties that distinguish them from naive CD8+ T cells. These properties may enable specialized microenvironmental and cell-cell interactions contributing to the fate of RTE in the periphery during the early post-thymic period. This phenotype will also facilitate the identification and isolation of RTE for further studies.

  View details for Web of Science ID 000221973100008

  View details for PubMedID 15187103

• An integrative biology approach for analysis of drug action in models of human vascular inflammation FASEB JOURNAL Kunkel, E. J., Dea, M., Ebens, A., Hytopoulos, E., Melrose, J., Nguyen, D., Ota, K. S., Plavec, I., Wang, Y., Watson, S. R., BUTCHER, E. C., Berg, E. L. 2004; 18 (9): 1279-?

  Abstract

  Unexpected drug activities discovered during clinical testing establish the need for better characterization of compounds in human disease-relevant conditions early in the discovery process. Here, we describe an approach to characterize drug function based on statistical analysis of protein expression datasets from multiple primary human cell-based models of inflammatory disease. This approach, termed Biologically Multiplexed Activity Profiling (BioMAP), provides rapid characterization of drug function, including mechanism of action, secondary or off-target activities, and insights into clinical phenomena. Using three model systems containing primary human endothelial cells and peripheral blood mononuclear cells in different environments relevant to vascular inflammation and immune activation, we show that BioMAP profiles detect and discriminate multiple functional drug classes, including glucocorticoids; TNF-alpha antagonists; and inhibitors of HMG-CoA reductase, calcineurin, IMPDH, PDE4, PI-3 kinase, hsp90, and p38 MAPK, among others. The ability of cholesterol lowering HMG-CoA reductase inhibitors (statins) to improve outcomes in rheumatic disease patients correlates with the activities of these compounds in our BioMAP assays. In addition, the activity profiles identified for the immunosuppressants mycophenolic acid, cyclosporin A, and FK-506 provide a potential explanation for a reduced incidence of posttransplant cardiovascular disease in patients receiving mycophenolic acid. BioMAP profiling can allow integration of meaningful human biology into drug development programs.

  View details for DOI 10.1096/fj.04-1538fje

  View details for Web of Science ID 000222327500004

• A central role for the chemokine MEC/CCL28 in regulating IgA mediated passive immunity of the neonate Experimental Biology 2004 Annual Meeting Wilson, E., Feng, N. G., Greenberg, H., Butcher, E. FEDERATION AMER SOC EXP BIOL. 2004: A818–A818

  View details for Web of Science ID 000220470700284

• Method for analyzing signaling networks in complex cellular systems PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Plavec, I., Sirenko, O., Privat, S., Wang, Y. K., Dajee, M., Melrose, J., Nakao, B., Hytopoulos, E., BERG, E. L., Butcher, E. C. 2004; 101 (5): 1223-1228

  Abstract

  Now that the human genome has been sequenced, the challenge of assigning function to human genes has become acute. Existing approaches using microarrays or proteomics frequently generate very large volumes of data not directly related to biological function, making interpretation difficult. Here, we describe a technique for integrative systems biology in which: (i) primary cells are cultured under biologically meaningful conditions; (ii) a limited number of biologically meaningful readouts are measured; and (iii) the results obtained under several different conditions are combined for analysis. Studies of human endothelial cells overexpressing different signaling molecules under multiple inflammatory conditions show that this system can capture a remarkable range of functions by a relatively small number of simple measurements. In particular, measurement of seven different protein levels by ELISA under four different conditions is capable of reconstructing pathway associations of 25 different proteins representing four known signaling pathways, implicating additional participants in the NF-kappaBorRAS/mitogen-activated protein kinase pathways and defining additional interactions between these pathways.

  View details for DOI 10.1073/pnas.0308221100

  View details for Web of Science ID 000188796800024

  View details for PubMedID 14745015

  View details for PubMedCentralID PMC337034

• RhoA and zeta PKC control distinct modalities of LFA-1 activation by chemokines: Critical role of LFA-1 affinity triggering in lymphocyte in vivo homing IMMUNITY Giagulli, C., Scarpini, E., Ottoboni, L., Narumiya, S., BUTCHER, E. C., Constantin, G., Laudanna, C. 2004; 20 (1): 25-35

  Abstract

  Chemokines regulate rapid leukocyte adhesion by triggering a complex modality of integrin activation. We show that the small GTPase RhoA and the atypical zeta PKC differently control lymphocyte LFA-1 high-affinity state and rapid lateral mobility induced by chemokines. Activation of LFA-1 high-affinity state and lateral mobility is controlled by RhoA through the activity of distinct effector regions, demonstrating that RhoA is a central point of diversification of signaling pathways leading to both modalities of LFA-1 triggering. In contrast, zeta PKC controls LFA-1 lateral mobility but not affinity triggering. Blockade of the 23-40 RhoA effector region prevents induction of LFA-1 high-affinity state as well as lymphocyte arrest in Peyer's patch high endothelial venules. Thus, RhoA controls the induction of LFA-1 high-affinity state by chemokines independently of zeta PKC, and this is critical to support chemokine-regulated homing of circulating lymphocytes.

  View details for Web of Science ID 000221442200004

  View details for PubMedID 14738762

• Chemokines in the systemic organization of immunity IMMUNOLOGICAL REVIEWS Campbell, D. J., Kim, C. H., BUTCHER, E. C. 2003; 195: 58-71

  Abstract

  Directed cellular migrations underlie immune system organization. Chemokines and their receptors (along with surface-adhesion molecules) are central to these migrations, targeting developing and mature leukocytes to tissues and microenvironments suitable for their differentiation and function. The chemokine CXCL12 and its receptor CXCR4 play a central role in the migration of hematopoietic stem cells, and several chemokine receptors are transiently expressed during distinct stages of B- and T-cell development. In the periphery, mature naïve B and T cells utilize the receptors CCR7, CXCR4, and CXCR5 to recirculate through specialized microenvironments within the secondary lymphoid tissues, while effector and memory lymphocytes express bewildering patterns of adhesion molecules and chemokine receptors that allow them to function within microenvironments and non-lymphoid tissues inaccessible to naïve cells. Here, we summarize the role of chemokines and their receptors in the spatial organization of the immune system and consider the implications for immune function.

  View details for Web of Science ID 000185274400005

  View details for PubMedID 12969310

• Plasma-cell homing NATURE REVIEWS IMMUNOLOGY Kunkel, E. J., BUTCHER, E. C. 2003; 3 (10): 822-829

  Abstract

  Recent studies indicate that chemoattractant cytokines (chemokines), together with tissue-specific adhesion molecules, coordinate the migration of antibody-secreting cells (ASCs) from their sites of antigen-driven differentiation in lymphoid tissues to target effector tissues. Developing ASCs downregulate the expression of receptors for lymphoid tissue chemokines and selectively upregulate the expression of chemokine receptors that might target the migration of IgA ASCs to mucosal surfaces, IgG ASCs to sites of tissue inflammation and both types of ASC to the bone marrow - an important site for serum antibody production. By directing plasma-cell homing, chemokines might help to determine the character and efficiency of mucosal, inflammatory and systemic antibody responses.

  View details for DOI 10.1038/nri1203

  View details for Web of Science ID 000185970700015

  View details for PubMedID 14523388

• Targeting T cell responses by selective chemokine receptor expression SEMINARS IN IMMUNOLOGY Campbell, D. J., Debes, G. F., Johnston, B., Wilson, E., BUTCHER, E. C. 2003; 15 (5): 277-286

  Abstract

  Immune responses require the orchestrated migration of T cells throughout the body. Conventional CD4+ and CD8+ alphabeta T cells undergo clonal expansion in the secondary lymphoid tissues, during which they are programmed to migrate into specific non-lymphoid tissues and other lymphoid effector sites such as B cell follicles. By contrast, T cell populations expressing receptors with limited diversity (i.e. gammadelta T cells and NK T cells) appear to be preprogrammed to localize in non-lymphoid tissues where they monitor tissue integrity or serve regulatory functions. By promoting chemotaxis and integrin activation, chemokines and their receptors (in conjunction with surface adhesion molecules) control these T cell homing events. Thus, expression of chemokine receptors defines T cells with tropism for particular tissues and/or microenvironments, and identifies T cell subsets with distinct functional properties.

  View details for DOI 10.1016/j.smim.2003.08.005

  View details for Web of Science ID 000187466100006

  View details for PubMedID 15001177

• Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets JOURNAL OF IMMUNOLOGY Johnston, B., Kim, C. H., Soler, D., Emoto, M., BUTCHER, E. C. 2003; 171 (6): 2960-2969

  Abstract

  NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRalphabeta NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Valpha14i-positive and approximately 50% of Valpha14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Valpha14i-negative than Valpha14i-positive NKT cells. In spleen only, subsets of Valpha14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-gamma/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4(-) Valpha14i-negative NKT cells that were L-selectin(high) and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Valpha14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1(+) Valpha14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Valpha14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4(+) or CD4(-)CD8(-) and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Valpha14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.

  View details for Web of Science ID 000185247300030

  View details for PubMedID 12960320

• Motility analysis of pancreatic adenocarcinoma cells reveals a role for the atypical zeta isoform of protein kinase C in cancer cell movement LABORATORY INVESTIGATION Laudanna, C., Sorio, C., Tecchio, C., BUTCHER, E. C., BONORA, A., Bassi, C., Scarpa, A. 2003; 83 (8): 1155-1163

  Abstract

  The acquisition of an invasive and metastatic phenotype is accompanied by profound alterations of intracellular mechanisms controlling cell movement. Analysis of quantitative parameters of cell motility in cancer cells may help in the identification of intracellular signaling events determining invasion and metastasis. Here we developed a novel procedure of quantification of cell motility based on time-lapse video microscopy and digital image analysis. Three kinetic parameters, including area change, plasma membrane remodeling, and speed of linear movement, are quantified and combined in one single, time-normalized value we defined motility score (MS). Through calculation of the MS for various human pancreatic adenocarcinoma cell subclones, we identified clones characterized by low or high spontaneous motility in vitro. Analysis of the signaling mechanisms involved in the regulation of pancreatic adenocarcinoma cell motility showed that the atypical zeta isozyme of the serine-threonine protein kinase C (PKC) plays a critical role in maintaining a high MS in motile subclones, as demonstrated by the inhibitory effect of cell permeable peptides with sequence corresponding to the pseudosubstrate inhibitory region of the atypical zeta PKC. Other PKC isozymes, either classic or novel, seem not involved. Furthermore, biochemical analysis showed that in motile cells, zeta PKC is constitutively associated with the plasma membrane, whereas in nonmotile cells, zeta PKC is totally excluded from the plasma membrane. These data suggest that the disregulation of the function of atypical zeta PKC might be involved in the acquisition of an invasive and metastatic phenotype in pancreatic adenocarcinoma cells.

  View details for DOI 10.1097/01.LAB.0000081390.92179.F3

  View details for Web of Science ID 000184887300006

  View details for PubMedID 12920244

• Dendritic cells support sequential reprogramming of chemoattractant receptor profiles during naive to effector T cell differentiation JOURNAL OF IMMUNOLOGY Kim, C. H., Nagata, K., BUTCHER, E. C. 2003; 171 (1): 152-158

  Abstract

  T cells undergo chemokine receptor switches during activation and differentiation in secondary lymphoid tissues. Here we present evidence that dendritic cells can induce changes in T cell expression of chemokine receptors in two continuous steps. In the first switch over a 4-5 day period, dendritic cells up-regulate T cell expression of CXCR3 and CXCR5. Additional stimulation leads to the second switch: down-regulation of lymphoid tissue homing related CCR7 and CXCR5, and up-regulation of Th1/2 effector tissue-targeting chemoattractant receptors such as CCR4, CCR5, CXCR6, and CRTH2. We show that IL-4 and IL-12 can determine the fate of the secondary chemokine receptor switch. IL-4 enhances the generation of CCR4(+) and CRTH2(+) T cells, and suppresses the generation of CXCR3(+) T cells and CCR7(-) T cells, while IL-12 suppresses the level of CCR4 in responding T cells. Furthermore, IL-4 has positive effects on generation of CXCR5(+) and CCR7(+) T cells during the second switch. Our study suggests that the sequential switches in chemokine receptor expression occur during naive T cell interaction with dendritic cells. The first switch of T cell chemokine receptor expression is consistent with the fact that activated T cells migrate within lymphoid tissues for interaction with B and dendritic cells, while the second switch predicts the trafficking behavior of effector T cells away from lymphoid tissues to effector tissue sites.

  View details for Web of Science ID 000183674400020

  View details for PubMedID 12816993

• CD8(+) T cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for P-selectin glycoprotein ligand-1 BLOOD Battistini, L., Piccio, L., Rossi, B., Bach, S., Galgani, S., Gasperini, C., Ottoboni, L., Ciabini, D., Caramia, M. D., Bernardi, G., Laudanna, C., Scarpini, E., McEver, R. P., BUTCHER, E. C., Borsellino, G., Constantin, G. 2003; 101 (12): 4775-4782

  Abstract

  Multiple sclerosis (MS) is considered an autoimmune inflammatory disease of the central nervous system. Under physiologic conditions, we compared the adhesiveness of CD4+ and CD8+ lymphocytes from nontreated patients with acute, relapsing-remitting multiple sclerosis (RRMS) and from healthy donors. We show that in patients with RRMS CD8+, but not with RRMS CD4+, T cells display increased rolling and arrest in inflamed murine brain venules. Moreover, CD8+, but not CD4+, lymphocytes from MS patients show increased rolling on P-selectin in vitro. Anti-P-selectin glycoprotein ligand-1 (PSGL-1) antibodies dramatically block the recruitment of CD8+ cells in brain vessels of patients with MS, suggesting that PSGL-1 represents a novel pharmaceutical target that may be exploited to block the selective entrance of CD8+ cells during early inflammation. Vascular cell adhesion molecule-1 (VCAM-1), but not PSGL-1, is critical for the adhesion of CD4+ cells in MS patients, highlighting a fundamental dichotomy in the mechanisms governing the recruitment of lymphocyte subsets in RRMS. Importantly, 7-color fluorescence-activated cell sorter (FACS) analysis, together with functional data, indicates that a large fraction of CD8+ cells from MS patients display the characteristics of memory-effector phenotype. In conclusion, our results show that CD8+, but not CD4+, T cells from patients with RRMS in the acute phase of the disease display increased ability to be recruited in inflamed brain venules.

  View details for Web of Science ID 000183481700028

  View details for PubMedID 12595306

• Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, alpha(4)beta(1) integrin/VCAM-1, and LFA-1 adhesion pathways JOURNAL OF EXPERIMENTAL MEDICINE Xu, B. H., Wagner, N., Pham, L. N., Magno, V., Shan, Z. Y., BUTCHER, E. C., Michie, S. A. 2003; 197 (10): 1255-1267

  Abstract

  Bronchus-associated lymphoid tissue (BALT) participates in airway immune responses. However, little is known about the lymphocyte-endothelial adhesion cascades that recruit lymphocytes from blood into BALT. We show that high endothelial venules (HEVs) in BALT express substantial levels of VCAM-1, in marked contrast to HEVs in other secondary lymphoid tissues. BALT HEVs also express the L-selectin ligand PNAd. Anti-L-selectin, anti-PNAd, and anti-LFA-1 mAbs almost completely block the homing of B and T lymphocytes into BALT, whereas anti-alpha4 integrin and anti-VCAM-1 mAbs inhibit homing by nearly 40%. alpha4beta7 integrin and MAdCAM-1 are not involved. Importantly, we found that mAbs against alpha4 integrin and VCAM-1 significantly block the migration of total T cells (80% memory phenotype) but not naive T and B cells to BALT. These results suggest that an adhesion cascade, which includes L-selectin/PNAd, alpha4beta1 integrin/VCAM-1, and LFA-1, targets specific lymphocyte subsets to BALT. This high level of involvement of alpha4beta1 integrin/VCAM-1 is unique among secondary lymphoid tissues, and may help unify lymphocyte migration pathways and immune responses in BALT and other bronchopulmonary tissues.

  View details for DOI 10.1084/jem.20010685

  View details for Web of Science ID 000183090200004

  View details for PubMedID 12756264

  View details for PubMedCentralID PMC2193791

• A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts JOURNAL OF IMMUNOLOGY Lazarus, N. H., Kunkel, E. J., Johnston, B., Wilson, E., Youngman, K. R., Butcher, E. C. 2003; 170 (7): 3799-3805

  Abstract

  IgA immunoblasts can seed both intestinal and nonintestinal mucosal sites following localized mucosal immunization, an observation that has led to the concept of a common mucosal immune system. In this study, we demonstrate that the mucosae-associated epithelial chemokine, MEC (CCL28), which is expressed by epithelia in diverse mucosal tissues, is selectively chemotactic for IgA Ab-secreting cells (ASC): MEC attracts IgA- but not IgG- or IgM-producing ASC from both intestinal and nonintestinal lymphoid and effector tissues, including the intestines, lungs, and lymph nodes draining the bronchopulmonary tree and oral cavity. In contrast, the small intestinal chemokine, TECK (CCL25), attracts an overlapping subpopulation of IgA ASC concentrated in the small intestines and its draining lymphoid tissues. Surprisingly, T cells from mucosal sites fail to respond to MEC. These findings suggest a broad and unifying role for MEC in the physiology of the mucosal IgA immune system.

  View details for Web of Science ID 000181754100043

  View details for PubMedID 12646646

• CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells JOURNAL OF CLINICAL INVESTIGATION Kunkel, E. J., Kim, C. H., Lazarus, N. H., Vierra, M. A., Soler, D., Bowman, E. P., BUTCHER, E. C. 2003; 111 (7): 1001-1010

  Abstract

  The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA(+)CD38(hi)CD19(int/-)CD20(-)), including circulating IgA(+) plasmablasts and almost all IgA(+) plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.

  View details for DOI 10.1172/JCI200317244

  View details for Web of Science ID 000181970600011

  View details for PubMedID 12671049

  View details for PubMedCentralID PMC152588

• Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Honjo, M., Nakamura, K., Yamashiro, K., Kiryu, J., Tanihara, H., McEvoy, L. M., Honda, Y., BUTCHER, E. C., Masaki, T., Sawamura, T. 2003; 100 (3): 1274-1279

  Abstract

  Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a major endothelial receptor for oxidized low-density lipoprotein, and is assumed to play a proatherogenic role in atherosclerosis. LOX-1 expression is induced by inflammatory cytokines as well as by proatherogenic stimuli. LOX-1 protein binds agedapoptotic cells, activated platelets, and bacteria, suggesting that it may have diverse activities in vivo. Here, we reveal a role for LOX-1 in endotoxin-induced inflammation. In a model of endotoxemia, injection of a high dose of endotoxin into rats induced leukopenia within 1 h and death of the animals within 24 h. Preadministration of anti-LOX-1 antibody reduced the degree of leukopenia and completely rescued the animals, whereas control IgG did not. In a model of low-dose endotoxin-induced uveitis, anti-LOX-1 antibody efficiently suppressed leukocyte infiltration and protein exudation. In situ videomicroscopic analyses of leukocyte interactions with retinal veins revealed that anti-LOX-1 antibody reduced the number of rolling leukocytes and increased the velocity of rolling, suggesting that LOX-1 functions as a vascular tethering ligand. The ability of LOX-1 to capture leukocytes under physiologic shear was confirmed in an in vitro flow model. Thus, LOX-1 is an adhesion molecule involved in leukocyte recruitment and may represent an attractive target for modulation of endotoxin-induced inflammation.

  View details for DOI 10.1073/pnas.0337528100

  View details for Web of Science ID 000180838100091

  View details for PubMedID 12538855

  View details for PubMedCentralID PMC298763

• Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha 4 beta 7 VIROLOGY Gonzalez, A. M., Jaimes, M. C., Cajiao, I., Rojas, O. L., Cohen, J., Pothier, P., Kohli, E., BUTCHER, E. C., Greenberg, H. B., Angel, J., Franco, M. A. 2003; 305 (1): 93-105

  Abstract

  In vivo replication of rotaviruses is generally limited to enterocytes. Because of this restriction, most blood circulating rotavirus-specific B cells are hypothesized to originate in Peyer's patches and should express the intestinal homing receptor alpha4beta7. To test this hypothesis in humans, we used a flow cytometry assay that identifies antigen-activated (IgD-) B cells (CD19+) that express surface rotavirus-specific immunoglobulin. With this assay we could detect rotavirus-specific B cells in both children and adults with an acute rotavirus (RV) infection. Staining with an anti-alpha4beta7 monoclonal antibody, we could determine that B cells that express rotavirus-specific surface immunoglobulin predominantly express alpha4beta7. The response of rotavirus-specific antibody-secreting cells in the peripheral blood of children and adults with acute rotavirus infection was also studied by ELISPOT. The antibody-secreting cells of children were mainly of the IgM isotype, while the antibody-secreting cells of adults were predominantly of the IgA and IgG isotype. alpha4beta7+ and alpha4beta7- subsets of peripheral blood mononuclear cells were purified using paramagnetic beads and then tested in the ELISPOT assay. Rotavirus-specific antibody-secreting cells were predominantly present in the alpha4beta7+ subpopulation. The flow cytometry assay we have described will permit future studies to characterize the phenotype of virus-specific B cells and could be useful in the study of the immunogenicity and protective efficacy of RV vaccines and the identification of markers of protective immunity.

  View details for DOI 10.1006/viro.2002.1708

  View details for Web of Science ID 000180214200010

  View details for PubMedID 12504544

• Liver-infiltrating lymphocytes in end-stage hepatitis C virus: Subsets, activation status, and chemokine receptor phenotypes JOURNAL OF HEPATOLOGY Boisvert, J., Kunkel, E. J., Campbell, J. J., Keeffe, E. B., BUTCHER, E. C., Greenberg, H. B. 2003; 38 (1): 67-75

  Abstract

  Hepatitis C virus (HCV) is a leading cause of chronic liver disease, yet little is known about the intrahepatic immune response in end-stage patients. Chemokines and their receptors are important regulators of immunity, particularly in the migration and localization of circulating leukocytes within peripheral tissues.This report provides a comprehensive comparison of the chemokine receptor and activation phenotype of the major leukocyte subsets present in end-stage HCV-infected and non-HCV infected livers.Lymphocytes were purified from homogenized explant liver tissue and analyzed by flow cytometry.NK cells are the predominant cell type, followed by T cells, B cells and NK-T cells, independent of HCV status. T cells displayed a memory phenotype and low levels of activation markers. CCR5, CXCR3 and CXCR6 were expressed on a large fraction of activated cells, while moderate to low expression of CCR2, CCR6 and CX(3)CR1 was observed. Several other tissue-specific and inflammatory chemokine receptors were absent from infiltrating lymphocytes.These results identify the chemokine receptors present on infiltrating lymphocytes during end-stage liver disease and suggest that such infiltration is predominantly controlled by non-tissue-specific inflammatory chemokines, a situation that may be distinct from liver homing pathways under normal conditions.

  View details for PubMedID 12480562

• Distinct subsets of human V alpha 24-invariant NKT cells: cytokine responses and chemokine receptor expression TRENDS IN IMMUNOLOGY Kim, C. H., BUTCHER, E. C., Johnston, B. 2002; 23 (11): 516-519

  Abstract

  CD1d-restricted T-cell receptor Valpha-invariant natural killer T (NKT) cells are important regulators of immune responses through their efficient secretion of T helper 1 (Th1) and Th2 cytokines. Recently, it has been shown that this NKT-cell population contains functionally distinct subsets, producing different sets of cytokines and cytotoxic effector molecules. NKT-cell subsets are also distinct from each other in their expression of adhesion molecules and chemokine receptors, suggesting that they might be targeted to different tissues and perform different immune functions.

  View details for Web of Science ID 000178938100003

  View details for PubMedID 12401396

• Bovine gamma delta T cell subsets express distinct patterns of chemokine responsiveness and adhesion molecules: A mechanism for tissue-specific gamma delta T cell subset accumulation JOURNAL OF IMMUNOLOGY Wilson, E., Hedges, J. F., BUTCHER, E. C., Briskin, M., Jutila, M. A. 2002; 169 (9): 4970-4975

  Abstract

  Subsets of gammadelta T cells localize to distinct tissue sites in the absence of exogenous Ag stimulation or development of effector/memory cells. Selective lymphocyte homing from the blood into tissues is controlled by a multistep process involving vascular and lymphocyte adhesion molecules, and G protein-linked chemokine receptors. The role of these mechanisms in the tissue tropism of gammadelta T cells is still poorly understood. In this study, we demonstrate that a subset of gammadelta T cells, most of which express an antigenically distinct TCR and are characterized by coexpression of CD8, selectively accumulated in tissues that expressed high levels of the mucosal vascular addressin, mucosal addressin cell adhesion molecule 1. These cells expressed higher levels of alpha(4)beta(7) integrins than other gammadelta T cell subsets and selectively migrated to the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21). Integrin activation by CCL21 selectively increased CD8(+)gammadelta T cell binding to recombinant mucosal addressin cell adhesion molecule 1. These results suggest that the tropism of circulating CD8(+)gammadelta T cells for mucosal tissues is due, at least in part, to selective developmental expression of adhesion molecules and chemokine receptors.

  View details for Web of Science ID 000178777400036

  View details for PubMedID 12391210

• Tropism of varicella-zoster virus for human tonsillar CD4(+) T lymphocytes that express activation, memory, and skin homing markers JOURNAL OF VIROLOGY Ku, C. C., Padilla, J. A., Grose, C., BUTCHER, E. C., Arvin, A. M. 2002; 76 (22): 11425-11433

  Abstract

  Varicella-zoster virus (VZV) is an alphaherpesvirus with the characteristic neurotropism of this group, but VZV also infects T cells productively and downregulates major histocompatibility complex (MHC) class I expression on infected T cells, as shown in the SCID-hu mouse model. T-cell tropism is likely to be critical for the cell-associated viremia associated with primary VZV infection. In these experiments, we found that VZV infects human tonsillar CD4(+) T cells in culture, with 15 to 25% being positive for VZV proteins as detected by polyclonal anti-VZV immunoglobulin G (IgG) staining and monitored by flow cytometry analysis. RNA transcripts for VZV gE, a late gene product, were detected in T-cell populations that expressed VZV surface proteins, but not in the VZV-negative cell fraction. Exposure to phorbol myristate acetate resulted in an increase in VZV-positive T cells, indicating that viral DNA was present within these cells and that VZV gene expression could be induced by T-cell activation. By immune scanning electron microscopy, VZV virions were detected in abundance on the surfaces of infected tonsillar T cells. The predominant CD4(+) T-lymphocyte subpopulations that became infected were activated CD69(+) T cells with the CD45RA(-) memory phenotype. Subsets of CD4(+) T cells that expressed skin homing markers, cutaneous leukocyte antigen, and chemokine receptor 4 were also infected with VZV. By chemotaxis assay, VZV-infected T cells migrated to SDF-1, demonstrating that skin migratory function was intact despite VZV infection. The susceptibility of tonsil T cells to VZV suggests that these cells may be important targets during the initial VZV infection of upper respiratory tract sites. Viral transfer to migrating T cells in the tonsils may facilitate cell-associated viremia, and preferential infection of CD4 T cells that express skin homing markers may enhance VZV transport to cutaneous sites of replication.

  View details for DOI 10.1128/JVI.76.22.11425-11433.2002

  View details for Web of Science ID 000178822400027

  View details for PubMedID 12388703

  View details for PubMedCentralID PMC136789

• Intestinal attraction: CCL25 functions in effector lymphocyte recruitment to the small intestine JOURNAL OF CLINICAL INVESTIGATION Campbell, D. J., BUTCHER, E. C. 2002; 110 (8): 1079-1081

  View details for DOI 10.1172/JCI200216946

  View details for Web of Science ID 000178793700006

  View details for PubMedID 12393843

  View details for PubMedCentralID PMC150808

• Cytokine control of memory B cell homing machinery JOURNAL OF IMMUNOLOGY Roy, M. P., Kim, C. H., BUTCHER, E. C. 2002; 169 (4): 1676-1682

  Abstract

  The germinal center (GC) is a pivotal site for the development of B cell memory. Whereas GC B cells do not chemotax to most chemokines and do not express the adhesion receptors L-selectin, alpha(4)beta(7), and cutaneous lymphocyte Ag (CLA), memory B cells respond to various chemotactic signals and express adhesion receptors. In this study, we show that CD40 ligand, IL-2, and IL-10 together drive this transition of GC B cells to memory phenotype in vitro, up-regulating memory B cell markers, chemotactic responses to CXC ligand (CXCL)12, CXCL13, and CCL19, and expression of adhesion receptors L-selectin, alpha(4)beta(7), and CLA. Moreover, addition of IL-4 modulates this transition, preventing chemotactic responses to CXCL12 and CXCL13 (but not to CCL19), and inhibiting the re-expression of L-selectin, but not of CLA or alpha(4)beta(7). CCR7 expression, responsiveness to CCL19, and L-selectin/alpha(4)beta(7) phenotype are coordinately regulated. Thus, IL-2/IL-10 and IL-4 play important and distinctive roles in developing the migratory capacities of memory B cells.

  View details for Web of Science ID 000177365800003

  View details for PubMedID 12165486

• Rapid leukocyte integrin activation by chemokines IMMUNOLOGICAL REVIEWS Laudanna, C., Kim, J. Y., Constantin, G., BUTCHER, E. C. 2002; 186: 37-46

  Abstract

  Chemokines control selective targeting of circulating leukocytes to the microvasculature by triggering inside-out signal transduction pathways leading to integrin-dependent adhesion. Integrin activation by chemokines is very rapid, is downmodulated within minutes and appears to involve both enhanced heterodimer lateral mobility on the plasma membrane, facilitating encounters with dispersed ligand, as well as induction of a high-affinity state. These two modalities of integrin activation by chemokines involve distinct signaling pathways in the cell, yet complement each other functionally, allowing binding of rolling cells under conditions of low as well as high ligand density. Recent data show that chemokines generate both pro- and anti-adhesive intracellular signaling events, whose equilibrium is likely to be relevant to the kinetics of adhesion and de-adhesion, and to cell movement during diapedesis and chemotaxis. Importantly, chemokines utilize different signaling mechanisms to modulate the activity of distinct integrin subtypes. These recent advances suggest that chemokines may regulate adhesive responses of immune cells based not only on patterns of chemokine receptor expression, but also on variable signaling pathways that can modulate the pro-adhesive responses of leukocytes as a function of their differentiated state, and of the local microenvironment.

  View details for Web of Science ID 000178043100004

  View details for PubMedID 12234360

• Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity BLOOD Kim, C. H., Johnston, B., BUTCHER, E. C. 2002; 100 (1): 11-16

  Abstract

  Natural killer T (NKT) cells are important regulators of the immune system, but their trafficking machinery, including expression of chemokine receptors, has been poorly defined. Unlike other conventional T-cell populations, we show that most NKT cells express receptors for extralymphoid tissue or inflammation-related chemokines (CCR2, CCR5, and CXCR3), while few NKT cells express lymphoid tissue-homing chemokine receptors (CCR7 and CXCR5). A population with homing potential for lymph nodes (L selectin(+) CCR7(+)) exists only within a small subset of CD4 NKT cells. We show differential expression of chemokine receptors among NKT cell subsets: CCR4 is mainly expressed by a high cytokine (interleukin-4/interleukin-2)-producing (CD4) NKT subset, while CCR1, CCR6, and CXCR6 are preferentially expressed by the low cytokine-producing CD8 and CD4(-)CD8(-) subsets. In line with this, TARC/CCL17 (a CCR4 ligand) induces preferential chemotaxis of the CD4 NKT subset, while chemotactic activities of LARC/CCL20 (a CCR6 ligand) and MIP-1 alpha/CCL3 (a CCR1 ligand) are focused on the CD8 and CD4(-)CD8(-) NKT cells. We conclude that, unlike conventional naive, memory, or effector T cells, the entire NKT cell population expresses nonlymphoid tissue homing chemokine receptors, yet NKT cell subsets differ considerably from each other by displaying distinct and reciprocal expression patterns of some chemokine receptors. Our results identify chemokine receptors that are potentially important for trafficking of human blood NKT cell subsets and reveal their function (cytokine production capacity)-dependent differential trafficking potentials.

  View details for Web of Science ID 000176477400002

  View details for PubMedID 12070001

• Hematopoietic stem cells are uniquely selective in their migratory response to chemokines JOURNAL OF EXPERIMENTAL MEDICINE Wright, D. E., Bowman, E. P., Wagers, A. J., BUTCHER, E. C., Weissman, I. L. 2002; 195 (9): 1145-1154

  Abstract

  Although hematopoietic stem cell (HSC) migration into and out of sites of active hematopoiesis is poorly understood, it is a critical process that underlies modern clinical stem cell transplantation and may be important for normal hematopoietic homeostasis. Given the established roles of chemotactic cytokine (chemokine)-directed migration of other leukocyte subsets, the migration of murine HSC to a large panel of CC and CXC chemokines was investigated. HSC migrated only in response to stromal derived factor-1alpha, the ligand for the CXC chemokine receptor 4 (CXCR4). CXCR4 expression by HSC was confirmed by reverse transcription polymerase chain reaction analysis. Surprisingly, HSC also expressed mRNA for CCR3 and CCR9, although they failed to migrate to the ligands for these receptors. The sharply restricted chemotactic responsiveness of HSC is unique among leukocytes and may be necessary for the specific homing of circulating HSC to bone marrow, as well as for the maintenance of HSC in hematopoietic microenvironments.

  View details for DOI 10.1084/jem.20011284

  View details for Web of Science ID 000176110700006

  View details for PubMedID 11994419

  View details for PubMedCentralID PMC2193709

• Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice AMERICAN JOURNAL OF PATHOLOGY Skovseth, D. K., Yamanaka, T., Brandtzaeg, P., BUTCHER, E. C., Haraldsen, G. 2002; 160 (5): 1629-1637

  Abstract

  To establish a model for adoptive transfer of endothelial cells, we transferred human umbilical vein endothelial cells (HUVECs) to immunodeficient mice (Rag 2(-/-)). HUVECs were suspended as single cells in Matrigel and injected subcutaneously in the abdominal midline. Within 10 days after injection, HUVECs expressed pseudopod-like extensions and began to accumulate in arrays. By day 20, we observed human vessels that contained erythrocytes, and on day 30 we observed perivascular cells that expressed smooth muscle actin, thus resembling mature vessels. Throughout the experimental period, HUVECs bound Ulex europaeus lectin and expressed CD31, VE-cadherin, von Willebrand factor, as well as ICAM-2. A fraction of the cells also expressed the proliferation marker Ki67. Moreover, the sialomucin CD34, which is rapidly down-regulated in cultured HUVECs, was reinduced in vivo. However, we found no reinduction of CD34 in cells cultured inside or on top of Matrigel in vitro. We also injected cells suspended in Matrigel around the catheter tip of implanted osmotic pumps. Delivery of recombinant human interferon-gamma by this route led to strong induction of MHC class II and ICAM-1 on the human vessels. In conclusion, isolated human endothelial cells can integrate with the murine vascular system to form functional capillaries and regain in vivo properties.

  View details for Web of Science ID 000175468200012

  View details for PubMedID 12000715

  View details for PubMedCentralID PMC1850884

• Chemokines in rapid leukocyte adhesion triggering and migration SEMINARS IN IMMUNOLOGY Johnston, B., BUTCHER, E. C. 2002; 14 (2): 83-92

  Abstract

  Leukocyte subsets are recruited from the blood to lymphoid and non-lymphoid tissues via a multi-step process that involves distinct adhesive and activation steps. Chemokines, a family of chemotactic cytokines that signal through G-protein-coupled receptors, play critical roles in regulating the leukocyte recruitment cascade. Chemokines can be transported and immobilized on the surface of vascular endothelial cells, where they activate leukocyte subsets expressing specific receptors. Activation signals induce firm adhesion of rolling leukocytes by rapidly upregulating integrin affinity and/or avidity. Chemokines can also direct migration of adherent cells across the endothelium, and control segregation of cells into specific microenvironments within tissues. The regulated expression of chemokines and their receptors is a critical determinant for homing of specialized lymphocyte subsets, and controls both tissue and inflammation-specific immune processes.

  View details for DOI 10.1006/smim.2001.0345

  View details for Web of Science ID 000177130900003

  View details for PubMedID 11978080

• Unique yet differential trafficking machinery among NKT cell subsets Kim, C. H., Johnston, B., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 2002: A1212–A1212

  View details for Web of Science ID 000174593902692

• Correlation of tissue distribution, developmental phenotype, and intestinal homing receptor expression of antigen-specific B cells during the murine anti-rotavirus immune response JOURNAL OF IMMUNOLOGY Youngman, K. R., Franco, M. A., Kuklin, N. A., Rott, L. S., BUTCHER, E. C., Greenberg, H. B. 2002; 168 (5): 2173-2181

  Abstract

  The intestinal homing receptor, alpha(4)beta(7), helps target lymphocytes to Peyer's patches (PP) and intestinal lamina propria (ILP). We have previously shown that protective immunity to rotavirus (RV), an intestinal pathogen, resides in memory B cells expressing alpha(4)beta(7). In this study, using a novel FACS assay, we have directly studied the phenotype of B cells that express surface RV-specific Ig during the in vivo RV immune response. During primary infection, RV-specific B cells first appear as large IgD(-)B220(low)alpha(4)beta(7)(-)and alpha(4)beta(7)(+) cells (presumptive extrafollicular, Ab-secreting B cells), and then as large and small IgD(-)B220(high)alpha(4)beta(7)(-)cells (presumptive germinal center B cells). The appearance of B cells with the phenotype of large IgD(-)B220(low)alpha(4)beta(7)(+) cells in PP and most notably in mesenteric lymph nodes coincides with the emergence of RV-specific Ab-secreting cells (ASC) in the ILP. Thus, these B lymphocytes are good candidates for the migratory population giving rise to the RV-specific ASC in the ILP. RV-specific long-term memory B cells preferentially accumulate in PP and express alpha(4)beta(7). Nine months after infection most RV-specific IgA ASC are found in PP and ILP and at lower frequency in bone marrow and spleen. This study is the first to follow changes in tissue-specific homing receptor expression during Ag-specific B cell development in response to a natural host, tissue-specific pathogen. These results show that alpha(4)beta(7) is tightly regulated during the Ag-specific B cell response to RV and is expressed concurrently with the specific migration of memory and effector B cells to intestinal tissues.

  View details for Web of Science ID 000173990200013

  View details for PubMedID 11859103

• Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: Critical roles for P-selectin glycoprotein ligand-1 and heterotrimeric G(i)-linked receptors JOURNAL OF IMMUNOLOGY Piccio, L., Rossi, B., Scarpini, E., Laudanna, C., Giagulli, C., Issekutz, A. C., VESTWEBER, D., BUTCHER, E. C., Constantin, G. 2002; 168 (4): 1940-1949

  Abstract

  Lymphocyte recruitment into the brain is a critical event in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. We developed a novel intravital microscopy model to directly analyze through the skull the interactions between lymphocytes and the endothelium in cerebral venules of mice. No adhesive interactions were observed between lymphocytes and the nonactivated endothelium in the cerebral microcirculation. When brain venules were activated by pretreating mice with TNF-alpha or LPS, proteolipid protein 139-151 autoreactive T lymphocytes rolled and arrested; notably, only a few peripheral lymph node cells rolled and firmly adhered. Abs anti-P-selectin glycoprotein ligand-1 and anti-E- and P-selectin blocked tethering and rolling of autoreactive lymphocytes, suggesting that P-selectin glycoprotein ligand-1/endothelial selectins are critical in the recruitment of lymphocytes in inflamed brain venules. E- and P-selectin were expressed on cerebral vessels upon in vivo activation and had a patchy distribution during the preclinical phase of active and passive experimental autoimmune encephalomyelitis. LFA-1/ICAM-1 and alpha(4) integrins/VCAM-1 supported rolling, but were not relevant to rolling velocity. Firm arrest was mainly mediated by LFA-1 and ICAM-1. Pretreatment of autoreactive lymphocytes with pertussis toxin blocked integrin-dependent arrest, implicating a requirement for G(i) protein-dependent signaling in vessels from nonlymphoid districts. In conclusion, our data unveils the molecular mechanisms controlling the recruitment of autoreactive lymphocytes in inflamed cerebral vessels and suggest new insights into the pathogenesis of autoimmune inflammatory diseases of the CNS.

  View details for Web of Science ID 000173771000057

  View details for PubMedID 11823530

• Alterations in the expression of homing-associated molecules at the maternal/fetal interface during the course of pregnancy BIOLOGY OF REPRODUCTION Kruse, A., Martens, N., Fernekorn, U., Hallmann, R., BUTCHER, E. C. 2002; 66 (2): 333-345

  Abstract

  One of the most fascinating immunologic questions is how the genetically distinct fetus is able to survive and develop within the mother without provoking an immune rejection response. The pregnant uterus undergoes rapid morphological and functional changes, and these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. We hypothesized that specialized mechanisms exist to control access of maternal leukocyte subsets to the decidua and that these mechanisms are modulated during the course of pregnancy. At the critical period of initial placenta development, the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns and with recruitment of specialized leukocyte subsets (monocytes, granulated metrial gland cells, and granulocytes) thought to support, modulate, and regulate trophoblast invasion. One of the most striking observations at this time of gestation is the almost complete exclusion of lymphocytes from the maternal/fetal interface. The second half of pregnancy is characterized by a partial loss of microenvironmental specialization and different switches in vascular specificity within the decidua basalis, paralleling dramatic changes in the populations of recruited leukocytes (e.g., a striking influx of lymphocytes, especially T cells). In the term pregnant uterus, the expression of all vascular addressins decreased dramatically; only weakly staining maternal vascular segments remained. These segments may define sites of extremely low residual traffic in the term decidua, which contains remarkably few maternal leukocytes overall. Our results suggest that the maternal/fetal interface represents a situation in which leukocyte trafficking is exquisitely regulated to allow entry of specialized leukocyte subsets that may play a fundamental role in immune regulation during pregnancy.

  View details for Web of Science ID 000173481900010

  View details for PubMedID 11804946

• The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells JOURNAL OF EXPERIMENTAL MEDICINE Bowman, E. P., Kuklin, N. A., Youngman, K. R., Lazarus, N. H., Kunkel, E. J., Pan, J. L., Greenberg, H. B., Butcher, E. C. 2002; 195 (2): 269-275

  Abstract

  Immunoglobulin A (IgA) provides protection against pathogens at mucosal surfaces. Chemotactic responses have been hypothesized to target IgA plasma cells involved in mucosal immune responses. We show here that thymus-expressed chemokine (TECK, CCL25) is a potent and selective chemoattractant for IgA antibody-secreting cells (ASC), efficiently recruiting IgA-producing cells from spleen, Peyer's patches, and mesenteric lymph node. Cells secreting IgA antibody in response to rotavirus, an intestinal pathogen, also respond well. In contrast, IgG- and IgM-ASC respond poorly. Epithelial cells in the small intestines, a principal site of IgA-ASC localization and IgA production in the body, highly and selectively express TECK. The migration of IgA-ASC to the intestinal epithelial cell chemokine TECK may help target IgA-producing cells to the gut wall, thus helping define and segregate the intestinal immune response.

  View details for Web of Science ID 000173759300013

  View details for PubMedID 11805153

• Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lmphoid tissues JOURNAL OF EXPERIMENTAL MEDICINE Campbell, D. J., BUTCHER, E. C. 2002; 195 (1): 135-141

  Abstract

  Effector and memory T cells can be subdivided based on their ability to traffic through peripheral tissues such as inflamed skin and intestinal lamina propria, a property controlled by expression of 'tissue-specific' adhesion and chemoattractant receptors. However, little is known about the development of these selectively homing T cell subsets, and it is unclear whether activation in cutaneous versus intestinal lymphoid organs directly results in effector/memory T cells that differentially express adhesion and chemoattractant receptors targeting them to the corresponding nonlymphoid site. We define two murine CD4(+) effector/memory T cell subsets that preferentially localize in cutaneous or intestinal lymphoid organs by their reciprocal expression of the adhesion molecules P-selectin ligand (P-lig) and alpha 4 beta 7, respectively. We show that within 2 d of systemic immunization CD4(+) T cells activated in cutaneous lymph nodes upregulate P-lig, and downregulate alpha 4 beta 7, while those responding to antigen in intestinal lymph nodes selectively express high levels of alpha 4 beta 7 and acquire responsiveness to the intestinal chemokine thymus-expressed chemokine (TECK). Thus, during an immune response, local microenvironments within cutaneous and intestinal secondary lymphoid organs differentially direct T cell expression of these adhesion and chemoattractant receptors, targeting the resulting effector T cells to the inflamed skin or intestinal lamina propria.

  View details for Web of Science ID 000173305600014

  View details for PubMedID 11781372

• Homeostatic chemokines and the targeting of regional immunity. Advances in experimental medicine and biology Kunkel, E., Butcher, E. 2002; 512: 65-72

  View details for PubMedID 12661574

• Homeostatic chemokines and the targeting of regional immunity (Reprinted from Immunity, vol 16, pg 1-4, 2002) 9th International Conference on Lymphocyte Activation and Immune Regulation Kunkel, E. J., BUTCHER, E. C. KLUWER ACADEMIC/PLENUM PUBL. 2002: 65–72

  View details for Web of Science ID 000179161600009

• Expression of the chemokine receptors CCR4, CCR5, and CXCR3 by human tissue-infiltrating lymphocytes AMERICAN JOURNAL OF PATHOLOGY Kunkel, E. J., Boisvert, J., Murphy, K., Vierra, M. A., Genovese, M. C., Wardlaw, A. J., Greenberg, H. B., Hodge, M. R., Wu, L. J., BUTCHER, E. C., Campbell, J. J. 2002; 160 (1): 347-355

  Abstract

  Differential expression of adhesion molecules and chemokine receptors has been useful for identification of peripheral blood memory lymphocyte subsets with distinct tissue and microenvironmental tropisms. Expression of CCR4 by circulating memory CD4(+) lymphocytes is associated with cutaneous and other systemic populations while expression of CCR9 is associated with a small intestine-homing subset. CCR5 and CXCR3 are also expressed by discrete memory CD4(+) populations in blood, as well as by tissue-infiltrating lymphocytes from a number of sites. To characterize the similarities and differences among tissue-infiltrating lymphocytes, and to shed light on the specialization of lymphocyte subsets that mediate inflammation and immune surveillance in particular tissues, we have examined the expression of CCR4, CXCR3, and CCR5 on CD4(+) lymphocytes directly isolated from a wide variety of normal and inflamed tissues. Extra-lymphoid tissues contained only memory lymphocytes, many of which were activated (CD69(+)). As predicted by classical studies, skin lymphocytes were enriched in CLA expression whereas intestinal lymphocytes were enriched in alpha(4)beta(7) expression. CCR4 was expressed at high levels by skin-infiltrating lymphocytes, at lower levels by lung and synovial fluid lymphocytes, but never by intestinal lymphocytes. Only the high CCR4 levels characteristic of skin lymphocytes were associated with robust chemotactic and adhesive responses to TARC, consistent with a selective role for CCR4 in skin lymphocyte homing. In contrast, CXCR3 and CCR5 were present on the majority of lymphocytes from each non-lymphoid tissue examined, suggesting that these receptors are unlikely to determine tissue specificity, but rather, may play a wider role in tissue inflammation.

  View details for Web of Science ID 000173231700038

  View details for PubMedID 11786428

• Chemokines and the tissue-specific migration of lymphocytes IMMUNITY Kunkel, E. J., BUTCHER, E. C. 2002; 16 (1): 1-4

  Abstract

  Tissue-selective trafficking of memory and effector T and B lymphocytes is mediated by unique combinations of adhesion molecules and chemokines. The discovery of several related epithelial-expressed chemokines (TECK/CCL25 in small intestine, CTACK/CCL27 in skin, and MEC/CCL28 in diverse mucosal sites) now highlights an important role for epithelial cells in controlling homeostatic lymphocyte trafficking, including the localization of cutaneous and intestinal memory T cells, and of IgA plasma cells. Constitutively expressed epithelial chemokines may help determine the character of local immune responses and contribute to the systemic organization of the immune system.

  View details for Web of Science ID 000173575700001

  View details for PubMedID 11825560

• CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin JOURNAL OF EXPERIMENTAL MEDICINE Reiss, Y., Proudfoot, A. E., Power, C. A., Campbell, J. J., BUTCHER, E. C. 2001; 194 (10): 1541-1547

  Abstract

  The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell-attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.

  View details for PubMedID 11714760

• Rules of chemokine receptor association with T cell polarization in vivo JOURNAL OF CLINICAL INVESTIGATION Kim, C. H., Rott, L., Kunkel, E. J., Genovese, M. C., Andrew, D. P., Wu, L. J., BUTCHER, E. C. 2001; 108 (9): 1331-1339

  Abstract

  Current concepts of chemokine receptor (CKR) association with Th1 and Th2 cell polarization and effector function have largely ignored the diverse nature of effector and memory T cells in vivo. Here, we systematically investigated the association of 11 CKRs, singly or in combination, with CD4 T cell polarization. We show that Th1, Th2, Th0, and nonpolarized T cells in blood and tissue can express any of the CKRs studied but that each CKR defines a characteristic pool of polarized and nonpolarized CD4 T cells. Certain combinations of CKRs define populations that are markedly enriched in major subsets of Th1 versus Th2 cells. For example, although Th0, Th1, and Th2 cells are each found among blood CD4 T cells coordinately expressing CXCR3 and CCR4, Th1 but not Th2 cells can be CXCR3(+)CCR4(-), and Th2 but only rare Th1 cells are CCR4(+)CXCR3(-). Contrary to recent reports, although CCR7(-) cells contain a higher frequency of polarized CD4 T cells, most Th1 and Th2 effector cells are CCR7(+) and thus may be capable of lymphoid organ homing. Interestingly, Th1-associated CKRs show little or no preference for Th1 cells except when they are coexpressed with CXCR3. We conclude that the combinatorial expression of CKRs, which allow tissue- and subset-dependent targeting of effector cells during chemotactic navigation, defines physiologically significant subsets of polarized and nonpolarized T cells.

  View details for PubMedID 11696578

• Nonpolarized memory T cells TRENDS IN IMMUNOLOGY Kim, C. H., Campbell, D. J., BUTCHER, E. C. 2001; 22 (10): 527-530

  View details for Web of Science ID 000171791100001

  View details for PubMedID 11574259

• Immune cell trafficking in uterus and early life is dominated by the mucosal addressin MAdCAM-1 in humans GASTROENTEROLOGY SALMI, M., Alanen, K., Grenman, S., Briskin, M., BUTCHER, E. C., JALKANEN, S. 2001; 121 (4): 853-864

  Abstract

  In adults, binding of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) to lymphocyte alpha4beta7 integrin directs cell trafficking to gut, whereas interaction of peripheral node addressins (PNAd) with lymphocyte L-selectin targets immune cells to peripheral lymph nodes (PLNs). Because nothing is known about these addressins during human development, we studied the expression and function of MAdCAM-1 (and PNAd for comparison) in fetuses and children.Series of human tissue samples obtained from fetuses (7-40 weeks), children (2 months-7 years), and adults were immunostained with monoclonal antibodies. The function of the addressins and their lymphocyte counter-receptors was tested in in vitro binding assays on fetal and adult tissues.Unlike in adults, MAdCAM-1 is widely expressed from embryonic week 7 onwards, and it only gradually becomes polarized to mucosal vessels after birth. In utero MAdCAM-1 functionally governs lymphocyte adhesion to vessels both in the gut and PLNs by binding to alpha4beta7 integrin. The later induction of PNAd gradually starts to dominate the binding of lymphocytes to PLNs during childhood.There are striking age-dependent switches and species-specific variation in the molecular mechanisms of lymphocyte migration. In utero and during early childhood, the mucosal addressin MAdCAM-1 plays a dominant role in lymphocyte-endothelial cell adhesion at mucosal and nonmucosal sites.

  View details for Web of Science ID 000171422800017

  View details for PubMedID 11606499

• Separable effector T cell populations specialized for B cell help or tissue inflammation NATURE IMMUNOLOGY Campbell, D. J., Kim, C. H., BUTCHER, E. C. 2001; 2 (9): 876-881

  Abstract

  We identified specialized B helper and tissue inflammatory CD4(+) T cell subsets that developed concurrently from common naïve precursors during the primary immune response. These separable populations were distinguishable by their expression of adhesion and chemoattractant receptors that directed their homing to the appropriate effector sites in vivo and also showed intrinsic differences in their ability to support B cell antibody production and produce effector cytokines in vitro. Thus, our data show a previously unappreciated functional specialization among CD4(+) effector T cells, further defining their diversity and role in adaptive immunity.

  View details for Web of Science ID 000170781200029

  View details for PubMedID 11526405

• Subspecialization of CXCR5(+) T cells: B helper activity is focused in a germinal center-localized subset of CXCR5(+) T cells JOURNAL OF EXPERIMENTAL MEDICINE Kim, C. H., Rott, J. S., Clark-Lewis, I., Campbell, D. J., Wu, L., BUTCHER, E. C. 2001; 193 (12): 1373-1381

  Abstract

  The T helper (Th) cell pool is composed of specialized cells with heterogeneous effector functions. Apart from Th1 and 2 cells, CXCR5+ T cells have been suggested to be another type of effector T cell specialized for B cell help. We show here that CXCR5+ T cells are heterogeneous, and we identify subsets of CXCR5+ CD4 T cells that differ in function and microenvironmental localization in secondary lymphoid tissues. CD57+CXCR5 T cells, hereafter termed germinal center Th (GC-Th) cells, are localized only in GCs, lack CCR7, and are highly responsive to the follicular chemokine B lymphocyte chemoattractant but not to the T cell zone EBI1-ligand chemokine. Importantly, GC-Th cells are much more efficient than CD57-CXCR5+ T cells or CXCR5- T cells in inducing antibody production from B cells. Consistent with their function, GC-Th cells produce elevated levels of interleukin 10 upon stimulation which, with other cytokines and costimulatory molecules, may help confer their B cell helper activity. Our results demonstrate that CXCR5+ T cells are functionally heterogeneous and that the GC-Th cells, a small subset of CXCR5+ T cells, are the key helpers for B cell differentiation and antibody production in lymphoid tissues.

  View details for Web of Science ID 000169475500006

  View details for PubMedID 11413192

• Unique subpopulations of CD56(+) NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire JOURNAL OF IMMUNOLOGY Campbell, J. J., Qin, S. X., Unutmaz, D., Soler, D., Murphy, K. E., Hodge, M. R., Wu, L. J., BUTCHER, E. C. 2001; 166 (11): 6477-6482

  Abstract

  CD56, an adhesion molecule closely related to neural cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLS: Although CD56(+) cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56(+) populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16(+) NK cells from CD16(-) NK cells. CD16(+) NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1beta. CD16(-) NK cells were the only CD56(+) population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.

  View details for Web of Science ID 000170948900002

  View details for PubMedID 11359797

• CXCR5 expression defines fundamental specialization of CD4 T cells preceding the conventional Th1/2 polarization Kim, C. H., Rou, L. S., Campbell, D. J., Clarke-Lewis, I., Wu, L. J., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 2001: A360–A360

  View details for Web of Science ID 000167438102061

• Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential JOURNAL OF CLINICAL INVESTIGATION Kim, C. H., Kunkel, E. J., Boisvert, J., Johnston, B., Campbell, J. J., Genovese, M. C., Greenberg, H. B., BUTCHER, E. C. 2001; 107 (5): 595-601

  Abstract

  Chemokine receptor expression is finely controlled during T-cell development. We show that newly identified chemokine receptor Bonzo/CXCR6 is expressed by subsets of Th1 or T-cytotoxic 1 (Tc1) cells, but not by Th2 or Tc2 cells, establishing Bonzo as a differential marker of polarized type 1 T cells in vitro and in vivo. Priming of naive T cells by dendritic cells induces expression of Bonzo on T cells. IL-12 enhances this dendritic cell-dependent upregulation, while IL-4 inhibits it. In blood, 35-56% of Bonzo+ CD4 T cells are Th1 cells, and 60-65% of Bonzo+ CD8 T cells are Tc1 cells, while few Bonzo+ cells are type 2 T cells. Almost all Bonzo+ Tc1 cells contain preformed granzyme A and display cytotoxic effector phenotype. Most Bonzo+ T cells lack L-selectin and/or CCR7, homing receptors for lymphoid tissues. Instead, Bonzo+ T cells are dramatically enriched among T cells in tissue sites of inflammation, such as rheumatoid joints and inflamed livers. Bonzo may be important in trafficking of effector T cells that mediate type 1 inflammation, making it a potential target for therapeutic modulation of inflammatory diseases.

  View details for Web of Science ID 000167337800009

  View details for PubMedID 11238560

  View details for PubMedCentralID PMC199429

• Expression of chemokine receptors by lung T cells from normal and asthmatic subjects JOURNAL OF IMMUNOLOGY Campbell, J. J., Brightling, C. E., Symon, F. A., Qin, S., Murphy, K. E., Hodge, M., Andrew, D. P., Wu, L. J., BUTCHER, E. C., Wardlaw, A. J. 2001; 166 (4): 2842-2848

  Abstract

  The lung is an important tertiary lymphoid organ with constant trafficking of T cells through the lung in both health and disease. T cell migration is controlled by a combination of adhesion receptors and chemokines expressed on vascular endothelium and in the tissue, often in an organ-specific manner. This leads to selective accumulation of different T cell subsets, a process called lymphocyte homing. There is evidence for a distinct lung-homing pathway, but no specific lung-homing receptors have been described. We analyzed the chemokine receptor profile of lung T cells to determine the extent to which lung T cells shared homing pathways with other organs such as the gut and skin. In addition, we compared expression of receptors in normal and asthmatic individuals to determine whether different pathways were used in health and disease. We observed that lung T cells expressed a profile of chemokine and adhesion receptors distinct from that of gut- and skin-homing T cells although no chemokine receptor specific for the lung was found. In particular, lung T cells expressed CCR5 and CXCR3, but not CCR9 or cutaneous lymphocyte Ag, and only low levels of CCR4 and alpha(4)beta(7). No differences were observed between lung T cells from normal vs asthmatic subjects. This study provides added support for the concept of a lung-homing pathway separate from other mucosal organs such as the gut and suggests that the chemokine pathways that control T cell migration in normal homeostasis and Th2-type inflammatory responses are similar.

  View details for Web of Science ID 000166882000086

  View details for PubMedID 11160352

• CCR7 expression and memory T cell diversity in humans JOURNAL OF IMMUNOLOGY Campbell, J. J., Murphy, K. E., Kunkel, E. J., Brightling, C. E., Soler, D., Shen, Z. M., Boisvert, J., Greenberg, H. B., Vierra, M. A., Goodman, S. B., Genovese, M. C., Wardlaw, A. J., BUTCHER, E. C., Wu, L. J. 2001; 166 (2): 877-884

  Abstract

  CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.

  View details for Web of Science ID 000166259600023

  View details for PubMedID 11145663

• C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential JOURNAL OF IMMUNOLOGY Andrew, D. P., Ruffing, N., Kim, C. H., Miao, W. Y., Heath, H., Li, Y., Murphy, K., Campbell, J. J., BUTCHER, E. C., Wu, L. J. 2001; 166 (1): 103-111

  Abstract

  CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4(+) thymocytes and on a major fraction of blood nonintestinal (alpha(4)beta(7)(-)) memory CD4 lymphocytes, including almost all skin memory CD4(+) cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4(+) memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4(+) CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4(+) memory CD4 lymphocytes (including both CLA(+) and CLA(-) fractions) readily express the Th1 cytokine IFN-gamma after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.

  View details for Web of Science ID 000166012400015

  View details for PubMedID 11123282

• alpha(4)beta(7) independent pathway for CD8+ T cell-mediated intestinal immunity to rotavirus JOURNAL OF CLINICAL INVESTIGATION Kuklin, N. A., Rott, L., Darling, J., Campbell, J. J., Franco, M., Feng, N. G., Muller, W., Wagner, N., Altman, J., BUTCHER, E. C., Greenberg, H. B. 2000; 106 (12): 1541-1552

  Abstract

  Rotavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin alpha(4)ss(7) is not essential for CD8(+) T cells to migrate to the intestine or provide immunity to RV. Mice deficient in ss7 expression (ss7(-/-)) and unable to express alpha(4)ss(7) integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8(+) T cells in ss7(-/-) animals prolonged viral shedding, and transfer of immune ss7(-/-) CD8(+) T cells into chronically infected Rag-2-deficient mice resolved RV infection as efficiently as wt CD8(+) T cells. Paradoxically, alpha(4)ss(7)(hi) memory CD8(+) T cells purified from wt mice that had been orally immunized cleared RV more efficiently than alpha(4)ss(7)(low) CD8(+) T cells. We explained this apparent contradiction by demonstrating that expression of alpha(4)ss(7) on effector CD8(+) T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8(+) T cells primarily of an alpha(4)ss(7)(hi) phenotype, but subcutaneous immunization yields both alpha(4)ss(7)(hi) and alpha(4)ss(7)(low) immune CD8(+) T cells with anti-RV effector capabilities. Thus, alpha(4)ss(7) facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8(+) T cell immunity.

  View details for PubMedID 11120761

• Expression of alpha 4 beta 7 and E-selectin ligand by circulating memory B cells: implications for targeted trafficking to mucosal and systemic sites JOURNAL OF LEUKOCYTE BIOLOGY Rott, L. S., Briskin, M. J., BUTCHER, E. C. 2000; 68 (6): 807-814

  Abstract

  We have examined the expression of homing receptors on circulating memory B cells subsets. Blood IgD+ (naive) B cells homogeneously express a high level of intestinal homing receptor, alpha4beta7, but IgD- (putative memory) B cells comprise distinct alpha4beta7+ and alpha4beta7- subsets. Naive and alpha4beta7+ memory B cells but not alpha4beta7- cells bind MAdCAM-1, suggesting that alpha4beta7 expression may predict B cell intestinal homing. In contrast, alpha4beta7+ and alpha4beta7- B cells bind well to VCAM-1, possibly allowing recruitment of both subsets to extra-intestinal sites, including those tissues of the "common mucosal immune system" characterized by vascular VCAM-1 expression. sIgA+ B cells, which are associated with mucosal immunity in the gut and elsewhere, are heterogeneous in homing receptor expression--with discrete subsets expressing alpha4beta7, L-selectin, and cutaneous lymphocyte antigen (CLA). sIgA+ CLA+ B cells are enriched by binding to E-selectin, suggesting that CLA may participate in B cell homing to nonintestinal mucosal tissues characterized by vascular E-selectin expression, such as chronically inflamed bronchial or oral mucosal. We conclude that circulating human peripheral blood memory B cells, like T cells, consist of discrete homing receptor-defined subsets. This diversity in homing phenotypes is apparent even among sIgA (presumptive mucosal) memory B cells, implying heterogeneity in trafficking mechanisms to different target mucosal surfaces.

  View details for Web of Science ID 000165751000004

  View details for PubMedID 11129647

• Chemokines trigger immediate beta 2 integrin affinity and mobility changes: Differential regulation and roles in lymphocyte arrest under flow IMMUNITY Constantin, G., Majeed, M., Giagulli, C., Piccio, L., Kim, J. Y., BUTCHER, E. C., Laudanna, C. 2000; 13 (6): 759-769

  Abstract

  Chemokines trigger rapid integrin-dependent lymphocyte arrest to vascular endothelium. We show that the chemokines SLC, ELC, and SDF-1alpha rapidly induce lateral mobility and transient increase of affinity of the beta2 integrin LFA-1. Inhibition of phosphatidylinositol 3-OH kinase (PI(3)K) activity blocks mobility but not affinity changes and prevents lymphocyte adhesion to ICAM-1 immobilized at low but not high densities, suggesting that mobility enhances the frequency of encounters between high-affinity integrin and ligand but that at higher ligand density affinity changes are sufficient for arrest. Thus, chemokines trigger, through distinct signaling pathways, both a high-affinity state and lateral mobility of LFA-1 that can coordinately determine the vascular arrest of circulating lymphocytes under physiologic conditions.

  View details for Web of Science ID 000166142300002

  View details for PubMedID 11163192

• A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues. Journal of immunology Pan, J., Kunkel, E. J., Gosslar, U., Lazarus, N., Langdon, P., Broadwell, K., Vierra, M. A., Genovese, M. C., BUTCHER, E. C., Soler, D. 2000; 165 (6): 2943-2949

  Abstract

  Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.

  View details for PubMedID 10975800

• Cutting edge: A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues JOURNAL OF IMMUNOLOGY Pan, J. L., Kunkel, E. J., Gosslar, U., Lazarus, N., Langdon, P., Broadwell, K., Vierra, M. A., Genovese, M. C., BUTCHER, E. C., Soler, D. 2000; 165 (6): 2943-2949

  Abstract

  Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.

  View details for Web of Science ID 000165938100004

• The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse JOURNAL OF IMMUNOLOGY Unutmaz, D., Xiang, W. K., Sunshine, M. J., Campbell, J., Butcher, E., Littman, D. R. 2000; 165 (6): 3284-3292

  Abstract

  Chemokines play necessary and important roles in regulating the trafficking of lymphocytes to intra- or interlymphoid tissues as well as to sites of inflammation. The complex migratory patterns of lymphoid lineage cells is governed by subset-specific expression of chemokine receptors and their access to specific ligands. Several chemokine receptors and chemokine receptor-like orphan receptors also serve, in conjunction with CD4, as coreceptors for infection by human and simian immunodeficiency viruses (HIV and SIV). Here we show that the expression pattern of Bonzo/STRL33, an orphan SIV/HIV coreceptor, is highly restricted to the memory subset of T cells and is up-regulated upon stimulation of these cells with IL-2 or IL-15. Both the pattern and the regulation of Bonzo expression closely paralleled that of CC family chemokine receptors CCR5 or CCR6 and inversely correlated with CXCR4 expression. However, in striking contrast to CCR5, Bonzo expression was not down-modulated by PMA or mitogen stimulation of T cells. Targeted replacement of the Bonzo gene with a gene encoding green fluorescent protein in mice revealed that the expression and cytokine regulation of mouse Bonzo are comparable to those of its human counterpart. The similar expression and regulation patterns of Bonzo and the HIV coreceptor CCR5 may have implications for understanding the role of HIV/SIV receptors in viral evolution and pathogenesis.

  View details for Web of Science ID 000165938100049

  View details for PubMedID 10975845

• Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: Epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity JOURNAL OF EXPERIMENTAL MEDICINE Kunkel, E. J., Campbell, J. J., Haraldsen, G., Pan, J. L., Boisvert, J., Roberts, A. I., Ebert, E. C., Vierra, M. A., Goodman, S. B., Genovese, M. C., Wardlaw, A. J., Greenberg, H. B., Parker, C. M., Butcher, E. C., Andrew, D. P., Agace, W. W. 2000; 192 (5): 761-767

  Abstract

  The immune system has evolved specialized cellular and molecular mechanisms for targeting and regulating immune responses at epithelial surfaces. Here we show that small intestinal intraepithelial lymphocytes and lamina propria lymphocytes migrate to thymus-expressed chemokine (TECK). This attraction is mediated by CC chemokine receptor (CCR)9, a chemoattractant receptor expressed at high levels by essentially all CD4(+) and CD8(+) T lymphocytes in the small intestine. Only a small subset of lymphocytes in the colon are CCR9(+), and lymphocytes from other tissues including tonsils, lung, inflamed liver, normal or inflamed skin, inflamed synovium and synovial fluid, breast milk, and seminal fluid are universally CCR9(-). TECK expression is also restricted to the small intestine: immunohistochemistry reveals that intense anti-TECK reactivity characterizes crypt epithelium in the jejunum and ileum, but not in other epithelia of the digestive tract (including stomach and colon), skin, lung, or salivary gland. These results imply a restricted role for lymphocyte CCR9 and its ligand TECK in the small intestine, and provide the first evidence for distinctive mechanisms of lymphocyte recruitment that may permit functional specialization of immune responses in different segments of the gastrointestinal tract. Selective expression of chemokines by differentiated epithelium may represent an important mechanism for targeting and specialization of immune responses.

  View details for PubMedID 10974041

• Chemokines in tissue-specific and microenvironment-specific lymphocyte homing CURRENT OPINION IN IMMUNOLOGY Campbell, J. J., BUTCHER, E. C. 2000; 12 (3): 336-341

  Abstract

  This review describes recent breakthroughs in our understanding of the roles played by chemokines in lymphocyte trafficking. These include the first demonstration that chemokines control lymphocyte/vascular recognition by shear-resistant rapid adhesion; the first example of specialized tissue-specific homing mediated by chemokines; and the implication that chemokines may control microenvironmental segregation within lymphoid organs.

  View details for PubMedID 10781407

• Intestinal homing receptor expression during the B cell response to murine rotavirus infection. Youngman, K. R., Franco, M. A., Kuklin, N. A., Rott, L. S., Greenberg, H. B., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 2000: A973–A973

  View details for Web of Science ID 000086643100361

• alpha 4 beta 7 independent pathway for CD8 T cell mediated intestinal immunity to rotavirus Kuklin, N. A., Rott, L., Darling, J., Campbell, J. J., Franco, M., Feng, N., Muller, W., Wagner, N., Altman, J. D., BUTCHER, E. C., Greenberg, H. B. FEDERATION AMER SOC EXP BIOL. 2000: A971–A971

  View details for Web of Science ID 000086643100350

• Developmental switches in chemokine response profiles during B cell differentiation and maturation JOURNAL OF EXPERIMENTAL MEDICINE Bowman, E. P., Campbell, J. J., Soler, D., Dong, Z. J., Manlongat, N., Picarella, D., Hardy, R. R., BUTCHER, E. C. 2000; 191 (8): 1303-1317

  Abstract

  Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre-pro-B cells (AA4.1(+)/natural killer 1.1(-) Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell-attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3beta, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3alpha and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell-derived factor 1alpha is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.

  View details for PubMedID 10770798

• Specialized patterns of vascular differentiation antigens in the pregnant mouse uterus and the placenta BIOLOGY OF REPRODUCTION Kruse, A., Hallmann, R., BUTCHER, E. C. 1999; 61 (6): 1393-1401

  Abstract

  The success of pregnancy depends on the ability of trophoblast cells to infiltrate the maternal decidua and breach uterine vessels. To ask whether the antigenic phenotype of maternal endothelial cells (EC) in the vascular zone and central decidua basalis may reflect a specialized programming of these vessels for interaction with the trophoblast, we did a survey of several mouse EC differentiation antigens, including MECA-32, MECA-99, and endoglin. Our results revealed striking differences in the phenotype of endothelial lining of vessels in the distinct compartments of the pregnant uterus during Day 9 of pregnancy and at midgestation. Vessels in the central decidua basalis and the vascular zone showed strong expression of MECA-99 but only weak expression of MECA-32, contrasting with the MECA-99(lo), MECA-32(hi) vessels in the capsularis. The vascular zone in addition stained brightly with anti-endoglin. Importantly, invading trophoblast as well as trophoblast cells lining maternal blood spaces were MECA-99(+), MECA-32(-), and endoglin(-), suggesting that the expression of MECA-99 may reflect a specialized co-programming of these trophoblast and EC for future interaction, but also that trophoblast cells may mimic selected antigenic characteristics of endothelium in association with their role in lining maternal blood spaces. In the term pregnant uterus the expression of all differentiation antigens decreased dramatically, suggesting that trophoblast cells as well as maternal EC lose their selected antigenic characteristics when the process of placentation is complete.

  View details for Web of Science ID 000083909700004

  View details for PubMedID 10569981

• Anti-CD43 monoclonal antibody L11 flocks migration of T cells to inflamed pancreatic islets and prevents development of diabetes in nonobese diabetic mice JOURNAL OF IMMUNOLOGY Johnson, G. G., MIKULOWSKA, A., BUTCHER, E. C., McEvoy, L. M., Michie, S. A. 1999; 163 (10): 5678-5685

  Abstract

  Nonobese diabetic mice are a well-known model for human insulin-dependent diabetes mellitus. These mice develop autoimmune-mediated inflammation of the pancreatic islets, followed by destruction of the insulin-producing beta cells and development of diabetes. Nonobese diabetic mice also have salivary gland inflammation, and serve as a model for human Sjogren's syndrome. T cells are a prominent component of the inflammatory infiltrate in these sites, and T cell recruitment from the blood is thought to be essential for the initiation and maintenance of pathologic tissue damage. A unique mAb to murine CD43, L11, has recently been shown to block the migration of T cells from blood into organized lymphoid tissues. Here we demonstrate that L11 significantly inhibits T cell migration from blood into inflamed islets and salivary glands. Treatment of nonobese diabetic mice with L11 from 1 to 4 or 8 to 12 wk of age led to significant protection against the development of diabetes. Moreover, protection was long-lived, with decreased incidence of diabetes even months after cessation of Ab administration. When treatment was started at 1 wk of age, L11 inhibited the development of inflammation in pancreatic islets and salivary glands. L11 treatment had no long-term effect on numbers or phenotypes of peripheral lymphocytes. These data indicate that anti-CD43 Abs that block T cell migration may be useful agents for the prevention or treatment of autoimmune diseases including insulin-dependent diabetes mellitus and Sjogren's syndrome.

  View details for Web of Science ID 000083638400063

  View details for PubMedID 10553098

• Human G protein-coupled receptor GPR-9-G/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis JOURNAL OF EXPERIMENTAL MEDICINE Zabel, B. A., Agace, W. W., Campbell, J. J., Heath, H. M., Parent, D., Roberts, A. I., Ebert, E. C., Kassam, N., Qin, S. X., Zovko, M., LaRosa, G. J., Yang, L. L., Soler, D., BUTCHER, E. C., Ponath, P. D., Parker, C. M., Andrew, D. P. 1999; 190 (9): 1241-1255

  Abstract

  TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

  View details for Web of Science ID 000083552800005

  View details for PubMedID 10544196

  View details for PubMedCentralID PMC2195678

• Cutting edge: Developmental switches in chemokine responses during T cell maturation JOURNAL OF IMMUNOLOGY Campbell, J. J., Pan, J. L., BUTCHER, E. C. 1999; 163 (5): 2353-2357

  Abstract

  We show that developmental transitions during thymocyte maturation are associated with dramatic changes in chemotactic responses to chemokines. Macrophage-derived chemokine, a chemokine expressed in the thymic medulla, attracts thymocytes only during a brief window of development, between the late cortical and early medullary stages. All medullary phenotypes (CD4 or CD8 single positive) but not immature thymocytes respond to the medullary stroma-expressed (and secondary lymphoid tissue-associated) chemokines secondary lymphoid-tissue chemokine and macrophage inflammatory protein-3beta. The appearance of these responses is associated with the phenotypic stage of cortex to medulla migration and with up-regulation of mRNA for the receptors CCR4 (for macrophage-derived chemokine and thymus and activation-regulated chemokine) and CCR7 (for secondary lymphoid-tissue chemokine and macrophage inflammatory protein-3beta). In contrast, most immature and medullary thymocytes migrate to thymus-expressed chemokine, an ability that is lost only with up-regulation of the peripheral homing receptor L-selectin during the latest stages of thymocyte maturation associated with export to the periphery. Developmental switches in chemokine responses may help regulate critical migratory events during T cell development.

  View details for Web of Science ID 000082125400001

  View details for PubMedID 10452965

• L11, a unique anti-CD43 monoclonal antibody, inhibits the adoptive transfer of diabetes and pancreatic islet, salivary gland, and lacrimal gland inflammation in NOD scid mice CELLULAR IMMUNOLOGY MIKULOWSKA, A., Johnson, G. G., Berberian, J. M., BUTCHER, E. C., McEvoy, L. M., Michie, S. A. 1999; 194 (1): 112-117

  Abstract

  L11 is an anti-murine CD43 monoclonal antibody that blocks the migration of T cells from blood into lymphoid tissues. We used a T-cell-mediated adoptive transfer model to evaluate the ability of L11 to inhibit inflammation and destruction in extranodal tissues in the nonobese diabetic (NOD) mouse. Splenocytes from diabetic NOD mice were transferred intravenously into NOD/scid mice. The host mice were treated with L11, negative control antibody, or saline for the first 8 days after transfer. L11 treatment significantly delayed the onset of diabetes and inhibited the development of inflammation in pancreatic islets, salivary gland, and lacrimal gland. These results suggest that L11 may be a useful immunotherapeutic tool for the prevention of T-cell-mediated autoimmune diseases.

  View details for Web of Science ID 000081028700014

  View details for PubMedID 10357887

• Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface EUROPEAN JOURNAL OF IMMUNOLOGY Kruse, A., Merchant, M. J., Hallmann, R., BUTCHER, E. C. 1999; 29 (4): 1116-1126

  Abstract

  In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal/fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well-defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E-selectin. Cells with the phenotype of monocytes but expressing alpha4beta7 integrin are localized in the blood vessels of the specialized "vascular zone", which display the unusual combination of P-selectin (partially associated with platelets) and the alpha4beta7 ligand mucosal vascular addressin-1 (MAdCAM-1). Granulated metrial gland cells (alpha4+beta7-, probably alpha4beta1+) constitute a well-defined cluster positioned in the central decidua basalis around venules prominently expressing the alpha4beta1 ligand VCAM-1 (but not MAdCAM-1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

  View details for Web of Science ID 000079849800008

  View details for PubMedID 10229078

• Inhibition of experimental autoimmune encephalomyelitis by a tyrosine kinase inhibitor JOURNAL OF IMMUNOLOGY Constantin, G., Laudanna, C., Brocke, S., BUTCHER, E. C. 1999; 162 (2): 1144-1149

  Abstract

  Migration of lymphocytes from the blood into the brain is a critical event in the pathogenesis of experimental autoimmune encephalomyelitis. Lymphocyte adhesion to brain endothelium is the first step in lymphocyte entry into the central nervous system, leading subsequently to myelin damage and paralysis. In this paper we show that the tyrosine kinase inhibitor, tyrphostin AG490, prevents binding of freshly isolated mouse lymph node cells and of in vivo activated lymphocytes to endothelium of inflamed brain in Stamper-Woodruff adhesion assays. Moreover, AG490 inhibits adhesion of encephalitogenic T cell lines to purified ICAM-1 and VCAM-1, molecules implicated in T cell recruitment into the central nervous system. In contrast, 2-h treatment of T cell lines with high doses of tyrphostin AG490 have no effect on the viability, intracellular calcium elevation induced by Con A or TCR cross-linking, proliferation, or TNF production by Ag-stimulated T cell lines. Systemic administration of AG490 prevents the accumulation of leukocytes in the brain and the development of experimental autoimmune encephalomyelitis induced by proteolipid protein, peptide 139-151-specific T cell lines in SJL/J mice. Blood leukocytes isolated from mice treated with tyrphostin AG490 are less adhesive on purified very late Ag-4 ligands compared with adhesion of leukocytes from control animals. Our results suggest that inhibition of signaling pathways involved in lymphocyte adhesion may represent a novel therapeutic approach for demyelinating diseases.

  View details for Web of Science ID 000077951400067

  View details for PubMedID 9916745

• L-selectin-mediated leukocyte adhesion in vivo: Microvillous distribution determines tethering efficiency, but not rolling velocity JOURNAL OF EXPERIMENTAL MEDICINE Stein, J. V., Cheng, G. Y., Stockton, B. M., Fors, B. P., BUTCHER, E. C., von Andrian, U. H. 1999; 189 (1): 37-49

  Abstract

  Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 micrometers in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.

  View details for Web of Science ID 000078077700004

  View details for PubMedID 9874562

  View details for PubMedCentralID PMC1887701

• Lymphocyte trafficking and regional immunity ADVANCES IN IMMUNOLOGY, VOL. 72 BUTCHER, E. C., Williams, M., Youngman, K., Rott, L., Briskin, M. 1999; 72: 209-253

  View details for Web of Science ID 000082270400006

  View details for PubMedID 10361577

• Human airway epithelial monolayers promote selective transmigration of memory T cells: A transepithelial model of lymphocyte migration into the airways AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Miller, L. A., BUTCHER, E. C. 1998; 19 (6): 892-900

  Abstract

  It has previously been demonstrated that T lymphocytes in the conducting airways express a pattern of adhesion molecules that are uniquely different from T lymphocytes found circulating in peripheral blood. To examine the role of airway epithelia in the determination of migratory capacity for human monocyte and lymphocyte populations in vivo, we have developed an in vitro transepithelial migration model using the human transformed bronchial epithelial cell line BEAS-2B S.6. In this study, we have demonstrated the preferential migration of human peripheral blood mononuclear cells (PBMC) across BEAS-2B S.6 cell monolayers in a physiologically appropriate direction (basal to apical epithelial cell surface). Stimulation of BEAS-2B S.6 cells with a combination of interferon-gamma and tumor necrosis factor-alpha upregulated basal-to-apical transepithelial migration by at least twofold. Monocytes migrated most efficiently, but subpopulations of CD19(+) B cells and CD2(+) cells were also recruited across epithelial cell monolayers. In the T lymphocyte subset of PBMC, CD45RO+ "memory" cells migrated preferentially. In addition, CD4(+) cells exhibited a significantly greater capacity to migrate across airway epithelium compared with CD8(+) cells. Migrated CD4(+) cells were predominantly CD29(high)/CD26(high), and within this subset uniformly expressed CD62L (L-selectin) at an intermediate level. PBMC migration across BEAS-2B S.6 cells was significantly inhibited by pertussis toxin; this result implicates a G protein signaling event as an important mediator of lymphocyte/monocyte transepithelial migration. On the basis of these data, we conclude that bronchial epithelium provides a unique microenvironment that supports the selective, G protein-dependent migration of memory T cells.

  View details for Web of Science ID 000077551000005

  View details for PubMedID 9843923

• Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis JOURNAL OF BIOLOGICAL CHEMISTRY Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G., BUTCHER, E. C. 1998; 273 (46): 30306-30315

  Abstract

  Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation.

  View details for PubMedID 9804792

• Tyrphostin AG490, a tyrosine kinase inhibitor, blocks actively induced experimental autoimmune encephalomyelitis EUROPEAN JOURNAL OF IMMUNOLOGY Constantin, G., Brocke, S., Izikson, A., Laudanna, C., BUTCHER, E. C. 1998; 28 (11): 3523-3529

  Abstract

  Migration of lymphocytes from blood into the brain is a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Previous observations made in our laboratory showed that protein tyrosine kinase inhibitors were able to block lymphocyte adhesion to brain endothelium and prevent the entry of encephalitogenic T cell lines into the brain of SJL/J mice. Here we show that systemic administration of the protein tyrosine kinase inhibitor, tyrphostin AG490, blocks the development of actively induced EAE in a dose-dependent manner. Administration of 1 mg of drug daily significantly decreased the severity of the disease, while 3 mg of AG490 daily totally blocked the disease in 62% of treated animals, and in those that developed the disease, paralysis was delayed and clinical score was significantly reduced. Blood leukocytes isolated from mice treated with tyrphostin AG490 were less adhesive on VCAM-1 and fibronectin, when compared with control animals. AG490 treatment had no effect on the proliferation by antigen-stimulated peripheral lymph nodes cells. Interestingly, cells obtained from draining lymph nodes in AG490-treated animals and stimulated with antigen secreted two times more IFN-gamma and four times more IL-10, when compared with control animals, whereas no difference was observed in TNF-alpha production. Our results suggest that tyrphostin AG490 may have therapeutic potential by blocking tyrosine kinase activities involved in key mechanisms leading to demyelinating diseases of the central nervous system.

  View details for Web of Science ID 000076985600013

  View details for PubMedID 9842895

• Regulation of chemotactic and proadhesive responses to chemoattractant receptors by RGS (Regulator of G-protein Signaling) family members JOURNAL OF BIOLOGICAL CHEMISTRY Bowman, E. P., Campbell, J. J., Druey, K. M., Scheschonka, A., Kehrl, J. H., BUTCHER, E. C. 1998; 273 (43): 28040-28048

  Abstract

  Serpentine Galphai-linked receptors support rapid adhesion and directed migration of leukocytes and other cell types. The intracellular mechanisms mediating and regulating chemoattractant-directed adhesion and locomotion are only now beginning to be explored. RGS (for regulator of G-protein signaling) proteins are a recently described family that regulate Galphai-stimulated pathways by acting as GTPase-activating proteins. Little is known about the GTPase activity of the Galphai proteins involved in adhesion and chemotaxis, or the significance of their regulation to these responses. Using transiently transfected lymphoid cells as a model system, we show that expression of RGS1, RGS3, and RGS4 inhibits chemoattractant-induced migration. In contrast, RGS2, a regulator of Galphaq activity, had no effect on cell migration to any chemoattractant. RGS1, RGS3, and RGS4 also reduced rapid chemoattractant-triggered adhesion, although the proadhesive response appears quantitatively less sensitive to RGS action than chemotaxis. The results suggest that the duration of the Galphai signal may be a particularly important parameter in the chemotactic responses of leukocytes, and demonstrate the potential for RGS family members to regulate cellular adhesive and migratory behaviors.

  View details for PubMedID 9774420

• The memory B cell subset responsible for the secretory IgA response and protective humoral immunity to rotavirus expresses the intestinal homing receptor, alpha(4)beta(7) JOURNAL OF IMMUNOLOGY Williams, M. B., Rose, J. R., Rott, L. S., Franco, M. A., Greenberg, H. B., BUTCHER, E. C. 1998; 161 (8): 4227-4235

  Abstract

  Infection of mice with murine rotaviruses induces life-long immunity, characterized by high levels of IgA in the intestine and large numbers of rotavirus (RV)-specific Ab-secreting cells in gut-associated lymphoid tissues. Lymphocyte trafficking into gut-associated lymphoid tissues is mediated by interaction of the alpha4beta7 integrin on lymphocytes with the vascular mucosal addressin cell adhesion molecule-1. To determine whether B cell memory for RV correlates with alpha4beta7 expression, we transferred sorted B220+ phenotypically defined memory (IgD- alpha4beta7(high) and IgD- alpha4beta7-) and naive (IgD+ alpha4beta7+) splenocytes into recombination-activating gene-2 knockout mice (B and T cell-deficient) that were chronically infected with RV. Only mice receiving alpha4beta7(high) memory (IgD-) B cells produced RV-specific IgA in the stool, cleared the virus, and were immune to reinfection. Alpha4beta7(high) (but not alpha4beta7-) memory B cells from donors boosted as much as 7 mo previously also cleared the virus, indicating that alpha4beta7(high) memory B cells maintain long term functional immunity to RV. Although only alpha4beta7(high) memory cells provided mucosal immunity, alpha4beta7- cells from recently boosted donor animals could generate RV-specific serum IgG, but, like naive (IgD+) B cells, were unable to induce viral clearance even 60 days after cell transfer. These data indicate that protective immunity for an intestinal pathogen, RV, resides in memory phenotype B cells expressing the intestinal homing receptor, alpha4beta7.

  View details for Web of Science ID 000076343300057

  View details for PubMedID 9780197

• 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3 beta receptor CCR7 JOURNAL OF CELL BIOLOGY Campbell, J. J., Bowman, E. P., Murphy, K., Youngman, K. R., Siani, M. A., Thompson, D. A., Wu, L. J., Zlotnik, A., BUTCHER, E. C. 1998; 141 (4): 1053-1059

  Abstract

  The beta chemokine known as 6-C-kine, secondary lymphoid-tissue chemokine (SLC), TCA4, or Exodus-2 (herein referred to as 6CK/SLC) can trigger rapid integrin-dependent arrest of lymphocytes rolling under physiological shear and is highly expressed by high endothelial venules, specialized vessels involved in lymphocyte homing from the blood into lymph nodes and Peyer's patches. We show that 6CK/SLC is an agonist for the lymphocyte chemoattractant receptor, CCR7 (EBI-1, BLR-2), previously described as a receptor for the related beta chemokine MIP-3beta (ELC or Exodus-3). Moreover, 6CK/SLC and MIP-3beta attract the same major populations of circulating lymphocytes, including naive and memory T cells > B cells (but not natural killer cells); desensitization to MIP-3beta inhibits lymphocyte chemotaxis to 6CK/SLC but not to the alpha chemokine SDF-1 (stromal cell-derived factor); and 6CK/SLC competes for MIP-3beta binding to resting mouse lymphocytes. The findings suggest that the majority of circulating lymphocytes respond to 6CK/SLC and MIP-3beta in large part through their common receptor CCR7 and that these molecules may be important mediators of physiological lymphocyte recirculation in vivo.

  View details for PubMedID 9585422

• Adhesion molecule phenotype of T lymphocytes in inflamed CNS JOURNAL OF NEUROIMMUNOLOGY Engelhardt, B., Martin-Simonet, M. T., Rott, L. S., BUTCHER, E. C., Michie, S. A. 1998; 84 (1): 92-104

  Abstract

  The phenotype of T cells in the central nervous system (CNS) in two models of chronic inflammation (experimental allergic encephalomyelitis and Corynebacterium parvum-induced inflammation) was compared to that of T cells in gut and chronically inflamed subcutaneous tissue and lung. CNS T cells display a similar phenotype in both inflammatory models, and are phenotypically unique compared to T cells from the other inflamed tissues. T cells from inflamed CNS are mainly CD4+ and are the only population examined that express a typical activated/memory phenotype: CD44high/LFA-1high/ICAM-1high/CD45RBlow. The CNS T cells are alpha4beta7-integrin(negative), but express alpha4-integrin and activated beta1 integrin, suggesting expression of the alpha4beta1-heterodimer in an activated state. In contrast, most T cells in gut express low levels of activated beta1 integrin. The CNS T cells lack expression of alpha6 and alphaE integrin chains and L-selectin. In inflamed CNS and inflamed subcutaneous tissue, approximately 50% of T cells express high affinity ligands for P-selectin while fewer than 10% express high affinity ligands for E-selectin. In summary, our data show that, independent of the inflammatory stimulus, T cells recruited into the inflamed CNS are phenotypically distinct from T cells in other inflamed tissues. This finding leads us to hypothesize the existence of a phenotypically distinct 'CNS-seeking' T lymphocyte population.

  View details for Web of Science ID 000073390900012

  View details for PubMedID 9600713

• Asp-698 and Asp-811 of the integrin alpha(4)-subunit are critical for the formation of a functional heterodimer JOURNAL OF BIOLOGICAL CHEMISTRY Zeller, Y., Lohr, J., Sammar, M., BUTCHER, E. C., Altevogt, P. 1998; 273 (12): 6786-6795

  Abstract

  The amino acid motif LDV is the principal binding site for alpha4 integrins in fibronectin, and homologous motifs are recognized in vascular cell adhesion molecule-1 and MAdCAM-1. Three conserved LDV motifs (LDV-1 to 3) occur in the ectodomain of the human and mouse alpha4-subunit, the functions of which are unknown. We demonstrate here that alpha4-transfected fibroblasts with mutation in LDV-1 (D489N) behaved like alpha4-wild type but that LDV-2 (D698N) and LDV-3 (D811N) mutants were impaired in binding and spreading on alpha4-specific substrates. On the RGD-containing fibronectin fragment FN-120 there was an inverse behavior; now the alpha4-wild type and the LDV-1 mutant could not adhere whereas the two other mutants could. The beta1 chain was critical for the differential integrin response. Biochemical analysis demonstrated that the LDV-2 and -3 mutations reduced the strength of the alpha4beta1 association, favored the formation of alpha5beta1, and prevented the expression of alpha4beta7 on the cell surface. Our results indicate that LDV-2 and LDV-3 are critical for the formation of a functional heterodimer. The presence of similar amino acid motifs in ligands and the alpha4-subunit suggest that metal coordination plays an important role in integrin-ligand binding as well as for heterodimer formation.

  View details for Web of Science ID 000072775900030

  View details for PubMedID 9506980

• Complexity and differential expression of carbohydrate epitopes associated with L-selectin recognition of high endothelial venules AMERICAN JOURNAL OF PATHOLOGY BERG, E. L., Mullowney, A. T., Andrew, D. P., Goldberg, J. E., BUTCHER, E. C. 1998; 152 (2): 469-477

  Abstract

  Carbohydrate ligands for lymphocyte L-selectin are expressed on high endothelial venules (HEVs) in peripheral lymph nodes and sites of chronic inflammation and mediate the recruitment of lymphocytes from the blood into these tissues. In the mouse, these ligands, collectively termed the peripheral lymph node addressin (PNAd), have been shown to contain fucose, sialic acid, and sulfate and to include several HEV glycoproteins including GlyCAM-1, CD34, and MAdCAM-1. Monoclonal antibody (MAb) MECA-79, which binds a sulfate-dependent epitope, recognizes PNAd in both mouse and man. In humans, only CD34 has been identified among the glycoprotein species that react with MECA-79. Although P-selectin is highly expressed in tonsil HEVs, it was not found to react with MECA-79 or to support L-selectin-mediated lymphocyte rolling. To further characterize human PNAd, MAbs were developed against purified PNAd immunoisolated from human tonsil. MAbs JG-1, JG-5, JG-9, and JG-10, like MECA-79, bind HEVs in human tonsil and react similarly in Western blots, and JG-9 and JG-10 also block lymphocyte rolling on purified PNAd. In addition, by competitive ELISA on purified tonsil PNAd, all MAbs were found to react with overlapping epitopes. However, JG-1, JG-5, JG-9, and JG-10 do not recognize mouse PNAd, and unlike MECA-79, they recognize determinants that are sensitive to neuraminidase. Strikingly, the epitope recognized by JG-1, although abundant in tonsil and peripheral lymph node, is absent from appendix HEVs or HEVs in some samples of chronically inflamed skin, even though these HEVs are MECA-79 reactive. Moreover, although JG-5 and JG-9 react well with tonsil, peripheral lymph node, and inflamed skin HEVs, they react only with occasional endothelial cells in appendix tissues. These findings point to significant diversity in the carbohydrate determinants expressed by HEVs and recognized by L-selectin and demonstrate their differential representation in different sites in vivo. These antibodies should be useful in probing the precise structure of human L-selectin ligands.

  View details for Web of Science ID 000071905500019

  View details for PubMedID 9466573

  View details for PubMedCentralID PMC1857953

• Molecular mechanisms of lymphocyte homing to peripheral lymph nodes JOURNAL OF EXPERIMENTAL MEDICINE Warnock, R. A., Askari, S., BUTCHER, E. C., von Andrian, U. H. 1998; 187 (2): 205-216

  Abstract

  To characterize the adhesion cascade that directs lymphocyte homing to peripheral lymph nodes (PLNs), we investigated the molecular mechanisms of lymphocyte interactions with the microvasculature of subiliac lymph nodes. We found that endogenous white blood cells and adoptively transferred lymph node lymphocytes (LNCs) tethered and rolled in postcapillary high endothelial venules (HEVs) and to a lesser extent in collecting venules. Similarly, firm arrest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear (PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequencies, but only MNLs arrested suggesting that the events downstream of primary rolling interactions critically determine the specificity of lymphocyte recruitment. Antibody inhibition studies revealed that L-selectin was responsible for attachment and rolling of LNCs, and that LFA-1 was essential for sticking. LFA-1-dependent arrest was also abolished by pertussis toxin, implicating a requirement for G alpha i--protein-linked signaling. alpha 4 integrins, which play a critical role in lymphocyte homing to Peyer's Patches, made no significant contribution to attachment, rolling, or sticking in resting PLNs. Velocity analysis of interacting LNCs revealed no detectable contribution by LFA-1 to rolling. Taken together, our results suggest that lymphocyte- HEV interactions within PLNs are almost exclusively initiated by L-selectin followed by a G protein-coupled lymphocyte-specific activation event and activation-induced engagement of LFA-1. These events constitute a unique adhesion cascade that dictates the specificity of lymphocyte homing to PLNs.

  View details for Web of Science ID 000071729000007

  View details for PubMedID 9432978

• Chemokines and the arrest of lymphocytes rolling under flow conditions SCIENCE Campbell, J. J., Hedrick, J., Zlotnik, A., Siani, M. A., Thompson, D. A., BUTCHER, E. C. 1998; 279 (5349): 381-384

  Abstract

  Circulating lymphocytes are recruited from the blood to the tissue by rolling along the endothelium until being stopped by a signaling event linked to the Gialpha subunit of a heterotrimeric GTP-binding protein; that event then triggers rapid integrin-dependent adhesion. Four chemokines are now shown to induce such adhesion to intercellular adhesion molecule-1 and to induce arrest of rolling cells within 1 second under flow conditions similar to those of blood. SDF-1 (also called PBSF), 6-C-kine (also called Exodus-2), and MIP-3beta (also called ELC or Exodus-3) induced adhesion of most circulating lymphocytes, including most CD4+ T cells; and MIP-3alpha (also called LARC or Exodus-1) triggered adhesion of memory, but not naïve, CD4+ T cells. Thus, chemokines can regulate the arrest of lymphocyte subsets under flowing conditions, which may allow them to control lymphocyte-endothelial cell recognition and lymphocyte recruitment in vivo.

  View details for PubMedID 9430588

• Expression of the mucosal homing receptor alpha(4)beta(7) correlates with the ability of CD8(+) memory T cells to clear rotavirus infection JOURNAL OF VIROLOGY Rose, J. R., Williams, M. B., Rott, L. S., BUTCHER, E. C., Greenberg, H. B. 1998; 72 (1): 726-730

  Abstract

  The integrin alpha4beta7 plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with alpha4beta7 expression. Alpha4beta7(hi) memory phenotype (CD44hi), alpha4beta7- memory phenotype, and presumptively naive (CD44(lo)) CD8+ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. Alpha4beta7(hi) memory phenotype CD8+ cells were highly efficient at clearing rotavirus infection, alpha4beta7- memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44(lo) cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor alpha4beta7.

  View details for Web of Science ID A1998YL01000086

  View details for PubMedID 9420279

• Multistep navigation and the combinatorial control of leukocyte chemotaxis JOURNAL OF CELL BIOLOGY Foxman, E. F., Campbell, J. J., BUTCHER, E. C. 1997; 139 (5): 1349-1360

  Abstract

  Cells migrating within tissues may encounter multiple chemoattractant signals in complex spatial and temporal patterns. To understand leukocyte navigation in such settings, we have explored the migratory behavior of neutrophils in model scenarios where they are presented with two chemoattractant sources in various configurations. We show that, over a wide range of conditions, neutrophils can migrate "down" a local chemoattractant gradient in response to a distant gradient of a different chemoattractant. Furthermore, cells can chemotax effectively to a secondary distant agonist after migrating up a primary gradient into a saturating, nonorienting concentration of an initial attractant. Together, these observations suggest the potential for cells' step-by-step navigation from one gradient to another in complex chemoattractant fields. The importance of such sequential navigation is confirmed here in a model system in which neutrophil homing to a defined domain (a) requires serial responses to agonists presented in a defined spatial array, and (b) is a function of both the agonist combination and the sequence in which gradients are encountered. We propose a multistep model of chemoattractant-directed migration, which requires that leukocytes display multiple chemoattractant receptors for successful homing and provides for combinatorial determination of microenvironmental localization.

  View details for PubMedID 9382879

• Anti-CD43 inhibits monocyte-endothelial adhesion in inflammation and atherogenesis BLOOD McEvoy, L. M., JUTILA, M. A., Tsao, P. S., Cooke, J. P., BUTCHER, E. C. 1997; 90 (9): 3587-3594

  Abstract

  Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti-L-selectin and anti-alpha4 and beta2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

  View details for Web of Science ID A1997YD10800034

  View details for PubMedID 9345042

• Chemoattractant receptor cross talk as a regulatory mechanism in leukocyte adhesion and migration EUROPEAN JOURNAL OF IMMUNOLOGY Campbell, J. J., Foxman, E. F., BUTCHER, E. C. 1997; 27 (10): 2571-2578

  Abstract

  Leukocytes express multiple chemoattractant receptors that can trigger adhesion and direct their migration. Regulation of such proadhesive and migratory responses must often occur in a complex cytokine milieu in vivo, in which multiple receptors may be engaged simultaneously or sequentially, Here we have examined the interplay between interleukin-8 (IL-8) receptor and formyl peptide receptor (fPR)-stimulation and its consequences for leukocyte adhesion and chemotactic responses. IL-8 has no significant effect on fMLP-stimulated adhesion and migration of human neutrophils, indicating that leukocytes have the potential to respond to sequential proadhesive and chemoattractant stimuli during homing and targeted migration. In contrast, fMLP at > or = 10 nM totally abrogated proadhesive and chemoattractant responses to IL-8, a trnas effect to which the fPR itself is relatively resistant. N-formyl peptides are released by invasive bacteria and lysed cells, and the dominance of the fPR may ensure that signals from these terminal phagocyte targets can override host-derived recruitment signaling through IL-8 and other chemokine receptors. Asymmetric inhibition of adhesion-triggering responses is also observed in lymphoid cells transfected with IL-8 receptor A and fPR, but in this cellular context chemotactic responses are bidirectionally abrogated, suggesting the potential for downstream desensitization of motility programs as well. Cross talk between chemoattractant receptors and their signaling pathways may help target leukocyte migration in the context of complex chemoattractant arrays in vivo.

  View details for PubMedID 9368612

• Elevation of intracellular cAMP inhibits RhoA activation and integrin-dependent leukocyte adhesion induced by chemoattractants JOURNAL OF BIOLOGICAL CHEMISTRY Laudanna, C., Campbell, J. J., BUTCHER, E. C. 1997; 272 (39): 24141-24144

  Abstract

  Chemoattractant receptors of the serpentine, heterotrimeric Galphai protein-linked family can activate leukocyte integrins and in this role regulate leukocyte traffic and cell-cell interactions in immune and inflammatory responses. Using a mouse lymphoid cell line transfected with human formyl peptide or interleukin-8 receptors and normal human neutrophils as models, we show that cAMP functions as a gating element on the chemoattractant-induced rho-dependent signaling pathway leading to leukocyte integrin activation and adhesion. cAMP, acting through protein kinase A, inhibits chemoattractant-triggered integrin-dependent leukocyte adhesion. cAMP also prevents guanine nucleotide exchange on RhoA, a small GTP-binding protein of the rho subfamily, which is activated in seconds by chemoattractants. In contrast, chemoattractant-triggered intracellular calcium elevation is unaffected by cAMP, and cAMP has no effect on rho-dependent adhesion and RhoA guanine nucleotide exchange triggered through the independent protein kinase C pathway. These data suggest that cAMP-induced inhibition of rho activation may be responsible for the anti-adhesive effect of cAMP and may contribute to the anti-inflammatory activity of cAMP elevating agonists and drugs. Moreover, the findings extend the concept of cyclic nucleotide gating as a broadly important mechanism in the regulation of intracellular signaling pathways and the cellular activities they control.

  View details for PubMedID 9305861

• Expression of mucosal homing receptor alpha 4 beta 7 by circulating CD4(+) cells with memory for intestinal rotavirus JOURNAL OF CLINICAL INVESTIGATION Rott, L. S., Rose, J. R., Bass, D., Williams, M. B., Greenberg, H. B., BUTCHER, E. C. 1997; 100 (5): 1204-1208

  Abstract

  The integrin alpha4beta7 mediates lymphocyte binding to mucosal addressin cell adhesion molecule-1, and its expression defines lymphocytes capable of trafficking through the intestines and the intestinal lymphoid tissues. We examined the ability of discrete alpha4beta7(hi) and alpha4beta7- subsets of circulating memory phenotype (CD45RA-) CD4+ T cells to proliferate in response to rotavirus, a ubiquitous intestinal pathogen. alpha4beta7(hi) memory (CD45RA-) CD4+ T cells displayed much greater reactivity to rotavirus than alpha4beta7- memory or naive (CD45RA+) CD4+ T cells. In contrast, alpha4beta7- memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. Our results are consistent with the conclusion that natural rotavirus infection, an enteric pathogen, results in a specific circulating memory CD4+ response that is largely limited to the gut-homing alpha4beta7+ subpopulation. This phenotype is not shared with memory cells elicited by intramuscular immunization (shown here) or by skin contact allergens. The results support the hypothesis that gut trafficking memory CD4+ T cells comprise cellular memory for intestinal antigens and suggest that regulated expression of alpha4beta7 helps target and segregate intestinal versus systemic immune response.

  View details for Web of Science ID A1997XV75300030

  View details for PubMedID 9276738

• Vascular adhesion protein 1 (VAP-1) mediates lymphocyte subtype-specific, selectin-independent recognition of vascular endothelium in human lymph nodes JOURNAL OF EXPERIMENTAL MEDICINE SALMI, M., Tohka, S., BERG, E. L., BUTCHER, E. C., JALKANEN, S. 1997; 186 (4): 589-600

  Abstract

  Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.

  View details for Web of Science ID A1997XT78700012

  View details for PubMedID 9254657

  View details for PubMedCentralID PMC2199032

• Homing of naive and memory T lymphocyte subsets to Peyer's patches, lymph nodes, and spleen JOURNAL OF IMMUNOLOGY Williams, M. B., BUTCHER, E. C. 1997; 159 (4): 1746-1752

  Abstract

  The specificity and efficiency of extravasation of subsets of memory and naive lymphocytes into organized lymphoid tissues has been the subject of recent controversy, but has not been directly assessed in physiologic systems. Here, we compare the lymphoid organ homing of naive and phenotypically defined memory T cells, focusing on memory subsets differentially expressing the integrin receptor alpha4beta7, which is implicated in homing to mucosal sites. Naive T cells (CD44(low), Thy1+) home to all secondary lymphoid organs. Alpha4beta7(high) memory-phenotype (CD44(high)) T cells home to Peyer's patches as efficiently as naive lymphocytes, whereas alpha4beta7- memory-phenotype T cells are essentially excluded from entry into these mucosal lymphoid organs. In contrast, alpha4beta7- memory-phenotype cells home approximately twice as efficiently as alpha4beta7(high) T cells to peripheral lymph nodes, but only approximately 20% as well as naive T cells. Interestingly, the spleen recruits all three identified subsets with nearly equal efficiency. The relative subset localization is similar 2.5 h after injection and after overnight trafficking, suggesting that memory cells can directly extravasate from blood into Peyer's Patches and lymph nodes. We conclude that subsets of memory T cells defined by patterns of homing receptor expression display differential homing to organized lymphoid tissues, an ability that may facilitate homeostatic interactions and target contributions to specialized regional immune responses.

  View details for Web of Science ID A1997XP43800022

  View details for PubMedID 9257836

• Structure-function analysis of the integrin beta(7) subunit - Identification of domains involved in adhesion to MAdCAM-1 JOURNAL OF IMMUNOLOGY Tidswell, M., Pachynski, R., Wu, S. W., Qiu, S. Q., Dunham, E., Cochran, N., Briskin, M. J., Kilshaw, P. J., Lazarovits, A. I., Andrew, D. P., BUTCHER, E. C., YEDNOCK, T. A., Erle, D. J. 1997; 159 (3): 1497-1505

  Abstract

  Beta 7 integrins serve special roles in mucosal immunity. Alpha 4 beta 7-mediated adhesion to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs lymphocyte homing to the gut, and alpha E beta 7 mediates binding of lymphocytes to E-cadherin on epithelial cells. Since alpha 4 beta 7 mediates adhesion to MAdCAM-1 but alpha 4 beta 1 does not, we used beta 7/beta 1 chimeras to directly assess the importance of specific regions of beta 7 in MAdCAM-1 binding. We found a region of beta 7 (residues 46-386) that accounts for specificity of alpha 4 beta 7 binding to MAdCAM-1. We also used human/mouse and human/rat chimeric beta 7 subunits to map epitopes recognized by fifteen anti-beta 7 mAbs. Six of seven Abs that block adhesion to MAdCAM-1 and E-cadherin (Fib 21, 22, 27, 30, 504; Act-1) mapped to amino acid residues 176-250. Residues 176-250 lie within the region of beta 7 that specifies MAdCAM-1 binding and also within a region that has a predicted structure homologous to the metal ion-dependent adhesion site (MIDAS) domains of the integrin subunits alpha L and alpha M. Three new Abs that recognize beta 7 in the presence of Mn2+, but not Ca2+, and promote adhesion to MAdCAM-1, mapped to amino acids 46-149. One blocking and five other Abs mapped to other regions (amino acids 387-725). We conclude that a MIDAS-like domain serves a critical role in beta 7 integrin-mediated adhesion.

  View details for Web of Science ID A1997XL78900055

  View details for PubMedID 9233649

• Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue AMERICAN JOURNAL OF PATHOLOGY Briskin, M., WinsorHines, D., Shyjan, A., Cochran, N., Bloom, S., Wilson, J., McEvoy, L. M., BUTCHER, E. C., Kassam, N., Mackay, C. R., NEWMAN, W., Ringler, D. J. 1997; 151 (1): 97-110

  Abstract

  Lymphocyte homing to normal tissues and recruitment to inflammatory tissue sites are controlled, in part, by the selective expression of chemokines, pro-inflammatory cytokines and mediators, and various adhesion proteins and molecules. In the mouse, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is selectively expressed on endothelium of high endothelial venules in gut and gut-associated lymphoid tissue. By interaction with its integrin ligand, alpha 4 beta 7, lymphocytes presumed to be involved in mucosal immunity are selectively recruited to these intestinal sites. After generating monoclonal antibodies against a murine cell line expressing recombinant human MAdCAM-1, we qualitatively and semiquantitatively assessed MAdCAM-1 expression in human tissue sections from various normal and inflammatory disorders. We found that human MAdCAM-1, as in the mouse, is expressed in a tissue-selective manner. In normal tissues, MAdCAM-1 is constitutively expressed to endothelium of venules of intestinal lamina propria. Interestingly, using computer-assisted morphometric analysis, the proportion of venular endothelium within lamina propria that expresses MAdCAM-1 is increased, compared with normal tissues, at inflammatory foci associated with ulcerative colitis and Crohn's disease. Moreover, for the most part, MAdCAM-1 is not detected in the majority of normal or inflamed extra-intestinal tissues, including those with mucosal surfaces. These results are consistent with a role, as originally defined in the mouse, for human MAdCAM-1 in the localization of alpha 4 beta 7+ lymphocytes in the gastrointestinal tract and associated lymphoid tissue. As such, the pathway defined by MAdCAM-1/alpha 4 beta 7 may be a relevant tissue-specific therapeutic target for the modulation of inflammatory bowel disease activity.

  View details for Web of Science ID A1997XH22700014

  View details for PubMedID 9212736

  View details for PubMedCentralID PMC1857942

• Novel vascular molecule involved in monocyte adhesion to aortic endothelium in models of atherogenesis JOURNAL OF EXPERIMENTAL MEDICINE McEvoy, L. M., Sun, H. L., Tsao, P. S., Cooke, J. P., Berliner, J. A., BUTCHER, E. C. 1997; 185 (12): 2069-2077

  Abstract

  Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.

  View details for Web of Science ID A1997XF79100005

  View details for PubMedID 9182678

• Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule-1 display JOURNAL OF IMMUNOLOGY SZABO, M. C., BUTCHER, E. C., McEvoy, L. M. 1997; 158 (12): 5584-5588

  Abstract

  Follicular dendritic cells (FDC) in the mucosal lymphoid organs, Peyer's patches (PP), display mucosal vascular addressin-cell adhesion molecule-1 (MAdCAM-1). MAdCAM-1 decorates interlacing dendritic cells throughout PP follicles and is accentuated on FDC of the germinal center (GC) light zone and on "junctional" dendritic cells overlapping the B/T border and subepithelial dome region, sites associated with microenvironmental homing decisions. In contrast, MAdCAM-1 is rarely displayed by coronal or junctional FDC in peripheral lymph node follicles and is largely confined to the GC after lymph node immunization. FDC-associated MAdCAM-1 plays a prominent role in lymphocyte adhesion to GC in PP frozen sections and participates significantly in binding to GC in chronically stimulated lymph nodes and spleen, but not to GC in lymph nodes after primary immunization (where binding is dominated by vascular cell adhesion molecule-1). Differential display of MAdCAM-1 by FDC could contribute to the specialization of mucosal vs nonmucosal immune responses.

  View details for Web of Science ID A1997XE74900002

  View details for PubMedID 9190904

• alpha 4 beta 7 integrin expression is associated with the leukemic evolution of human and murine T-cell lymphoblastic lymphomas AMERICAN JOURNAL OF PATHOLOGY Dolcetti, R., Giardini, R., Doglioni, C., Cariati, R., Pomponi, F., DOrazi, C., Rao, S., Lazarovits, A. I., BUTCHER, E. C., Boiocchi, M. 1997; 150 (5): 1595-1605

  Abstract

  We have previously shown that the in vivo coordinated expression of individual alpha 4 and beta 7 integrin chains correlated with the leukemic potential displayed by cell lines derived from murine lymphoblastic T-cell lymphomas (T-LBLs) when transplanted subcutaneously into syngeneic AKR mice. In the present study, by using immunofluorescence and immunocytochemical analyses, we have confirmed that the in vivo up-regulation of the alpha 4 beta 7 heterodimeric complex is associated with the leukemic behavior of AKR T-LBLs. In addition, when compared with the parental, highly leukemic NQ22 cells, the variant cell line NQ22V exhibited a reduced leukemic potential that was invariably associated with a delayed alpha 4 beta 7 up-regulation in vivo Moreover, the leukemic cell line SJ-1, derived from a spontaneous T-LBL of the SJL strain, also displayed high levels of alpha 4 beta 7 expression with a pattern of tissue distribution similar to that of NQ22 cells from leukemic AKR animals. Of note, in most of the tissues involved by murine T-LBL dissemination, and particularly in liver, kidney, and lung, alpha 4 beta 7-positive leukemic cells were always located around strongly VCAM-1-positive vascular spaces. These findings are consistent with a possible role of alpha 4 beta 7/VCAM-1 interactions in the extravasation and, consequently, in the leukemic dissemination of murine T-LBL cells. Immunocytochemical analysis carried out in 11 human T-LBLs showed that pathological lymph nodes from all 7 cases with bone marrow infiltration at presentation carried alpha 4 beta 7-positive cells, whereas all 4 aleukemic T-LBLs were repeatedly alpha 4 beta 7 negative, also in metachronous lesions. These findings suggest that alpha 4 beta 7-positive human T-LBLs may represent a distinct clinicopathological entity. In addition, alpha 4 beta 7 expression was significantly more prevalent in younger patients (< 11 years; P = 0.02), further supporting such a hypothesis. Moreover, as in murine T-LBLs, the pattern of alpha 4 beta 7 positivity in involved lymph nodes was mainly focal, whereas nearly all neoplastic cells infiltrating bone marrow expressed this integrin, suggesting a possible role for alpha 4 beta 7 in the leukemic dissemination also of human T-LBLs.

  View details for Web of Science ID A1997WX14100010

  View details for PubMedID 9137086

  View details for PubMedCentralID PMC1858193

• Anti-CD43 inhibition of T cell homing JOURNAL OF EXPERIMENTAL MEDICINE McEvoy, L. M., Sun, H. L., Frelinger, J. G., BUTCHER, E. C. 1997; 185 (8): 1493-1498

  Abstract

  The homing of lymphocytes from the blood is controlled by specialized processes of lymphocyte-endothelial cell interaction. Interference with these processes offers the potential to manipulate lymphocyte traffic, and thus to modulate normal and pathologic immune and inflammatory responses. We selected antilymphocyte monoclonal antibodies (mAbs) for inhibition of lymphocyte binding in vitro to lymph node high endothelial venules (HEV), specialized vessels that support lymphocyte recruitment into lymph nodes. mAb L11 blocks T cell binding to lymph node and Peyer's patch HEV and inhibits T cell extravasation from the blood into organized secondary lymphoid tissues. In contrast, L11 has no effect on lymphocyte binding to purified vascular ligands for L-selectin, alpha4beta7, or LFA-1, suggesting that it inhibits by a novel mechanism. The L11 antigen is CD43, a sialomucin implicated in vitro in regulation of lymphocyte activation, whose expression is often dysregulated in the Wiskott-Aldrich syndrome. CD43 represents a novel target for experimental and therapeutic manipulation of lymphocyte traffic and may help regulate T cell distribution in vivo.

  View details for Web of Science ID A1997WW04700011

  View details for PubMedID 9126930

• Novel method for following lymphocyte traffic in mice using [H-3]glycerol labeling JOURNAL OF IMMUNOLOGICAL METHODS Constantin, G., Laudanna, C., BUTCHER, E. C. 1997; 203 (1): 35-44

  Abstract

  We describe a new method based on radioactive metabolic labeling with [3H]glycerol to study the lymphocyte trafficking in mice. Lymphocyte labeling with [3H]glycerol is time- and dose-dependent. Radioactive leaking is less significant than in 51Cr-labeled cells. Lymphocytes, labeled with [3H]glycerol, with 51Cr, or with both labels together show the same pattern of homing to Peyer's patches (PP), peripheral and mesenteric lymph nodes and spleen and homing shows the expected dependence on pertussis toxin (PTX)-sensitive signaling, suggesting that the labeling procedure with [3H]glycerol does not affect lymphocyte trafficking properties. Tissue accumulation can be readily assessed by scintillation counting of sonicated samples obtained after perfusion of the vasculature with saline to remove blood. Moreover, we show that cell labeling with [3H]glycerol provides improved sensitivity in assessing the accumulation of small numbers of labeled cells in non-lymphoid organs, and permits identification of homed leukocytes in histologic sections.

  View details for Web of Science ID A1997WU65700004

  View details for PubMedID 9134028

• Differential expression of tissue-specific adhesion molecules on human circulating antibody-forming cells after systemic, enteric, and nasal immunizations - A molecular basis for the compartmentalization of effector B cell responses 7th International Congress of Mucosal Immunology QUIDINGJARBRINK, M., Nordstrom, I., GRANSTROM, G., Kilander, A., Jertborn, M., BUTCHER, E. C., Lazarovits, A. I., Holmgren, J., Czerkinsky, C. AMER SOC CLINICAL INVESTIGATION INC. 1997: 1281–86

  Abstract

  Expression of the adhesion molecules CD44, L-selectin (CD62L), and integrin alpha 4 beta 7 by antibody-secreting cells (ASC) was examined in human volunteers after oral, rectal, intranasal, or systemic immunization with cholera toxin B subunit. Almost all blood ASC, irrespective of immunization route, isotype (IgG and IgA), and immunogen, expressed CD44. On the other hand, marked differences were observed between systemically and intestinally induced ASC with respect to expression of integrin alpha 4 beta 7 and L-selectin, adhesion molecules conferring tissue specificity for mucosal tissues and peripheral lymph nodes, respectively. Thus, most ASC induced at systemic sites expressed L-selectin, whereas only a smaller proportion of ASC expressed alpha 4 beta 7. In contrast, virtually all IgA- and even IgG-ASC detected after peroral and rectal immunizations expressed alpha 4 beta 7, with only a minor fraction of these cells expressing L-selectin. Circulating ASC induced by intranasal immunization displayed a more promiscuous pattern of adhesion molecules, with a large majority of ASC coexpressing L-selectin and alpha 4 beta 7. These results demonstrate that circulating ASC induced by mucosal and systemic immunization express different sets of adhesion molecules. Furthermore, these findings provide for the first time evidence for differential expression of adhesion molecules on circulating ASC originating from different mucosal sites. Collectively, these results may explain the anatomical division of mucosal and systemic immune responses in humans as well as the compartmentalization of mucosal immune responses initiated in the upper vs. the lower aerodigestive tract.

  View details for Web of Science ID A1997WQ61300021

  View details for PubMedID 9077537

  View details for PubMedCentralID PMC507943

• RANTES stimulation of T lymphocyte adhesion and activation: Role for LFA-1 and ICAM-3 EUROPEAN JOURNAL OF IMMUNOLOGY SZABO, M. C., BUTCHER, E. C., MCINTYRE, B. W., Schall, T. J., Bacon, K. B. 1997; 27 (5): 1061-1068

  Abstract

  The chemokine RANTES is a potent chemoattractant and activator of T lymphocytes. Mechanisms underlying the RANTES-induced activation of T lymphocytes leading to adhesion and migration have not been fully analyzed. We investigate here the function of RANTES in the regulation of T cell adhesion, specifically the induction of homotypic aggregation. RANTES induced the expression of many important cell surface adhesion and activation receptors in a normal human T cell clone and peripheral blood T lymphocytes, including members of the beta 1 and beta 2 integrin family, CD44, CD50, and CD28. Up-regulation of these markers correlated with RANTES-stimulated homotypic adhesion of T cells. This homotypic aggregation event was RANTES dose-dependent, prolonged, and pertussis toxin-independent, but herbimycin A-sensitive, suggesting that it involves signaling through alternative (G alpha i protein-independent) pathways. Using specific monoclonal antibodies, the homotypic aggregation event was shown to be lymphocyte function-associated antigen-1 (LFA-1)-dependent, with no observable interaction through alpha 4 or beta 1 integrins. Intercellular adhesion molecule-3 (ICAM-3) and possibly ICAM-1 participate as LFA-1 ligands. Additionally, RANTES phosphorylated the beta chain of LFA-1 1-2 min following stimulation. These results imply a specific role for the chemokine RANTES in T cell activation and intercellular adhesion.

  View details for Web of Science ID A1997WZ36900003

  View details for PubMedID 9174593

• Monoclonal antibodies specific for beta(7) integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RB(high) CD4(+) T cells JOURNAL OF IMMUNOLOGY Picarella, D., Hurlbut, P., Rottman, J., Shi, X. J., Butcher, E., Ringler, D. J. 1997; 158 (5): 2099-2106

  Abstract

  Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is an adhesion protein expressed on endothelium in mucosal tissues that has been shown to play an important role in the selective homing of lymphocytes to intestinal mucosa and associated lymphoid tissue. To determine whether MAdCAM-1 or its ligand alpha 4 beta 7 would be appropriate targets for therapeutic intervention in gut-associated inflammation, we tested the ability of rat mAbs specific for beta 7 integrin and MAdCAM-1 to inhibit chronic colonic inflammation in scid mice reconstituted with CD4+ T cells enriched for the CD45RBhigh subpopulation. Abs specific for beta 7 and MAdCAM-1 blocked recruitment of lymphocytes to the colitic colon, and more importantly, these Abs significantly reduced the severity of colonic inflammatory disease in this animal model. Therefore, the adhesive interactions mediated by alpha 4 beta 7 and MAdCAM are intimately involved in leukocyte recruitment to gut in chronic inflammatory disease and may be relevant therapeutic targets for patients with inflammatory bowel disease.

  View details for Web of Science ID A1997WJ66000015

  View details for PubMedID 9036954

• Function of lymphocyte homing-associated adhesion molecules on human natural killer and lymphokine-activated killer cells JOURNAL OF IMMUNOLOGY Uksila, J., SALMI, M., BUTCHER, E. C., TARKKANEN, J., JALKANEN, S. 1997; 158 (4): 1610-1617

  Abstract

  We have analyzed the ability of fresh and rIL-2-activated human NK cells to interact with high endothelial venules (HEV) that are known to support physiologic lymphocyte extravasation, and examined the role of different adhesion molecules in this process. In in vitro HEV-binding assays, NK cells bound to both peripheral lymph node (pLN) and mucosal HEV. Activation by rIL-2 slightly decreased adherence to pLN HEV, but increased adherence to mucosal high endothelium. Markedly fewer NK cells than PBL expressed L-selectin, and the expression was diminished further upon treatment with rIL-2. Inhibition studies showed, however, that L-selectin was the most important single molecule to mediate adhesion to pLN HEV. Binding to mucosal HEV was mediated mainly by CD44 and alpha 4 integrin, and the expression level of these molecules was increased by rIL-2, paralleling the results in HEV-binding assays. Higher m.w. forms of CD44, representing differentially glycosylated/variant forms of CD44, were more abundant on large granular lymphocytes than on unseparated PBL. We conclude that, despite weak recirculatory capacity, NK cells or a subpopulation of NK cells with the correct adhesion molecules can interact with and bind to high endothelial cells. Lymphokines can modulate the expression of adhesion molecules that NK cells utilize for HEV binding. Our results suggest that activation of NK cells with IL-2 may facilitate the extravasation of lymphokine-activated killer cells, especially to mucosal sites, whereas homing to peripheral lymphoid tissues may be diminished. This should be taken into consideration when procedures for lymphokine-activated killer cell immunotherapy are planned.

  View details for Web of Science ID A1997WG30400014

  View details for PubMedID 9029096

• Homing potentials of circulating lymphocytes in humans depend on the site of activation - Oral, but not parenteral, typhoid vaccination induces circulating antibody-secreting cells that all bear homing receptors directing them to the gut JOURNAL OF IMMUNOLOGY KANTELE, A., Kantele, J. M., Savilahti, E., Westerholm, M., ARVILOMMI, H., Lazarovits, A., BUTCHER, E. C., MAKELA, P. H. 1997; 158 (2): 574-579

  Abstract

  Specific Ab-secreting cells (ASC) appear in the human blood as a response to oral and parenteral vaccination. The actual contribution of these cells to the defense of the body depends on their final effector site. The homing potentials of mucosally and parenterally induced ASC were compared by examining the homing receptor (HR) expression of circulating specific ASC in the blood of volunteers vaccinated orally or parenterally with the same Ag, Salmonella typhi Ty21a. Circulating lymphocytes were separated into receptor-positive and -negative populations, and the numbers of specific ASC were assayed. The alpha4 beta7 integrin, which acts as a gut HR, was expressed on all (99%) of the mucosally activated ASC, but on only 58% of the parenterally induced ASC or 58% of all Ig-secreting cells of the unvaccinated controls. L-selectin, the peripheral lymph node HR, showed an inverse distribution; it was found on 42% of mucosally activated ASC and on 86% of parenterally induced ASC. These results reveal that all of the circulating ASC after oral vaccination are committed to migrate to the mucosal compartment of the immune system, strongly arguing for a recirculation of activated mucosal cells in humans. By contrast, ASC induced by parenteral vaccination with the same Ag are mostly directed to the systemic compartment, yet a part of them has mucosal homing attitudes as well. These differences indicate that the site of Ag encounter determines the homing potential of the cell.

  View details for Web of Science ID A1997WC63800006

  View details for PubMedID 8992970

• Genetic defect in T lymphocyte-specific homing into peripheral lymph nodes EUROPEAN JOURNAL OF IMMUNOLOGY Nakano, H., Tamura, T., Yoshimoto, T., Yagita, H., Miyasaka, M., BUTCHER, E. C., Nariuchi, H., Kakiuchi, T., Matsuzawa, A. 1997; 27 (1): 215-221

  Abstract

  Lymphocytes circulating in the bloodstream home into lymph nodes (LN). T cells predominate in peripheral LN (PLN) and B cells in spleen or mucosal tissue, e.g. Peyer's patches (PP). DDD/1 mice are unique in marked paucity of LN cells, especially T cells. T cell frequency in PLN was 20-40% in this strain, compared to 60-80% in others. Immunohistochemistry confirmed the low density of T cells in the subcortical area but normal colonization of B cells in cortical area in PLN of DDD/1. In contrast, the T cell content of peripheral blood and spleen was higher in DDD/1 but that in PP was not significantly different compared to other strains. It was thus concluded that this abnormality in DDD/1 results from a homing defect of T cells into PLN but not from lymphopenia. Genetical analysis showed that the defect in T cell-specific homing was regulated by a single autosomal recessive gene, tentatively designated plt (paucity of lymph node T cells). Reciprocal bone marrow transplantation indicated that the plt phenotype may arise from some defect in PLN stroma but not in lymphocytes. An in vivo homing assay using fluorescence-labeled lymphocytes demonstrated that the homing defect was specific for T cells but not for B cells. A Stamper-Woodruff assay revealed that the binding between lymphocytes and PLN high endothelial venules was normal and that L-selectin and its ligand, peripheral node vascular addressin (PNAd), were expressed and functioned normally in DDD/1. These results taken together indicate that the T cell-specific homing into PLN is disturbed at a post-adhesion stage in DDD/1. The product of the plt locus may play a pivotal role at this stage.

  View details for Web of Science ID A1997WD56200031

  View details for PubMedID 9022021

• Phenotype, and migration properties of three major subsets of tissue homing T cells in sheep EUROPEAN JOURNAL OF IMMUNOLOGY Mackay, C. R., Andrew, D. P., Briskin, M., Ringler, D. J., BUTCHER, E. C. 1996; 26 (10): 2433-2439

  Abstract

  T cells show a bias in their migration pathways: some T cells preferentially migrate to peripheral lymph nodes (LN), some to mucosal tissues, and some to peripheral tissues such as skin. These recirculation pathways were examined in sheep by collecting lymph draining into and out of peripheral and intestinal LN, and using fluorescent dyes to trace the recirculation of the lymph cells. Monoclonal antibodies (mAb) to alpha 4, beta 1, and beta 7 integrins, and L-selectin, were used to define three major populations of recirculating T cells. Naive-type T cells (L-selectin+, alpha 4 beta 1lo beta 7lo) migrated preferentially through peripheral LN. Two memory populations could be defined: alpha 4 beta 1hi beta 7- and alpha 4 beta 7hi beta 1lo. alpha 4 beta 1hi beta 7- T cells were present in lymph draining from the skin. T cells migrating preferentially through intestinal LN were alpha 4 beta 7hi beta 1lo. Consistent with this migration pattern, the endothelial receptor for alpha 4 beta 7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was detected on high endothelial venules within intestinal LN and Peyer's patches, but only weakly on high endothelial venules within peripheral LN. Thus, there are at least three easily definable subsets of T cells, based on integrin expression, which show distinct migration preferences.

  View details for Web of Science ID A1996VM20000024

  View details for PubMedID 8898957

• A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+CD3- cells to colonize lymph nodes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mebius, R. E., STREETER, P. R., Michie, S., BUTCHER, E. C., Weissman, I. L. 1996; 93 (20): 11019-11024

  Abstract

  IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

  View details for Web of Science ID A1996VL33300091

  View details for PubMedID 8855301

• Functional cloning of the cDNA for a human hyaluronan synthase JOURNAL OF BIOLOGICAL CHEMISTRY Shyjan, A. M., Heldin, P., BUTCHER, E. C., Yoshino, T., Briskin, M. J. 1996; 271 (38): 23395-23399

  Abstract

  Hyaluronan is a constituent of the extracellular matrix of connective tissue and is actively synthesized during wound healing and tissue repair to provide a framework for ingrowth of blood vessels and fibroblasts. Changes in the serum concentration of hyaluronan are associated with inflammatory and degenerative arthropathies such as rheumatoid arthritis. In addition, hyaluronan has been implicated as an important substrate for migration of adhesion of leukocytes during inflammation. A human hyaluronan synthase (HuHAS1) cDNA was isolated by a functional expression cloning approach. Transfection of CHO cells conferred hyaluronidase-sensitive adhesiveness of a mucosal T cell line via the lymphocyte hyaluronan receptor, CD44, as well as increased hyaluronan levels in the cultures of transfected cells. The HuHAS1 amino acid sequence shows considerable homology to the hasA gene product of Streptococcus pyogenes, a glycosaminoglycan synthetase from Xenopus laevis (DG42), and is the human homolog of a recently described murine hyaluronan synthase.

  View details for Web of Science ID A1996VH76800070

  View details for PubMedID 8798544

• Peripheral lymphoid tissue-like adhesion molecule expression in nodular infiltrates in inflammatory myopathies NEUROMUSCULAR DISORDERS DeBleecker, J. L., Engel, A. G., BUTCHER, E. C. 1996; 6 (4): 255-260

  Abstract

  Non-granulomatous nodular accumulations of inflammatory cells in inflammatory myopathies were studied to characterize adhesion mechanisms used for leukocyte recruitment. The nodules had a B-cell-rich center surrounded by a helper T-cell-rich peripheral zone, resembling lymph nodes. The T-cell-rich zones harbored high-walled venules resembling high endothelial venules (HEV), whose endothelia frequently expressed ICAM-1, VCAM-1, and less constantly E-selectin. This endothelial adhesion molecule profile differs from that found in polymyositis, inclusion body myositis, or dermatomyositis, but resembles that in lymphoid tissues. Also, the peripheral lymph node addressin, a vascular addressin specific for peripheral lymphoid tissue HEV, was present on many HEV. This adhesion system is probably responsible for the excessive lymphocyte recruitment. The similar cellular organization and lymphocyte recirculation mechanisms of the nodular infiltrates in muscle and of lymph nodes suggest that the former may also produce antibodies.

  View details for Web of Science ID A1996VK75400006

  View details for PubMedID 8887954

• Biology of chemokine and classical chemoattractant receptors: Differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells JOURNAL OF CELL BIOLOGY Campbell, J. J., Qin, S. X., Bacon, K. B., Mackay, C. R., BUTCHER, E. C. 1996; 134 (1): 255-266

  Abstract

  Several chemoattractant receptors can support agonist-induced, integrin-dependent arrest of rolling neutrophils in inflamed venules in vivo, as well as subsequent crawling into tissues. It has been hypothesized that receptors of the Galpha(i)-linked chemoattractant subfamilies, especially receptors for chemokines, may mediate parallel activation-dependent arrest of homing lymphocyte subsets. However, although several chemokines can attract subsets of B or T cells, robust chemoattractant triggering of resting lymphocyte adhesion to vascular ligands has not been observed. To study the biology of individual leukocyte chemoattractant receptors in a defined lymphoid environment, mouse L1/2 pre-B cells and/or human Jurkat T cells were transfected with alpha (IL-8 receptor A) or beta (MIP-1alpha/CC-CKR-1) chemokine receptors, or with the classical chemoattractant C5a (C5aR) or formyl peptide receptors (fPR). All receptors supported robust agonist-dependent alpha4beta1 integrin-mediated adhesion of lymphocytes to VCAM-1. L1/2 cells cotransfected with fPR and beta7 integrin were also induced to bind MAdCAM-1, suggesting common mechanisms coupling chemoattractant receptors to activation of distinct integrins. Adhesion was rapid but transient, with spontaneous reversion to unstimulated levels within 5 min after peak binding. When observed under flow conditions, alpha4beta1-mediated arrest occurred within seconds after initiation of contact and rolling of IL-8RA transfectants on VCAM-1/IL-8 co-coated surface; and arrest reversed spontaneously after a mean of 5 min with a return to rolling behavior. Each of the receptors also conferred agonist-specific chemotaxis; however, whereas strong adhesion required simultaneous occupancy of many receptors with maximal responses above the Kd, chemotaxis in each case was suppressed at high agonist concentrations. The findings indicate that alpha and beta chemokine as well as classical chemoattractant receptors can trigger robust adhesion as well as directed migration of lymphoid cells, but that the requirements for and kinetics of adhesion triggering and chemotaxis are distinct, thus permitting their independent regulation. They suggest that the discordance between proadhesive and chemoattractant responses of circulating lymphocytes to many chemokines may reflect quantitative aspects of receptor expression and/or coupling rather than qualitative differences in receptor signaling.

  View details for PubMedID 8698820

• ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro AMERICAN JOURNAL OF PATHOLOGY Steffen, B. J., Breier, G., BUTCHER, E. C., Schulz, M., Engelhardt, B. 1996; 148 (6): 1819-1838

  Abstract

  The expression of cell adhesion molecules (CAMs) in the choroid plexus was studied in normal brain and during experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse during inflammation induced by intracerebral injection of killed Corynebacterium parvum in the C3H/He mouse. Both ICAM-1 and VCAM-1, but not MAdCAM-1, were constitutively expressed on choroid plexus epithelium but not on the fenestrated capillary endothelial cells within the choroid plexus. During EAE, we observed an up-regulation of ICAM-1 and VCAM-1 and de novo expression of MAdCAM-1 on choroid plexus epithelial cells. In contrast, endothelial cells in the choroid plexus were not induced to express any of the investigated CAMs. In in situ hybridization analysis we demonstrated that ICAM-1, VCAM-1, and MAdCAM-1 were locally synthesized and that the amount of their mRNAs increased in the inflamed choroid plexus. In vitro, primary choroid plexus epithelial cells could be induced to express ICAM-1, VCAM-1, and MAdCAM-1 on their surface after treatment with proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, interferon-gamma, and lipopolysaccharide. To investigate the functional status of the expressed CAMs we performed Stamper-Woodruff binding assays on frozen sections of inflamed and naive brains. ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelial cells mediated binding of lymphocytes via their known ligands LFA-1 and alpha4-integrin, respectively. The expression of ICAM-1, VCAM-1, and MAdCAM-1 on choroid plexus epithelial cells together with the lack of their expression on the fenestrated choroid plexus endothelium raises the possibility that the epithelial blood-cerebrospinal-fluid barrier plays an important role in the immunosurveillance of the central nervous system.

  View details for Web of Science ID A1996UN46800013

  View details for PubMedID 8669469

• A fundamental subdivision of circulating lymphocytes defined by adhesion to mucosal addressin cell adhesion molecule-1 - Comparison with vascular cell adhesion molecule-1 and correlation with beta(7) integrins and memory differentiation JOURNAL OF IMMUNOLOGY Rott, L. S., Briskin, M. J., Andrew, D. P., BERG, E. L., BUTCHER, E. C. 1996; 156 (10): 3727-3736

  Abstract

  The leukocyte integrin alpha 4 beta 7 is a receptor for the vascular mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Most circulating B and T lymphocytes in man are alpha 4+, and on these cells the regulated display of the beta 7 integrin chain determines the expression of alpha 4 beta 7 and, in large part, binding to MAdCAM-1. Among CD4+ T cells, beta 7 high memory cells (including the L-selectin+ subset) bind MAdCAM-1 better than beta 7int naive cells; whereas beta 7- memory cells,including skin homing lymphocytes, interact poorly if at all. Circulating alpha E beta 7+ T cells are alpha 4 beta 7high and also bind MAdCAM-1 well. B cells are also subdivided by beta 7 expression, and beta 7+ B cells bind MAdCAM-1 better than the beta 7low/- subset. The related vascular ligand vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelium primarily in nonmucosal sites of inflammation, interacts with blood lymphocytes (including beta 7high T cells) almost exclusively via alpha 4 beta 1 and binds beta 7low/-(beta 1high) better than beta 7+ B cells and memory cells better than naive CD4+ cells. beta 7-(beta 1high) memory T cells are somewhat enriched over beta 7high memory cells at low (but not at high) VCAM-1 densities. Interestingly, CD56+ NK cells, which express both alpha 4 beta 7 and alpha 4 beta 1, bind well to VCAM-1 but poorly to MAdCAM-1. The findings indicate that the display and function of alpha 4 beta 7 determine integrin-dependent blood lymphocyte interactions with MAdCAM-1, thus delineating discrete mucosal vs nonmucosal lymphocyte populations in vivo; that alpha 4 beta 1 dominates blood lymphocyte interactions with VCAM-1; and that quantitative and qualitative regulation of MAdCAM-1 vs VCAM-1 can critically control the recruitment of specialized lymphocyte subsets during inflammation.

  View details for PubMedID 8621908

• Differential expression of homing molecules on recirculating lymphocytes from sheep gut, peripheral, and lung lymph JOURNAL OF IMMUNOLOGY Abitorabi, M. A., Mackay, C. R., Jerome, E. H., Osorio, O., BUTCHER, E. C., Erle, D. J. 1996; 156 (9): 3111-3117

  Abstract

  The adhesion molecules integrin alpha 4 beta 7 and L-selectin have been hypothesized to help direct selective migration (homing) of lymphocytes to the gut and peripheral lymph nodes, respectively. An important prediction of current models is that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands. To test this prediction, we directly examined the expression of cell adhesion molecules on lymphocytes recirculating through the gut, the periphery, and the lung. Integrin beta 7 was highly expressed on CD4+CD45R- "memory/effector" T cells recirculating through the gut (mesenteric efferent and lower thoracic duct lymph). In contrast, cells recirculating through the periphery (prescapular efferent lymph) and the lung (caudal mediastinal efferent lymph) had much less beta 7 expression. A similar pattern of organ-specific beta 7 expression was seen on B cells. Beta 7 expression corresponded with adhesion to the gut mucosal addressin, MAdCAM-1, in vitro. The peripheral lymph node homing receptor, L-selectin, was expressed at higher levels on CD4+CD45R- T cells from peripheral lymph than on cells from gut or lung lymph. These results provide additional strong support for alpha 4 beta 7 and L-selectin involvement in lymphocyte homing to the gut and to peripheral lymph nodes, respectively. Lymphocytes emigrating from the lung expressed low levels of both homing receptors and likely utilize molecules other than alpha 4 beta 7 and L-selectin for migration to the lung and associated lymphoid tissue.

  View details for Web of Science ID A1996UF74200005

  View details for PubMedID 8617931

• CD34-deficient mice have reduced eosinophil accumulation after allergen exposure and show a novel crossreactive 90-kD protein BLOOD Suzuki, A., Andrew, D. P., Gonzalo, J. A., Fukumoto, M., Spellberg, J., Hashiyama, M., Takimoto, H., Gerwin, N., Webb, I., Molineux, G., Amakawa, R., Tada, Y., Wakeham, A., Brown, J., McNiece, I., Ley, K., BUTCHER, E. C., Suda, T., GUTIERREZRAMOS, J. C., Mak, T. W. 1996; 87 (9): 3550-3562

  Abstract

  CD34 is expressed on the surface of hematopoietic stem/progenitor cells, stromal cells, and on the surface of high-endothelial venules (HEV). CD34 binds L-selectin, an adhesion molecule important for leukocyte rolling on venules and lymphocyte homing to peripheral lymph nodes (PLN). We generated CD34-deficient mutant animals through the use of homologous recombination. Wild-type and mutant animals showed no differences in lymphocyte binding to PLN HEV, in leukocyte rolling on venules or homing to PLN, in neutrophil extravasation into peritoneum in response to inflammatory stimulus, nor in delayed type hypersensitivity. Anti-L-selectin monoclonal antibody (MEL-14) also inhibited these immune responses similarly in both CD34-deficient and wild-type mice. However, eosinophil accumulation in the lung after inhalation of a model allergen, ovalbumin, is several-fold lower in mutant mice. We found no abnormalities in hematopoiesis in adult mice and interactions between mutant progenitor cells and a stromal cell line in vitro were normal. No differences existed in the recovery of progenitor cells after 5-fluorouracil treatment, nor in the mobilization of progenitor cells after granulocyte colony-stimulating factor treatment compared with wild-type animals. Surprisingly, although CD34 was not expressed in these mice, a portion of its 90-kD band crossreactive with MECA79 remained after Western blot. Thus, we have identified an additional molecule(s) that might be involved in leukocyte trafficking. These results indicate that CD34 plays an important role in eosinophil trafficking into the lung.

  View details for Web of Science ID A1996UH14200003

  View details for PubMedID 8611677

• Lymphocyte homing and homeostasis SCIENCE BUTCHER, E. C., Picker, L. J. 1996; 272 (5258): 60-66

  Abstract

  The integration and control of systemic immune responses depends on the regulated trafficking of lymphocytes. This lymphocyte "homing" process disperses the immunologic repertoire, directs lymphocyte subsets to the specialized microenvironments that control their differentiation and regulate their survival, and targets immune effector cells to sites of antigenic or microbial invasion. Recent advances reveal that the exquisite specificity of lymphocyte homing is determined by combinatorial "decision processes" involving multistep sequential engagement of adhesion and signaling receptors. These homing-related interactions are seamlessly integrated into the overall interaction of the lymphocyte with its environment and participate directly in the control of lymphocyte function, life-span, and population dynamics. In this article a review of the molecular basis of lymphocyte homing is presented, and mechanisms by which homing physiology regulated the homeostasis of immunologic resources are proposed.

  View details for Web of Science ID A1996UD59700037

  View details for PubMedID 8600538

• Distribution of alpha(4)beta(7) and alpha(E)beta(7) integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes EUROPEAN JOURNAL OF IMMUNOLOGY Andrew, D. P., Rott, L. S., Kilshaw, P. J., BUTCHER, E. C. 1996; 26 (4): 897-905

  Abstract

  Beta 7 is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of beta 7. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express alpha 4 beta 7 and alpha E beta 7 using mAb against alpha E, beta 7 and mAb DATK32 which recognizes a combinatiorial epitope on alpha 4 beta 7+ thymocytes have a mature phenotype: TcR+, CD11a(hi)CD44(hi)HSA(dull). Small subsets of double-negative CD4-CD8-, single-positive CD4+ and CD8+ thymocytes express beta 7, while double-positive CD4+CD8+ thymocytes are beta 7-. However, two integrins alpha E beta 7 and alpha 4 beta 7 recognized by anti-beta 7 are not expressed on an identical subpopulation of thymocytes, as alpha E beta 7+ alpha 4 beta 7-, alpha E beta 7 + alpha 4 beta 7+ and alpha E beta 7- alpha 4 beta 7+ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of alpha E beta 7 but little alpha 4 beta 7. In the spleen, Peyer's patches and lymph nodes, alpha 4 beta 7 is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4+ and CD8+ lymphocytes, express high levels of beta 7 in the form of alpha 4 beta 7 and alpha E beta 7, although, as observed with lymphocytes, not all alpha 4 beta 7 hi CD4+ lymphocytes expressed alpha 4 beta 7. The population of alpha 4 beta 7 hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4+ lymphocyte population, which can be further subdivided on the basis of alpha E beta 7, L-selectin and alpha 4 expression. Therefore, memory CD4+ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.

  View details for PubMedID 8625986

• Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: Correlation with transendothelial chemotactic potential EUROPEAN JOURNAL OF IMMUNOLOGY Qin, S. X., LaRosa, G., Campbell, J. J., SmithHeath, H., Kassam, N., Shi, X. J., Zeng, L., BUTCHER, E. C., Mackay, C. R. 1996; 26 (3): 640-647

  Abstract

  The differential expression of chemokine receptors may be an important mechanism for the regulation of T cell migration. To test this, we examined the expression and function of the monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 receptors on various population of T cells. Using a simple and reliable transendothelial chemotaxis assay, both MCP-1 and IL-8 were shown to be chemotactic for subsets of blood T cells, although the relative response varied from donor to donor. To examine receptor expression and correlate it with chemotaxis of T cell subsets, monoclonal antibodies (mAb) to the receptors were produced by immunizing mice either with synthetic peptides (MCP-1 receptor), or with receptor transfectants (IL-8 receptors A and B). A flow cytometric analysis of blood T cells with an anti-MCP-1 receptor mAb revealed low expression on the CD26hi subset and undetectable expression on other T cells. Staining of T cells with anti-Il-8RA and anti-IL-8RB showed much higher levels of expression, but only on a subset of CD3+ cells which were CD8+ and CD56+. That IL-8 and MCP-1 attracted distinct subsets of T cells was best illustrated using the CD26 marker, since IL-8R+ T cells were CD26-, whereas T cells expressing detectable MCP-1R or which responded to MCP-1 in chemotaxis assays were CD26hi. T cells activated in vitro with anti-CD3 up-regulated expression of the MCP-1 receptor, but not the IL-8 receptors, and were attracted to MCP-1 much more efficiently than resting T cells. These results show that there is a clear distinction between the IL-8 and MCP-1-responsive T cell populations and that chemokine receptor expression on T cells may be regulated with respect to linkage as well as cellular activation.

  View details for Web of Science ID A1996UJ76800019

  View details for PubMedID 8605932

• Role of Rho in chemoattractant-activated leukocyte adhesion through integrins SCIENCE Laudanna, C., Campbell, J. J., BUTCHER, E. C. 1996; 271 (5251): 981-983

  Abstract

  Heterotrimeric guanine nucleotide binding protein (G protein)-linked receptors of the chemoattractant subfamily can trigger adhesion through leukocyte integrins, and in this role they are thought to regulate immune cell-cell interactions and trafficking. In lymphoid cells transfected with formyl peptide or interleukin-8 receptors, agonist stimulation activated nucleotide exchange on the small guanosine triphosphate-binding protein RhoA in seconds. Inactivation of Rho by C3 transferase exoenzyme blocked agonist-induced lymphocyte alpha4beta1 adhesion to vascular cell adhesion molecule-1 and neutrophil beta2 integrin adhesion to fibrinogen. These findings suggest that Rho participates in signaling from chemoattractant receptors to trigger rapid adhesion in leukocytes.

  View details for PubMedID 8584934

• Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor alpha(4)beta(7) - Common themes among integrin-Ig family interactions JOURNAL OF IMMUNOLOGY Briskin, M. J., Rott, L., BUTCHER, E. C. 1996; 156 (2): 719-726

  Abstract

  The mucosal vascular addressin, MAdCAM-1, is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in mucosal lymphoid tissues and lamina propria. MAdCAM-1 binds the lymphocyte homing receptor for Peyer's patches, the integrin alpha 4 beta 7. We describe a point mutation within the first Ig domain of MAdCAM-1 that abolishes activation-independent alpha 4 beta 7 binding. This point mutation resides within an eight-amino acid motif with homology to sequences important for the integrin binding ability of the related vascular Ig family members, ICAM-1 and VCAM-1. To understand the contributions of the individual MAdCAM-1 domains, chimeric exchanges with the beta 1 integrin ligand, ICAM-1, were made. The two N-terminal domains of MAdCAM-1 are sufficient to confer alpha 4 beta 7 binding comparable to that of native MAdCAM-1. A chimera containing only the N-terminal Ig domain (domain 1) of MAdCAM-1 can also bind (to a lesser extent) alpha 4 beta 7, but only after integrin (to a lesser extent) alpha 4 beta 7, but only after integrin activation. Conversely, the first domain of ICAM-1 appears sufficient to bind activated LFA-1. These data suggest that the first domain of MAdCAM-1 is sufficient for interaction with alpha 4 beta 7, but that sequences within the second domain support this interaction, either by providing additional contact points for integrin binding or by contributing to the conformation or presentation of the N-terminal domain. The second domain of MAdCAM-1 can also support activation-dependent LFA-1 binding to domain 1 of ICAM-1. The findings parallel studies of VCAM-1 binding to alpha 4 beta 1 and suggest that structural differences exist between vascular Ig-like ligands for alpha 4 vs beta 2 integrins.

  View details for Web of Science ID A1996TP36600038

  View details for PubMedID 8543825

• Generalized and compartmentalized mucosal immune responses in humans: Cellular and molecular aspects 8th International Congress of Mucosal Immunology QUIDINGJARBRINK, M., Eriksson, K., Lakew, M., Butcher, E., Banchereau, J., Lazarovits, A., Holmgren, J., Czerkinsky, C. ACADEMIC PRESS INC. 1996: 477–487

  View details for Web of Science ID A1996BG31B00034

• INHIBITION OF T-CELL COSTIMULATION BY VCAM-1 PREVENTS MURINE GRAFT-VERSUS-HOST DISEASE ACROSS MINOR HISTOCOMPATIBILITY BARRIERS JOURNAL OF IMMUNOLOGY Schlegel, P. G., Vaysburd, M., Chen, Y. F., BUTCHER, E. C., Chao, N. J. 1995; 155 (8): 3856-3865

  Abstract

  Activation of T cells leading to graft-vs-host disease (GVHD) requires two signaling events: the Ag-specific signal generated through the engagement of the TCR/CD3 complex with antigenic peptide fragments presented by MHC molecules on APCs and the second signal provided through additional costimulatory ligands. T cells have preferential costimulatory requirements depending on their state of activation-induced maturation. In the present study, we investigated the role of the receptor-ligand pair VLA-4 (alpha 4 beta 1) and VCAM-1 in allogeneic T cell responses in vitro and in vivo. Anti-VCAM-1 mAb effectively inhibited mixed lymphocyte culture (MLC) across several major MHC barriers and secondary MLC across minor histocompatibility Ags (95.5% and 90.0% inhibition, respectively). In contrast, anti-VLA-4 mAb inhibited a CD8(+)-mediated primed CTL response in vitro by 100%, yet had little effect on proliferative responses. In the B10.D2/nSnJ-->BALB/c (both H-2d) system of GVHD, BALB/c received anti-VCAM-1, or anti-VLA-4 or controls NS-1 or Y13-259 for the first 5 wk after transplant. Anti-VLA-4 mAb delayed the onset of GVHD, but failed to reduce incidence, severity or GVHD-related mortality. In contrast, anti-VCAM-1 reduced the incidence of GVHD from 100% (18/18) in control animals to 53.3% (8/15) (p < 0.01) on day 70 post-transplant and significantly decreased GVHD-related mortality. Sixty percent (9/15) of anti-VCAM-1 recipients survived more than 180 days after transplant. Long-term engraftment of allogeneic bone marrow was documented in all transplanted mice by PCR analysis of a microsatellite region in the IL-1 beta gene.

  View details for Web of Science ID A1995RY58100022

  View details for PubMedID 7561092

• HEAT-STABLE ANTIGEN (MOUSE CD24) SUPPORTS MYELOID CELL-BINDING TO ENDOTHELIAL AND PLATELET P-SELECTIN INTERNATIONAL IMMUNOLOGY Aigner, S., Ruppert, M., Hubbe, M., Sammar, M., Sthoeger, Z., BUTCHER, E. C., VESTWEBER, D., Altevogt, P. 1995; 7 (10): 1557-1565

  Abstract

  P-selectin is a Ca(2+)-dependent lectin that participates in leukocyte adhesion to vascular endothelium and platelets. Myeloid cells and a subset of T lymphocytes express carbohydrate ligands at the cell surface. Previously, we suggested that heat stable antigen (HSA/mouse CD24), an extensively glycosylated cell surface molecule on many mouse cells, is a ligand for P-selectin. Here we show that HSA mediates the binding of monocytic cells and neutrophils to P-selectin. The monocytic cell lines ESb-MP and J774, peritoneal exudate cells, and bone marrow neutrophils could bind to lipopolysaccharide-activated bend3 endothelioma cells under rotation-induced shear forces and this binding was inhibited by mAb to P-selectin and HSA. Blocking was weak at room temperature but more efficient at 4 degrees C when integrin-mediated binding was decreased. Also the adhesion of neutrophils to stimulated platelets expressing P-selectin was blocked by HSA- and P-selectin-specific mAb. Latex beads coated with purified HSA from myeloid cells bound to activated endothelioma cells or platelets, and the binding was similarly blocked by mAb to P-selectin and HSA respectively. The HSA-coated beads were stained with P-selectin-IgG, very weakly with L-selectin-IgG but not with E-selectin-IgG. The staining was dependent on divalent cations and treatment with endoglycosidase F or neuraminidase indicated that sialylated N-linked glycans were recognized. The presence of these glycans was confirmed by biosynthetic labeling studies. Our data suggest that HSA, in addition to the recently identified 160 kDa glycoprotein ligand on mouse neutrophils, belongs to a group of monospecific P-selectin ligands on myeloid cells.

  View details for Web of Science ID A1995TB41300003

  View details for PubMedID 8562500

• A CENTRAL ROLE FOR MICROVILLOUS RECEPTOR PRESENTATION IN LEUKOCYTE ADHESION UNDER FLOW CELL VONANDRIAN, U. H., HASSLEN, S. R., Nelson, R. D., Erlandsen, S. L., BUTCHER, E. C. 1995; 82 (6): 989-999

  Abstract

  Leukocyte adhesion to endothelium requires specialized mechanisms for contact initiation under flow. L-selectin (CD62L), an efficient initiator of adhesion, is clustered on the tips of leukocyte microvilli. To test whether microvillous presentation is critical for contact formation ("tethering"), we transfected lymphoid cells with chimeras of L-selectin and CD44, an adhesion molecule that is excluded from microvilli. CD44 transmembrane and intracellular (TM-IC) domains targeted the L-selectin ectodomain to the planar body, whereas L-selectin TM-IC segments conferred CD44 ectodomain clustering on microvilli. Wild-type and chimeric transfectants bound similarly to anti-ectodomain MAbs in static assays, but MAb binding under flow was much more efficient in the context of microvillous presentation. Similarly, wild-type and chimeric L-selectin possessed equivalent lectin activity, but microvillous presentation dramatically enhanced contact initiation on a native ligand. These findings demonstrate a critical role for receptor topography in leukocyte adhesion and suggest a novel regulatory mechanism of leukocyte trafficking.

  View details for Web of Science ID A1995RW69300015

  View details for PubMedID 7553859

• SOLUBLE L-SELECTIN (SCD62L) UMBILICAL-CORD PLASMA-LEVELS INCREASE WITH GESTATIONAL-AGE PEDIATRIC RESEARCH Buhrer, C., Stibenz, D., Graulich, J., GERNHOLD, U., BUTCHER, E. C., Dudenhausen, J. W., Obladen, M. 1995; 38 (3): 336-341

  Abstract

  L-Selectin (CD62L) is a leukocyte surface membrane glycoprotein involved in extravasation and homotypic aggregation which is rapidly cleaved off after cellular activation. From culture supernatants and body fluids, soluble L-selectin (sCD62L) has been recovered with its functional activity retained. We devised a sensitive enzyme-linked immunoassay for quantitation of sCD62L which was used to measure sCD62L in umbilical cord plasma of 255 human newborns with a gestational age (GA) of 23-43 wk (median 38 wk). sCD62L levels ranged from 1.14-13.8 pmol/mL (median 7.2 pmol/mL) and showed strong correlations with GA (r = 0.71, p < 0.001), birth weight (r = 0.66, p < 0.001), and absolute neutrophil cell counts (ANC) (r = 0.62, p < 0.001) obtained from a peripheral vein within the first 6 h of life (n = 153), whereas there was a weak inverse correlation with absolute normoblast counts (r = -0.27, p < 0.001). In multiple regression analysis, only GA and ANC retained a significant association with sCD62L levels (p < 0.001). Decreased sCD62L levels were found to be associated with multiple gestation (4.8 +/- 2.4 pmol/mL versus 7.7 +/- 2.3 pmol/mL, p < 0.05) also when considering GA and ANC as covariates. In contrast, increased sCD62L levels in infants born from meconium-stained amniotic fluid, and decreased levels in newborns with acute bacterial infection could be fully attributed to differences in GA and ANC.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1995RP60800011

  View details for PubMedID 7494656

• EXPRESSION OF THE MUCOSAL VASCULAR ADDRESSIN, MADCAM-1 - ON SINUS-LINING CELLS IN THE SPLEEN AMERICAN JOURNAL OF PATHOLOGY Kraal, G., Schornagel, K., STREETER, P. R., Holzmann, B., BUTCHER, E. C. 1995; 147 (3): 763-771

  Abstract

  The MAdCAM-1 adhesion molecule is involved in lymphocyte homing into mucosal sites and is expressed on high endothelial venules of Peyer's patches and mesenteric lymph nodes. In the spleen, where high endothelial venules are absent, expression can be found on cells in the marginal zone between red and white pulp. By immunohistochemistry and electron microscopy it was demonstrated that in the spleen cells expressing MAdCAM-1 belong to the population of sinus-lining cells and that the expression is restricted to the sinus-lining cells closest to the lymphoid white pulp. Lymphocytes that migrate from the blood into this white pulp area will have to pass through the rim of cells expressing MAdCAM-1. A functional role for MAdCAM-1 or its lymphocyte ligand, the alpha 4 beta 7 integrin complex, was investigated by in vivo short-term homing experiments with anti-MAdCAM-1 and anti-alpha 4 beta 7 antibodies, but no direct role for this receptor-ligand interaction could be demonstrated.

  View details for Web of Science ID A1995RU16000023

  View details for PubMedID 7677187

  View details for PubMedCentralID PMC1870972

• ORGANIZATION, REGULATORY SEQUENCES, AND ALTERNATIVELY SPLICED TRANSCRIPTS OF THE MUCOSAL ADDRESSIN CELL-ADHESION MOLECULE-1 (MADCAM-1) GENE JOURNAL OF IMMUNOLOGY SAMPAIO, S. O., Li, X., Takeuchi, M., Mei, C., FRANCKE, U., BUTCHER, E. C., Briskin, M. J. 1995; 155 (5): 2477-2486

  Abstract

  The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is expressed selectively at venular sites of lymphocyte extravasation into mucosal lymphoid tissues and lamina propria, where it directs local lymphocyte trafficking. MAdCAM-1 is a multifunctional type l transmembrane adhesion molecule comprising two distal lg domains involved in alpha 4 beta 7 integrin binding, a mucin-like region able to display L-selectin-binding carbohydrates, and a membrane-proximal lg domain homologous to lgA. We show in this work that the MAdCAM-1 gene is located on chromosome 10 and contains five exons. The signal peptide and each one of the three lg domains are encoded by a distinct exon, whereas the transmembrane, cytoplasmic tail, and 3'-untranslated region of MAdCAM-1 are combined on a single exon. The mucin-like region and the third lg domain are encoded together on exon 4. An alternatively spliced MAdCAM-1 mRNA is identified that lacks the mucin/lgA-homologous exon 4-encoded sequences. This short variant of MAdCAM-1 may be specialized to support alpha 4 beta 7-dependent adhesion strengthening, independent of carbohydrate-presenting function. Sequences 5' of the transcription start site include tandem nuclear factor-kappa B sites; AP-1, AP-2, and signal peptide-1 binding sites; and an estrogen response element. Our findings reinforce the correspondence between the multidomain structure and versatile functions of this vascular addressin, and suggest an additional level of regulation of carbohydrate-presenting capability, and thus of its importance in lectin-mediated vs alpha 4 beta 7-dependent adhesive events in lymphocyte trafficking.

  View details for Web of Science ID A1995RP74300021

  View details for PubMedID 7650378

• TABS, A T-CELL ACTIVATION ANTIGEN THAT INDUCES LFA-1-DEPENDENT AGGREGATION JOURNAL OF IMMUNOLOGY Andrew, D. P., Yoshino, T., GUH, L., MARTINSIMONET, M. T., BUTCHER, E. C. 1995; 155 (4): 1671-1684

  Abstract

  We describe here a mAb, DATK44, which induces homotypic aggregation of TK1 cells (a CD8 lymphoma). The glycoprotein recognized by DATK44 is of approximate m.w. 50 kDa and is expressed by monocytes, neutrophils, and subsets of lymphocytes, as well as on the high endothelial venule in peripheral and mesenteric lymph nodes. We named this Ag TABS (T cell activation B cell subset Ag), as TABS appears on T lymphocyte activation and is expressed at low and high levels by B cells. TABS is differentially regulated during T lymphocyte development, CD4+veCD8+ve thymocytes being TABShigh, while single positive CD4+ve and CD8+ve thymocytes are TABSdull CD4-veCD8-ve thymocytes are clearly split into dull and bright populations by the mAb. On exit from the thymus, T lymphocytes cease to express TABS, but T lymphocyte activation results in re-expression of TABS. TABS also shows tight coregulation with heat stable Ag on resting lymphocytes, but coexpression of these two molecules is lost upon lymphocyte activation. DATK44-induced aggregation of TK1 cells is temperature sensitive and blocked by pretreatment of the cells with metabolic inhibitors, genestein, dibutyl cAMP or cytochalasin B, while colchicine, staurosporin, sphingosine, okadaic acid, and W7 are without effect. DATK44-induced TK1 cell aggregation appears to be mediated by the LFA-1 pathway, as aggregation is blocked by anti-LFA-1 and anti-ICAM-1 mAbs but not by Abs capable of blocking CD44 and alpha 4 beta 7-mediated adhesion. Thus, TABS appears to be an adhesion inducer that selectively activates LFA-1-mediated lymphocyte aggregation.

  View details for PubMedID 7543529

• THE ALPHA-4 INTEGRIN CHAIN IS A LIGAND FOR ALPHA-4-BETA-7 AND ALPHA-4-BETA-1 JOURNAL OF EXPERIMENTAL MEDICINE Altevogt, P., Hubbe, M., Ruppert, M., Lohr, J., VONHOEGEN, P., Sammar, M., Andrew, D. P., McEvoy, L., Humphries, M. J., BUTCHER, E. C. 1995; 182 (2): 345-355

  Abstract

  The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

  View details for Web of Science ID A1995RL09800010

  View details for PubMedID 7629498

  View details for PubMedCentralID PMC2192118

• L-SELECTIN AND ALPHA(4)BETA(7) INTEGRIN HOMING RECEPTOR PATHWAYS MEDIATE PERIPHERAL LYMPHOCYTE TRAFFIC TO AKR MOUSE HYPERPLASTIC THYMUS AMERICAN JOURNAL OF PATHOLOGY Michie, S. A., STREETER, P. R., BUTCHER, E. C., Rouse, R. V. 1995; 147 (2): 412-421

  Abstract

  Before the development of thymic lymphoma, AKR mice undergo a striking lymphoid hyperplasia of the thymic medulla. We have previously shown that there is a marked increase in traffic of B and T lymphocytes from the periphery into the preneoplastic, hyperplastic thymuses of these mice, in contrast to the scant traffic of such cells to normal thymuses. The traffic of lymphocytes to lymph nodes and Peyer's patches is controlled in part by the interaction of lymphocyte adhesion molecules called homing receptors with their tissue-selective endothelial ligands known as vascular addressins. We have investigated the roles of homing receptors and vascular addressins in the traffic of lymphocytes to the AKR hyperplastic thymus. We demonstrate that development of hyperplasia is accompanied by an increase in the number of thymic medullary blood vessels with high endothelial venule morphology and expression of the peripheral node addressin (PNAd) and the mucosal addressin (MAdCAM-1). In vitro and in vivo functional assays show that the addressin/homing receptor pairs PNAd/L-selectin and MAdCAM-1/alpha 4 beta 7 are involved in lymphocyte traffic to the hyperplastic thymus. These results indicate that molecular adhesion mechanisms involved in tissue-selective migration of lymphocytes to peripheral lymph node and to mucosal lymphoid tissues play a role in the recruitment of B and T lymphocytes to the AKR thymus and thus in the pathogenesis of thymic hyperplasia.

  View details for Web of Science ID A1995RN76400018

  View details for PubMedID 7543735

  View details for PubMedCentralID PMC1869832

• SPATIAL-DISTRIBUTION OF L-SELECTIN (CD62L) HUMAN-LYMPHOCYTES AND TRANSFECTED MURINE L1-2 CELLS HISTOCHEMICAL JOURNAL HASSLEN, S. R., VONANDRIAN, U. H., BUTCHER, E. C., Nelson, R. D., Erlandsen, S. L. 1995; 27 (7): 547-554

  Abstract

  We have examined the topographical distribution of L-selectin on surface membrane domains of human lymphocytes and murine L1-2 cells transfected to express human L-selectin. L-selectin was immunolocalized using murine monoclonal DREG 200 Fab antibody and a 12 nm colloidal gold-conjugated secondary antibody. Cell surface morphology and surface distribution of gold-labelled L-selectin were visualized using backscatter electron images obtained by high-resolution, field emission scanning electron microscopy. The topographical morphologies of lymphocytes of both types were complex. The surface of human lymphocytes was composed of both microvilli and ruffles; that of the murine cells was composed of long microvilli and few, if any, ruffles. L-selectin on human lymphocytes was observed primarily as focal clusters on the apical surfaces of ruffles and microvilli. Similarly, on the transfected murine cells, L-selectin was detected predominantly on the apical surface of microvilli. We conclude that L-selectin has a common spatial distribution and clustered organization on all leukocytes examined to-date, and that these features of receptor expression likely facilitate rolling of circulating leukocytes on the endothelial surface.

  View details for Web of Science ID A1995RK68200006

  View details for PubMedID 7591847

• DISTINCT ROLES OF L-SELECTIN AND INTEGRINS ALPHA-4-BETA-7 AND LFA-1 IN LYMPHOCYTE HOMING TO PEYERS PATCH-HEV IN-SITU - THE MULTISTEP MODEL CONFIRMED AND REFINED IMMUNITY BARGATZE, R. F., JUTILA, M. A., BUTCHER, E. C. 1995; 3 (1): 99-108

  Abstract

  Circulating lymphocytes home to the mucosal lymphoid organs, Peyer's patches (PP), through high endothelial venules (HEV). In situ analyses revealed that transfused lymph node cells (LNCs) interact with PP-HEV in a series of overlapping adhesion events: L-selectin (CD62L) > alpha 4 beta 7 initiates interaction, L-selectin and alpha 4 beta 7 both participate in rolling, and G alpha i-linked activation triggers arrest that requires both alpha 4 beta 7 and LFA-1. alpha 4 beta 7 dramatically reduces rolling velocity, and appears to be required for engagement of LFA-1. In contrast with resting LNC, preactivated LNC or alpha 4 beta 7hi lymphoma cells require only alpha 4 beta 7 for arrest in PP-HEV. The predominant PP-HEV ligand for alpha 4 beta 7 but also apparently for L-selectin is the mucosal addressin MAd-CAM-1. These results validate the concept of multimolecular adhesion/decision cascades in physiologic lymphocyte-endothelial recognition, define a novel role for alpha 4 integrins as a "bridge" between selectin and beta 2 integrin-dependent events, and reemphasize the potential for direct adhesion through preactivated alpha 4 integrins alone.

  View details for Web of Science ID A1995RK86800011

  View details for PubMedID 7542550

• PRIMITIVE HUMAN HEMATOPOIETIC PROGENITORS ADHERE TO P-SELECTIN (CD62P) BLOOD Zannettino, A. C., Berndt, M. C., Butcher, C., BUTCHER, E. C., Vadas, M. A., Simmons, P. J. 1995; 85 (12): 3466-3477

  Abstract

  P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P-selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX-1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells.

  View details for Web of Science ID A1995RD28800012

  View details for PubMedID 7540063

• LAMININ MEDIATES AGGREGATION OF CD31-TRANSFECTED CELLS Zehnder, J. L., BUTCHER, E. C., Leung, L. L. SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN. 1995: 1153–53

  View details for Web of Science ID A1995RP38500966

• ROLLING OF LYMPHOCYTES AND NEUTROPHILS ON PERIPHERAL NODE ADDRESSIN AND SUBSEQUENT ARREST ON ICAM-1 IN SHEAR-FLOW EUROPEAN JOURNAL OF IMMUNOLOGY Lawrence, M. B., BERG, E. L., BUTCHER, E. C., Springer, T. A. 1995; 25 (4): 1025-1031

  Abstract

  We studied leukocyte interactions in shear flow with peripheral lymph node addressin (PNAd), a mixture of glycoproteins expressed on high endothelial venules (HEV) that is required for lymphocyte homing and has been shown to contain a ligand for L-selectin. T lymphocytes and neutrophils tether and roll on plastic-immobilized PNAd and E-selectin at 1.8 dyn/cm2 wall shear stress, but fail to interact with immobilized ICAM-1, a ligand for LFA-1 and Mac-1, at the same flow rate. Cells roll faster on PNAd than on P-selectin or E-selectin. L-selectin mAb inhibit T lymphocyte and neutrophil tethering to PNAd, but do not inhibit T lymphocyte tethering to purified E-selectin. If allowed to interact with ICAM-1 under static conditions, phorbol ester-treated T lymphocytes, but not resting T lymphocytes, are able to form stationary adhesions that withstand the detachment force generated by 36 dyn/cm2 wall shear stress. In contrast, a wall shear stress of 7.3 dyn/cm2 detaches 50% of resting T lymphocytes bound to PNAd. Incubating T lymphocytes on PNAd and ICAM-1 does not result in adhesion strengthening, suggesting that adhesion through PNAd by L-selectin does not stimulate lymphocyte LFA-1 avidity for ICAM-1. Chemoattractant stimulation of neutrophils or phorbol ester stimulation of lymphoblasts rolling on coimmobilized PNAd and ICAM-1 results in rapid arrest and firm sticking, extending the model of sequential selectin-mediated rolling and subsequent integrin-mediated firm arrest to lymphocytes and ligands expressed on HEV.

  View details for Web of Science ID A1995QY50800023

  View details for PubMedID 7537667

• NOVEL MOUSE ENDOTHELIAL-CELL SURFACE MARKER IS SUPPRESSED DURING DIFFERENTIATION OF THE BLOOD-BRAIN-BARRIER DEVELOPMENTAL DYNAMICS Hallmann, R., MAYER, D. N., BERG, E. L., Broermann, R., BUTCHER, E. C. 1995; 202 (4): 325-332

  Abstract

  Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

  View details for Web of Science ID A1995QU44800001

  View details for PubMedID 7626790

• LYMPHOCYTES INFILTRATING THE CNS DURING INFLAMMATION DISPLAY A DISTINCTIVE PHENOTYPE AND BIND TO VCAM-1 BUT NOT TO MADCAM-1 INTERNATIONAL IMMUNOLOGY Engelhardt, B., CONLEY, F. K., Kilshaw, P. J., BUTCHER, E. C. 1995; 7 (3): 481-491

  Abstract

  The nature of inflammatory lymphocytes recruited to the CNS has been studied in a model of chronic inflammation. Injection of killed Corynebacterium parvum into the cortex of the mouse brain produces a circumscribed inflammatory cellular infiltrate around the injection site, and recruited mononuclear inflammatory cells (IC) can be isolated for flow cytometric analysis. The majority of IC were T cells. In comparison with the predominant naive population of mesenteric lymph node T cells, IC T cells express much higher levels of CD44, LFA-1 and ICAM-1, and lower levels of CD45RB, features commonly associated with memory (previously activated) cells. In addition, in contrast to the L-selectin+ alpha 6-integrinlow phenotype of naive lymph node T cells, IC T cells lacked L-selectin and were alpha 6-integrin-. Mac-1, recently proposed as another marker of memory T cell differentiation, was not displayed by IC T cells, suggesting that Mac-1 expression may be heterogeneous among memory T cell subsets. A subset of mesenteric lymph node (MLN) T cells, probably representing activated T cells undergoing the naive to memory transition, but not of IC T cells, expressed high levels of alpha 6-, beta 7- and alpha E-integrin. IC and MLN naive T cells expressed comparable levels of alpha 4-integrin, but IC T cells stain poorly with anti-beta 7 mAbs and with mAb DATK 32, specific for the alpha 4 beta 7 heterodimeric lymphocyte homing receptor for the mucosal addressin MAdCAM-1, suggesting that these inflammatory cells express more alpha 4 beta 1 than alpha 4 beta 7. Consistent with this, in in vitro adhesion assays, brain IC bound better than MLN cells to the alpha 4 beta 1 integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adhered very poorly to the alpha 4 beta 7 ligand MAdCAM-1. These findings are consistent with and extend previous immunohistological studies of T cells in murine experimental autoimmune encephalomyelitis, and demonstrate a distinctive phenotype for lymphocytes being present in the chronically inflamed brain.

  View details for Web of Science ID A1995QQ04100014

  View details for PubMedID 7540864

• ALPHA-4 INTEGRINS MEDIATE LYMPHOCYTE ATTACHMENT AND ROLLING UNDER PHYSIOLOGICAL FLOW CELL Berlin, C., BARGATZE, R. F., Campbell, J. J., VONANDRIAN, U. H., SZABO, M. C., HASSLEN, S. R., Nelson, R. D., BERG, E. L., Erlandsen, S. L., BUTCHER, E. C. 1995; 80 (3): 413-422

  Abstract

  Of the several families of adhesion receptors involved in leukocyte-endothelial cell interactions, only the selectins have been shown to initiate leukocyte interaction under physiologic shear; indeed, beta 2 (CD18) intergrins responsible for neutrophil arrest are unable to engage without prior selectin-mediated rolling. In contrast, alpha 4 (CD49d) integrins are shown here to initiate lymphocyte contract ("tethering") in vitro under shear and in the absence of a selectin contribution. The alpha 4 integrin ligands MAdCAM-1 and VCAM-1 support loose reversible interactions including rolling, as well as rapid sticking and arrest that is favored following integrin activation. Moreover, alpha 4 beta 7 mediates L-selectin (CD62L)-independent attachment of blood-borne lymphocytes to lamina propria venules in situ. Scanning electron microscopy of alpha 4 beta 7hi lymphoid cells reveals that, like L-selectin, alpha 4 beta 7 is highly concentrated on microvillous sites of initial cellular contact, whereas the beta 2 integrin LFA-1 is excluded from villi. Thus, alpha 4 but not beta 2 integrins can initiate leukocyte adhesion under flow, a capacity that may be in part a function of topographic presentation on microvilli.

  View details for Web of Science ID A1995QG47000007

  View details for PubMedID 7532110

• HUMAN CIRCULATING SPECIFIC ANTIBODY-FORMING-CELLS AFTER SYSTEMIC AND MUCOSAL IMMUNIZATIONS - DIFFERENTIAL HOMING COMMITMENTS AND CELL-SURFACE DIFFERENTIATION MARKERS EUROPEAN JOURNAL OF IMMUNOLOGY QUIDINGJARBRINK, M., Lakew, M., Nordstrom, I., Banchereau, J., Butcher, E., Holmgren, J., Czerkinsky, C. 1995; 25 (2): 322-327

  Abstract

  Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and alpha 4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.

  View details for Web of Science ID A1995QY50600002

  View details for PubMedID 7533081

• DUAL BINDING-CAPACITY OF MUCOSAL IMMUNOBLASTS TO MUCOSAL AND SYNOVIAL ENDOTHELIUM IN HUMANS - DISSECTION OF THE MOLECULAR MECHANISMS JOURNAL OF EXPERIMENTAL MEDICINE SALMI, M., Andrew, D. P., BUTCHER, E. C., JALKANEN, S. 1995; 181 (1): 137-149

  Abstract

  Lymphocytes continuously migrate throughout the body in search of antigens. Virgin lymphocytes recirculate freely between the blood and different lymphatic organs, whereas immunoblasts extravasate preferentially into sites similar to those where they initially responded to antigen. Tissue-specific extravasation of lymphocytes is largely controlled by distinct lymphocyte surface receptors that mediate lymphocyte binding to high endothelial venules (HEV). In the present study, the molecular mechanisms determining the specificity of human mucosal (lamina propria) lymphocyte binding to different endothelial recognition systems were analyzed. Mucosal immunoblasts adhered five times better than small mucosal lymphocytes to mucosal HEV. Importantly, mucosal immunoblasts also bound to synovial HEV almost as efficiently as to mucosal HEV, but they did not adhere to peripheral lymph node HEV. To study the impact of different homing-associated molecules in this dual endothelial binding, we used a gut-derived T cell line and freshly isolated mucosal immunoblasts. Both cell types expressed integrins alpha 4, beta 1, beta 7, and lymphocyte function associated antigen 1 (LFA-1), and were CD44 positive, but practically L-selectin negative. Binding of mucosal immunoblasts to mucosal HEV was almost completely abolished by pretreatment with anti-beta 7 monoclonal antibodies, but it was independent of alpha 4/beta 1 function. In contrast, alpha 4/beta 1 partially mediated immunoblast adherence to synovial HEV, whereas alpha 4/beta 7 had only a minor role in adherence of blasts at this site. CD44 and LFA-1 contributed to HEV-binding both in mucosa and synovium. Taken together, this is the first report that demonstrates a critical role for alpha 4/beta 7 in the binding of gut lymphocytes to mucosal venules in humans. Moreover, a hitherto unknown interaction between mucosal effector cells and synovial endothelial cells was shown to be only partially mediated by the currently known homing receptors. The dual endothelial binding capacity of mucosal blasts may help to explain the pathogenesis of reactive arthritis not uncommonly associated with inflammatory and infectious bowel disease.

  View details for Web of Science ID A1995QA04200015

  View details for PubMedID 7528765

  View details for PubMedCentralID PMC2191840

• Phenotypic characterization of antibody-secreting cells after systemic immunizations in humans 7th International Congress of Mucosal Immunology Lakew, M., Nordstrom, I., QUIDING, M., Holmgren, J., Butcher, E., Czerkinsky, C. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1995: 1451–1453

  View details for Web of Science ID A1995BD67X00312

• Induction of specific immunity at mucosal surfaces: prospects for vaccine development. Advances in experimental medicine and biology Czerkinsky, C., QUIDING, M., Eriksson, K., Nordström, I., Lakew, M., Wenerås, C., Kilander, A., Björck, S., Svennerholm, A. M., Butcher, E. 1995; 371B: 1409-1416

  Abstract

  We have demonstrated the feasibility of studying antigen-specific immune responses in a variety of mucosal tissues in humans after vaccination and during infection. In this respect, we have documented the usefulness of both oral cholera and ETEC vaccines for assessing in functional terms specific subpopulations of B- and T-cell immunocytes during an immune response initiated and/or expressed in human mucosal tissues. Circulating specific IgA antibody-secreting cells in blood appear to reflect recent or ongoing antigen exposure of mucosal surfaces. This implies that the detection of such cells in blood, the most accessible lymphoid compartment in humans, represents the simplest way to assess the immunogenicity of mucosal vaccines and to supplement the diagnostic and monitoring of active mucosal infections. Our studies indicate that while the concept of an integrated mucosal immune network is clearly operational in humans (at least in regards to induction of secretory antibody responses), its generalization appears somewhat simplistic as illustrated by the compartmentalization of immune responses initiated in certain mucosal organs such as the small intestine and the tonsils. Finally, the potential of the cholera toxin B subunit as a carrier for delivery of chemically or genetically linked foreign epitopes for induction of disseminated mucosal immune responses raises hope for the development of broadly applicable vaccines to control mucosal infections.

  View details for PubMedID 7502829

• Induction of specific immunity at mucosal surfaces: Prospects for vaccine development 7th International Congress of Mucosal Immunology Czerkinsky, C., QUIDING, M., Eriksson, K., Nordstrom, I., Lakew, M., WENERAS, C., Kilander, A., Bjorck, S., Svennerholm, A. M., Butcher, E., Holmgren, J. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1995: 1409–1416

  View details for Web of Science ID A1995BD67X00306

• Phenotypic characterization of circulating antibody-secreting cells after mucosal and systemic immunizations in humans. Advances in experimental medicine and biology Lakew, M., Nordström, I., QUIDING, M., Holmgren, J., Butcher, E., Czerkinsky, C. 1995; 371B: 1451-1453

  Abstract

  We have developed a simple approach for immunophenotyping of functional lymphoid cell subpopulations. Using this approach we have partly characterized circulating antigen-specific ASC after systemic versus mucosal immunization with respect to maturational and activation stages, and to their anatomic commitment(s). Comparative analyses of mucosally versus systemically activated circulating ASC reveal differences with respect not only to utilization of homing receptor molecules but also to certain activation markers, and especially cell surface MHC class II molecules. We have now extended these studies to several other markers including early and late B-cell maturation markers, and we are currently examining whether differential expression of HLA-DQ molecules on the majority of mucosally activated blood ASC may explain the relative inability of these cells to present soluble antigens to class II-restricted T-cells.

  View details for PubMedID 7502835

• SULFATION-DEPENDENT RECOGNITION OF HIGH ENDOTHELIAL VENULES (HEV)-LIGANDS BY L-SELECTIN AND MECA-79, AN ADHESION-BLOCKING MONOCLONAL-ANTIBODY JOURNAL OF EXPERIMENTAL MEDICINE Hemmerich, S., BUTCHER, E. C., ROSEN, S. D. 1994; 180 (6): 2219-2226

  Abstract

  L-selectin is a lectin-like receptor that mediates the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes during the process of lymphocyte recirculation. Two sulfated, mucin-like glycoproteins known as Sgp50/GlyCAM-1 and Sgp90/CD34 have previously been identified as HEV-associated ligands for L-selectin. These proteins were originally detected with an L-selectin/Ig chimera called LEC-IgG. GlyCAM-1 and CD34 are also recognized by an antiperipheral node addressin (PNAd) mAb called MECA 79, which blocks L-selectin-dependent adhesion and selectively stains lymph node HEV. The present study compares the requirements for the binding of MECA 79 and LEC-IgG to HEV-ligands. Whereas desialylation of GlyCAM-1 and CD34 drastically reduced binding to LEC-IgG, this treatment enhanced the binding of GlyCAM-1 to MECA 79. In contrast, the binding of both MECA 79 and LEC-IgG to GlyCAM-1 and CD34 was greatly decreased when the sulfation of these ligands was reduced with chlorate, a metabolic inhibitor of sulfation. Because MECA 79 stains HEV-like vessels at various sites of inflammation, recognition by L-selectin of ligands outside of secondary lymphoid organs may depend on sulfation. In addition to their reactivity with GlyCAM-1 and CD34, both MECA 79 and LEC-IgG recognize an independent molecule of approximately 200 kD in a sulfate-dependent manner. Thus, this molecule, which we designate Sgp200, is an additional ligand for L-selectin.

  View details for Web of Science ID A1994PU36100022

  View details for PubMedID 7525849

  View details for PubMedCentralID PMC2191797

• L-SELECTIN AND VERY LATE ANTIGEN-4 INTEGRIN PROMOTE EOSINOPHIL ROLLING AT PHYSIOLOGICAL SHEAR RATES IN-VIVO JOURNAL OF IMMUNOLOGY Sriramarao, P., VONANDRIAN, U. H., BUTCHER, E. C., Bourdon, M. A., Broide, D. H. 1994; 153 (9): 4238-4246

  Abstract

  Adherence of eosinophils to vascular endothelium and their accumulation at sites of allergen challenge are hallmarks of allergic inflammation. However, the molecular mechanisms mediating eosinophil adhesion under conditions of blood flow are not well understood. The present studies were performed to identify the receptors on human eosinophils involved in initiating adhesion to activated endothelium at physiologic shear rates in vivo. We have compared the relative contribution of L-selectin, VLA-4 (CD49d), and CD18 integrins in mediating eosinophil adhesion to microvascular endothelial cells in the rabbit mesentery by using intravital video microscopy. Eosinophils were found to roll in venules, but not arterioles, and this rolling could be stimulated by activation of endothelium with IL-1. In contrast to neutrophil rolling, which is predominantly L-selectin-dependent, eosinophil rolling was mediated by L-selectin, and also VLA-4. mAbs to L-selectin and VLA-4 alpha, but not CD18, significantly inhibited eosinophil rolling in vivo. The inhibition of VLA-4-mediated eosinophil rolling was not caused by modulation of eosinophil L-selectin or CD18 expression. This inhibition also was not caused by nonspecific inhibitory effect of the Abs studied, because the anti-VLA-4 mAbs inhibited eosinophil (VLA-4+) but not neutrophil (VLA-4-) rolling in the mesenteric venules. These results demonstrate that early events of eosinophil adhesion, i.e., rolling, are mediated by multiple adhesion receptors, including L-selectin and VLA-4, at physiologic shear rates in vivo.

  View details for Web of Science ID A1994PN07100040

  View details for PubMedID 7523519

• NEUTROPHILS ROLL ON ADHERENT NEUTROPHILS BOUND TO CYTOKINE-INDUCED ENDOTHELIAL-CELLS VIA L-SELECTIN ON THE ROLLING CELLS JOURNAL OF EXPERIMENTAL MEDICINE BARGATZE, R. F., Kurk, S., BUTCHER, E. C., JUTILA, M. A. 1994; 180 (5): 1785-1792

  Abstract

  Specific arrest of neutrophils in venules is central to their rapid accumulation during local inflammatory responses. Initial neutrophil rolling on endothelium is mediated by leukocyte L-selectin and the inducible vascular adhesion proteins P- and E-selectin. This rolling is a prerequisite for endothelial-dependent neutrophil arrest. Here we describe rolling of neutrophils on the surface of previously arrested neutrophils and demonstrate that this interaction involves L-selectin exclusively on rolling cells. The adherent neutrophil support of L-selectin-dependent neutrophil rolling in vivo can promote continuous and augmented leukocyte recruitment at sites of previous neutrophil accumulation.

  View details for Web of Science ID A1994PP54300021

  View details for PubMedID 7525838

  View details for PubMedCentralID PMC2191733

• DISTINCT BUT OVERLAPPING EPITOPES ARE INVOLVED IN ALPHA(4)BETA(7)-MEDIATED ADHESION TO VASCULAR CELL-ADHESION MOLECULE-1, MUCOSAL ADDRESSIN-1, FIBRONECTIN, AND LYMPHOCYTE AGGREGATION JOURNAL OF IMMUNOLOGY Andrew, D. P., Berlin, C., Honda, S., Yoshino, T., Hamann, A., Holzmann, B., Kilshaw, P. J., BUTCHER, E. C. 1994; 153 (9): 3847-3861

  Abstract

  The mouse CD8+ T cell lymphoma TK1 expresses high levels of alpha 4 beta 7 integrin, which it can use to interact with multiple ligands including mucosal addressin-1 (MAdCAM-1), VCAM-1, and fibronectin. In addition, alpha 4 beta 7 can support TK1 cell aggregation. Here we have produced and characterized a panel of mAbs against alpha 4 beta 7 to define antigenic and functional epitopes associated with its distinct functions. One mAb, DATK32, is unique in recognizing an epitope specific to the alpha 4 beta 7 heterodimer. Furthermore, DATK32 induces TK1 cell aggregation yet inhibits TK1 cell adhesion to MAdCAM-1, VCAM-1, and fibronectin. Considered as a whole, the panel of anti-alpha 4 beta 7 mAbs studied define unique patterns of inhibition for alpha 4 beta 7 binding to each of its defined molecular ligands. We conclude that alpha 4 beta 7 interactions with MAdCAM-1, VCAM-1, and fibronectin can be modulated by Ab binding to distinct epitopes and thus probably involve functionally separable, although physically overlapping binding sites on this multifunctional integrin. These findings are consistent with the general observation that integrins use distinct, potentially differentially regulated interaction sites for adhesion to multiple ligands. Extension of these concepts to alpha 4 beta 7 has important considerations for understanding the roles of this integrin in lymphocyte homing to mucosal sites and in cell-cell interactions during the immune response.

  View details for PubMedID 7523506

• E-SELECTIN MEDIATES LEUKOCYTE ROLLING IN INTERLEUKIN-1-TREATED RABBIT MESENTERY VENULES BLOOD Olofsson, A. M., Arfors, K. E., Ramezani, L., WOLITZKY, B. A., BUTCHER, E. C., VONANDRIAN, U. H. 1994; 84 (8): 2749-2758

  Abstract

  The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class-matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

  View details for Web of Science ID A1994PL35900041

  View details for PubMedID 7522640

• EXPRESSION AND FUNCTION OF THE MADCAM-1 RECEPTOR, INTEGRIN ALPHA-4-BETA-7, ON HUMAN-LEUKOCYTES JOURNAL OF IMMUNOLOGY Erle, D. J., Briskin, M. J., BUTCHER, E. C., GARCIAPARDO, A., Lazarovits, A. I., Tidswell, M. 1994; 153 (2): 517-528

  Abstract

  Recirculation of mouse lymphocytes to the gut involves binding of the lymphocyte integrin alpha 4 beta 7 to the mucosal vascular addressin, MAdCAM-1. In humans, indirect evidence suggests that CD4+ T cells that express high levels of alpha 4 beta 7 migrate selectively to the gut. We now report that human adult blood CD8+ T cells and B cells, like CD4+ T cells, have heterogeneous expression of alpha 4 beta 7. In contrast, NK cells, eosinophils, and newborn blood T and B cells have relatively homogeneous expression of alpha 4 beta 7. CD4+ and CD8+ T cell expression of alpha 4 beta 7 was related to age, CD45RA expression, and integrin beta 1 (CD29) expression, suggesting that alpha 4 beta 7 expression changes after primary activation of CD4+ and CD8+ T cells in vivo. To directly determine whether human alpha 4 beta 7 mediates adhesion to MAdCAM-1, we performed in vitro adhesion assays with two alpha 4 beta 7+ human lymphoma cell lines. The results indicate that human alpha 4 beta 7 is a receptor for MAdCAM-1, whereas alpha 4 beta 1 is not. Adhesion of HUT 78 cells to MAdCAM-1 required Mn2+, whereas adhesion of RPMI 8866 cells did not, suggesting that alpha 4 beta 7 may have at least two distinct functional states. The ability of lymphocytes to bind to MAdCAM-1 and recirculate to mucosal organs is likely to be influenced both by the level of alpha 4 beta 7 expression and by the functional state of the alpha 4 beta 7 molecule.

  View details for Web of Science ID A1994NW18300006

  View details for PubMedID 7517418

• EVIDENCE FOR INVOLVEMENT OF ICAM-1 AND VCAM-1 IN LYMPHOCYTE INTERACTION WITH ENDOTHELIUM IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS IN THE CENTRAL-NERVOUS-SYSTEM IN THE SJL/J MOUSE AMERICAN JOURNAL OF PATHOLOGY Steffen, B. J., BUTCHER, E. C., Engelhardt, B. 1994; 145 (1): 189-201

  Abstract

  We have investigated the expression of vascular adhesion molecules during the first stage of chronic inflammation in experimental autoimmune encephalomyelitis in the SJL/J mouse. Immunocytochemical analysis of frozen sections of inflamed versus noninflamed brains and spinal cords showed that the vascular endothelium in brains and spinal cords from diseased animals expressed high levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) but no detectable mucosal addressin or peripheral lymph node addressin. In frozen section assays, anti-alpha 4 integrin and anti-VCAM-1 monoclonal antibodies inhibited binding of mouse peripheral lymphocytes to inflamed brains at both 4 C and 20 C. Antilymphocyte function-associated antigen-1 and anti-ICAM-1 monoclonal antibodies inhibited binding of mouse peripheral lymphocytes to inflamed brains at 20 C. These results are consistent with an important role for the vascular adhesion molecules VCAM-1 and ICAM-1 and for their lymphocytes receptors in lymphocyte recruitment to the central nervous system.

  View details for Web of Science ID A1994NV86100024

  View details for PubMedID 7518194

• ENHANCED ENDOTHELIAL ADHESIVENESS IN HYPERCHOLESTEROLEMIA IS ATTENUATED BY L-ARGININE CIRCULATION Tsao, P. S., McEvoy, L. M., Drexler, H., BUTCHER, E. C., Cooke, J. P. 1994; 89 (5): 2176-2182

  Abstract

  We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine normalizes NO-dependent vasodilation and markedly inhibits atherogenesis in a hypercholesterolemic rabbit model. We hypothesized that this antiatherogenic effect is due to modulation of endothelial adhesiveness by endothelium-derived NO.New Zealand White rabbits were fed normal chow (Cont), a high-cholesterol diet (Chol), a high-cholesterol diet supplemented with L-arginine (Arg), or a normal diet supplemented with the NO synthase antagonist L-nitroarginine (L-NA) for 2 weeks. In additional studies, some animals receiving L-NA were also treated with hydralazine to normalize blood pressure. After 2 weeks, thoracic aortas were harvested, opened longitudinally, and placed in a culture dish with the endothelial surface exposed to medium containing WEHI 78/24 cells, a monocytoid cell line. After incubation with the monocytoid cells for 30 minutes on a rocking platform, the aortic segments were washed repeatedly to remove nonadherent cells and adherent cells counted by epifluorescent microscopy. Monocytoid cell binding to aortic endothelium was significantly increased in Chol (P < .001 versus Cont); binding was markedly reduced in arginine-fed hypercholesterolemic animals (P < .05, Arg versus Chol). Monocytoid cell binding to aortic endothelium was also significantly increased in L-NA (P < .05); hydralazine normalized blood pressure but did not reduce monocytoid cell binding. To confirm that alterations in NO activity modulate endothelial cell-monocyte interaction, the release of nitrogen oxides (NOx) by thoracic aortas was assessed by a chemiluminescent technique. The concentration of NOx in the conditioned medium from segments of Arg thoracic aortas was significantly greater than that from Cont aortas, whereas that from L-NA aortas was significantly less.Hypercholesterolemia enhances the adhesiveness of aortic endothelium for monocytes; this effect is attenuated by dietary L-arginine. Conversely, inhibition of NO synthesis enhances monocyte binding. The results suggest that endothelium-derived NO plays an important role in regulating the endothelial adhesiveness for monocytes. Alterations in NO activity may play a critical role in atherogenesis.

  View details for Web of Science ID A1994NL67500034

  View details for PubMedID 8181143

• CELL-ADHESION MOLECULES ON VESSELS DURING INFLAMMATION IN THE MOUSE CENTRAL-NERVOUS-SYSTEM JOURNAL OF NEUROIMMUNOLOGY Engelhardt, B., CONLEY, F. K., BUTCHER, E. C. 1994; 51 (2): 199-208

  Abstract

  The expression of endothelial cell adhesion molecules (CAMs) in the central nervous system (CNS) of the mouse was examined during an inflammation induced by intracerebral injection of killed Corynebacterium parvum (C. parvum). We showed that injection of killed C. parvum produced an inflammatory cellular infiltrate limited to the injected brain hemisphere. However, the upregulation of ICAM-1 and VCAM-1 on brain endothelium occurred starting 2 days after C. parvum injection throughout the entire CNS and was not restricted to vessels surrounded by a cellular infiltrate. In contrast to the systemic upregulation of ICAM-1 and VCAM-1, cerebral vessels located in the center of the cellular infiltrate started to express the MECA-32 antigen, suggesting an altered functional status of the endothelial cells, as this antigen is suppressed during development of the blood-brain barrier (BBB). Binding assays performed on frozen sections of inflamed brains are consistent with an important role for endothelial VCAM-1 in the recruitment of lymphocytes during inflammation in the CNS of the mouse.

  View details for Web of Science ID A1994NL35800010

  View details for PubMedID 7514186

• LIGAND-INDUCED ADHESION TO ACTIVATED ENDOTHELIUM AND TO VASCULAR CELL-ADHESION MOLECULE-1 IN LYMPHOCYTES TRANSFECTED WITH THE N-FORMYL PEPTIDE RECEPTOR JOURNAL OF IMMUNOLOGY Honda, S., Campbell, J. J., Andrew, D. P., Engelhardt, B., Butcher, B. A., Warnock, R. A., Ye, R. D., BUTCHER, E. C. 1994; 152 (8): 4026-4035

  Abstract

  Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

  View details for PubMedID 7511663

• ROLE OF ALPHA(4)-INTEGRINS IN LYMPHOCYTE HOMING TO MUCOSAL TISSUES IN-VIVO JOURNAL OF IMMUNOLOGY Hamann, A., Andrew, D. P., JABLONSKIWESTRICH, D., Holzmann, B., BUTCHER, E. C. 1994; 152 (7): 3282-3293

  Abstract

  Lymphocyte recirculation through different organs is thought to be regulated by adhesion molecules ("homing receptors") recognizing tissue-specific vascular addressins on endothelium. Here we show that the alpha 4/beta 7-integrin has a key role in the migration of mouse lymphocytes to mucosal sites. Homing to Peyer's patches but not to peripheral lymph nodes is inhibited by Fab fragments of mAb PS/2 against the alpha 4-integrin chain, by mAb DATK32 recognizing a combinatorial epitope on the alpha 4/beta 7-integrin, and by mAb FIB30 against the beta 7-chain. The Abs significantly reduce homing of lymphocytes to the intestine, as well. The migration of immunoblasts to gut and gut-associated lymphoid tissue also involves the alpha 4/beta 7-integrin heterodimer. Another anti-alpha 4 Ab, R1-2, which blocks lymphocyte binding to Peyer's patches in the Stamper-Woodruff frozen section assay and lymphocyte adhesion to VCAM-1 and fibronectin, has only minor effects on lymphocyte traffic in vivo. Anti-VCAM-1 Ab as well as the fibronectin peptide CS-1 are without influence on the migration to Peyer's patches or intestine, in contrast to Ab against the mucosal addressin MAdCAM-1. Thus, homing to gut-associated sites is regulated by the alpha 4/beta 7-integrin heterodimer interacting with the vascular addressin, MAdCAM-1, and not with fibronectin or VCAM-1 as counterstructures. Inhibition of homing to Peyer's patches and intestine by the anti-integrin Abs studied was only partial. L-selectin also participates in the homing of small lymphocytes to mucosal sites, especially Peyer's patches, but does not contribute substantially to the localization of blasts into the intestinal wall. The results support a major, but not exclusive role of the alpha 4/beta 7-integrin in lymphocyte traffic to mucosal sites.

  View details for Web of Science ID A1994NC60900005

  View details for PubMedID 7511642

• CLONING AND EXPRESSION OF A CDNA-ENCODING MOUSE ENDOGLIN, AN ENDOTHELIAL-CELL TGF-BETA LIGAND GENE GE, A. Z., BUTCHER, E. C. 1994; 138 (1-2): 201-206

  Abstract

  The rat monoclonal antibody, MJ7/18, which reacts selectively with the endothelium of blood vessels in mouse was used to screen a cDNA library derived from a transformed mouse brain endothelial cell line. The sequence of a cDNA encoding the cell surface MJ7/18 antigen revealed homology to human endoglin, a homodimeric transforming growth factor-beta (TGF-beta)-binding cell-surface glycoprotein expressed predominantly on vascular endothelial cells. Northern blot analysis shows a 3.4-kb single transcript of the mouse endoglin. The mouse endoglin is a type-I integral membrane protein of 653 amino acids (aa). The human and mouse sequences display 71% aa sequence identity with almost identical transmembrane and cytoplasmic domains. Like its human counterpart, mouse endoglin displays significant sequence homology to the type-III TGF-beta receptor in two extracellular domains, as well as striking similarity in the transmembrane and cytoplasmic regions. One of the extracellular regions of homology with TGF-beta receptor III represents a truncated version of a homology unit defining a novel gene family including uromodulin, the pancreatic granule protein gp2, and zona pellucida receptors for sperm. However, unlike its human counterpart, mouse endoglin does not contain an RGD tripeptide which has been suggested as a ligand of integrins.

  View details for Web of Science ID A1994NA66100033

  View details for PubMedID 8125301

• EXPRESSION OF ENDOTHELIAL-CELL ADHESION MOLECULES IN JOINTS AND HEART DURING BORRELIA-BURGDORFERI INFECTION OF MICE CELL ADHESION AND COMMUNICATION Schaible, U. E., VESTWEBER, D., BUTCHER, E. G., Stehle, T., Simon, M. M. 1994; 2 (6): 465-479

  Abstract

  The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both peripheral- (PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.

  View details for Web of Science ID A1994QD61400001

  View details for PubMedID 7538017

• L-SELECTIN-MEDIATED LYMPHOCYTE ROLLING ON MADCAM-1 NATURE BERG, E. L., McEvoy, L. M., Berlin, C., BARGATZE, R. F., BUTCHER, E. C. 1993; 366 (6456): 695-698

  Abstract

  The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.

  View details for Web of Science ID A1993MM26500072

  View details for PubMedID 7505053

• THE HUMAN PERIPHERAL LYMPH-NODE VASCULAR ADDRESSIN - AN INDUCIBLE ENDOTHELIAL ANTIGEN INVOLVED IN LYMPHOCYTE HOMING AMERICAN JOURNAL OF PATHOLOGY Michie, S. A., STREETER, P. R., BOLT, P. A., BUTCHER, E. C., Picker, L. J. 1993; 143 (6): 1688-1698

  Abstract

  The extravasation of blood-borne lymphocytes into organized lymphoid tissues and sites of chronic inflammation is directed in part by interactions of lymphocyte surface adhesion molecules, known as homing receptors, with tissue-selective endothelial ligands called vascular addressins. In mice and humans, lymphocyte L-selectin and the peripheral lymph node addressin (PNAd) form a homing receptor-endothelial ligand pair involved in lymphocyte traffic to peripheral lymph node (PLN). We have examined the tissue distribution and function of human PNAd, using monoclonal antibody MECA-79 and in vitro assays of L-selectin-dependent lymphocyte binding. We demonstrate that PNAd is expressed by human high endothelial venules (HEV) in lymphoid tissues which support lymphocyte adhesion via a PLN-associated recognition system. MECA-79 inhibits adhesion to these HEV of a cell line that binds predominantly via the PLN-homing receptor, L-selectin, but has no effect on adhesion by a mucosal HEV-binding cell line. Furthermore, MECA-79 blocks binding of human peripheral blood mononuclear cells to both PLN and tonsil HEV, but not significantly to HEV in the appendix. In addition, we demonstrate PNAd induction on venules at chronic inflammatory sites in humans, particularly sites with severe or long-standing chronic inflammatory involvement. These results confirm that PNAd functions as a PLN vascular addressin in humans, and that in addition to directing normal lymphocyte recirculation to lymph nodes and tonsils, this addressin likely participates in lymphocyte recruitment to sites of chronic inflammation.

  View details for Web of Science ID A1993ML23400020

  View details for PubMedID 8256856

  View details for PubMedCentralID PMC1887255

• THE PEYER PATCH HIGH ENDOTHELIAL RECEPTOR FOR LYMPHOCYTES, THE MUCOSAL VASCULAR ADDRESSIN, IS INDUCED ON A MURINE ENDOTHELIAL-CELL LINE BY TUMOR-NECROSIS-FACTOR-ALPHA AND IL-1 JOURNAL OF IMMUNOLOGY Sikorski, E. E., Hallmann, R., BERG, E. L., BUTCHER, E. C. 1993; 151 (10): 5239-5250

  Abstract

  The specificity of lymphocyte homing from the blood into a tissue is determined in part by complementary pairs of adhesion receptors on lymphocytes and endothelial cells termed homing receptors and vascular addressins, respectively. The mucosal vascular addressin involved in lymphocyte homing to Peyer's patches is a 66-kDa glycoprotein, MAdCAM-1. Investigation of the regulation and molecular genetics of MAdCAM-1 have been hampered by the lack of a murine cell line expressing this adhesion molecule. We show herein using indirect immunofluorescence studies that MAdCAM-1 can be induced on a murine endothelial cell line, bEnd.3, by cytokines and LPS. Western blot analysis of MAdCAM-1 purified by affinity column chromatography from TNF-alpha-treated bEnd.3 cells demonstrates a 66-kDa protein that comigrates in SDS-PAGE with the MAdCAM-1 constitutively found on high endothelial venules in murine mesenteric lymph nodes. Comparison of MAdCAM-1 expression on the bEnd.3 cells was made to the expression of adhesion molecules ICAM-1 and VCAM-1. MAdCAM-1 and VCAM-1 are not constitutively expressed on the bEND.3 surface but can be induced in a concentration-dependent manner by LPS, TNF-alpha, and IL-1. ICAM-1 is constitutively expressed on the endothelioma surface and expression is increased by TNF-alpha, IL-1, LPS, and IFN-gamma. Surface expression of MAdCAM-1 peaks 12 to 18 h after exposure to TNF-alpha and remains elevated at 48 h, whereas expression of VCAM-1 peaks at 4 h and inducible ICAM-1 peaks between 4 and 18 h. Interestingly, IFN-gamma has differential effects on expression of these three adhesion receptors. IFN-gamma alone induces VCAM-1 and enhances ICAM-1 expression, but does not induce MAdCAM-1. Furthermore, although, preincubation of bEND.3 cells with IFN-gamma modestly increases the induction of ICAM-1 and VCAM-1 in response to TNF-alpha and IL-1, it dramatically reduces the TNF-alpha, IL-1, and LPS-induced expression of MAdCAM-1. MAdCAM-1 on bEnd.3 cells is functional as the murine T lymphoma TK1, known to bind MAdCAM-1, also binds to TNF-alpha-stimulated endothelioma but not to unstimulated cells. This binding is blocked by the antibodies against MAdCAM-1 and against the alpha 4-chain of its integrin receptor, alpha 4 beta 7, on TK1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1993MF99500013

  View details for PubMedID 7693807

• SPECIFICITY OF LEUKOCYTE-ENDOTHELIAL INTERACTIONS AND DIAPEDESIS - PHYSIOLOGICAL AND THERAPEUTIC IMPLICATIONS OF AN ACTIVE DECISION-PROCESS RESEARCH IN IMMUNOLOGY BUTCHER, E. C. 1993; 144 (9): 695-698

  Abstract

  In situ studies have revealed that an active multistep process regulates leukocyte-endothelial cell recognition and diapedesis in vivo. The requirement for sequential engagement of receptors mediating transient adhesion, leukocyte activation, activation-dependent sticking and diapedesis implies that leukocyte recruitment can be regulated at any of these 4 steps, and provides a combinatorial mechanism to explain the remarkable specificity and diversity of leukocyte homing events in vivo. The model will help direct therapeutic approaches to controlling leukocyte traffic.

  View details for Web of Science ID A1993MW20400005

  View details for PubMedID 8159870

• ALPHA-4-BETA-7-INTEGRIN MEDIATES LYMPHOCYTE BINDING TO THE MUCOSAL VASCULAR ADDRESSIN MADCAM-1 CELL Berlin, C., BERG, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A., BUTCHER, E. C. 1993; 74 (1): 185-195

  Abstract

  The mucosal vascular addressin, MAdCAM-1, is an immunoglobulin superfamily adhesion molecule for lymphocytes that is expressed by mucosal venules and helps direct lymphocyte traffic into Peyer's patches (PP) and the intestinal lamina propria. We demonstrate that the lymphocyte integrin alpha 4 beta 7, also implicated in homing to PP, is a receptor for MAdCAM-1. Certain antibodies to alpha 4 and beta 7 integrin chains but not to the beta 2 integrin LFA-1 inhibit lymphocyte binding to purified MAdCAM-1 and to MAdCAM-1 transfectants. Lymph node lymphocytes, alpha 4 beta 7+ TK1 lymphoma cells, and a beta 7-transfected variant of an alpha 4+ B cell line, 38C13, bind constitutively to MAdCAM-1. Binding is enhanced by Mn(++)-induced integrin activation. The related integrin alpha 4 beta 1 supports efficient binding to VCAM-1 but not to MAdCAM-1, even after integrin activation, indicating that MAdCAM-1 is a preferential ligand for alpha 4 beta 7. Alpha 4 beta 7 can also bind VCAM-1, but this requires greater integrin activation than binding to MAdCAM-1. The findings imply a selective role for the interaction of alpha 4 beta 7 and MAdCAM-1 lymphocyte in homing to mucosal sites.

  View details for PubMedID 7687523

• RAPID G-PROTEIN-REGULATED ACTIVATION EVENT INVOLVED IN LYMPHOCYTE BINDING TO HIGH ENDOTHELIAL VENULES JOURNAL OF EXPERIMENTAL MEDICINE BARGATZE, R. F., BUTCHER, E. C. 1993; 178 (1): 367-372

  Abstract

  The homing of blood borne lymphocytes into lymph nodes and Peyer's patches is mediated in part by recognition and binding to specialized high endothelial venules (HEV). Here we demonstrate that a rapid pertussis toxin-sensitive lymphocyte activation event can participate in lymphocyte recognition of HEV. In situ video microscopic analyses of lymphocyte interactions with HEV in exteriorized mouse Peyer's patches reveal that pertussis toxin has no effect on an initial "rolling" displayed by many lymphocytes, but inhibits an activation-dependent "sticking" event required for lymphocyte arrest. This is the first demonstration that physiologic lymphocyte-endothelial interactions can involve sequential rolling, activation, and activation-dependent arrest, previously shown only for neutrophils. The inhibitory effect of the toxin is dependent on its G protein-modifying ADP-ribosyltransferase activity and can be reversed by phorbol myristic acetate, which bypasses cell surface receptors to trigger activation-dependent adhesion. Lymphocyte sticking can occur within 1-3 s after initiation of rolling. We conclude that a rapid receptor-mediated activation event involving G protein signaling can trigger stable lymphocyte attachment to HEV in vivo, and may play a critical role in regulating lymphocyte homing.

  View details for Web of Science ID A1993LH54900036

  View details for PubMedID 8315393

• L-SELECTIN MEDIATES NEUTROPHIL ROLLING IN INFLAMED VENULES THROUGH SIALYL LEWIS(X)-DEPENDENT AND LEWIS(X)-INDEPENDENT RECOGNITION PATHWAYS BLOOD VONANDRIAN, U. H., Chambers, J. D., BERG, E. L., Michie, S. A., Brown, D. A., KAROLAK, D., Ramezani, L., Berger, E. M., Arfors, K. E., BUTCHER, E. C. 1993; 82 (1): 182-191

  Abstract

  The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L-selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1-2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L-selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.

  View details for Web of Science ID A1993LL36600025

  View details for PubMedID 7686786

• IN-VIVO DISTRIBUTION AND CHARACTERIZATION OF 2 NOVEL MONONUCLEAR PHAGOCYTE DIFFERENTIATION ANTIGENS IN MICE JOURNAL OF LEUKOCYTE BIOLOGY JUTILA, M. A., BERG, E. L., Kroese, F. G., Rott, L., Perry, V., BUTCHER, E. C. 1993; 54 (1): 30-39

  Abstract

  Flow cytometry, immunohistology, and SDS-PAGE Western blot analysis were used to characterize two novel anti-mouse mononuclear phagocyte differentiation antigens defined by monoclonal antibodies. One antibody, Monts-4, recognized an 80-100 kd cell-surface protein expressed on resident macrophages in the peritoneum and stained macrophages in the splenic white pulp and marginal zone, liver, lymph node paracortical regions, Peyers patches, and cortex of the thymus. Monts-4 did not stain blood monocytes or monocyte-derived inflammatory macrophages in the peritoneal cavity. Macrophages that were Monts-4 positive became Monts-4 negative within 24-72 h after culturing in vitro. Monoclonal antibody SK39 recognized a 180 kd cell-surface molecule expressed on inflamed peritoneal monocytes/macrophages and stained macrophages in the splenic red pulp, cortex of the thymus, subcapsule and medullary regions of lymph nodes, and in sites of acute and chronic inflammation. No differences were seen in the expression of these antigens in the immunodeficient SCID versus normal BALB/c mouse. A comparison of the distribution and molecular weights of the Monts-4 and SK39 antigens with other described mononuclear phagocyte-specific antigens, including F4/80 and those defined by the M1/70, ERTR9, MOMA1, MOMA2, and SER4 monoclonal antibodies, shows these to be novel mononuclear phagocyte-specific differentiation antigens.

  View details for Web of Science ID A1993LP12900005

  View details for PubMedID 8336077

• MADCAM-1 HAS HOMOLOGY TO IMMUNOGLOBULIN AND MUCIN-LIKE ADHESION RECEPTORS AND TO IGA1 NATURE Briskin, M. J., McEvoy, L. M., BUTCHER, E. C. 1993; 363 (6428): 461-464

  Abstract

  Tissue-specific homing of lymphocytes is regulated by interactions with the endothelium of specialized venules, such as the high endothelial venules (HEV) in lymph nodes and mucosal lymphoid tissues. The mucosal vascular addressin, a 58-66K glycoprotein adhesion receptor for lymphocytes, is selectively expressed on HEV of mucosal lymphoid organ and on lamina propria venules and helps direct lymphocyte traffic to these mucosal tissues. We now report the isolation of a complementary DNA that, on transfection into COS cells, encodes immunoreactive addressin that specifically binds the mucosal HEV-binding T-cell lymphoma TK1. The predicted amino-acid sequence defines the mucosal addressin as a novel immunoglobulin family member, MAdCAM-1, with two amino-terminal domains that display strong homology to previously described vascular adhesion receptors for leukocytes, ICAM-1 (ref. 6) and VCAM-1 (ref. 7). The membrane proximal domain is homologous to the third domain (C alpha 2) of another mucosa-associated immunoglobulin family member, IgA1 (refs 8, 9). In addition to the immunoglobulin domains, there is a serine/threonine-rich region which may serve as a backbone to present carbohydrate ligands to lymphocytes. MAdCAM-1 is thus a complex multidomain receptor displaying several structural motifs that may participate in lymphocyte homing interactions.

  View details for Web of Science ID A1993LE93800062

  View details for PubMedID 8502297

• PMN EXTRAVASATION IN ACUTE-INFLAMMATION - A ROLE FOR SELECTIN INTERACTION IN INITIAL PMN-ENDOTHELIAL CELL RECOGNITION 2ND INTERNATIONAL CONF ON STRUCTURE AND FUNCTION OF MOLECULES INVOLVED IN LEUKOCYTE ADHESION II Picker, L. J., VONANDRIAN, U. H., Burns, A. R., Doerschuk, C. M., Arfors, K. E., BUTCHER, E. C. SPRINGER-VERLAG. 1993: 151–167

  View details for Web of Science ID A1993BX28M00012

• AN ANTIBODY THAT PROMOTES ADHESION EVENTS MEDIATED BY BOTH LFA-1 AND CR3 2ND INTERNATIONAL CONF ON STRUCTURE AND FUNCTION OF MOLECULES INVOLVED IN LEUKOCYTE ADHESION II Robinson, M. K., Andrew, D., Rosen, H., Brown, D., Ortlepp, S., Stephens, P., BUTCHER, E. C. SPRINGER-VERLAG. 1993: 25–33

  View details for Web of Science ID A1993BX28M00003

• NEUTROPHIL AGGREGATION IS BETA(2)-INTEGRIN-DEPENDENT AND L-SELECTIN-DEPENDENT IN BLOOD AND ISOLATED CELLS JOURNAL OF IMMUNOLOGY Simon, S. I., Chambers, J. D., Butcher, E., Sklar, L. A. 1992; 149 (8): 2765-2771

  Abstract

  Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.

  View details for Web of Science ID A1992JT98200033

  View details for PubMedID 1383326

• L-SELECTIN FUNCTION IS REQUIRED FOR BETA-2-INTEGRIN-MEDIATED NEUTROPHIL ADHESION AT PHYSIOLOGICAL SHEAR RATES INVIVO AMERICAN JOURNAL OF PHYSIOLOGY VONANDRIAN, U. H., Hansell, P., Chambers, J. D., Berger, E. M., Torres, I., BUTCHER, E. C., Arfors, K. E. 1992; 263 (4): H1034-H1044

  Abstract

  In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

  View details for Web of Science ID A1992JU80600007

  View details for PubMedID 1384360

• H-CAM EXPRESSION IN THE HUMAN NERVOUS-SYSTEM - EVIDENCE FOR A ROLE IN DIVERSE GLIAL INTERACTIONS JOURNAL OF NEUROCYTOLOGY Vogel, H., BUTCHER, E. C., Picker, L. J. 1992; 21 (5): 363-373

  Abstract

  H-CAM (CD44/Hermes antigen) is an 85-95 kDa widely-distributed cell surface adhesion molecule that participates in diverse cellular interactions. It is an important cell surface receptor of hyaluronate, and has been implicated in the binding of circulating lymphocytes of endothelial cells in the process of lymphocyte homing. Here we define the immunohistological distribution of H-CAM in the human nervous system as a means of assessing its possible participation in nervous system ontogeny and function. H-CAM is widely expressed in human CNS white matter by subsets of glial cells, and within the neuropil of several grey matter structures. Neurons appear uniformly negative. H-CAM+ cells and processes are first detected at 20 weeks gestation in a diffuse subependymal pattern, and staining of the anchoring processes but not the cortical extensions of radial glia is seen by 24 weeks. Beginning at 26 weeks, H-CAM+ astrocytes also demarcate fascicles of axons in developing white matter tracts, becoming diffusely distributed in all CNS white matter by full term gestation. In the mature CNS, fibrous and subpial astrocytes, glial outlines within the glomeruli of the cerebellar granule cell layer, Bergmann glia, and extraneuronal grey matter matrix in certain locations are H-CAM+. In reactive gliosis occurring in foetal and developed brains, H-CAM is strongly and uniformly expressed by GFAP+ astroglial cells. In the PNS, dorsal roots express substantially higher levels of H-CAM than ventral roots, and there is an accompanying inverse staining pattern displayed by weakly immunoreactive posterior horns and positive anterior horns. Also, there is an abrupt cessation of H-CAM expression at the junction of the central and peripheral segments of cranial nerves. These findings indicate the dynamic regulation of H-CAM expression in the developing human nervous system, and suggest the hyaluronate-binding activity and potentially other cell-cell or cell-matrix adhesive functions of H-CAM may play an important role in development of the nervous system.

  View details for Web of Science ID A1992HU16500005

  View details for PubMedID 1607880

• COMPARISON OF L-SELECTIN AND E-SELECTIN LIGAND SPECIFICITIES - THE L-SELECTIN CAN BIND THE E-SELECTIN LIGANDS SIALYL LEX AND SIALYL LEA BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS BERG, E. L., Magnani, J., Warnock, R. A., Robinson, M. K., BUTCHER, E. C. 1992; 184 (2): 1048-1055

  Abstract

  The L- and E-selectins are leukocyte and endothelial cell surface molecules which mediate leukocyte-endothelial cell adhesion by interacting with carbohydrate ligands. In the present study we find that L-selectin, like E-selectin, can interact with synthetic neoglycoproteins containing Sialyl Le(x) (Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta-R), or Sialyl Le(a) (Neu5Ac-alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta-R). Additionally, both the E-selectin and L-selectin can bind the peripheral lymph node addressin, a high endothelial venule ligand for L-selectin. Despite overlapping interactions, the L- and E-selectins discriminate between their native ligands. The peripheral lymph node addressin is a preferential ligand for L-selectin; and furthermore, L-selectin expressing cells do not interact detectably with the cutaneous lymphocyte antigen, a native glycoprotein ligand for E-selectin found on a subset of lymphocytes associated with the skin.

  View details for Web of Science ID A1992HR52600073

  View details for PubMedID 1374233

• ROLE OF INTEGRIN ALPHA-4-BETA-7/ALPHA-4-BETA-P IN LYMPHOCYTE ADHERENCE TO FIBRONECTIN AND VCAM-1 AND IN HOMOTYPIC CELL CLUSTERING JOURNAL OF CELL BIOLOGY Ruegg, C., Postigo, A. A., Sikorski, E. E., BUTCHER, E. C., Pytela, R., Erle, D. J. 1992; 117 (1): 179-189

  Abstract

  Integrins are heterodimeric cell surface proteins that mediate both cell-cell and cell-extracellular matrix interactions. We and others recently identified cDNAs encoding a novel integrin beta subunit, beta 7, in lymphocytes. We have now detected beta 7 mRNA in mouse TK-1 T lymphoma cells, which are known to express the putative Peyer's patch homing receptor alpha 4 beta P. We used an anti-peptide antiserum and a novel mAb against the beta 7 subunit to show that TK-1 cells express beta 7 as the only subunit associated with alpha 4. We conclude that beta 7 and beta P are identical. We also show that activated peripheral blood T cells express alpha 4 beta 7. We studied the function of alpha 4 beta 7/alpha 4 beta P in TK-1 cells, which do not express very late antigen (VLA)-4 (alpha 4 beta 1). Cells adhered to intact fibronectin and to a fibronectin fragment containing the CS-1 region, but not to a fragment containing the RGD sequence. Adhesion to fibronectin was inhibited by antibodies to alpha 4, suggesting that alpha 4 beta 7 is a fibronectin receptor. We confirmed that alpha 4 beta 7 binds to the CS-1 region of fibronectin using affinity chromatography. TK-1 cell adhesion to the vascular cell adhesion molecule VCAM-1 was also inhibited by antibodies to alpha 4, implying that alpha 4 beta 7 also plays a role in the adherence of lymphocytes to endothelial cells. TK-1 cell binding to fibronectin and VCAM-1 is markedly increased by brief PMA stimulation. We also found that mAbs against alpha 4 and beta 7 induce homotypic clustering of TK-1 cells. Taken together these results suggest that alpha 4 beta 7/alpha 4 beta P recognizes some or all of the same widely distributed ligands recognized by VLA-4 (alpha 4 beta 1) and that the role of alpha 4 beta 7/alpha 4 beta P may not be restricted to lymphocyte homing.

  View details for Web of Science ID A1992HL81800016

  View details for PubMedID 1372909

  View details for PubMedCentralID PMC2289398

• ADHESION-MECHANISMS INVOLVED IN INFLAMMATION IN THE CENTRAL-NERVOUS-SYSTEM (CNS) IN THE MOUSE Engelhardt, B., CONLEY, F. K., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 1992: A1894–A1894

  View details for Web of Science ID A1992HH27101703

• A MONOCLONAL-ANTIBODY AGAINST THE BETA-7 INTEGRIN SUBUNIT MEDIATES CELL-ADHESION ACTIVITIES Sikorski, E. E., Martin, M. T., Postigo, A. A., Erle, D. J., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 1992: A1143–A1143

  View details for Web of Science ID A1992HG71901196

• ANTIBODY AGAINST THE LEU-CAM BETA-CHAIN (CD18) PROMOTES BOTH LFA-1-DEPENDENT AND CR3-DEPENDENT ADHESION EVENTS JOURNAL OF IMMUNOLOGY Robinson, M. K., Andrew, D., Rosen, H., Brown, D., Ortlepp, S., Stephens, P., BUTCHER, E. C. 1992; 148 (4): 1080-1085

  Abstract

  The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.

  View details for Web of Science ID A1992HD97500015

  View details for PubMedID 1371129

• PHYSIOLOGICAL AND MOLECULAR MECHANISMS OF LYMPHOCYTE HOMING ANNUAL REVIEW OF IMMUNOLOGY Picker, L. J., BUTCHER, E. C. 1992; 10: 561-591

  Abstract

  The lymphoid system is functionally compartmentalized in vivo into discrete primary, secondary, and tertiary lymphoid organs. Primary lymphoid tissues--the bone marrow and thymus--are responsible for the production of mature "virgin" lymphocytes. Secondary lymphoid tissues--lymph nodes, the spleen, and gut-associated lymphoid tissues--are specialized for the accumulation and presentation of antigen to both virgin and memory lymphocyte subsets. The remainder of the body's tissues may be considered "tertiary" lymphoid tissues, in that they normally contain only a few lymphoid elements, but in the setting of inflammation can be induced to recruit unique subsets of primarily memory lymphocytes. Each lymphoid tissue is further subdivided into discrete microenvironments, each characterized by a distinct complement of lymphocyte subsets and stromal cells. Lymphocyte homing comprises the physiologic processes by which lymphocytes seek out and localize to particular tissues and to specific microenvironments therein. Homing mechanisms play a major role in the maintenance of these specialized microenvironments and are critical for the dispersal and targeting of naive and memory lymphocyte populations that are required for effective immune surveillance. Here, we provide a brief overview of mechanisms thought to control the homing of lymphocyte populations in vivo, focusing in particular on the adhesive interactions involved in lymphocyte-endothelial cell recognition and in the selective extravasation of lymphocyte populations into secondary and tertiary lymphoid tissues.

  View details for Web of Science ID A1992HN01200020

  View details for PubMedID 1590996

• LEUKOCYTE-ENDOTHELIAL CELL-ADHESION AS AN ACTIVE, MULTISTEP PROCESS - A COMBINATORIAL MECHANISM FOR SPECIFICITY AND DIVERSITY IN LEUKOCYTE TARGETING 4TH INTERNATIONAL CONF ON LYMPHOCYTE ACTIVATION AND IMMUNE REGULATION BUTCHER, E. C. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1992: 181–194

  View details for Web of Science ID A1992BX11S00023

  View details for PubMedID 1283049

• LEUKOCYTE-ENDOTHELIAL CELL RECOGNITION - 3 (OR MORE) STEPS TO SPECIFICITY AND DIVERSITY CELL BUTCHER, E. C. 1991; 67 (6): 1033-1036

  View details for Web of Science ID A1991GX16400002

  View details for PubMedID 1760836

• THE CUTANEOUS LYMPHOCYTE ANTIGEN IS A SKIN LYMPHOCYTE HOMING RECEPTOR FOR THE VASCULAR LECTIN ENDOTHELIAL CELL-LEUKOCYTE ADHESION MOLECULE-1 JOURNAL OF EXPERIMENTAL MEDICINE BERG, E. L., Yoshino, T., Rott, L. S., Robinson, M. K., Warnock, R. A., Kishimoto, T. K., Picker, L. J., BUTCHER, E. C. 1991; 174 (6): 1461-1466

  Abstract

  A skin-associated population of memory T lymphocytes, defined by expression of the cutaneous lymphocyte antigen (CLA), binds selectively and avidly to the vascular lectin endothelial cell-leukocyte adhesion molecule 1 (ELAM-1), an interaction that may be involved in targeting of CLA+ T cells to cutaneous sites of chronic inflammation. Here we present evidence that CLA itself is the (or a) lymphocyte homing receptor for ELAM-1. Antigen isolated with anti-CLA monoclonal antibody HECA-452 from human tonsillar lysates avidly binds ELAM-1 transfected mouse cells. Anti-CLA antibody blocks T lymphocyte binding to ELAM-1 transfectants. HECA-452 and ELAM-1 binding to lymphocytes or to isolated tonsillar HECA-452 antigen is abrogated by neuraminidase treatment implying a prominent role for sialic acid in CLA structure and function. The dominant form of CLA on T cells is immunologically distinct from the major neutrophil ELAM-1 ligand, the sialyl Lewis x (sLex) antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc), which is absent, weakly expressed, or masked on T cells. However, neuraminidase treatment of CLA+ T cells, but not of CLA- T cells, reveals Lewis x (CD15) structures. In combination with the known requirement for terminal NeuAc alpha 2-3Gal and fucose residues attached to N-acetylglucosamine for ELAM-1 and HECA-452 binding, this finding suggests that CLA may comprise an additionally sialylated or otherwise modified form of sLex. The identification of a lymphocyte homing receptor for skin may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders.

  View details for Web of Science ID A1991GU64400019

  View details for PubMedID 1720810

• DISSECTION OF MURINE LYMPHOCYTE-ENDOTHELIAL CELL-INTERACTION MECHANISMS BY SV-40-TRANSFORMED MOUSE ENDOTHELIAL-CELL LINES - NOVEL MECHANISMS MEDIATING BASAL BINDING, AND ALPHA-4-INTEGRIN-DEPENDENT CYTOKINE-INDUCED ADHESION EXPERIMENTAL CELL RESEARCH Harder, R., Uhlig, H., KASHAN, A., Schutt, B., Duijvestijn, A., BUTCHER, E. C., Thiele, H. G., Hamann, A. 1991; 197 (2): 259-267

  Abstract

  Lymph node-derived endothelial cells were immortalized by infection with SV40 virus and subclones expressing the marker MECA 325 specific for high-endothelial venules (HEV) were selected. These transformed mouse endothelial (TME-) cell lines grow permanently without requirement for special growth factors. Staining of the selected clones with endothelium-specific antibodies and with anti-von Willebrand factor antiserum and uptake of acetylated low-density lipoprotein provide evidence for their endothelial origin. The vascular addressins identified by mAbs MECA 79 and MECA 367 on HEV are not detectable, indicating that the phenotype of the cells differs from that of HEV-type endothelium. The TME cells display a constitutive capacity to bind lymphocytes. An additional binding component is induced by treatment of the TME cells with TNF alpha. Antibodies against the homing receptor LECAM-1 (lectin-related leucocyte-endothelial cell adhesion molecule 1), alpha 4-integrins, vascular addressins, LFA-1, or ICAM-1 known to block lymphocyte interaction with particular types of HEV were unable to inhibit the basal adhesion to TME cells, indicating that a further binding mechanism in mice is displayed by this cell type. The adhesion component induced by TNF alpha is mediated by alpha 4-integrins since enhanced binding could be blocked by an antibody against mouse alpha 4 (lymphocyte-Peyer's patch adhesion molecule 1/2). TME cell lines therefore seem to be a useful model for the dissection and analysis of hitherto poorly characterized murine lymphocyte/endothelial cell interaction mechanisms.

  View details for Web of Science ID A1991GQ96300018

  View details for PubMedID 1720392

• THE NEUTROPHIL SELECTIN LECAM-1 PRESENTS CARBOHYDRATE LIGANDS TO THE VASCULAR SELECTINS ELAM-1 AND GMP-140 CELL Picker, L. J., Warnock, R. A., Burns, A. R., Doerschuk, C. M., BERG, E. L., BUTCHER, E. C. 1991; 66 (5): 921-933

  Abstract

  LECAM-1 (leukocyte-endothelial cell adhesion molecule 1), the lymphocyte lectin ("selectin") homing receptor for peripheral lymph nodes (PLNs), participates in the earliest interactions of polymorphonuclear neutrophilic leukocytes (PMNs) with inflamed venules. Here, we present evidence that LECAM-1 mediates this function through a novel mechanism--presentation of oligosaccharide ligands to the inducible vascular selectins endothelial-leukocyte adhesion molecule (ELAM-1) and granule membrane protein 140 (GMP-140). PMN, but not lymphocyte, LECAM-1 is modified with the vascular selectin ligand sialyl Lewis x (sLex) and specifically binds ELAM-1-transfected cells. Although only a small fraction of total cell surface sLex, LECAM-1-associated sLex appears to play a prominent role in PMN interactions with cell-associated ELAM-1 and GMP-140, as anti-LECAM-1 monoclonal antibodies or selective removal of cell surface LECAM-1 inhibits PMN binding to vascular selectin transfectants by up to 70%. The enhanced function of LECAM-1-associated sLex may reflect the striking concentration, shown here, of LECAM-1 on PMN surface microvilli, the site of initial cellular contact.

  View details for Web of Science ID A1991GE46000011

  View details for PubMedID 1716182

• LATE TREATMENT WITH ANTI-LFA-1 (CD11A) ANTIBODY PREVENTS CEREBRAL MALARIA IN A MOUSE MODEL EUROPEAN JOURNAL OF IMMUNOLOGY Falanga, P. B., BUTCHER, E. C. 1991; 21 (9): 2259-2263

  Abstract

  CBA/Ca mice injected with Plasmodium berghei develop cerebral malaria (CM) characterized by ataxia and progressive paralysis leading to death 7-9 days after experimental infection. The development of cerebral symptoms is a function of the immune response in susceptible strains, and depends on cell-cell interactions involving T helper cells and mononuclear phagocytes. Here we ask whether antibodies to cell adhesion receptors of the immune system can influence the development of CM in this mouse model. When administrated on day 6 after infection, antibody to the leukocyte integrin leukocyte function-antigen-1 (LFA-1) but not antibodies to MAC-1, LECAM-1 (the MEL-14 antigen), alpha 4 integrin or ICAM-1 dramatically reduced the incidence of CM, leading to survival of most mice until the later onset of anemia. Anti-LFA-1 treatment did not result in a substantial decrease in the monocyte accumulation observed in cerebral vessels of susceptible mice. Its efficacy may be related to the broader roles of LFA-1 in cell-cell interactions important in the later pathogenic stages of the immune response to the parasite. Perturbation of immune cell function through interference with cell adhesion mechanisms may offer an important therapeutic tool in acute, life-threatening immune-mediated disorders.

  View details for Web of Science ID A1991GF65500037

  View details for PubMedID 1679716

• 2-STEP MODEL OF LEUKOCYTE ENDOTHELIAL-CELL INTERACTION IN INFLAMMATION - DISTINCT ROLES FOR LECAM-1 AND THE LEUKOCYTE BETA-2 INTEGRINS INVIVO PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA VONANDRIAN, U. H., Chambers, J. D., McEvoy, L. M., BARGATZE, R. F., Arfors, K. E., BUTCHER, E. C. 1991; 88 (17): 7538-7542

  Abstract

  The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the beta 2 integrins, particularly Mac-1 (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed while Mac-1 expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-1 monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall. Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. These results support a two-step model of leukocyte-endothelial cell interactions: reversible rolling mediated in part by LECAM-1 facilitates leukocyte recruitment by the local microenvironment and precedes activation-dependent firm attachment involving beta 2 integrins.

  View details for Web of Science ID A1991GC99200018

  View details for PubMedID 1715568

  View details for PubMedCentralID PMC52336

• A CARBOHYDRATE DOMAIN COMMON TO BOTH SIALYL LE(A) AND SIALYL LE(X) IS RECOGNIZED BY THE ENDOTHELIAL-CELL LEUKOCYTE ADHESION MOLECULE ELAM-1 JOURNAL OF BIOLOGICAL CHEMISTRY BERG, E. L., Robinson, M. K., MANSSON, O., BUTCHER, E. C., Magnani, J. L. 1991; 266 (23): 14869-14872

  Abstract

  The specificity of endothelial cell leukocyte adhesion molecule-1, ELAM-1, for binding to a panel of carbohydrate structures was determined by a sensitive cell binding assay with immobilized synthetic glycoconjugates. ELAM-1 cDNA transfectants were found to bind Sialyl Lea (sialylated lacto-N-fucopentaose II) or sialylated Lewis a antigen (NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc), as well as or slightly better than Sialyl Lex (sialylated lacto-N-fucopentaose III) or sialylated Lewis X antigen (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 ligands in addition to those containing Sialyl Lex, was also found to bind both Sialyl Lex and Sialyl Lea. Hard sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conformations indicate that Sialyl Lea and Sialyl Lex show a high degree of similarity in both the nonreducing and reducing termini. As Lea and Lex show much weaker reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and fucose residues which on one face of both Sialyl Lex and Sialyl Lea can be similarly positioned. The finding that Sialyl Lea is a potent ligand for ELAM-1 is important, as circulating Sialyl Lea and Sialyl Lex containing mucins which are elevated in the serum of many cancer patients may block leukocyte interactions with ELAM-1 and may contribute to the pathological immunodepression observed in these patients.

  View details for Web of Science ID A1991GB09700011

  View details for PubMedID 1714447

• ANTIBODIES AGAINST HUMAN NEUTROPHIL LECAM-1 (LAM-1/LEU-8/DREG-56 ANTIGEN) AND ENDOTHELIAL-CELL ELAM-1 INHIBIT A COMMON CD18-INDEPENDENT ADHESION PATHWAY INVITRO BLOOD Kishimoto, T. K., Warnock, R. A., JUTILA, M. A., BUTCHER, E. C., Lane, C., Anderson, D. C., Smith, C. W. 1991; 78 (3): 805-811

  Abstract

  Neutrophil adhesion to interleukin-1 (IL-1)-stimulated human umbilical vein endothelial cells (HUVEC) involves the CD18 family of leukocyte integrins (lymphocyte function-associated antigen-1 [LFA-1], Mac-1, and p150,95) and LECAM-1 (DREG-56/LEU-8/LAM-1 antigen) on neutrophils and intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1) on the endothelium. In this study, we compare CD18-independent adhesion pathways mediated by neutrophil LECAM-1 and endothelial ELAM-1 and find that these two pathways overlap in a variety of assays: (1) anti-LECAM-1 and anti-ELAM-1 monoclonal antibody (MoAb) inhibit neutrophil binding to HUVEC, and the inhibitory effect is not additive; (2) anti-LECAM-1 MoAb, like anti-ELAM-1 MoAb, inhibits neutrophil binding to HUVEC stimulated for 3 hours with IL-1, but not to HUVEC stimulated for 8 hours, by which time ELAM-1 expression is downregulated; (3) anti-ELAM-1 MoAb has no effect on transendothelial migration, a CD18-dependent, LECAM-1-independent neutrophil function. Interestingly, anti-ELAM MoAb has a reduced but significant inhibitory effect on the adhesion of activated neutrophils that have shed their cell-surface LECAM-1. We also show that neutrophil binding to ELAM-1-transfected L cells is inhibited not only by anti-ELAM-1 but also by anti-LECAM-1 MoAb. These results suggest that LECAM-1 and ELAM-1 can operate in the same adhesion pathway, possibly as a receptor-counterreceptor pair. LECAM-1 and ELAM-1 are likely to interact with other ligands as well, perhaps through carbohydrate determinants that modify more than one glycoprotein.

  View details for Web of Science ID A1991FZ56600036

  View details for PubMedID 1713515

• THE HUMAN PERIPHERAL LYMPH-NODE VASCULAR ADDRESSIN IS A LIGAND FOR LECAM-1, THE PERIPHERAL LYMPH-NODE HOMING RECEPTOR JOURNAL OF CELL BIOLOGY BERG, E. L., Robinson, M. K., Warnock, R. A., BUTCHER, E. C. 1991; 114 (2): 343-349

  Abstract

  The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.

  View details for Web of Science ID A1991FW73900016

  View details for PubMedID 1712790

• PERIPHERAL LYMPH-NODE HOMING RECEPTOR (LECAM-1) IMMUNOLOGY TODAY Anderson, D. C., BUTCHER, E. C., Gallatin, M., Rosen, S., Kishimoto, K., Lasky, L., Miyasaka, M., Scollay, R., Smith, C. W., Haskard, D. 1991; 12 (7): 216-216

  View details for Web of Science ID A1991FU06500003

  View details for PubMedID 1888429

• EXPRESSION OF VASCULAR ADDRESSINS AND ICAM-1 BY ENDOTHELIAL-CELLS IN THE SPINAL-CORD DURING CHRONIC RELAPSING EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN THE BIOZZI AB/H MOUSE IMMUNOLOGY ONEILL, J. K., Butter, C., Baker, D., Gschmeissner, S. E., Kraal, G., BUTCHER, E. C., TURK, J. L. 1991; 72 (4): 520-525

  Abstract

  The expression of adhesion molecules on central nervous system (CNS) endothelia was examined during chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Biozzi AB/H mouse. Active disease episodes (acute and relapse) were associated with the up-regulation of MALA-2, the murine homologue of intercellular adhesion molecule-1 (ICAM-1), on CNS endothelia and the infiltration of ICAM-1-positive mononuclear cells. In addition, the high endothelial venule (HEV)-associated MECA-325 antigen was evident in perivascular lesions, particularly in relapsing disease. The peripheral lymph node HEV-associated vascular addressin defined by MECA-79 antibody was not detectable in the CNS during CREAE. However, the mucosal HEV addressin was evident in lesions, which ultrastructurally was found to be expressed on the surface of endothelial cells by immunoelectron microscopy. The expression of adhesion molecules, such as ICAM-1, may provide a means by which both the initial neuroantigen-specific and the subsequent antigen-non specific cells extravasate into the CNS. Such infiltration may induce the expression of the vascular addressins which may then provide a means of site-selective cellular recruitment leading to disease progression.

  View details for Web of Science ID A1991FJ18100011

  View details for PubMedID 1674735

  View details for PubMedCentralID PMC1384371

• ELAM-1 IS AN ADHESION MOLECULE FOR SKIN-HOMING T-CELLS NATURE Picker, L. J., Kishimoto, T. K., Smith, C. W., Warnock, R. A., BUTCHER, E. C. 1991; 349 (6312): 796-799

  Abstract

  Endothelial cell leukocyte adhesion molecule-1 (ELAM-1) has been described as an inducible endothelial cell-adhesion molecule for neutrophils, and is believed to have a key role in the extravasation of these cells at sites of acute inflammation. Here we report that ELAM-1-transfected COS cells also bind a unique skin-associated subset of circulating memory T cells defined by the expression of the cutaneous lymphocyte-associated antigen. T cells expressing this antigen bind at least as well as neutrophils to expressed ELAM-1, whereas other lymphocytes in the peripheral blood bind poorly, or not at all. Immunohistological survey of chronically inflamed tissue specimens revealed that vascular expression of ELAM-1 occurs at cutaneous sites in preference to noncutaneous sites. We conclude that at sites of chronic inflammation, ELAM-1 may function as a skin vascular addressin, a tissue-selective endothelial cell-adhesion molecule for skin-homing memory T lymphocytes.

  View details for Web of Science ID A1991EZ66600058

  View details for PubMedID 1705666

• CHEMOTACTIC FACTORS REGULATE LECTIN ADHESION MOLECULE 1 (LECAM-1)-DEPENDENT NEUTROPHIL ADHESION TO CYTOKINE-STIMULATED ENDOTHELIAL-CELLS INVITRO JOURNAL OF CLINICAL INVESTIGATION Smith, C. W., Kishimoto, T. K., ABBASS, O., Hughes, B., Rothlein, R., McIntire, L. V., Butcher, E., Anderson, D. C. 1991; 87 (2): 609-618

  Abstract

  Monoclonal antibodies recognizing CD18, CD11a, CD11b, and neutrophil lectin adhesion molecule 1 (LECAM-1), i.e., the human homologue of the murine MEL-14 antigen, were used to assess the relative contribution of these glycoproteins to neutrophil-endothelial adhesion. Under static conditions, the adhesion of neutrophils to IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers was inhibited by antibodies to CD18, CD11a, and the neutrophil LECAM-1, and the effect of combining anti-LECAM-1 and anti-CD11a was almost additive. Under flow at a wall shear stress 1.85 dyn/cm2, a condition where CD18-dependent adhesion is minimal, anti-LECAM-1 inhibited adhesion by greater than 50%. Chemotactic stimulation of neutrophils induced a rapid loss of LECAM-1 from the neutrophil surface, and the level of neutrophil surface LECAM-1 was closely correlated with adhesion under flow. Neutrophils contacting the activated endothelial cells for 30 min lost much of their surface LECAM-1, a phenomenon induced by a soluble factor or factors released into the medium by the stimulated monolayers, and a high percentage migrated through the HUVEC monolayer. This migration was almost completely inhibited by anti-CD18, but was unaffected by antibodies to neutrophil LECAM-1. These results support the concept that LECAM-1 is a neutrophil adhesion molecule that participates in the adherence of unstimulated neutrophils to cytokine-stimulated endothelial cells under conditions of flow, and is then lost from the neutrophil surface coincident with the engagement of CD18-dependent mechanisms leading to transendothelial migration.

  View details for Web of Science ID A1991EW29500031

  View details for PubMedID 1991844

  View details for PubMedCentralID PMC296350

• THE PERIPHERAL LYMPH-NODE HOMING RECEPTOR, LECAM-1, IS INVOLVED IN CD18-INDEPENDENT ADHESION OF HUMAN NEUTROPHILS TO ENDOTHELIUM BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hallmann, R., JUTILA, M. A., Smith, C. W., Anderson, D. C., Kishimoto, T. K., BUTCHER, E. C. 1991; 174 (1): 236-243

  Abstract

  The binding of polymorphonuclear granulocytes (PMN) to activated vascular endothelium is a crucial step in the recruitment of PMN to an inflammatory site. Studies employing cytokine-activated endothelium in culture have shown that PMN binding involves the CD18 family of leukocyte integrins, but also CD18-independent adhesion mechanism(s) on PMN that have not been defined. We unify here two previously disparate approaches to study cell adhesion events between endothelial cells and leukocytes. We show that antibodies to human LECAM-1, the peripheral lymph node homing receptor that is also expressed on PMN, partially inhibit the adhesion of human PMN not only to HEV in frozen sections of lymph node tissue, but also to cytokine-activated human umbilical vein endothelium in vitro. Inhibition with anti-LECAM-1 antibodies and anti-CD18 antibodies is additive. Furthermore, the anti-LECAM-1 antibodies inhibit the adhesion of CD18-deficient PMN to cytokine activated human endothelial cells. These findings indicate that LECAM-1 and CD18-mediated binding mechanisms are independent, and act coordinately or sequentially to mediate PMN attachment to cytokine activated endothelium.

  View details for Web of Science ID A1991ET20700037

  View details for PubMedID 1671206

• RAT B-CELL SUBSETS IDENTIFIED BY FLOW-CYTOMETRY AND THEIR ANATOMICAL COUNTERPARTS 10TH INTERNATIONAL CONF ON LYMPHATIC TISSUES AND GERMINAL CENTRES IN IMMUNE REACTIONS Kroese, F. G., Hermans, M. H., DEBOER, N. K., BUTCHER, E. C., Herzenberg, L. A. MARCEL DEKKER. 1991: 397–402

  View details for Web of Science ID A1991BV18C00067

• THE HYALURONATE RECEPTOR IS A MEMBER OF THE CD44 (H-CAM) FAMILY OF CELL-SURFACE GLYCOPROTEINS JOURNAL OF CELL BIOLOGY Culty, M., Miyake, K., Kincade, P. W., SILORSKI, E., BUTCHER, E. C., Underhill, C. 1990; 111 (6): 2765-2774

  Abstract

  The present study was undertaken to determine the relationship between the hyaluronate receptor and CD44 (H-CAM), cell-surface glycoproteins of similar molecular weights that have been implicated in cell adhesion. In initial experiments, a panel of monoclonal antibodies directed against CD44 were tested for their ability to cross react with the hyaluronate receptor. These antibodies immunoprecipitated [3H]hyaluronate binding activity from detergent extracts of both mouse and human cells, indicating that the hyaluronate receptor is identical to CD44. In addition, one of these antibodies (KM-201 to mouse CD44) directly blocked the binding of labeled hyaluronate to the receptor and inhibited hyaluronate dependent aggregation of SV-3T3 cells. CD44 has also been implicated in lymphocyte binding to high endothelial venules during lymphocyte homing. Interestingly, the monoclonal antibody Hermes-3, which blocks lymphocyte binding to the high endothelial venules of mucosal lymphoid tissue, had no effect on the binding of labeled hyaluronate. Furthermore, the binding of lymphocytes to high endothelial cells of lymph nodes and mucosal lymphoid tissue was not significantly affected by treatment with agents that block the binding of hyaluronate (hyaluronidase, excess hyaluronate and specific antibodies). Thus, CD44 appears to have at least two distinct functional domains, one for binding hyaluronate and another involved in interactions with mucosal high endothelial venules.

  View details for Web of Science ID A1990ET17600050

  View details for PubMedID 1703543

  View details for PubMedCentralID PMC2116369

• IDENTIFICATION OF MESSENGER-RNA THAT ENCODES AN ALTERNATIVE FORM OF H-CAM(CD44) IN LYMPHOID AND NONLYMPHOID TISSUES IMMUNOGENETICS GOLDSTEIN, L. A., BUTCHER, E. C. 1990; 32 (6): 389-397

  Abstract

  The cluster of differentiation 44 (CD44), hereafter referred to as H-CAM(CD44), represents a novel class of polymorphic (Mr 80,000-215,000) cell adhesion molecules that are involved in cell-cell and cell-matrix adhesion events in a variety of organ systems. We report the detection of distinct mRNAs, in both hematopoietic and nonhematopoietic human cell lines, that encode H-CAM(CD44) with different cytoplasmic domains. Genomic Southern blot analyses indicate that the exons encoding these two cytoplasmic domains are located on the same approximately 16 kilobase (kb) Eco RI restriction fragment. Restriction endonuclease and Southern blot analyses performed on polymerase chain reaction (PCR) amplification copies of these mRNAs confirm that their sequences correspond with previously reported cDNA sequences. A consensus splice donor site which is conserved in human, baboon, and mouse mRNAs that encode a molecule with an elongated cytoplasmic domain (H-CAM-L) is utilized to generate a distinct but low-abundance mRNA species that encodes H-CAM(CD44) with a truncated cytoplasmic domain of only three amino acids (H-CAM-S). Estimations of the relative abundance of these mRNA species in B-lymphoblastoid cells using the PCR amplification technique exhibit average H-CAM-L/H-CAM-S ratios ranging between 100 and 200. Therefore, H-CAM (CD44)-mediated adhesive events may be regulated through a differential capacity of H-CAM-L and H-CAM-S to interact with the cytoskeleton and to participate in intracellular signaling events.

  View details for Web of Science ID A1990EN84900003

  View details for PubMedID 2272659

• DIFFERENTIAL EXPRESSION OF HOMING-ASSOCIATED ADHESION MOLECULES BY T-CELL SUBSETS IN MAN JOURNAL OF IMMUNOLOGY Picker, L. J., Terstappen, L. W., Rott, L. S., STREETER, P. R., Stein, H., BUTCHER, E. C. 1990; 145 (10): 3247-3255

  Abstract

  The ability of lymphocyte populations to recognize and bind high endothelial venules during homing into lymphoid tissues and sites of chronic inflammation is critically dependent on their expression of certain homing-associated adhesion molecules known as homing receptors (HR). In animal models, certain lymphocyte populations, particularly subsets of memory or previously activated lymphocytes, demonstrate tissue-selective homing behavior, and it has been hypothesized that differential expression of HR accounts for this selective migration. In this study, we analyzed expression of human HR--the Dreg 56/Leu 8-defined peripheral lymph node (PLN) HR (also known as LECAM-1), H-CAM (CD44), and alpha 4-integrins (CD49d; VLA-4)--among subsets of thymocytes and peripheral blood T cells to identify populations with differential homing potential. In the thymus, these three HR classes are differentially regulated relative to phenotypically defined maturational stages, but are all expressed on the mature, surface CD3high subset. In the peripheral blood, virgin T cells (LFA-3/CD58low) show uniform high expression of the PLN HR, uniform relatively low expression of H-CAM and alpha 4-integrin, and lack markers of tissue association--the mucosal and cutaneous lymphocyte associated Ag (MLA and CLA Ags) defined by mAb Ber ACT8 and HECA-452, respectively. In contrast, circulating memory T cells (LFA-3/CD58high) are bimodal with respect to PLN HR expression, show uniform high expression of H-CAM and alpha 4-integrin, and contain essentially all the CLA and MLA Ag-bearing T cells. The circulating skin-associated T cell subset (CLA Ag+; 10 to 15% of total T cells) is predominantly PLN HR+, and shows high levels of both the alpha 4- and beta 1-integrin chains. The distinct mucosa-associated T cell subset (MLA Ag+; 1 to 3% of peripheral blood T cells) is predominantly PLN HR-; and is alpha 4high, but beta 1low. These findings indicate the independent regulation of homing-associated adhesion molecules among populations of memory/previously activated T cells, and suggest that the expression patterns of these molecules contribute to, or perhaps determine, the tissue distribution of these subsets.

  View details for Web of Science ID A1990EG88900014

  View details for PubMedID 1700003

• THE RAT B-CELL SYSTEM - THE ANATOMICAL LOCALIZATION OF FLOW CYTOMETRY-DEFINED B-CELL SUBPOPULATIONS EUROPEAN JOURNAL OF IMMUNOLOGY Kroese, F. G., BUTCHER, E. C., Lalor, P. A., STALL, A. M., Herzenberg, L. A. 1990; 20 (7): 1527-1534

  Abstract

  Two-color flow cytometrical (FCM) analysis of rat peripheral lymphoid organs shows two distinct IgM/IgD-defined B cell subpopulations, similar to those of the mouse: a major population of cells expressing little IgM and high levels of IgD (population I) and a minor population of cells expressing high levels of IgM but little IgD (population III). In peripheral lymphoid organs population III cells are mainly found in spleen where they represent about 25% of the B cells; population III cells are almost absent from lymph nodes and Peyer's patches. In adult bone marrow and in neonatal spleen the majority of IgM/IgD-defined B cells (greater than 70%) are population III cells, similar to what is observed in the mouse. In contrast with mice, only a low proportion of the cells (1%) recovered from the peritoneal cavity are B cells, and most of them belong to population I. Previously defined monoclonal antibodies (HIS22 and HIS24) to B cell forms of the leukocyte common antigen (CD45R) in combination with staining for surface IgM and surface IgD demonstrates a further heterogeneity of rat B cells by three-color FCM analyses. HIS22 labels most population I cells; population III cells and a small subset (about one third) of population I express only very low levels of the HIS22 determinant. HIS24 reacts with population I cells and subdivides population III into two subsets: about one third of splenic population III cells are brightly stained with this antibody whereas fluorescence of the remaining two-thirds is lower. The HIS24bright population III cells likely are newly formed B cells since cells with this phenotype are the predominant surface Ig population found in adult bone marrow and neonatal spleen. In tissue sections of lymphoid organs, HIS22- and HIS24-positive cells are mainly found in lymphoid follicles; splenic marginal zones are almost unstained. Combining immunohistological analysis with the FCM data, we therefore conclude that the small follicular B cells are in population I and marginal zone B cells are found in the HIS24dull population III. The in situ localization of HIS24bright population III cells and the HIS22dull population I cells is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1990DV21200017

  View details for PubMedID 2143728

• REGULATION AND LECTIN ACTIVITY OF THE HUMAN NEUTROPHIL PERIPHERAL LYMPH-NODE HOMING RECEPTOR BLOOD JUTILA, M. A., Kishimoto, T. K., BUTCHER, E. C. 1990; 76 (1): 178-183

  Abstract

  We characterize the nature and regulation of a human neutrophil cell surface antigen recognized by monoclonal antibodies (the DREG series) against a human lymphocyte peripheral lymph node homing receptor. Human neutrophils express high levels of the DREG antigen, whose expression is downregulated after treatment with phorbol myristate acetate, or the chemotactic factors C5a and FMLP. Interestingly, C5a treatment also downregulated the monocyte DREG antigen, but had no effect on expression of the lymphocyte molecule. Within 3 minutes after treatment with C5a, greater than 80% of neutrophil DREG antigen expression is lost, and essentially the molecule is completely removed from the cell surface by 5 minutes. The human neutrophil DREG antigen is 10 Kd larger than the lymphocyte molecule. These features are similar to those of the mouse neutrophil MEL-14 antigen (murine peripheral lymph node homing receptor). The mannose-6-phosphate rich phosphomannan (PPME) binds human lymphocytes via the DREG antigen. PPME also binds neutrophils, but little difference in binding is seen between unactivated and activated cells. We show that PPME binding to unactivated neutrophils is mediated primarily by a cation- and DREG antigen-dependent mechanism, whereas activated neutrophil-PPME binding is DREG antigen- and cation-independent, and may be due to the translocation of lysosomal mannose-6-phosphate receptors to the cell surface. The DREG antibodies offer powerful tools for analyzing the role of homing receptors in human neutrophil-endothelial cell interactions, and also may prove valuable in the clinical assessment of neutrophil activation.

  View details for Web of Science ID A1990DM15500025

  View details for PubMedID 2194589

• A UNIQUE PHENOTYPE OF SKIN-ASSOCIATED LYMPHOCYTES IN HUMANS - PREFERENTIAL EXPRESSION OF THE HECA-452 EPITOPE BY BENIGN AND MALIGNANT T-CELLS AT CUTANEOUS SITES AMERICAN JOURNAL OF PATHOLOGY Picker, L. J., Michie, S. A., Rott, L. S., BUTCHER, E. C. 1990; 136 (5): 1053-1068

  Abstract

  It has been proposed that the skin is a functionally unique compartment of the immune system, although little direct evidence supporting this hypothesis has been presented. Here we show that lymphocyte populations at cutaneous sites can be differentiated from otherwise similar populations at noncutaneous sites by their preferential expression of an epitope defined by the MAb HECA-452. This MAb recognizes a predominantly 200-kd cell-surface glycoprotein present on about 16% of peripheral blood T cells, including both CD4+ and CD8+ T cells (17% and 11% HECA-452+, respectively), as well as TCR-delta-bearing T cells (32%+). Most thymocytes (99%) lacked HECA-452 antigen expression, and essentially all the HECA-452+ peripheral blood T cells were found in the adhesion molecule high, CD45R low putative memory cell subset, findings suggesting that HECA-452 expression develops peripherally as a consequence of antigenic stimulation. However, the HECA-452 antigen is not a conventional activation antigen because it was not upregulated with mitogen stimulation of peripheral blood T cells. Most significantly, among 54 diverse specimens of normal/reactive lymphoid tissues and sites of chronic inflammation, there was a clear association of lymphocyte HECA-452 expression and cutaneous location. In extracutaneous sites (n = 38) only about 5% of lymphocytes within the T-cell areas of these tissues expressed this antigen, whereas in inflammatory skin lesions (n = 16), 85% were HECA-452+. The association of HECA-452 expression and cutaneous location was also seen in a series of T-cell lymphomas. The malignant cells of 16 of 18 cases of epidermotropic (patch/plaque) stage mycosis fungoides were HECA-452+, as well as 2 of 7 nonmycosis fungoides peripheral T-cell lymphomas in skin. In contrast, this antigen was not expressed in thymic (lymphoblastic) lymphomas (n = 14), nonepidermotropic (tumor) stage mycosis fungoides (n = 5), and noncutaneous peripheral T-cell lymphomas (n = 15). Among lymphocytes, the preferential expression of the HECA-452 determinant by cutaneous T cells supports the hypothesis that the skin constitutes a immunologically unique lymphoid tissue and suggests that this molecule may play a role in either lymphocyte homing to skin or in lymphocyte interactions with the epidermis.

  View details for Web of Science ID A1990DG37000007

  View details for PubMedID 1693467

• EXPRESSION OF LOW-LEVELS OF PERIPHERAL LYMPH NODE-ASSOCIATED VASCULAR ADDRESSIN IN MUCOSAL LYMPHOID-TISSUES - POSSIBLE RELEVANCE TO THE DISSEMINATION OF PASSAGED AKR LYMPHOMAS JOURNAL OF CELLULAR BIOCHEMISTRY BARGATZE, R. F., STREETER, P. R., BUTCHER, E. C. 1990; 42 (4): 219-227

  Abstract

  Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1990CZ85900004

  View details for PubMedID 2187888

• IDENTIFICATION OF A HUMAN PERIPHERAL LYMPH-NODE HOMING RECEPTOR - A RAPIDLY DOWN-REGULATED ADHESION MOLECULE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kishimoto, T. K., JUTILA, M. A., BUTCHER, E. C. 1990; 87 (6): 2244-2248

  Abstract

  Lymphocyte migration to lymphoid organs involves organ-specific homing receptors. The mouse peripheral lymph node homing receptor, defined by the MEL-14 monoclonal antibody (mAb), is a lectin-like cell surface protein, which is rapidly down-regulated upon cell activation with phorbol 12-myristate 13-acetate. We have raised mAbs against rapidly shed molecules released from the cell surface of activated human leukocytes. Five mAbs, DREG-55, -56, -110, -152, and -200, define an 80- to 85-kDa molecule involved in human lymphocyte recognition of peripheral lymph node (PLN) high endothelial venules (HEVs). The DREG-56 mAb specifically inhibits greater than 90% of binding of human lymphocytes to HEVs within frozen sections of peripheral but not mucosal lymphoid tissue. Furthermore, the gp80 antigen is expressed on lymphoid cell lines that are capable of binding to PLN HEVs. The DREG-56 mAb also inhibits lymphocyte binding of the phosphomannan monoester core from Hansenula hostii Y-2448, an activity associated with human and mouse lymphocyte recognition of PLN HEVs. Finally, all five DREG mAbs specifically stain COS cells transfected with LAM-1 cDNA, a putative human homologue of mouse MEL-14 antigen. These results demonstrate that the DREG mAbs define a human lymphocyte homing receptor for PLN HEVs and indicate that this human antigen is homologous to the MEL-14-defined murine lymphocyte homing receptor.

  View details for Web of Science ID A1990CU85200041

  View details for PubMedID 2179952

• HEMATOPOIETIC PROGENITOR-CELL EXPRESSION OF THE H-CAM (CD44) HOMING-ASSOCIATED ADHESION MOLECULE BLOOD LEWINSOHN, D. M., Nagler, A., Ginzton, N., Greenberg, P., BUTCHER, E. C. 1990; 75 (3): 589-595

  Abstract

  We explored the expression of a lymphocyte homing-associated cell adhesion molecule (H-CAM, CD44) on hematopoietic progenitors. We demonstrate that immature myeloid and erythroid leukemic cell lines stain intensely with monoclonal antibodies Hermes-1 and Hermes-3, which define distinct epitopes on lymphocyte surface H-CAM, a glycoprotein involved in lymphocyte interactions with endothelial cells. Using fluorescence-activated cell sorting (FACS), human marrow cells were fractionated into Hermeshi, Hermesmed, and Hermeslo populations according to the expression of both the Hermes-1 and Hermes-3 epitopes. Granulocyte-macrophage colony-forming unit and erythroid burst-forming unit precursors were found predominantly in the brightly positive fractions. Two-color FACS analysis confirmed that the My10 (CD34) positive populations of cells in bone marrow, which contain most of the progenitor cell activity, are brightly positive for Hermes-1. Finally, we demonstrate that among bone marrow cells, the highest levels of H-CAM are expressed on myeloid and erythroid progenitors as well as mature granulocytes and lymphocytes. Thus we provide evidence that molecules related or identical to the H-CAM homing receptor are expressed on marrow progenitor cells. H-CAM may contribute to progenitor cell interactions with marrow endothelial and stromal cell elements important to the maintenance and regulation of hematopoiesis.

  View details for PubMedID 1688719

• Warner-Lambert/Parke-Davis Award lecture. Cellular and molecular mechanisms that direct leukocyte traffic. American journal of pathology BUTCHER, E. C. 1990; 136 (1): 3-11

  View details for PubMedID 2404417

• HOMING RECEPTORS IN LYMPHOCYTE, NEUTROPHIL, AND MONOCYTE INTERACTION WITH ENDOTHELIAL-CELLS 1ST INTERNATIONAL CONF ON STRUCTURE, FUNCTION AND REGULATION OF MOLECULES INVOLVED IN LEUKOCYTE ADHESION JUTILA, M. A., LEWINSOHN, D. M., BERG, E. L., BUTCHER, E. C. SPRINGER-VERLAG. 1990: 227–235

  View details for Web of Science ID A1990BR23P00017

• LYMPHOCYTE HOMING RECEPTORS AND VASCULAR ADDRESSINS SYMP ON GLYCOBIOLOGY BERG, E. L., GOLDSTEIN, L. A., Picker, L. J., STREETER, P. R., JUTILA, M. A., BARGATZE, R. F., ZHOU, D. F., BUTCHER, E. C. WILEY-LISS, INC. 1990: 91–105

  View details for Web of Science ID A1990BR58W00007

• FUNCTION AND REGULATION OF THE NEUTROPHIL MEL-14 ANTIGEN INVIVO - COMPARISON WITH LFA-1 AND MAC-1 JOURNAL OF IMMUNOLOGY JUTILA, M. A., Rott, L., BERG, E. L., BUTCHER, E. C. 1989; 143 (10): 3318-3324

  Abstract

  The CD11/18 (LFA-1, Mac-1) molecules participate in neutrophil adhesion to cultured endothelium in vitro and are critical for effective neutrophil localization into inflamed tissues in vivo. More recently, the MEL-14 Ag, which was first defined as a lymphocyte homing receptor, has also been implicated in inflammatory neutrophil extravasation. Here we compare the regulation and function of these adhesion molecules on neutrophils during the in vivo inflammatory response. The MEL-14 Ag is expressed at high levels on bone marrow and peripheral blood neutrophils, but is lost on neutrophils isolated from the thioglycollate-inflamed peritoneal cavity. In contrast, Mac-1 is up-regulated on inflammatory neutrophils and little change is seen in the level of LFA-1 expression. In vitro activation of bone marrow neutrophils with PMA or leukotriene B4 results in a dose dependent increase in Mac-1 and decrease in MEL-14 Ag expression within 1 h after treatment, thus reflecting what is found during inflammation in vivo. Neutrophils activated in vitro or in vivo (MEL-14Low, Mac-1Hi) do not home to inflammatory sites in vivo, correlating with the loss of the MEL-14 Ag and the increased Mac-1 expression. Anti-LFA-1, anti-Mac-1, or MEL-14 antibody given i.v. suppress neutrophil accumulation within the inflamed peritoneum (38%, 30%, and 37% of medium control, respectively) without affecting the levels of circulating neutrophils. However, when FITC-labeled cells are precoated with the mAb and injected i.v., only MEL-14 inhibits extravasation into the inflamed peritoneum (25% of medium control). Finally, in ex vivo adhesion assays of neutrophil binding to high endothelial venules in inflamed-lymph node frozen sections MEL-14 inhibits greater than 90%. anti-LFA-1 20 to 30% and anti-Mac-1 less than 10% of the binding of bone marrow neutrophils to inflamed-lymph node high endothelial venules. These results confirm that both the MEL-14 antigen and Mac-1/LFA-1 are important in neutrophil localization to inflamed sites in vivo, but suggest that their roles in endothelial cell interactions are distinct.

  View details for Web of Science ID A1989AZ28800029

  View details for PubMedID 2553811

• MOLECULAR-CLONING AND EXPRESSION OF PGP-1 - THE MOUSE HOMOLOG OF THE HUMAN H-CAM (HERMES) LYMPHOCYTE HOMING RECEPTOR JOURNAL OF IMMUNOLOGY ZHOU, D. F., Ding, J. F., Picker, L. J., BARGATZE, R. F., BUTCHER, E. C., Goeddel, D. V. 1989; 143 (10): 3390-3395

  Abstract

  Mouse phagocytic glycoprotein-1 (Pgp-1; Ly-24) is a 95-kDa glycoprotein of unknown function that has served as an important T cell/leukocyte differentiation marker. Recent work has suggested that it may be related to a human 85- to 95-kDa glycoprotein (termed variously the Hermes Ag/lymphocyte homing receptor, ECMRIII, P80, and CD44) that is involved in lymphocyte binding to high endothelial venules in the process of lymphocyte homing, and has been implicated in other cell adhesion events. The widespread expression of this molecular class in diverse organ systems suggests a broad role in cellular adhesion, and has led to the unifying designation homing-cellular adhesion molecule (H-CAM). By using human H-CAM cDNA probes, we have isolated a full-length cDNA for the mouse homolog. Comparison of the human and mouse sequences reveals that an N-terminal domain homologous to cartilage proteoglycan core and link proteins, as well as the C-terminal transmembrane and cytoplasmic sequences, are highly conserved (89% and 86% identity, respectively). In contrast, a proximal extracellular domain thought to serve as a target for O-glycosylation and chondroitin sulfate attachment has undergone substantial divergence (only 42% identity). Transient expression of the cDNA in CHO cells followed by immunologic staining confirms that this mouse H-CAM cDNA encodes Pgp-1.1, one of two known Pgp-1 alloantigens.

  View details for Web of Science ID A1989AZ28800040

  View details for PubMedID 2681416

• INDUCIBLE EXPRESSION OF AN ENDOTHELIAL-CELL ANTIGEN ON MURINE MYOCARDIAL VASCULATURE IN ASSOCIATION WITH INTERSTITIAL CELLULAR INFILTRATION TRANSPLANTATION LEPPINK, D. M., Bishop, D. K., Sedmak, D. D., Henry, M. L., Ferguson, R. M., STREETER, P. R., BUTCHER, E. C., Orosz, C. G. 1989; 48 (5): 874-877

  View details for Web of Science ID A1989CB15000032

  View details for PubMedID 2683264

• INFLAMMATION-INDUCED ENDOTHELIAL-CELL ADHESION TO LYMPHOCYTES, NEUTROPHILS, AND MONOCYTES - ROLE OF HOMING RECEPTORS AND OTHER ADHESION MOLECULES TRANSPLANTATION JUTILA, M. A., BERG, E. L., Kishimoto, T. K., Picker, L. J., BARGATZE, R. F., Bishop, D. K., Orosz, C. G., WU, N. W., BUTCHER, E. C. 1989; 48 (5): 727-731

  Abstract

  Adhesion to the vascular endothelium precedes or is a necessary prelude to leukocyte migration into the underlying tissue. Constitutive lymphocyte trafficking through lymphoid organs is controlled by tissue-specific interactions between molecules expressed on the surface of the lymphocyte (homing receptors) and ligands (vascular addressins) expressed on endothelial cells (HEV) within lymphoid tissues. Preliminary evidence suggests that lymphocytes may employ related but distinct interactions in their entry into some chronic sites of inflammation. Other leukocytes, such as neutrophils and monocytes, express molecules related or identical to lymphocyte homing receptors, and these molecules are exquisitely regulated by chemotactic factors and appear to be involved in the homing of these cells to inflamed tissues. In addition, inflammation in vivo induces increased endothelial cell adhesiveness for leukocytes that undoubtedly plays a key role in regulating leukocyte extravasation. Tissue- and inflammation-specific leukocyte/endothelial cell adhesion molecules constitute attractive targets for suppression or manipulation of the early stages of tissue inflammation.

  View details for Web of Science ID A1989CB15000001

  View details for PubMedID 2683262

• NEUTROPHIL MAC-1 AND MEL-14 ADHESION PROTEINS INVERSELY REGULATED BY CHEMOTACTIC FACTORS SCIENCE Kishimoto, T. K., JUTILA, M. A., BERG, E. L., BUTCHER, E. C. 1989; 245 (4923): 1238-1241

  Abstract

  The neutrophil Mac-1 and gp100MEL-14 adhesion proteins are involved in neutrophil extravasation during inflammation. Both the expression and activity of Mac-1 are greatly increased after neutrophil activation. In contrast, neutrophils shed gp100MEL-14 from the cell surface within 4 minutes after activation with chemotactic factors or phorbol esters, releasing a 96-kilodalton fragment of the antigen into the supernatant. Immunohistology showed that gp100MEL-14 was downregulated on neutrophils that had extravasated into inflamed tissue. The gp100MEL-14 adhesion protein may participate in the binding of unactivated neutrophils to the endothelium; rapid shedding of gp100MEL-14 may prevent extravasation into and damage of normal tissues by activated neutrophils.

  View details for Web of Science ID A1989AP68700037

  View details for PubMedID 2551036

• MONOCLONAL-ANTIBODIES TO HUMAN-LYMPHOCYTE HOMING RECEPTORS DEFINE A NOVEL CLASS OF ADHESION MOLECULES ON DIVERSE CELL-TYPES JOURNAL OF CELL BIOLOGY Picker, L. J., NAKACHE, M., BUTCHER, E. C. 1989; 109 (2): 927-937

  Abstract

  A 90-kD lymphocyte surface glycoprotein, defined by monoclonal antibodies of the Hermes series, is involved in lymphocyte recognition of high endothelial venules (HEV). Lymphocyte gp90Hermes binds in a saturable, reversible fashion to the mucosal vascular addressin (MAd), a tissue-specific endothelial cell adhesion molecule for lymphocytes. We and others have recently shown that the Hermes antigen is identical to or includes CD44 (In[Lu]-related p80), human Pgp-1, and extracellular matrix receptor III-molecules reportedly expressed on diverse cell types. Here, we examine the relationship between lymphoid and nonlymphoid Hermes antigens using serologic, biochemical, and, most importantly, functional assays. Consistent with studies using mAbs to CD44 or Pgp-1, mAbs against five different epitopes on lymphocyte gp90Hermes reacted with a wide variety of nonhematolymphoid cells in diverse normal human tissues, including many types of epithelium, mesenchymal elements such as fibroblasts and smooth muscle, and a subset of glia in the central nervous system. To ask whether these non-lymphoid molecules might also be functionally homologous to lymphocyte homing receptors, we assessed their ability to interact with purified MAd using fluorescence energy transfer techniques. The Hermes antigen isolated from both glial cells and fibroblasts--which express a predominant 90-kD form similar in relative molecular mass, isoelectric point, and protease sensitivity to lymphocyte gp90Hermes--was able to bind purified MAd. In contrast, a 140-160-kD form of the Hermes antigen isolated from squamous epithelial cells lacked this capability. Like lymphocyte binding to mucosal HEV, the interaction between glial gp90Hermes and MAd is inhibited by mAb Hermes-3, but not Hermes-1, suggesting that similar molecular domains are involved in the two binding events. The observation that the Hermes/CD44 molecules derived from several nonlymphoid cell types display binding domains homologous to those of lymphocyte homing receptors suggests that these glycoproteins represent a novel type of cell adhesion/recognition molecule (H-CAM) potentially mediating cell-cell or cell-matrix interactions in multiple tissues.

  View details for Web of Science ID A1989AK53200042

  View details for PubMedID 2474557

• FLOW CYTOMETRIC ANALYSIS OF THE HERMES HOMING-ASSOCIATED ANTIGEN ON HUMAN-LYMPHOCYTE SUBSETS BLOOD DELOSTOYOS, J., JALKANEN, S., BUTCHER, E. C. 1989; 74 (2): 751-760

  Abstract

  The homing of lymphocytes is controlled by interactions with high endothelial venules (HEV), specialized vessels that define sites of lymphocyte extravasation into lymph nodes and inflamed tissues. In humans, lymphocyte-HEV binding involves a lymphocyte surface glycoprotein (GP) of 85 to 95 kd (CD44, H-CAM), defined by monoclonal antibody (MoAb) Hermes-1. To define the expression of this homing-associated adhesion molecule during human lymphocyte development, we performed two-color immunofluorescence analyses of human bone marrow (BM), thymus, peripheral blood (PB), and tonsillar lymphocytes. The highest levels of Hermes-1 antigen are displayed by circulating B and T cells in the blood, which are uniformly positive and bear roughly twice the level of antigen present on mature lymphocytes within organized lymphoid tissues and BM. "Immature" (CD4+, CD8+) T cells in the thymus are Hermes-1lo to-, whereas thymocytes of mature phenotype (CD4+ or CD8+) are positive. The Hermes-1 antigen is present at high levels on the same population of thymocytes that bears high surface levels of CD3, a component of the T-cell antigen receptor complex, suggesting that levels of T-cell homing and antigen receptors characteristic of mature peripheral T cells appear coordinately during thymocyte maturation/selection. Essentially all T cells in the periphery are Hermes-1hi, including T blasts, and the homing-associated antigen is maintained at high levels on T cells stimulated in vitro by phytohemagglutinin (PHA) and on interleukin-2 (IL-2) maintained T-cell clones and lines. In contrast, although most resting IgD+ B cells are positive a significant fraction of B cells in tonsils are Hermes-1lo to-; these cells are predominantly PNAhi, IgD-, and CD20hi, a phenotype characteristic of sessile, activated B cells in germinal centers. In all lymphocyte populations examined, there is a linear correlation in staining for Hermes-1 and for Hermes-3, an antibody that defines a distinct functionally important epitope on this molecule. The results demonstrate a precise regulation of this homing-associated antigen during lymphocyte differentiation.

  View details for Web of Science ID A1989AH17500033

  View details for PubMedID 2473804

• STRUCTURAL HOMOLOGY BETWEEN LYMPHOCYTE RECEPTORS FOR HIGH ENDOTHELIUM AND CLASS-III EXTRACELLULAR-MATRIX RECEPTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gallatin, W. M., Wayner, E. A., Hoffman, P. A., STJOHN, T., BUTCHER, E. C., Carter, W. G. 1989; 86 (12): 4654-4658

  Abstract

  We have identified extensive structural homology between one type of heterotypic adhesion receptor (HAR) involved in lymphocyte interactions with high endothelium in lymphoid organs and a collagen-binding protein, termed class III extracellular matrix receptor (ECMRIII), expressed on most nucleated cell types. Both receptors have been described as heterogeneous 90-kDa transmembrane glycoproteins, referred to here as gp90. Monoclonal anti-HAR antibodies, Hermes-1 and Hutch-1, and monoclonal anti-ECMRIII antibodies, P1G12 and P3H9, were utilized to compare the two receptors. (i) All these monoclonal antibodies immunoprecipitated major gp90 components as well as uncharacterized additional higher molecular mass antigens of 120-200 kDa in human and macaque fibroblasts and peripheral blood mononuclear cells. (ii) Competitive binding analyses with the antibodies identified distinct epitopes present on gp90. (iii) Enzymatic and chemical digestions generated identical peptide fragments from all the antigens in human and macaque fibroblasts and peripheral blood mononuclear cells. (iv) Sequential immunoprecipitation with P1G12 followed by the other monoclonal antibodies indicated that all gp90 species reactive with Hermes-1 and Hutch-1 also expressed the P1G12 defined epitope. In reciprocal experiments, Hermes-1 and Hutch-1 immunoprecipitation did not completely remove all P1G12-reactive gp90 from cellular extracts. One inference from these data would be that gp90 is serologically heterogeneous, encompassing HARs as a major subset of this broadly expressed class of molecules.

  View details for Web of Science ID A1989AB24900067

  View details for PubMedID 2471973

  View details for PubMedCentralID PMC287329

• HOMING RECEPTORS AND VASCULAR ADDRESSINS - CELL-ADHESION MOLECULES THAT DIRECT LYMPHOCYTE TRAFFIC IMMUNOLOGICAL REVIEWS BERG, E. L., GOLDSTEIN, L. A., JUTILA, M. A., NAKACHE, M., Picker, L. J., STREETER, P. R., WU, N. W., Zhou, D., BUTCHER, E. C. 1989; 108: 5-18

  View details for Web of Science ID A1989U687100001

  View details for PubMedID 2670744

• A HUMAN-LYMPHOCYTE HOMING RECEPTOR, THE HERMES ANTIGEN, IS RELATED TO CARTILAGE PROTEOGLYCAN CORE AND LINK PROTEINS CELL GOLDSTEIN, L. A., ZHOU, D. F., Picker, L. J., MINTY, C. N., BARGATZE, R. F., Ding, J. F., BUTCHER, E. C. 1989; 56 (6): 1063-1072

  Abstract

  Lymphocyte interactions with high endothelial venules (HEV) during extravasation into lymphoid tissues involve an 85-95 kd class of lymphocyte surface glycoprotein(s), gp90Hermes (CD44). We report here the cloning of cDNA for gp90Hermes expressed in a mucosal HEV-binding B lymphoblastoid cell line, KCA. Northern hybridization revealed the presence of three invariant RNA bands at 1.5, 2.2, and 4.5 kb in mucosal HEV-, lymph node HEV-, or dual-binding cells. The deduced amino acid sequence predicts a mature protein with a C-terminal cytoplasmic tail, a hydrophobic transmembrane domain of 23 amino acids, and an N-terminal extracellular region of 248 amino acids. A proximal extracellular domain is the probable region of O-glycosylation and chondroitin sulfate linkage and displays at least two of the three immunodominant epitope clusters of native gp90Hermes. A distal region contains the majority of potential N-glycosylation sites and cysteines, and exhibits a striking homology to tandemly repeated domains of the cartilage link and proteoglycan core proteins. No significant similarities were found to the immunoglobulin, integrin, or cadherin gene families. Thus gp90Hermes represents a novel class of integral membrane protein involved in lymphocyte-endothelial cell interactions and lymphocyte homing.

  View details for Web of Science ID A1989T944600020

  View details for PubMedID 2466576

• MONOCLONAL-ANTIBODIES AGAINST THE CD44 [IN(LU)-RELATED P80], AND PGP-1 ANTIGENS IN MAN RECOGNIZE THE HERMES CLASS OF LYMPHOCYTE HOMING RECEPTORS JOURNAL OF IMMUNOLOGY Picker, L. J., TOYOS, J. D., Telen, M. J., HAYNES, B. F., BUTCHER, E. C. 1989; 142 (6): 2046-2051

  Abstract

  An 85- to 95 kDa class of lymphocyte surface molecules, defined in man by antibodies of the Hermes series, is involved in lymphocyte binding to high endothelial venules and is likely of central importance in the process of lymphocyte homing. In this report, we have examined the relationship between these Hermes-defined "homing-receptors" and two other 80 to 95 kDa lymphocyte surface molecules that have been extensively studied--CD44 [In(Lu)-related p80] defined by mAb A1G3 and A3D8, and Pgp-1 defined by antibody IM7. Our findings indicate that, in man, similar or identical glycoprotein(s) are recognized by these independently and diversely obtained antibodies. All antibodies showed identical immunohistologic patterns of reactivity on a variety of lymphoid and nonlymphoid human tissues, and demonstrated similar bands on Western blots of both crude tonsil lymphocyte lysates and highly purified Hermes-1 Ag preparations. Similarly, purified CD44/p80 Ag from RBC and human serum bound Hermes-1. Preclearing of tonsil lysates with the Hermes-1 antibody removed antigenic activity for all antibodies. Cross-blocking experiments demonstrated that A3D8, IM7 (anti-Pgp-1), and Hermes-2 antibodies recognize overlapping epitopes. Finally, expression of the epitopes defined by the Hermes-1, Hermes-3, H2-7, and H3-61 antibodies on RBC was shown to be regulated by the In(Lu) gene. These findings unify several different lines of investigation, and suggest the possibility that the CD44/Pgp-1/Hermes class of molecules may serve as cell-cell or cell-substrate adhesion/recognition elements for both hematolymphoid and non-hematolymphoid cell types.

  View details for Web of Science ID A1989T588500040

  View details for PubMedID 2646376

• THE MUCOSAL VASCULAR ADDRESSIN IS A TISSUE-SPECIFIC ENDOTHELIAL-CELL ADHESION MOLECULE FOR CIRCULATING LYMPHOCYTES NATURE NAKACHE, M., BERG, E. L., STREETER, P. R., BUTCHER, E. C. 1989; 337 (6203): 179-181

  Abstract

  Tissue position-dependent or address-dependent expression of cell adhesion molecules has been proposed to play a part in cellular positioning in a variety of systems, for example during neural development, the metastasis of neoplasms, and the tissue-specific homing of lymphocytes. The extravasation of blood-borne lymphocytes is regulated by interactions with the endothelium of specialised venules, such as the high endothelial venules (HEV) in organized lymph nodes and mucosal lymphoid tissues. At least three separate lymphocyte-HEV recognition systems have been described, one mediating tissue-selective lymphocyte interactions with HEV in peripheral lymph nodes, another in mucosal lymphoid organs, and a third in inflamed synovium. We have previously identified a tissue-specific 'vascular addressin' in the mouse which is selectively expressed by venules mediating lymphocyte-homing into mucosal tissues. To determine whether this addressin is a specific adhesion molecule for lymphocytes, we have isolated it by monoclonal antibody-affinity chromatography and inserted it into supported phospholipid planar membranes. Lymphocytes bind to membranes containing the addressin, but not to phospholipid bilayers or to control glycophorin-reconstituted membranes. Only those lymphocytes and lymphoma cell lines capable of binding to mucosal HEV adhere well to the isolated addressin; peripheral lymph node HEV-specific or HEV-non-binding cell lines do not bind. Binding is blocked by anti-addressin antibody MECA-367. We conclude that the mucosal vascular addressin is a tissue-specific endothelial cell-adhesion molecule for lymphocytes, and suggest that it could regulate lymphocyte traffic into mucosal tissues by mediating attachment of blood-borne cells to endothelium.

  View details for Web of Science ID A1989R738500062

  View details for PubMedID 2911352

• HUMAN LAMINA PROPRIA LYMPHOCYTES BEAR HOMING RECEPTORS AND BIND SELECTIVELY TO MUCOSAL LYMPHOID HIGH ENDOTHELIUM EUROPEAN JOURNAL OF IMMUNOLOGY JALKANEN, S., NASH, G. S., DELOSTOYOS, J., MacDermott, R. P., BUTCHER, E. C. 1989; 19 (1): 63-68

  Abstract

  It has been hypothesized that the selective recognition of tissue-specific endothelial cell molecules helps determine the in vivo distribution of lymphoid effector cells by controlling the extravasation of their circulating precursors. Here we report (a) immunofluorescence studies of the cell surface phenotype of human lamina propria lymphocytes (LPL), including staining with monoclonal antibody Hermes-1, which defines a 90-kDa lymphocyte surface glycoprotein involved in recognition of high endothelial venules (HEV); and (b) functional analyses of the ability of LPL to bind to HEV in frozen sections of mucosal lymphoid tissues (appendix or Peyer's patch) vs. peripheral lymph nodes. Essentially all LPL bear the Hermes-1 antigen, over 90% at levels comparable to those of circulating PBL. As a population, LPL display a quantitative preference for adherence to mucosal HEV, binding 0.8-1.5 times as well as PBL to mucosal HEV, but only 0.1-0.5 times as well to HEV in peripheral lymph nodes. Of particular interest was the behavior of the lymphoblast fraction, which typically constituted 3-7% of LPL. These cells, defined by size, consisted of a mixture of T cells and surface IgA+ blasts. One hundred percent were Hermes-1 bright, and they bound 4-8 times more efficiently to mucosal HEV than PBL while failing to bind detectably to lymph node HEV. LPL binding to mucosal HEV involves the gp90 Hermes, since the monoclonal anti-gp90 antibody, Hermes-3, and a polyclonal anti-gp90 antiserum inhibit the binding of small LPL and of LP blasts. The remarkable efficiency and specificity of binding by LP blasts may reflect retention of homing properties of the blood-borne precursors of these blasts and is discussed in relation to the capacity of immunoblasts in mesenteric nodes and in thoracic duct lymph to traffic selectively to mucosal lymphoid and extralymphoid sites. The demonstration of organ-specific endothelial cell recognition by LP lymphoblasts provides considerable support for the concept that selective interactions with endothelium play an important role in directing the distribution of activated lymphocyte subsets in vivo.

  View details for Web of Science ID A1989T963000010

  View details for PubMedID 2465905

• Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity INTERNATIONAL IMMUNOLOGY Kroese, F. G., Butcher, E. C., Stall, A. M., Lalor, P. A., Adams, S., Herzenberg, L. A. 1989; 1 (1): 75-84

  Abstract

  Long term B lineage chimeras are used here to study the origin of plasma cells in the mouse. Chimeric mice are constructed by reconstituting lethally irradiated mice with peritoneal cells (PerC) and bone marrow cells from congenic pairs of mice differing in Igh-C allotype. All conventional B cells in these mice express the allotype of the bone marrow donor and nearly all Ly-1 B lineage cells express the allotype of the PerC donor. FACS analysis and immunohistology of these mice shows that virtually all (sig+) B cells in peripheral lymphoid organs are derived from the bone marrow donor. However, despite this overwhelming number of bone marrow-derived B cells in these animals, immunohistological staining of lymphoid organs and gut shows that nearly half of the IgM, IgG, and IgA plasma cells derive from the PerC donor. These data demonstrate that the peritoneal cavity contains a major reservoir of self-replenishing cells that play a significant role in the mucosal immune response. The possibility that these are B cells that belong to the Ly-1 B lineage is discussed.

  View details for DOI 10.1093/intimm/1.1.75

  View details for Web of Science ID 000208919900010

• Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. International immunology Kroese, F. G., BUTCHER, E. C., STALL, A. M., Lalor, P. A., Adams, S., Herzenberg, L. A. 1989; 1 (1): 75-84

  Abstract

  Long term B lineage chimeras are used here to study the origin of plasma cells in the mouse. Chimeric mice are constructed by reconstituting lethally irradiated mice with peritoneal cells (PerC) and bone marrow cells from congenic pairs of mice differing in Igh-C allotype. All conventional B cells in these mice express the allotype of the bone marrow donor and nearly all Ly-1 B lineage cells express the allotype of the PerC donor. FACS analysis and immunohistology of these mice shows that virtually all (sig+) B cells in peripheral lymphoid organs are derived from the bone marrow donor. However, despite this overwhelming number of bone marrow-derived B cells in these animals, immunohistological staining of lymphoid organs and gut shows that nearly half of the IgM, IgG, and IgA plasma cells derive from the PerC donor. These data demonstrate that the peritoneal cavity contains a major reservoir of self-replenishing cells that play a significant role in the mucosal immune response. The possibility that these are B cells that belong to the Ly-1 B lineage is discussed.

  View details for PubMedID 2487677

• A MAJOR PERITONEAL RESERVOIR OF PRECURSORS FOR INTESTINAL IGA PLASMA-CELLS IMMUNOLOGICAL INVESTIGATIONS Kroese, F. G., BUTCHER, E. C., STALL, A. M., Herzenberg, L. A. 1989; 18 (1-4): 47-58

  Abstract

  Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.

  View details for Web of Science ID A1989U349300008

  View details for PubMedID 2786500

• LY-6C IS A MONOCYTE MACROPHAGE AND ENDOTHELIAL-CELL DIFFERENTIATION ANTIGEN REGULATED BY INTERFERON-GAMMA EUROPEAN JOURNAL OF IMMUNOLOGY JUTILA, M. A., Kroese, F. G., Jutila, K. L., STALL, A. M., Fiering, S., Herzenberg, L. A., BERG, E. L., BUTCHER, E. C. 1988; 18 (11): 1819-1826

  Abstract

  Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.

  View details for Web of Science ID A1988R780600024

  View details for PubMedID 2849552

• EVOLUTIONARY CONSERVATION OF TISSUE-SPECIFIC LYMPHOCYTE ENDOTHELIAL-CELL RECOGNITION MECHANISMS INVOLVED IN LYMPHOCYTE HOMING JOURNAL OF CELL BIOLOGY WU, N. W., JALKANEN, S., STREETER, P. R., BUTCHER, E. C. 1988; 107 (5): 1845-1851

  Abstract

  Tissue-specific interactions with specialized high endothelial venules (HEV) direct the homing of lymphocytes from the blood into peripheral lymph nodes, mucosal lymphoid organs, and tissue sites of chronic inflammation. These interactions have been demonstrated in all mammalian species examined and thus appear highly conserved. To assess the degree of evolutionary divergence in lymphocyte-HEV recognition mechanisms, we have studied the ability of lymphocytes to interact with HEV across species barriers. By using an in vitro assay of lymphocyte binding to HEV in frozen sections of lymphoid tissues, we confirm that mouse, guinea pig, and human lymphocytes bind to xenogeneic as well as homologous HEV. In addition, we show that mouse and human lymphoid cell lines that bind selectively to either peripheral lymph node or mucosal vessels (Peyer's patches, appendix) in homologous lymphoid tissues exhibit the same organ specificity in binding to xenogeneic HEV. Furthermore, monoclonal antibodies that recognize lymphocyte "homing receptors" and block homologous lymphocyte binding to peripheral lymph node or to mucosal HEV, also inhibit lymphocyte interactions with xenogeneic HEV in a tissue-specific fashion. Similarly, anti-HEV antibodies against organ-specific mouse high endothelial cell "addressins" involved in lymphocyte homing to peripheral lymph node or mucosal lymphoid organs, not only block the adhesion of mouse lymphocytes but also of human lymphocytes to target mouse HEV. The results illustrate a remarkable degree of functional conservation of elements mediating these cell-cell recognition events involved in organ-specific lymphocyte homing.

  View details for Web of Science ID A1988Q766200024

  View details for PubMedID 3182939

• IMMUNOHISTOLOGIC AND FUNCTIONAL-CHARACTERIZATION OF A VASCULAR ADDRESSIN INVOLVED IN LYMPHOCYTE HOMING INTO PERIPHERAL LYMPH-NODES JOURNAL OF CELL BIOLOGY STREETER, P. R., ROUSE, B. T., BUTCHER, E. C. 1988; 107 (5): 1853-1862

  Abstract

  The tissue localization or "homing" of circulating lymphocytes is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. In peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches and appendix), and sites of chronic inflammation, for example, lymphocytes leave the blood by adhering to and migrating between those endothelial cells lining postcapillary high endothelial venules (HEV). Functional analyses of lymphocyte interactions with HEV have shown the lymphocytes can discriminate between HEV in different tissues, indicating that HEV express tissue-specific determinants or address signals for lymphocyte recognition. We recently described such a tissue-specific "vascular addressin" that is selectively expressed by endothelial cells supporting lymphocyte extravasation into mucosal tissues and that appears to be required for mucosa-specific lymphocyte homing (Streeter, P. R., E. L. Berg, B. N. Rouse, R. F. Bargatze, and E. C. Butcher. 1988. Nature (Lond.). 331:41-46). Here we document the existence and tissue-specific distribution of a distinct HEV differentiation antigen. Defined by monoclonal antibody MECA-79, this antigen is expressed at high levels on the lumenal surface and in the cytoplasm of HEV in peripheral lymph nodes. By contrast, although MECA-79 stains many HEV in the mucosal Peyer's patches, expression in most cases is restricted to the perivascular or ablumenal aspect of these venules. In the small intestine lamina propria, a mucosa-associated site that supports the extravasation of lymphocytes, venules do not stain with MECA-79. Finally, we demonstrate that MECA-79 blocks binding of both normal lymphocytes and a peripheral lymph node-specific lymphoma to peripheral lymph node HEV in vitro and that it also inhibits normal lymphocyte homing to peripheral lymph nodes in vivo without significantly influencing lymphocyte interactions with Peyer's patch HEV in vitro or in vivo. Thus, MECA-79 defines a novel vascular addressin involved in directing lymphocyte homing to peripheral lymph nodes.

  View details for Web of Science ID A1988Q766200025

  View details for PubMedID 2460470

• BIOCHEMICAL-PROPERTIES OF GLYCOPROTEINS INVOLVED IN LYMPHOCYTE RECOGNITION OF HIGH ENDOTHELIAL VENULES IN MAN JOURNAL OF IMMUNOLOGY JALKANEN, S., JALKANEN, M., BARGATZE, R., TAMMI, M., BUTCHER, E. C. 1988; 141 (5): 1615-1623

  Abstract

  Lymphocyte interactions with high endothelial venules (HEV) are important to the in vivo migration of normal and neoplastic lymphocyte populations. We have previously described an 85- to 95-kDa lymphocyte surface glycoprotein(s) defined by mAb Hermes-1, that is involved in the recognition of HEV by human lymphocytes: antibodies against distinct epitopes of the Hermes-1 Ag differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial HEV. Here we characterize further the Hermes-1-defined glycoproteins. No well defined differences were observed between the Hermes-1 Ag immunoprecipitated from PBL and from mucosa- vs lymph HEV-specific cell lines. The Ag is an acidic (isoelectric point = 4.2) sulfated molecule bearing both O-linked and (3,4) N-linked oligosaccharide side chains. A subset of the Hermes-1-immunoprecipitated species is modified by covalent linkage to chondroitin sulfate, yielding a Mr of approximately 180 to 200 kDa. Pulse-chase labeling reveals a major precursor of 76 kDa that appears to be processed either to the 85- to 95-kDa form or, by addition of chondroitin sulfate, to a 180- to 200-kDa form. The potential role of these structural modifications, and particularly of chondroitin sulfate, in the function of the putative adhesion molecules is discussed.

  View details for Web of Science ID A1988P798700030

  View details for PubMedID 3137259

• MEMORY B-CELLS EXPRESS A PHENOTYPE CONSISTENT WITH MIGRATORY COMPETENCE AFTER SECONDARY BUT NOT SHORT-TERM PRIMARY IMMUNIZATION CELLULAR IMMUNOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1988; 115 (1): 78-87

  Abstract

  The cell surface phenotype of dinitrophenol (DNP)-specific memory B cells, defined by their capacity to transfer IgG responses into syngeneic irradiated recipients, was assessed using two markers of relevance to lymphocyte migratory properties: (i) peanut agglutinin, which binds to terminal galactosyl residues expressed at high levels by several nonmigrating lymphocyte subsets and, among lymph node B cells, is highly specific for germinal center cells; and (ii) MEL-14, a monoclonal antibody specific for lymphocyte surface receptors required for migration from the blood into peripheral lymph nodes. At various times after primary or secondary immunization with DNP-keyhole limpet hemocyananin (KLH), lymph node B cells were separated by fluorescence-activated cell sorting on the basis of staining with PNA and/or MEL-14, and the presence of B-memory cells in each fraction was assessed by adoptive transfer with antigen (DNP-KLH) and helper T cells. One week after immunization, most of the memory sorted in the PNAhi population, confirming a previous report by R. F. Coico, B. S. Bhogal, and G. J. Thorbecke (J. Immunol. 131, 2254, 1983) that early memory B cells or their precursors are contained within the germinal center cell population, a population which is known to be MEL-14- and migratory-incompetent. Six weeks after primary stimulation, however, the bulk of memory cells, unlike germinal center cells, were MEL-14hi. After secondary immunization, memory was still predominantly MEL-14+ and PNAlo, although in some experiments adoptive responses were transferred by all sorted fractions. The results are consistent with the hypothesis that antigen-specific B cells initially undergo local (sessile) differentiation and proliferation in germinal centers, where they develop the capacity for adoptive transfer of antigen-specific secondary responses, but that with continued development their long-lived memory-containing progeny express a phenotype permitting their reentry into the recirculating lymphocyte pool.

  View details for Web of Science ID A1988P804100007

  View details for PubMedID 3261207

• A FLUOROMETRIC APPROACH TO THE QUANTITATION OF CELL NUMBER WITH APPLICATION TO A CELL-ADHESION ASSAY JOURNAL OF IMMUNOLOGICAL METHODS LEWINSOHN, D. M., Nickoloff, B. J., BUTCHER, E. C. 1988; 110 (1): 93-100

  Abstract

  We describe a fluorometric approach to the determination of cell number. Cells are covalently labeled with fluorescein isothiocyanate (FITC) under physiologic conditions. Cells are lysed with detergent, and released fluorochrome is assessed quantitatively with a fluorescence spectrophotometer. Fluorescence varies linearly with regard to cell number over a wide range of concentrations, and allows detection of as few as 8 X 10(3) cells/ml. We describe the effect of different time and temperature incubations of FITC-labeled cells on fluorescence intensity, and we use the method to analyze the binding of peripheral blood mononuclear leukocytes to interferon-gamma-treated keratinocytes. Finally, we demonstrate that the results in an adhesion assay are comparable to those obtained with 51Cr-labeled cells. Quantitative fluorescence analysis of cell number offers a safe, inexpensive, rapid, and accurate method of determining cell number without the biological hazard and waste disposal problems associated with radioactive labeling.

  View details for Web of Science ID A1988N445300012

  View details for PubMedID 3131438

• SPIROCHETAL ANTIGENS AND LYMPHOID-CELL SURFACE-MARKERS IN LYME SYNOVITIS - COMPARISON WITH RHEUMATOID SYNOVIUM AND TONSILLAR LYMPHOID-TISSUE ARTHRITIS AND RHEUMATISM STEERE, A. C., Duray, P. H., BUTCHER, E. C. 1988; 31 (4): 487-495

  Abstract

  Using monoclonal antibodies to spirochetal antigenes and lymphoid cell surface markers, we examined the synovial lesions of 12 patients with Lyme disease, and compared them with rheumatoid synovium and tonsillar lymphoid tissue. The synovial lesions of Lyme disease patients and rheumatoid arthritis patients were similar and often consisted of the elements found in normal organized lymphoid tissue. In both diseases, T cells, predominantly of the helper/inducer subset, were distributed diffusely in subsynovial lining areas, often with nodular aggregates of tightly intermixed T and B cells. IgD-bearing B cells were scattered within the aggregates, and a few follicular dendritic cells and activated germinal center B cells were sometimes present. Outside the aggregates, many plasma cells, high endothelial venules, scattered macrophages, and a few dendritic macrophages were found. HLA-DR and DQ expression was intense throughout the lesions. In 6 of the 12 patients with Lyme arthritis, but in none of those with rheumatoid arthritis, a few spirochetes and globular antigen deposits were seen in and around blood vessels in areas of lymphocytic infiltration. Thus, in Lyme arthritis, a small number of spirochetes are probably the antigenic stimulus for chronic synovial inflammation.

  View details for Web of Science ID A1988N169200005

  View details for PubMedID 3258751

• SELECTIVE RECOGNITION OF MUCOSAL LYMPHOID HIGH ENDOTHELIUM BY GUT INTRAEPITHELIAL LEUKOCYTES GASTROENTEROLOGY Schmitz, M., Nunez, D., BUTCHER, E. C. 1988; 94 (3): 576-581

  Abstract

  Circulating precursors of mucosal immunoglobulin A plasma cells and T-cell immunoblasts migrate selectively into mucosal sites from the blood, but the mechanisms controlling this selective trafficking have not been determined. One possibility is that the site-specific extravasation of circulating effector cell populations is determined by organ-specific endothelial cell recognition mechanisms. Here we have assessed the ability of isolated mouse gut intraepithelial lymphocytes to recognize and bind to mucosal versus nonmucosal lymphoid organ high endothelial venules, vessels that support high levels of lymphocyte traffic in vivo. In an in vitro assay of lymphocyte interaction with high endothelial venules in frozen sections, intraepithelial leukocytes bind well to high endothelial venules in Peyer's patches but, unlike most circulating B and T lymphocytes, are unable to interact with peripheral lymph node high endothelial venules. Furthermore, we show by in situ immunohistology and in cell suspension immunofluorescence studies that intraepithelial leukocytes fail to stain with a monoclonal antibody, MEL-14, against putative lymphocyte receptors for lymph node high endothelial venules. Thus, they lack a cell surface glycoprotein required for homing to peripheral nodes. The demonstration of organ-specific recognition of endothelial cells by a normal mucosal effector lymphocyte population suggests that selective interactions with endothelium may play an important role in controlling the distribution of effector cells in vivo. The utilization of organ-specific endothelial cell recognition mechanisms by circulating precursors of mucosal effector cells could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from nonmucosal immune mechanisms.

  View details for Web of Science ID A1988M312500003

  View details for PubMedID 3338630

• EXPRESSION OF LYMPHOCYTE HOMING RECEPTOR ANTIGEN IN NON-HODGKINS LYMPHOMA AMERICAN JOURNAL OF PATHOLOGY Picker, L. J., Medeiros, L. J., Weiss, L. M., Warnke, R. A., BUTCHER, E. C. 1988; 130 (3): 496-504

  Abstract

  In man, lymphocyte binding to high endothelial venules (HEVs) involves specific 85-95 kd cell surface glycoprotein(s) recognized by the monoclonal antibodies Hermes-1 and Hermes-3. These putative "homing receptor" molecule(s) are believed to play an important role in the normal regulation of lymphocyte circulation. To investigate the possibility that homing receptors also play a role in the biology of lymphoid malignancies, the authors studied over 300 cases of non-Hodgkin's lymphoma by immunohistologic staining with Hermes-1 and -3, antibodies that define two distinct epitopes on the gp 85-95 putative homing receptor molecules. Furthermore, they directly compared expression of the Hermes-3 antigen with clinical extent of disease in 57 patients with diffuse large cell lymphoma. They found that staining of the various subtypes of lymphoma was heterogeneous, and in general correlated with patterns of expression seen in benign lymphoid populations. Essentially all normal lymphoid populations examined, except germinal center B cells and most cortical thymocytes, bear a high level of homing receptor antigen. Similarly, nearly all peripheral T-cell lymphomas, diffuse small cell lymphomas of B lineage, and plasma cell tumors were positive for homing receptor antigen (95%, 97%, and 100%, respectively). Small noncleaved cell, follicular, and diffuse large cell lymphomas of B lineage, tumors having morphologic or immunologic features resembling germinal center cells, frequently failed to express Hermes-defined epitopes (81%, 41%, 25% Hermes-3-, respectively). Antigen expression in T-lymphoblastic lymphomas strongly correlated with immunophenotypic subtypes: only 8% of CD4+/CD8+ were Hermes-1+ versus 86% of CD4-/CD8- and 43% of CD4+/CD8-. Hermes-3 expression by cases of diffuse, large cell lymphoma which showed generalized lymph node involvement (a pattern strongly suggestive of HEV-mediated spread; 100% Hermes-3+, mean intensity 3.4) was higher than that of cases with localized or multifocal, contiguous involvement (consistent with lymphatic spread; 69% Hermes-3+, mean intensity 2.2), but these differences did not achieve statistical significance. The results indicate that homing receptor antigen expression, although perhaps necessary for wide-spread blood-borne lymphoma dissemination to lymphoid sites, is not in and of itself sufficient to predict such behavior in this subtype of lymphoid malignancy.

  View details for Web of Science ID A1988M609900010

  View details for PubMedID 2450463

• REGULATION OF LYMPHOCYTE HOMING .1. ALTERATIONS IN HOMING RECEPTOR EXPRESSION AND ORGAN-SPECIFIC HIGH ENDOTHELIAL VENULE BINDING OF LYMPHOCYTES UPON ACTIVATION JOURNAL OF IMMUNOLOGY Hamann, A., JABLONSKIWESTRICH, D., SCHOLZ, K. U., Duijvestijn, A., BUTCHER, E. C., Thiele, H. G. 1988; 140 (3): 737-743

  Abstract

  Upon activation, lymphocytes display profound alterations in their in vivo migration behavior. In an attempt to understand some of the cellular mechanisms responsible for this altered behavior, in vitro stimulated lymphocytes have been analyzed for their expression of a putative homing receptor (HOR) (defined by mAb MEL-14) and for their ability to bind to specialized lymphoid organ high endothelial venules (HEV) in vitro. The results indicate that signals related to lymphocyte activation induce complex alterations in HOR expression and organ-specificity of HEV-binding: 1) submitogenic stimuli induce an increase in MEL-14 antigen expression. This applies to almost all lymphocytes in autologous cultures, for the fraction of cells in periodate, LPS- or Con A-treated cultures not fully activated and for cultures stimulated with suboptimal doses of Con A. 2) Full blast transformation is associated with a decrease or complete loss of MEL-14 antigen expression on the majority of blasts in all activating systems used, but a subset of up to 30 to 40% of fully activated cells may nonetheless express very high levels of the MEL-14 antigen. 3) Functional assays reveal that Con A and periodate stimulation lead to a selective, nearly complete suppression of the lymphocytes binding to HEV of Peyer's patches, even under conditions where overall binding to peripheral node HEV is increased. This indicates a differential regulation of the two respective receptors, with the mucosa system-specific HOR being more prone to down-regulation during in vitro activation by these mitogens.

  View details for Web of Science ID A1988M021200009

  View details for PubMedID 3276778

• EVIDENCE FOR AN ACCESSORY ROLE OF LFA-1 IN LYMPHOCYTE-HIGH ENDOTHELIUM INTERACTION DURING HOMING JOURNAL OF IMMUNOLOGY Hamann, A., JABLONSKIWESTRICH, D., Duijvestijn, A., BUTCHER, E. C., Baisch, H., Harder, R., Thiele, H. G. 1988; 140 (3): 693-699

  Abstract

  In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.

  View details for Web of Science ID A1988M021200002

  View details for PubMedID 3276776

• A TISSUE-SPECIFIC ENDOTHELIAL-CELL MOLECULE INVOLVED IN LYMPHOCYTE HOMING NATURE STREETER, P. R., BERG, E. L., ROUSE, B. T., BARGATZE, R. F., BUTCHER, E. C. 1988; 331 (6151): 41-46

  Abstract

  An endothelial cell surface molecule that is selectively expressed in mucosal organs is required for lymphocyte homing to mucosal lymphoid tissues. This 'vascular addressin' appears to function as a tissue-specific marker or address signal for recognition by lymphocytes circulating in the blood.

  View details for Web of Science ID A1988L550500044

  View details for PubMedID 3340147

• Expression and regulation of an antigen specific for endothelium involved in human lymphocyte homing. Advances in experimental medicine and biology HORST, E., PALS, S. T., DUIJVESTIJN, A. M., von Mourik, J., Kraal, G., BUTCHER, E. C., Meijer, C. J. 1988; 237: 477-484

  View details for PubMedID 3151039

• Lymphocyte migration molecules. Advances in experimental medicine and biology JALKANEN, S., BARGATZE, R., JALKANEN, M., Lewinsohn, D., STREETER, P., LAKEY, E., BUTCHER, E. C. 1988; 237: 21-29

  View details for PubMedID 2855387

• Regulation of functional and morphological aspects of high endothelium in mouse. Advances in experimental medicine and biology DUIJVESTIJN, A. M., Rep, M., BUTCHER, E. C., Hendriks, H. R., Kraal, G. 1988; 237: 491-497

  View details for PubMedID 3254061

• LYMPHOCYTE AND ENDOTHELIAL-CELL RECOGNITION ELEMENTS THAT CONTROL LYMPHOCYTE TRAFFIC TO MUCOSA-ASSOCIATED LYMPHATIC TISSUES MONOGRAPHS IN ALLERGY JALKANEN, S., STREETER, P., LAKEY, E., BARGATZE, R., BUTCHER, E. C. 1988; 24: 144-149

  View details for Web of Science ID A1988N156500023

  View details for PubMedID 2834636

• HOMING RECEPTORS AND METASTASIS ADVANCES IN CANCER RESEARCH SHER, B. T., BARGATZE, R., Holzmann, B., Gallatin, W. M., MATTHEWS, D., Wu, N., PICKER, L., BUTCHER, E. C., Weissman, I. L. 1988; 51: 361-390

  Abstract

  As discussed in the preceding sections, there are several indications that the lymphocyte homing receptors involved in the normal process of lymphocyte recirculation are also relevant to the behavior of metastatic cells. Cell fusion experiments indicate that previously nonmetastatic cells can acquire metastatic capacity from fusion with normal lymphocytes. Murine T lymphomas that bear high levels of functional homing receptors can metastasize to peripheral lymphoid organs, whereas those lymphomas lacking homing receptors cannot. Virtually all lymph node metastases of lymphomas contain a high proportion of MEL-14hi cells, even if the primary tumor has been selected to be relatively deficient in these cells. Further investigations of the biology of lymphocyte homing receptors will reveal whether or not there are additional lymphocyte homing receptors and will clarify the role of lymphocyte homing receptors in metastasis. Antibodies against three lymphocyte homing receptors could therefore be useful for diagnosis and treatment of metastatic disease.

  View details for Web of Science ID A1988Q449700007

  View details for PubMedID 3066147

• HIGH ENDOTHELIAL DIFFERENTIATION IN HUMAN LYMPHOID AND INFLAMMATORY TISSUES DEFINED BY MONOCLONAL-ANTIBODY HECA-452 AMERICAN JOURNAL OF PATHOLOGY DUIJVESTIJN, A. M., HORST, E., PALS, S. T., ROUSE, B. N., STEERE, A. C., Picker, L. J., Meijer, C. J., BUTCHER, E. C. 1988; 130 (1): 147-155

  Abstract

  Lymphocyte traffic into lymph nodes and into mucosa-associated lymphoid tissues is regulated by specialized postcapillary high endothelial venules (HEVs). The authors have produced a rat monoclonal antibody, HECA-452, that detects a human endothelial cell differentiation antigen selectively expressed on high endothelium. In immunoperoxidase studies, HECA-452 intensely stains all HEVs within lymphoid organs. In normal nonlymphoid tissues the antibody stains no vascular endothelium. The antibody, in addition to reacting with high endothelium, cross-reacts with a set of monocytic cells. In pathologic states such as autoimmune thyroiditis and Crohn's disease, known for the development of dense, frequently organized, lymphocytic infiltrates, HECA-452 detects HEV-like vessels containing luminal and intramural lymphocytes, presumably in the process of extravasating. The antigen was not expressed at detectable levels by venules in less heavily infiltrated chronic inflammatory sites nor in acutely inflamed tissues. In lymphoid malignancies, the only vessels stained were morphologically characteristic HEVs in association with areas of residual normal lymphoid tissue or reactive lymphocytic infiltrates. The specificity of HECA-452 for high endothelial cells confirms the highly specialized nature of these vessels and will permit studies of the regulation of high endothelial cell differentiation in vivo and in vitro. The HECA-452 antigen is preserved in paraffin sections of sublimate formaldehyde- and also routinely formalin-fixed tissues. Thus, HECA-452 will be widely applicable for the immunohistologic detection of endothelium specialized for the support of highly increased lymphocyte extravasation in inflammatory sites.

  View details for Web of Science ID A1988L934000016

  View details for PubMedID 3276207

• RECOMBINANT GAMMA-INTERFERON INCREASES THE BINDING OF PERIPHERAL-BLOOD MONONUCLEAR LEUKOCYTES AND A LEU-3+ LYMPHOCYTE-T CLONE TO CULTURED KERATINOCYTES AND TO A MALIGNANT CUTANEOUS SQUAMOUS CARCINOMA CELL-LINE THAT IS BLOCKED BY ANTIBODY AGAINST THE LFA-1 MOLECULE JOURNAL OF INVESTIGATIVE DERMATOLOGY Nickoloff, B. J., LEWINSOHN, D. M., BUTCHER, E. C., KRENSKY, A. M., CLAYBERGER, C. 1988; 90 (1): 17-22

  Abstract

  Because keratinocytes (KCs) express HLA-DR in a wide variety of skin diseases in which mononuclear leukocytes are observed in close apposition to KCs (i.e., graft-versus-host disease), and since gamma interferon (IFN-gamma) induces HLA-DR expression on KCs, we asked whether IFN-gamma treatment of KCs would influence the adherence of mononuclear leukocytes. When allogeneic peripheral blood mononuclear leukocytes (PBML) and a Leu-3+ T cell clone were coincubated with IFN-gamma-treated KCs (300 U/ml, 3 days), there was a marked increase in binding compared with nontreated KCs. Similar binding results were obtained using a cutaneous squamous carcinoma cell line (SCL-1) after IFN-gamma treatment. The IFN effect was relatively specific for IFN-gamma, as neither IFN-alpha nor -beta had any effect. Tumor necrosis factor exposure (500 U/ml, 3 days) increased the binding of the Leu-3+ T cell clone to both KCs and SCL-1 cells. Neutrophils displayed a less marked (but statistically significant) increase in binding to IFN-gamma-treated KCs. Using the Leu-3+ cell clone and SCL-1 cells, detailed kinetic analysis of the effect of IFN-gamma on binding was performed. The increased adherence between the cells began to appear after only 7 hours of treatment with r-IFN-gamma (300 U/ml) and reached a plateau at 48 hours, with significantly enhanced binding continuing for at least 48 hours after removal of IFN-gamma. The mechanism of binding was explored by preincubation of the PBML/Leu-3+ T cells with anti-LFA-1 (lymphocyte function-associated antigen) antibody (0.6-6.0 micrograms/ml), which totally inhibited the binding with no effect by anti-LFA-2 or -3 or class I or II antibodies despite documented binding of these antibodies to the cells. These results suggest that, after exposure to IFN-gamma, the ability of KCs to bind mononuclear leukocytes is strongly enhanced, and this adherence may be important in leukocyte trafficking in the skin as well as contributing to altered KC-leukocyte interaction, which may be of fundamental importance in a variety of skin disease.

  View details for Web of Science ID A1988L481700004

  View details for PubMedID 2447190

• ENHANCED BINDING OF PERIPHERAL-BLOOD MONONUCLEAR LEUKOCYTES TO GAMMA-INTERFERON-TREATED CULTURED KERATINOCYTES AMERICAN JOURNAL OF DERMATOPATHOLOGY Nickoloff, B. J., LEWINSOHN, D. M., BUTCHER, E. C. 1987; 9 (5): 413-418

  Abstract

  Dermatopathologists observe mononuclear leukocytes in close apposition to keratinocytes (KCs) in graft versus host disease and in other lymphocyte-mediated skin diseases, such as lichen planus, erythema multiforme, and lupus erythematosus. Since the KCs are Class II histocompatibility antigen (HLA-DR) positive in these diseases (indicating local production of gamma interferon, IFN-gamma, by activated T-cells), we sought to determine whether IFN-gamma treatment of KCs would influence the ability of allogeneic peripheral blood mononuclear leukocytes (PBMLs) to adhere to cultured KCs in vitro. The adherence of PBMLs to KC monolayers was determined by the three following methods: (a) methanol fixation of the washed KCs (after PBML incubation), followed by hematoxylin-eosin staining and direct counting of adherent PBMLs; (b) fluorescein isothiocyanate (FITC) labeling of PBML, followed by measuring the amount of FITC-PBML bound to KCs after washing either by direct visualization with a fluorescence microscope; or by (c) quantitative fluorescence spectroscopy following lysis of the adherent cells. While untreated KCs bound allogeneic PBMLs minimally 15-120 min at 37 degrees C, pretreatment of the KCs with IFN-gamma (300 U/ml, 3 days) produced significantly increased binding of the PBMLs by approximately fivefold. By contrast, IFN-alpha and IFN-beta (10(3) U/ml) had no effect. Also, despite the induction of HLA-DR on cultured human fibroblasts, no increased binding of PBMLs after IFN-gamma treatment was observed. The selective ability of IFN-gamma to produce a marked increase in adherence between KCs and PBMLs suggests a new role for IFN-gamma in the immunobiology of the skin.

  View details for Web of Science ID A1987K335200007

  View details for PubMedID 2446518

• HIGH ENDOTHELIAL VENULE BINDING AS A PREDICTOR OF THE DISSEMINATION OF PASSAGED MURINE LYMPHOMAS JOURNAL OF EXPERIMENTAL MEDICINE BARGATZE, R. F., WU, N. W., Weissman, I. L., BUTCHER, E. C. 1987; 166 (4): 1125-1131

  Abstract

  It has long been postulated that normal lymphocyte homing mechanisms help determine the metastatic spread of lymphoid neoplasms. The traffic of normal lymphocytes is controlled in part by the regulated expression of surface receptors for high endothelial venules (HEV), specialized venules that mediate the extravasation of circulating lymphocytes from the blood into lymphoid organs and sites of chronic inflammation. Here we have compared the in vivo growth patterns of HEV-binding vs. nonbinding murine lymphomas passaged intramuscularly into syngeneic recipients. We report that lymphomas that bind well to HEV (as assessed in a quantitative in vitro assay) disseminate widely via the blood, involving all lymph node groups symmetrically. Although both HEV-binding and nonbinding lymphomas gain access to the blood, gross involvement of lymph nodes by nonbinding lymphomas is limited to nodes draining local tumor at the site of injection, a prominent feature of these lymphomas; distant lymph nodes are not enlarged. The results suggest that the expression of functional receptors for HEV either controls the hematogenous dissemination of malignant lymphocyte populations to HEV-bearing organs, or is coregulated with factors determining this metastatic behavior. The findings support the concept that normal lymphocyte homing mechanisms are important to the spread of leukemias and lymphomas.

  View details for Web of Science ID A1987K464000024

  View details for PubMedID 3655655

• LYMPHOCYTE RECOGNITION OF HIGH ENDOTHELIUM - ANTIBODIES TO DISTINCT EPITOPES OF AN 85-95-KD GLYCOPROTEIN ANTIGEN DIFFERENTIALLY INHIBIT LYMPHOCYTE BINDING TO LYMPH-NODE, MUCOSAL, OR SYNOVIAL ENDOTHELIAL-CELLS JOURNAL OF CELL BIOLOGY JALKANEN, S., BARGATZE, R. F., DELOSTOYOS, J., BUTCHER, E. C. 1987; 105 (2): 983-990

  Abstract

  The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.

  View details for Web of Science ID A1987J819800034

  View details for PubMedID 2442176

  View details for PubMedCentralID PMC2114763

• LEUKOCYTE-ENDOTHELIAL CELL RECOGNITION - EVIDENCE OF A COMMON MOLECULAR MECHANISM SHARED BY NEUTROPHILS, LYMPHOCYTES, AND OTHER LEUKOCYTES JOURNAL OF IMMUNOLOGY LEWINSOHN, D. M., BARGATZE, R. F., BUTCHER, E. C. 1987; 138 (12): 4313-4321

  Abstract

  The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.

  View details for Web of Science ID A1987H683200040

  View details for PubMedID 3584977

• RECEPTORS INVOLVED IN LYMPHOCYTE HOMING - RELATIONSHIP BETWEEN A CARBOHYDRATE-BINDING RECEPTOR AND THE MEL-14 ANTIGEN JOURNAL OF CELL BIOLOGY YEDNOCK, T. A., BUTCHER, E. C., Stoolman, L. M., ROSEN, S. D. 1987; 104 (3): 725-731

  Abstract

  Blood-borne lymphocytes extravasate in large numbers within peripheral lymph nodes (PN) and other secondary lymphoid organs. It has been proposed that the initiation of extravasation is based upon a family of cell adhesion molecules (homing receptors) that mediate lymphocyte attachment to specialized high endothelial venules (HEV) within the lymphoid tissues. A putative homing receptor has been identified by the monoclonal antibody, MEL-14, which recognizes an 80-90-kD glycoprotein on the surface of mouse lymphocytes and blocks the attachment of lymphocytes to PN HEV. In a companion study we characterize a carbohydrate-binding receptor on the surface of mouse lymphocytes that also appears to be involved in the interaction of lymphocytes with PN HEV. This receptor selectively binds to fluorescent beads derivatized with PPME, a polysaccharide rich in mannose-6-phosphate. In this report we examine the relationship between this carbohydrate-binding receptor and the putative homing receptor identified by the MEL-14 antibody. We found that: MEL-14 completely and selectively blocks the activity of the carbohydrate-binding receptor on mouse lymphocytes; the ability of six lymphoma cell lines to bind PPME beads correlates with cell-surface expression of the MEL-14 antigen, as well as PN HEV-binding activity; selection of lymphoma cell line variants for PPME-bead binding by fluorescence-activated cell sorting (FACS) produces highly correlated (r = 0.974, P less than 0.001) and selective changes in MEL-14 antigen expression. These results show that the carbohydrate-binding receptor on lymphocytes and the MEL-14 antigen, which have been independently implicated as receptors involved in PN-specific HEV attachment, are very closely related, if not identical, molecules.

  View details for Web of Science ID A1987G207900037

  View details for PubMedID 2950122

  View details for PubMedCentralID PMC2114538

• LYMPHOID TISSUE-SPECIFIC AND INFLAMMATION-SPECIFIC ENDOTHELIAL-CELL DIFFERENTIATION DEFINED BY MONOCLONAL-ANTIBODIES JOURNAL OF IMMUNOLOGY DUIJVESTIJN, A. M., KERKHOVE, M., BARGATZE, R. F., BUTCHER, E. C. 1987; 138 (3): 713-719

  Abstract

  Endothelial cells play an essential role in immune responses by regulating the entry of leukocytes into lymphoid tissues and sites of inflammation. As an initial approach to analyzing endothelial cell specialization in relation to such immune function, we have produced monoclonal antibodies (MAB) against mouse lymph node endothelium. Three antibodies were selected: MECA-20, recognizing the endothelium of all blood vessels in lymphoid as well as non-lymphoid organs; MECA-217, which stains the endothelium lining large elastic arteries, but among small vessels is specific for post-capillary venules within lymphoid organs and tissues exposed to exogenous antigen, such as skin and uterus; and MECA-325, an antibody that demonstrates specificity for the specialized high endothelial venules (HEV) that control lymphocyte homing into lymph nodes and Peyer's patches. MECA-325 failed to stain vessels in any non-lymphoid organs tested. Immunoperoxidase studies of HEV in lymph node frozen sections, and of isolated high endothelial cells in suspensions, demonstrated that the antigens recognized by all three antibodies are expressed at the cell surface; those defined by MECA-20 and MECA-325 are also present in the cytoplasm. To study the regulation of the antigens defined by these MAB in relation to extra-lymphoid immune reactions, we assessed their expression in induced s.c. granulomas as a model for chronic inflammation. Small vessels in the granulomas were already stained by MECA-217 in the first days of development. In contrast MECA-325 detected postcapillary venules (which frequently displayed the morphologic characteristics of HEV) only from approximately 1 wk, in parallel with the development of a persistent mononuclear cell infiltrate including numerous lymphocytes. The selective appearance of the MECA-325 antigen on vascular endothelium supporting lymphocyte traffic in both lymphoid and extra-lymphoid sites suggests that this antigen may play an important role in the process of lymphocyte extravasation. The demonstration of lymphoid organ- and inflammation-specific microvascular antigens offers direct evidence for a complex specialization of endothelium in relation to immune stimuli, and supports the concept that microvascular differentiation may play an important role in local immune responses.

  View details for Web of Science ID A1987F910000009

  View details for PubMedID 3543117

• THE CORRELATION OF LECTIN-STIMULATED PROLIFERATION AND CYTOTOXICITY IN MURINE THYMOCYTES WITH EXPRESSION OF THE MEL-14-DEFINED HOMING RECEPTOR JOURNAL OF IMMUNOLOGY Wilson, A., Scollay, R., REICHERT, R. A., BUTCHER, E. C., Weissman, I. L., Shortman, K. 1987; 138 (2): 352-357

  Abstract

  The relationship between the expression of the MEL-14-defined lymphocyte homing receptor and the proliferation and functional differentiation of thymocytes in response to lectin stimulation was examined. Two-color fluorescent staining with MEL-14 in various combinations with PNA and anti-Ly-2 and anti-L3T4 was used to separate thymocyte populations for functional analysis. A high cloning-efficiency limit-dilution culture system was used to determine the frequency of all cells responsive to concanavalin A (PTL-p) or of all precursors of lectin-enhanced cytolytic lymphocytes (CTL-p). As expected from earlier studies, PTL-p and CTL-p were concentrated in the PNA- thymocytes, PTL-p were in both the Ly-2- L3T4+ and the Ly-2+ L3T4- subpopulations, and CTL-p were predominantely in the Ly-2+ L3T4- subpopulation. Within the PNA- thymocytes, two distinct peaks of PTL-p were found in cells stained with MEL-14, corresponding to MEL-14- and MEL-14medium-to-high cells, whereas the CTL-p frequency increased in fractions showing increasing expression of the MEL-14-defined antigen. Within the Ly-2- L3T4+ subpopulation, two distinct peaks of PTL-p were found corresponding to groups of MEL-14- and MEL-14medium-to-high cells, with the intermediate fraction of MEL-14low cells displaying a very low PTL-p frequency. The Ly-2+ L3T4- subpopulation included fewer MEL-14- cells and more MEL-14high cells than the Ly-2- L3T4+ subpopulation. Within the Ly-2+ L3T4- subpopulation, the few MEL-14- cells expressed a relatively low but definite frequency of CTL-p and PTL-p. The more numerous MEL-14+, Ly-2+ L3T4- cells included a high frequency of CTL-p and PTL-p, which did not vary over the medium-to-high MEL-14 expression range. These results indicate that the correlation of MEL-14 expression with CTL-p frequency among thymocytes is largely a consequence of the relative frequency of Ly-2+ L3T4- cells in the separated fractions, rather than a direct link between MEL-14 expression and function. Nevertheless, MEL-14 does define significant heterogeneity in both the Ly-2+ L3T4- and the Ly-2- L3T4+ subpopulations. In particular, there is a reduced functional response among the small subgroup of Ly-2+ L3T4- MEL-14- cells, suggesting this population includes either immature cells or cells of a different functional type.

  View details for Web of Science ID A1987F617900003

  View details for PubMedID 3491850

• HUMAN-LYMPHOCYTE AND LYMPHOMA HOMING RECEPTORS ANNUAL REVIEW OF MEDICINE JALKANEN, S., Wu, N., BARGATZE, R. F., BUTCHER, E. C. 1987; 38: 467-476

  Abstract

  The migration of normal and malignant lymphocytes is controlled in part by selective lymphocyte recognition of high endothelial venules (HEV) at sites of lymphocyte exit from the blood. Recirculating lymphocytes appear to utilize structurally related, yet functionally distinct, 90-kD receptors to interact in an organ-specific manner with HEV in peripheral lymph nodes, in mucosa-associated lymphoid tissues (Peyer's patches, appendix), and in inflamed joint tissue (synovium). These lymphocyte "homing receptors" constitute a family of glycoprotein endothelial cell recognition elements that regulate the extravasation of circulating normal and neoplastic lymphocytes into different organs of the body, and thus play an important role in determining the characteristics of local immune responses and the patterns of dissemination of lymphoid neoplasms.

  View details for Web of Science ID A1987G775700038

  View details for PubMedID 3555307

• INTERFERON-GAMMA REGULATES AN ANTIGEN SPECIFIC FOR ENDOTHELIAL-CELLS INVOLVED IN LYMPHOCYTE TRAFFIC PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA DUIJVESTIJN, A. M., SCHREIBER, A. B., BUTCHER, E. C. 1986; 83 (23): 9114-9118

  Abstract

  One of the most striking examples of localized vascular differentiation is exhibited by specialized lymphoid organ venules that mediate the extravasation of circulating lymphocytes from the blood. These vessels are characterized by cuboidal or "high" endothelial cell morphology and are unique in their functional capacity to interact with migrating lymphocytes, regulating both the rate and specificity of lymphocyte traffic through particular regions of the body. We describe here a monoclonal antibody, MECA-325, that defines an endothelial cell differentiation antigen selectively expressed on high endothelium in the mouse. Thus an antigen defining a specific functional subset of endothelial cells has been found. Furthermore, we demonstrate that the MECA-325 antigen can be induced in mouse lung or bone marrow-derived endothelial cell lines in vitro by interferon-gamma but not by interferon-beta, interleukin-1, or endothelial cell mitogens. The results define a unique marker associated with differentiated endothelial cells mediating lymphocyte traffic from the blood, and they provide evidence that the specialized phenotype of these high endothelial cells may be induced and controlled by local factors associated with immune activity.

  View details for Web of Science ID A1986F147100062

  View details for PubMedID 3097642

  View details for PubMedCentralID PMC387085

• A LYMPHOID-CELL SURFACE GLYCOPROTEIN INVOLVED IN ENDOTHELIAL-CELL RECOGNITION AND LYMPHOCYTE HOMING IN MAN EUROPEAN JOURNAL OF IMMUNOLOGY JALKANEN, S. T., BARGATZE, R. F., Herron, L. R., BUTCHER, E. C. 1986; 16 (10): 1195-1202

  Abstract

  We describe a 90-kDa lymphocyte surface glycoprotein, recognized by the monoclonal antibody Hermes-1, that is involved in endothelial cell recognition and lymphocyte trafficking in man. This molecule is selectively expressed on normal or transformed lymphoid cells that are able to recognize and bind to high endothelial venules (HEV, specialized vessels that mediate lymphocyte exit from the blood into lymphoid organs); appears to be linked to HEV recognition function since, in fluorescence-activated cell sorting of variants of a cloned cell line, HEV binding ability co-selects with the Hermes-1 antigen; bears the predominant cell surface epitopes recognized by heterologous anti-human lymphocyte antibodies able to interfere with lymphocyte binding to HEV; and is structurally similar to a previously described mouse lymphocyte surface receptor for HEV. These findings demonstrate that the molecule defined by Hermes-1 either functions as a specific lymphocyte surface receptor for HEV, or is both precisely coregulated and physically and/or functionally associated with such receptors. The expression of this putative receptor for HEV on normal human lymphocyte populations parallels, and thus presumably helps determine, their migratory status in vivo. Hermes-1 should be a powerful tool for analyzing the role of endothelial cell recognition in the traffic of normal and neoplastic human lymphocytes.

  View details for Web of Science ID A1986E839100002

  View details for PubMedID 2429846

• A DISTINCT ENDOTHELIAL-CELL RECOGNITION SYSTEM THAT CONTROLS LYMPHOCYTE TRAFFIC INTO INFLAMED SYNOVIUM SCIENCE JALKANEN, S., STEERE, A. C., FOX, R. I., BUTCHER, E. C. 1986; 233 (4763): 556-558

  Abstract

  Lymphocytes are essential mediators of normal tissue inflammatory reactions and of pathologic tissue damage in, for example, rheumatoid arthritis and other autoimmune diseases. In a study of the mechanisms controlling lymphocyte entry into sites of inflammation from the blood, the function and specificity of lymphocyte-endothelial interactions were examined in inflamed joint tissue (synovium) from patients with rheumatoid arthritis. Synovial high endothelial venules (HEV) supported the binding of normal peripheral blood lymphocytes in vitro. The characteristics of this binding, which were similar to those of lymphocyte-HEV interactions controlling lymphocyte migration into organized lymphoid tissues, included a requirement for calcium ions, a dependence on metabolic activity, and a preferential adherence of circulating lymphocytes as opposed to immature thymocytes. However, the binding of lymphocytes to synovial HEV was not inhibited by a monoclonal antibody to lymphocyte receptors for lymph node HEV, and synovial HEV failed to bind either lymph node HEV-specific or mucosal HEV-specific B lymphoblastoid cells. The results suggest that a lymphocyte-endothelial cell recognition system that is distinct from such systems in organized lymphoid tissues directs the extravasation of normal lymphocytes as well as pathologically important effector cells into inflamed synovium.

  View details for Web of Science ID A1986D339100030

  View details for PubMedID 3726548

• ANTIGEN-INDUCED CHANGES IN B-CELL SUBSETS IN LYMPH-NODES - ANALYSIS BY DUAL FLUORESCENCE FLOW CYTOFLUOROMETRY EUROPEAN JOURNAL OF IMMUNOLOGY Kraal, G., Hardy, R. R., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1986; 16 (7): 829-834

  Abstract

  Changes in the representation and surface phenotype of defined B cell subsets in murine lymph nodes stimulated with keyhole limpet hemocyanin or sheep red blood cells have been analyzed by two-color immunofluorescence fluorocytometric analysis. Shortly after immunization with either antigen there is a dramatic increase in both the frequency and absolute number of IgM+, IgD+ B cells, which is followed by the formation of germinal centers. Germinal center cells, as soon as they appear on day 3 after primary immunization, bind high levels of peanut agglutinin, bear low levels of surface IgM but no detectable surface IgD, and are characterized by lack of staining with MEL-14, a monoclonal antibody which recognizes a lymphocyte surface receptor involved in lymphocyte homing. The level of I-A and H-2K region-encoded surface antigens on early germinal center cells is higher than on PNAlo B cells. During the first 7 days of the germinal centers there is a progressive decrease in the average level of H-2K but not of Ia antigens. A similar decrease was observed for ThB. It is confirmed that the germinal center cell population contains the majority of antigen-binding cells in the stimulated lymph node. These findings indicate that B cells are recruited nonspecifically to antigen-stimulated lymph nodes, and that the antigen-specific cells then selectively participate in the formation of germinal centers where they undergo specific differentiation events.

  View details for Web of Science ID A1986D318200017

  View details for PubMedID 2873041

• DUAL IMMUNOFLUORESCENCE STUDIES OF CORTISONE-INDUCED THYMIC INVOLUTION - EVIDENCE FOR A MAJOR CORTICAL COMPONENT TO CORTISONE-RESISTANT THYMOCYTES JOURNAL OF IMMUNOLOGY REICHERT, R. A., Weissman, I. L., BUTCHER, E. C. 1986; 136 (10): 3529-3534

  Abstract

  Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.

  View details for Web of Science ID A1986C223100004

  View details for PubMedID 3084633

• PHENOTYPIC ANALYSIS OF THYMOCYTES THAT EXPRESS HOMING RECEPTORS FOR PERIPHERAL LYMPH-NODES JOURNAL OF IMMUNOLOGY REICHERT, R. A., Weissman, I. L., BUTCHER, E. C. 1986; 136 (10): 3521-3528

  Abstract

  Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.

  View details for Web of Science ID A1986C223100003

  View details for PubMedID 3084632

• ONTOGENY OF LYMPHOCYTE HOMING RECEPTOR EXPRESSION IN THE MOUSE THYMUS JOURNAL OF IMMUNOLOGY REICHERT, R. A., Jerabek, L., Gallatin, W. M., BUTCHER, E. C., Weissman, I. L. 1986; 136 (10): 3535-3542

  Abstract

  The monoclonal antibody MEL-14 has been used in conjunction with immunohistology and multiparameter immunofluorescence to identify and characterize homing receptor-bearing thymocytes at various stages of embryonic and neonatal development. MEL-14hi thymocytes first appear at day 14 of gestation and come to represent about 40% of day 15 fetal thymocytes. Thereafter, the proportion of MEL-14hi thymocytes rapidly declines such that by birth (usually the 20th day of embryonic development) only about 2% of thymocytes are MEL-14hi. Although newborn thymocytes resemble adult thymocytes in this respect, the phenotypic characteristics of fetal and neonatal MEL-14hi thymocytes suggest that this unique subset undergoes a gradual transition from containing exclusively phenotypically immature cells in early gestation to containing predominantly phenotypically mature cells by young adulthood. Thus, virtually none of day 15 MEL-14hi fetal thymocytes are peanut agglutinin (PNA)lo, Ly-1hi, or either Lyt-2-/L3T4+ or Lyt-2+/L3T4-, whereas in the weeks that follow a steadily greater proportion of MEL-14hi thymocytes come to express this mature pattern (roughly 70% at 4 wk of age). Most day 15 MEL-14hi fetal thymocytes appear to express the functional homing receptor molecule, since day 15 fetal thymocytes bind to peripheral lymph node high endothelial venules about 40 to 50% as well as do adult mesenteric node lymphocytes, whereas adult thymocytes bind only about 5% as well. We have also identified a population of outer cortical MEL-14hi Lyt-2-/L3T4- lymphoblasts that appears during thymus regeneration 5 to 6 days after the administration of hydrocortisone. These lymphoblasts express the same phenotype as cells that constitute 40% of the day 15 fetal thymus and only 0.4% of normal adult thymocytes, implying that this particular subset may make up a significant fraction of thymocytes whenever there is a requirement for rapid expansion of the intrathymic and/or peripheral T cell pools. Taken together, these results are consistent with the notion that expression of the MEL-14-defined homing receptor may be closely linked to important intrathymic events that may occur early in T cell development and yet still have an overriding impact on the selection of those thymocytes that will serve as precursors of thymus emigrants.

  View details for Web of Science ID A1986C223100005

  View details for PubMedID 3084634

• LYMPHOCYTE HOMING RECEPTORS CELL Gallatin, M., STJOHN, T. P., Siegelman, M., Reichert, R., BUTCHER, E. C., Weissman, I. L. 1986; 44 (5): 673-680

  View details for Web of Science ID A1986A542800001

  View details for PubMedID 3004743

• INTERACTIONS BETWEEN ENDOTHELIAL-CELLS AND LEUKOCYTES JOURNAL OF CELLULAR BIOCHEMISTRY BUTCHER, E. C., Lewinsohn, D., Duijvestijn, A., BARGATZE, R., Wu, N., JALKANEN, S. 1986; 30 (2): 121-131

  Abstract

  We present evidence that specific receptors are utilized by neutrophils to control their interaction with endothelial cells at sites of acute inflammation and that these receptors are related if not identical to lymphocyte "homing receptors" for lymphoid tissue high endothelium. We speculate that such receptors play a fundamental but not exclusive role in controlling the extravasation and tissue localization of all bone marrow-derived nucleated cells. In addition, we emphasize the active role of endothelial cells in the process of lymphocyte migration and leukocyte extravasation. By the expression of as yet unidentified organ-specific determinants for lymphocyte recognition, endothelial cells control the exit of particular lymphocyte subsets into mucosal versus nonmucosal sites, thus helping to determine the unique features of mucosal versus nonmucosal immune responses. Furthermore, we argue that endothelial cells are exquisitely responsive to local immune reactivity and present evidence that specific lymphokines, including gamma-interferon, play an important role in inducing postcapillary venules to express differentiated features required for the support of lymphocyte traffic into lymphoid organs and into sites of chronic inflammation. Leukocytes, endothelial cells, and probably other tissue cell classes appear to interact at multiple levels by a variety of mechanisms to regulate the local extravasation of immune effector cells.

  View details for Web of Science ID A1986A436100003

  View details for PubMedID 3517023

• THE REGULATION OF LYMPHOCYTE TRAFFIC CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY BUTCHER, E. C. 1986; 128: 85-122

  View details for Web of Science ID A1986E409700003

  View details for PubMedID 3533449

• HOMING RECEPTORS AND THE CONTROL OF LYMPHOCYTE MIGRATION IMMUNOLOGICAL REVIEWS JALKANEN, S., REICHERT, R. A., Gallatin, W. M., BARGATZE, R. F., Weissman, I. L., BUTCHER, E. C. 1986; 91: 39-60

  Abstract

  The traffic of lymphocytes is controlled in part by the selective interaction of circulating lymphocytes with specialized high endothelial venule (HEV) cells at sites of lymphocyte exit from the blood. At least three independent receptor systems are responsible for controlling lymphocyte traffic to different lymphoid organs or to sites of inflammation: one mediates lymphocyte interaction with HEV in peripheral lymph nodes, another in mucosa-associated lymphoid tissues, and a third in inflamed synovium. The receptors mediating lymphocyte recognition of HEV in different organs appear to be structurally related yet antigenically and functionally distinct 90 kD glycoproteins. Receptors for lymph node HEV can function as mammalian lectins, and probably interact with specific carbohydrate ligands on high endothelial cells. Mouse and human homing receptors share both antigenic and structural features, indicating a high conservation of lymphocyte-endothelial recognition systems during evolution. They play an essential part in the immune process by controlling lymphocyte traffic during B- and T-cell differentiation, and by segregating effector cells derived from stimulation in different tissues, thus simultaneously increasing the efficiency of organ-specific immune responses and decreasing possibilities for autoimmune crossreactions. Homing receptors are also expressed by many mouse and human lymphoid neoplasms, and appear to play a role in lymphoma metastasis. Related if not identical receptors are expressed by other leukocyte types, including polymorphonuclear leukocytes, monocytes, and large granular lymphocytes (natural killer cells). Thus lymphocyte homing receptors are members of a family of glycoprotein receptors for endothelium that control the extravasation of lymphocytes as well as other leukocytes, and help regulate both non-specific and specific immune responses in vivo.

  View details for Web of Science ID A1986D137400002

  View details for PubMedID 2426181

• PROSPECTS FOR UNDERSTANDING THE ROLE OF LYMPHOID MICROENVIRONMENTS IN B-CELL DIFFERENTIATION INVIVO ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY BUTCHER, E. C. 1985; 186: 3-8

  View details for Web of Science ID A1985APB7200001

  View details for PubMedID 3876704

• Lymphocyte and lymphoma receptors utilized in differentiation, in homing, and in lymphomagenesis. Haematology and blood transfusion Weissman, I. L., McGrath, M. S., REICHERT, R. A., Gallatin, W. M., Ezine, S., Fink, P., BUTCHER, E. C., Marian, J., O'Neill, H. C. 1985; 29: 449-454

  View details for PubMedID 4029738

• GERMINAL CENTER CELLS - ANTIGEN-SPECIFICITY, HEAVY-CHAIN CLASS EXPRESSION AND EVIDENCE OF MEMORY ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1985; 186: 145-151

  View details for Web of Science ID A1985APB7200018

  View details for PubMedID 3876700

• HUMAN T-CELL CLONES EXPRESS FUNCTIONAL HOMING RECEPTORS REQUIRED FOR NORMAL LYMPHOCYTE TRAFFICKING JOURNAL OF EXPERIMENTAL MEDICINE NAVARRO, R. F., JALKANEN, S. T., HSU, M., SOENDERSTRUPHANSEN, G., GORONZY, J., WEYAND, C., Fathman, C. G., CLAYBERGER, C., KRENSKY, A. M., BUTCHER, E. C. 1985; 162 (3): 1075-1080

  Abstract

  To function efficiently in vivo, lymphocytes must circulate from the blood into lymphoid tissues and other sites of immune reaction. Herein, we show that human cytotoxic and helper T cell clones and lines, maintained in vitro with IL-2, express the functional capacity to recognize and bind to high endothelial venules (HEV), a capacity essential for lymphocyte exit from the blood, and hence for normal lymphocyte trafficking. The expression of functional homing receptors distinguishes human T cell clones from their murine counterparts, which uniformly lack receptors for HEV and are unable to migrate normally from the blood in vivo. The results raise the possibility that human T cell clones may be more effective in mediating in vivo immune responses than is suggested by murine models.

  View details for Web of Science ID A1985AQH0300021

  View details for PubMedID 3875680

  View details for PubMedCentralID PMC2187804

• INVITRO ANALYSIS OF THE HOMING PROPERTIES OF HUMAN-LYMPHOCYTES - DEVELOPMENTAL REGULATION OF FUNCTIONAL RECEPTORS FOR HIGH ENDOTHELIAL VENULES BLOOD JALKANEN, S. T., BUTCHER, E. C. 1985; 66 (3): 577-582

  Abstract

  Circulating lymphocytes leave the blood by binding to specialized high endothelial cells lining postcapillary venules in lymphoid organs or sites of chronic inflammations, migrating through the vessel wall into the surrounding tissue. The capacity of lymphocytes to recognize and bind to high endothelial venules (HEVs) is thus central to the overall process of lymphocyte traffic and recirculation. We show that viable human lymphocytes bind selectively to HEVs in frozen sections of normal human lymph nodes, thus defining a simple in vitro model for the study of human lymphocyte homing properties. Optimal conditions for the quantitative analysis of lymphocyte-HEV interaction are described. Furthermore, by using this assay, we demonstrate that the ability of human lymphocyte populations to bind to HEVs parallels their presumed migratory status in vivo. Thus, thymocytes and bone marrow cells, which are sessile in vivo, bind poorly to HEVs in comparison with mature circulating lymphocytes in peripheral blood or in peripheral lymphoid tissues. These results indicate that HEV-binding ability is a regulated property of mature lymphocytes and, as demonstrated previously in animal models, probably plays a fundamental role in controlling lymphocyte traffic in humans. The in vitro model of lymphocyte-HEV interaction thus provides a unique means to assay the migratory properties of normal and neoplastic human lymphocyte subsets, to analyze the role of lymphocyte traffic mechanisms in normal and pathologic inflammatory reactions, and to define some of the molecular mechanisms responsible for the control of lymphocyte migration and positioning in humans.

  View details for Web of Science ID A1985AQK6200015

  View details for PubMedID 4027380

• HUMAN LYMPHOCYTE-HIGH ENDOTHELIAL VENULE INTERACTION - FUNCTIONAL AND MOLECULAR CHARACTERIZATION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY JALKANEN, S., BARGATZE, R., HERRON, L., BUTCHER, E. C. 1985; 186: 615-620

  View details for Web of Science ID A1985APB7200075

  View details for PubMedID 2996314

• ENRICHMENT OF MURINE AND HUMAN LANGERHANS CELLS WITH SOLID-PHASE IMMUNOABSORPTION USING PAN-LEUKOCYTE MONOCLONAL-ANTIBODIES JOURNAL OF INVESTIGATIVE DERMATOLOGY Wood, G. S., Kosek, J., BUTCHER, E. C., Morhenn, V. B. 1985; 84 (1): 37-40

  Abstract

  Using a solid phase immunoabsorption (panning) technique, we have employed pan-leukocyte monoclonal antibodies to enrich and deplete murine and human Langerhans cells from cell suspensions of normal skin. Langerhans cell-enriched fractions contained 80-99% mononuclear cells, almost all of which had the ultrastructural features of Langerhans cells. These results are comparable to those achieved by panning for human Langerhans cells with anti-Leu-6(T6) antibody. Similarly, less than 1% of these cells were detectable in Langerhans cell-depleted fractions and such fractions were incapable of stimulating allogeneic lymphocytes in the skin cell-lymphocyte reaction. We conclude that panning with pan-leukocyte antibodies is an effective means of enriching or depleting Langerhans cells from heterogeneous skin cell suspensions and can yield results similar to those achieved with more Langerhans cell-specific reagents such as anti-Leu-6(T6). These findings are of particular significance to the enrichment and depletion of murine Langerhans cells since they express no known correlate of the human Leu-6(T6) antigen.

  View details for Web of Science ID A1985TZ98100010

  View details for PubMedID 3880795

• HOMING RECEPTOR-BEARING THYMOCYTES, AN IMMUNOCOMPETENT CORTICAL SUBPOPULATION NATURE Fink, P. J., Gallatin, W. M., REICHERT, R. A., BUTCHER, E. C., Weissman, I. L. 1985; 313 (5999): 233-235

  Abstract

  Much of the differentiation of murine T cells takes place in the thymus, perhaps influenced by the operation of stringent selection mechanisms whose existence has been inferred from the high rate of thymocyte turnover in the absence of extensive emigration. The origin of those 1% of total thymocytes which leave the thymus and seed the peripheral lymphoid organs is obscure. Recent thymic emigrants are functionally and phenotypically mature, and the purported greater maturity of medullary relative to cortical thymocytes is often cited a evidence for the medullary origin of thymic emigrants, a suggestion not without its critics. To approach this question, we have now isolated a a subpopulation of thymocytes expressing high levels of a receptor that mediates the homing of blood-borne lymphocytes into peripheral lymph nodes. Surprisingly, this population of cells (1-3% of total thymocytes) is both cortical and immunocompetent, containing approximately half of all thymic cytolytic T-lymphocyte precursors. The combination of homing receptor expression and immunocompetence makes this cortical population ideally suited for emigration to peripheral lymphoid organs.

  View details for Web of Science ID A1985AAB1100056

  View details for PubMedID 3871507

• T-CELL CLONES - A MODEL OF A NON-RECIRCULATING PHASE OF T-CELL DIFFERENTIATION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Dailey, M. O., Gallatin, W. M., Kraal, G., Butcher, E., Weissman, I. 1985; 186: 621-627

  View details for Web of Science ID A1985APB7200076

  View details for PubMedID 2996315

• A HOMING RECEPTOR-BEARING CORTICAL THYMOCYTE SUBSET - IMPLICATIONS FOR THYMUS-CELL MIGRATION AND THE NATURE OF CORTISONE-RESISTANT THYMOCYTES CELL REICHERT, R. A., Gallatin, W. M., BUTCHER, E. C., Weissman, I. L. 1984; 38 (1): 89-99

  Abstract

  The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.

  View details for Web of Science ID A1984TF99100012

  View details for PubMedID 6432340

• LOCALIZATION OF LYMPHOCYTE SUBPOPULATIONS IN PERIPHERAL LYMPHOID ORGANS - DIRECTED LYMPHOCYTE MIGRATION AND SEGREGATION INTO SPECIFIC MICROENVIRONMENTS AMERICAN JOURNAL OF ANATOMY Rouse, R. V., REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1984; 170 (3): 391-405

  Abstract

  The distribution of lymphocytes in the peripheral lymphoid organs is controlled by recirculatory and microenvironmental factors. Specific interactions between recirculating lymphocytes and high endothelial venules in various lymphoid organs determine the presence and proportions of the various lymphoid sets and subsets in those organs. Separate endothelial determinants on peripheral node and Peyer's patch endothelium along with complementary lymphocyte receptors mediate this organ specificity. B and T cells also exhibit nonrandom organization within lymphoid tissues; after entry via high endothelial venules they segregate into their respective domains, which appear to be determined by distinct types of nonlymphoid stromal cells. Antigenic stimulation results in changes in lymphocyte phenotype as well as in the lymphoid microenvironment. The response to most complex antigens is the formation of germinal centers (GC) composed primarily of proliferating B cells; the phenotype of the few T cells therein is supportive of the GC as a site of B-T interaction. The phenotype of the B cells in GCs suggest a role for GCs in immunoglobulin class switching and the determination of subsequent homing specificity.

  View details for Web of Science ID A1984TC39700012

  View details for PubMedID 6383006

• LANGERHANS CELLS REACT WITH PAN-LEUKOCYTE MONOCLONAL-ANTIBODY - ULTRASTRUCTURAL DOCUMENTATION USING A LIVE CELL-SUSPENSION IMMUNOPEROXIDASE TECHNIQUE JOURNAL OF INVESTIGATIVE DERMATOLOGY Wood, G. S., Morhenn, V. B., BUTCHER, E. C., Kosek, J. 1984; 82 (4): 322-325

  Abstract

  Langerhans cells are generally regarded as members of an Ia+ dendritic cell system capable of potent accessory cell function in immune responses. While it has been shown that murine Langerhans cells are bone marrow-derived, the ontogenic relationships among human Langerhans cells, other dendritic cells, macrophages, and leukocytes in general have yet to be fully clarified. Recently, several pan-leukocyte monoclonal antibodies have been produced which react with the human leukocyte common antigen. This antigen resembles the murine T200 antigen and is expressed by all leukocyte subtypes but not by nonhematopoietic cells. Using an immunoperoxidase technique for staining suspensions of live skin cells, we have documented Langerhans cell reactivity with pan-leukocyte monoclonal antibody L3B12 at the ultrastructural level. Reactivity with this highly sensitive and specific pan-leukocyte marker supports the concept of the human Langerhans cell as a specialized form of bone marrow-derived mononuclear leukocyte and defines an immunologic feature common to dendritic cells, macrophages, and leukocytes that is not shared by other cell types. This finding is discussed in the context of other recent data concerning the immunologic phenotype of Langerhans cells. Since the immunoultrastructural method employed does not require cell fixation of any kind prior to immunologic staining, it should prove particularly useful for studying cell surface antigens that are adversely affected by fixation.

  View details for Web of Science ID A1984SM05400006

  View details for PubMedID 6368698

• The role of carbohydrate in heterotypic cell-cell recognition: lymphocyte-high endothelial cell interaction as a model system. Biology of the cell BUTCHER, E. C. 1984; 51 (2): 248-250

  Abstract

  The studies reviewed here demonstrate that the interaction of lymphocytes with HEV is one of the most approachable models available for the study of heterotypic cell-cell recognition mechanisms. Lymphocyte-HEV interaction is mediated by specific lymphocyte surface receptors recognizing, by as yet unknown mechanisms, determinants expressed by specialized high endothelial cells in lymphoid tissues and sites of chronic inflammation. Both the lymphocyte and endothelial cell surface elements of the interaction are precisely regulated, controlling the traffic of lymphocyte subsets through particular lymphoid organs and into sites of inflammation. Considerable progress, reviewed here, has been made in defining and characterizing the lymphocyte surface molecules mediating this cellular interaction. By contrast, the nature of the endothelial cell determinants recognized by migrating lymphocytes remains a mystery. Future experiments must be designed to identify these endothelial cell determinants, and to examine critically a proposed role of carbohydrate in lymphocyte-HEV interaction.

  View details for PubMedID 6240308

• Cellular traffic cues in vivo. Immunology today Butcher, E. 1983; 4 (11): 309

  View details for DOI 10.1016/0167-5699(83)90187-1

  View details for PubMedID 25290710

• DIFFERENCES IN INVIVO DISTRIBUTION AND HOMING OF T-CELL SUBSETS TO MUCOSAL VS NONMUCOSAL LYMPHOID ORGANS JOURNAL OF IMMUNOLOGY Kraal, G., Weissman, I. L., BUTCHER, E. C. 1983; 130 (3): 1097-1102

  Abstract

  The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.

  View details for Web of Science ID A1983QC72100018

  View details for PubMedID 6600472

• DUAL FLUORESCENCE STUDIES OF LYMPHOCYTE MIGRATION AND DIFFERENTIATION BUTCHER, E. C., Weissman, I. L. MARCEL DEKKER INC. 1983: 105–

  View details for Web of Science ID A1983QG07800075

• A CELL-SURFACE MOLECULE INVOLVED IN ORGAN-SPECIFIC HOMING OF LYMPHOCYTES NATURE Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1983; 304 (5921): 30-34

  Abstract

  Lymphocytes migrate from the bloodstream by recognizing and binding to specialized endothelial cells lining the high endothelial venules (HEV) in lymph nodes and Peyer's patches. We describe here a monoclonal antibody, MEL-14, specific for a lymphocyte surface molecule that appears to mediate recognition of lymph node HEV, and to be required for lymphocyte homing into lymph nodes in vivo.

  View details for Web of Science ID A1983QX44800035

  View details for PubMedID 6866086

• GERMINAL CENTER B-CELLS LACK HOMING RECEPTORS NECESSARY FOR NORMAL LYMPHOCYTE RECIRCULATION JOURNAL OF EXPERIMENTAL MEDICINE REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. 1983; 157 (3): 813-827

  Abstract

  Germinal center B cells (GCLC) are a discrete population of antigen-activated lymphoblasts that lack surface IgD and express abundant cell surface binding sites for peanut agglutinin (PNA). These phenotypic features render GCLC easily distinguishable from nearly all plasma cells, T cells, and unstimulated B cells, and have enabled us to identify and isolate GCLC from antigen-stimulated murine lymphoid organs. We have examined the migratory properties of these lymphoblasts in (a) short-term in vivo homing studies, and (b) an in vitro assay of lymphocyte binding to post-capillary, high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral lymph nodes. In the in vivo experiments, intravenously injected GCLC failed to migrate in significant numbers to peripheral lymphoid organs in comparison with T cells or IgD+ B cells. In the in vitro binding assay, GCLC did not adhere to HEV in either Peyer's patch or peripheral node sections. A variety of factors, such as preferential sequestration in the liver, may operate in vivo to influence the localization of these cells. However, their nearly total failure to migrate into lymphoid organs can best be explained by their inability to recognize and adhere to the specialized HEV which normally mediate the emigration of recirculating lymphocytes from the blood into these sites. The concept that GCLC fail to express functional homing receptors for HEV has been further supported by studies using MEL-14, a monoclonal antibody that appears to recognize the lymphocyte surface receptor for peripheral node HEV: In contrast to most peripheral lymphocytes, GCLC fail to bind MEL-14. These migratory and endothelial-recognition properties of GCLC, when viewed in the context of the possible role of these cells as precursors of plasma cells and/or memory B cells, have led us to propose that the inability of GCLC to recognize HEV may be transient and related to a phase of sessile B cell differentiation.

  View details for Web of Science ID A1983QH26700001

  View details for PubMedID 6339668

  View details for PubMedCentralID PMC2186964

• GENETIC-CONTROL OF T-CELL SUBSET REPRESENTATION IN INBRED MICE IMMUNOGENETICS Kraal, G., Weissman, I. L., BUTCHER, E. C. 1983; 18 (6): 585-592

  Abstract

  Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.

  View details for Web of Science ID A1983RW97000003

  View details for PubMedID 6606619

• SURFACE PHENOTYPE AND MIGRATORY CAPABILITY OF PEYERS PATCH GERMINAL CENTER CELLS ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY BUTCHER, E. C., REICHERT, R. A., Coffman, R. L., Nottenburg, C., Weissman, I. L. 1982; 149: 765-772

  Abstract

  Peanut agglutinin (PNA) binds selectively to germinal center cells in mouse peripheral lymphoid organs. Using PNA as a marker, we have determined that Peyer's patch germinal center cells are B cells with a unique phenotype---they express a low level of surface immunoglobulin (about 85% Ig+), predominantly of the IgA class (70% alpha+), with only 10% bearing surface IgM, and few if any expressing IgD. This phenotype identifies murine Peyer's patch germinal center cells as fairly late cells in B cell differentiation, and suggests that they may be precursors of IgA-secreting plasma cells in the gut wall. In addition, we have described a means of purifying PNA+ Peyer's patch lymphocytes, and have demonstrated that these cells lack functional receptors for high endothelial venules and fail to migrate to lymphoid organs in vivo. It is speculated that PNA may be a general marker for nonmigratory lymphocyte populations undergoing local differentiation.

  View details for Web of Science ID A1982PL44700107

  View details for PubMedID 6983239

• SURFACE PHENOTYPE AND HOMING PROPERTIES OF ACTIVATED LYMPHOCYTES AND T-CELL CLONES Dailey, M. O., Gallatin, W. M., Butcher, E., Weissman, I. ELSEVIER GMBH, URBAN & FISCHER VERLAG. 1982: 240–40

  View details for Web of Science ID A1982PR60800210

• ANTIGEN-SPECIFICITY OF GERMINAL CENTER CELLS AND CHANGES IN ISOTYPE EXPRESSION Kraal, G., Weissman, I. L., BUTCHER, E. C. FEDERATION AMER SOC EXP BIOL. 1982: 418–18

  View details for Web of Science ID A1982NG28200898

• GERMINAL CENTER B-CELLS ARE LYMPHOCYTES UNDERGOING A SESSILE PHASE OF DIFFERENTIATION REICHERT, R. A., Gallatin, W. M., Weissman, I. L., BUTCHER, E. C. ELSEVIER GMBH, URBAN & FISCHER VERLAG. 1982: 248–49

  View details for Web of Science ID A1982PR60800225

• DIFFERENCES IN THE MIGRATION OF LYMPHOCYTE-B AND LYMPHOCYTE-T - ORGAN-SELECTIVE LOCALIZATION INVIVO AND THE ROLE OF LYMPHOCYTE-ENDOTHELIAL CELL RECOGNITION JOURNAL OF IMMUNOLOGY Stevens, S. K., Weissman, I. L., BUTCHER, E. C. 1982; 128 (2): 844-851

  Abstract

  The migration of B and T lymphocytes in the mouse has been studied by using 1) short-term in vivo homing studies, and 2) an in vitro assay of lymphocyte binding to specialized lymphoid organ venules (post-capillary, high endothelial venules (HEV)) in frozen sections of lymph nodes and Peyer's patches. The homing characteristics of B and T cell populations are largely independent of their organ of origin. B cells from any source distribute preferentially to Peyer's patches, whereas T cells home preferentially to peripheral lymph nodes. This organ specificity of migration appears to be determined at the site of lymphocyte exit from the blood by selective recognition of organ-specific determinants on the endothelial cells of HEV. In addition, the in vivo tendency of B cells to migrate preferentially to the spleen, and of T cells to localize better in lymph nodes is confirmed. The results indicate that, in a hypothetical situation in which an equal number of B and T lymphocytes localized in peripheral lymph nodes (or bound in vitro to peripheral node HEV), there would be about 2.5 B cells for every T cell in the mesenteric node, four to six B cells per T cell in Peyer's patches, and seven to nine B cells per T cell in the spleen. Comparison of these homing preferences with the distribution of B and T lymphocyte populations in situ suggests that selective lymphocyte migration may help determine the proportions of functionally distinct lymphocyte classes in particular lymphoid organs or sites of chronic inflammation, and thus may serve to influence the character of local immune responses.

  View details for Web of Science ID A1982MZ49700059

  View details for PubMedID 6976385

• ABNORMAL MIGRATION OF LYMPHOCYTE-T CLONES JOURNAL OF IMMUNOLOGY Dailey, M. O., Fathman, C. G., BUTCHER, E. C., Pillemer, E., Weissman, I. 1982; 128 (5): 2134-2136

  Abstract

  Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.

  View details for Web of Science ID A1982NL17700035

  View details for PubMedID 6460817

• SURFACE PHENOTYPE OF PEYERS PATCH GERMINAL CENTER CELLS - IMPLICATIONS FOR THE ROLE OF GERMINAL-CENTERS IN B-CELL DIFFERENTIATION JOURNAL OF IMMUNOLOGY BUTCHER, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R., Weissman, I. L. 1982; 129 (6): 2698-2707

  Abstract

  The surface phenotype of Peyer's patch germinal center lymphoid cells in the mouse is described. It is confirmed that most germinal center lymphocytes bind high levels of peanut agglutination (PNA), a lectin with specificity for terminal galactosyl residues. It is shown that germinal center lymphocytes can be identified in cell suspensions as a discrete PNAhi population distinct from other B cells, plasma cells, and most T cells, which bind only low levels of PNA. Using fluorescence-labeled PNA as a marker in dual fluorescence studies, we found that the majority of Peyer's patch germinal center cells are B lymphocytes: PNAhi Peyer's patch cells express B220, the B lineage-specific form of the T200 family of molecules, as well as low levels of surface Ig. They do not express the T cell-lineage antigens Thy-1, Lyt-1, or Lyt-2 (only 1 to 3% positive). They bear lower levels of H2-K than PNAlo B cells, but two to three times the level of surface I-A-encoded determinants. A discrete but variable subpopulation of PNAhi Peyer's patch cells bear ThB in AKR/c mice, but BALB/c PNAhi lymphocytes are ThB-. About 10 to 30% bear surface IgM or IgG, but in contrast to essentially all PNAlo B lymphocytes in this site, they express no detectable surface IgD. The majority of Peyer's patch germinal center cells bear surface IgA, and this IgA is allelically excluded in F1 mice, indicating it is synthesized by the germinal center cells themselves. In fact, germinal centers contain most of the IgA-bearing cells in Peyer's patches (70 to 85%). These findings lend considerable support to the concept that germinal centers in Peyer's patches are the site of generation of precursors of the IgA-secreting plasma cells that characterize mucosal immune responses, and also suggest that germinal centers may play an important role in the process of heavy chain class switching.

  View details for Web of Science ID A1982PR36900070

  View details for PubMedID 6982940

• SELECTIVE MIGRATION OF MURINE LYMPHOCYTES AND LYMPHOBLAST POPULATIONS AND THE ROLE OF ENDOTHELIAL-CELL RECOGNITION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY BUTCHER, E. C., Kraal, G., Stevens, S. K., Weissman, I. L. 1982; 149: 199-206

  View details for Web of Science ID A1982PL44700028

  View details for PubMedID 6983214

• GERMINAL CENTER B-CELLS - ANTIGEN-SPECIFICITY AND CHANGES IN HEAVY-CHAIN CLASS EXPRESSION NATURE Kraal, G., Weissman, I. L., BUTCHER, E. C. 1982; 298 (5872): 377-379

  Abstract

  Germinal centres are histologically defined aggregates of blast cells that occur in B-cell areas of lymphoid tissues after antigenic stimulation. They are believed to be associated with the development of B-cell memory and plasma cell (especially secondary, IgG and IgA) responses. Recent studies of murine lymphoid tissues have defined cell-surface markers that distinguish germinal centre B cells from other mature B cells, permitting their identification and characterization in cell suspensions. Here we have used these markers to define and study germinal centre cells in lympho nodes, and have found that they constitute a unique population of B cells which (1) arises in response to antigenic stimulation, (2) contains nearly all of the demonstrably antigen-specific B cells in the stimulated organ, (3) bears surface IgM after primary stimulation and (4) as a population, demonstrates isotype switching to a predominant population, demonstrates isotype switching to a predominant surface IgG phenotype after secondary stimulation with specific surface IgG phenotype after secondary stimulation with specific antigen. These findings demonstrate that germinal centres are a major site of proliferation and differentiation of antigen-specific B cells in vivo, and suggest that the germinal centre microenvironment may have an important role in heavy chain class switching during B-cell responses.

  View details for Web of Science ID A1982NY37400044

  View details for PubMedID 6806671

• NORMAL AND NEOPLASTIC LYMPHOCYTE MATURATION JOURNAL OF SUPRAMOLECULAR STRUCTURE AND CELLULAR BIOCHEMISTRY Weissman, I. L., McGrath, M. S., Pillemer, E., Hollander, N., Rouse, R. V., Jerabek, L., Stevens, S. K., SCOLLAY, R. G., BUTCHER, E. C. 1981; 15 (3): 303-314

  View details for Web of Science ID A1981MA98800006

  View details for PubMedID 6790720

• Cellular, genetic, and evolutionary aspects of lymphocyte interactions with high-endothelia venules. Ciba Foundation symposium BUTCHER, E. C., Weissman, I. L. 1980; 71: 265-286

  View details for PubMedID 6899991

• T-CELL MATURATION - THYMOCYTE AND THYMUS MIGRANT SUB-POPULATIONS DEFINED WITH MONOCLONAL-ANTIBODIES TO MHC REGION ANTIGENS JOURNAL OF IMMUNOLOGY Scollay, R., Jacobs, S., Jerabek, L., Butcher, E., Weissman, I. 1980; 124 (6): 2845-2853

  Abstract

  The maturation sequences of thymocytes is known to some extent: A generative layer of subcapsular large lymphoblasts gives rise to a major population of small cortical thymocytes and a minor population of midsize medullary thymocytes. The relative contribution of these three populations to the peripheral T cell populations is not yet known. In this study, subcapsular lymphoblasts, cortical small cells, medullary cells, and thymic emigrant cells have all been analyzed by immunofluorescence for expression of the antigens H-2D, I-A, H-2K, and TL. H-2D is expressed brightly on all subcapsular large cells, dimly on cortical small cells, and brightly on all migrants, cortisone-resistant thymocytes (CRT), and peripheral T cells. I-A can be detected at low levels on 30 to 50% of cells in all the thymic subpopulations, and on 30 to 50% of migrants and peripheral T cells. Fifty to 80% of small cortical cells do not express detectable H-2K, but all the other subpopulations, both inside and outside the thymus, stain uniformly quite brightly. TL3 is expressed on 70 to 80% of subcapsular and cortical thymocytes, 30 to 40% of CRT, is undetectable on migrants but can be seen at low levels on 10 to 20% of spleen and lymph node T cells. The possibility that some or all of these antigens represent stable markers of separate lineages rather than unstable, stage-specific markers is discussed.

  View details for Web of Science ID A1980JT75200051

  View details for PubMedID 6154739

• ORGAN SPECIFICITY OF LYMPHOCYTE MIGRATION - MEDIATION BY HIGHLY SELECTIVE LYMPHOCYTE INTERACTION WITH ORGAN-SPECIFIC DETERMINANTS ON HIGH ENDOTHELIAL VENULES EUROPEAN JOURNAL OF IMMUNOLOGY BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1980; 10 (7): 556-561

  Abstract

  Evidence is presented that the organ specificity of lymphocyte migration is determined by selective interaction of lymphocytes with specialized endothelial cells. Mouse Peyer's patch and lymph node lymphocytes bind preferentially to high endothelial venules (HEV) in frozen sections of Peyer's patches and peripheral nodes, respectively, and this in vitro binding preference accurately predicts their differential segregation in vivo 30 min after i.v. injection. Both in vivo and in vitro, about 1.4 times as many as many Peyer's patch as lymph node lymphocytes bind HEV in Peyer's patches, and, conversely, twice as many lymph node cells interact with HEV in nonmesenteric lymph nodes. Even greater specificity is shown by certain homogeneous lymphocyte populations, i.e. thymic lymphomas. Some lymphomas bind with remarkable selectivity to HEV in Peyer's patches, and others interact almost exclusively with those in lymph nodes indicating that the mechanisms mediating selective recognition of HEV are capable of nearly absolute discrimination. Mesenteric node HEV are unique in that they allow both Peyer's patch- and lymph node-specific cells to bind. It is proposed that lymphocyte surface receptors specific for organ-restricted endothelial cell determinants mediate the antigen-independent organ specificity of lymphocyte migration. According to this model, there are at least 2 sets of complementary lymphocyte and endothelial cell receptors, one mediating lymphocyte-HEV adherence in Peyer's patches, the other in lymph nodes.

  View details for Web of Science ID A1980KG04100012

  View details for PubMedID 6157544

• DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE .1. TECHNICAL ASPECTS JOURNAL OF IMMUNOLOGICAL METHODS BUTCHER, E. C., Weissman, I. L. 1980; 37 (2): 97-108

  Abstract

  A rapid and simple method of cell labeling by stable conjugation with fluorescein or rhodamine is described. Viable cells are incubated under benign conditions (near physiologic pH in normal media) with free fluorescein or tetramethyl rhodamine isothiocyanate, and are adequately separated from unreacted fluorochrome by washing or centrifugation through fetal calf serum. The effects of the pH, the time and temperature of incubation, and the concentration of cells, fluorochrome, and free protein in the media are described. The method labels all cell types, although to different degrees. Fluorescence microscopy reveals fluorescence throughout the cell, although chromatin appears relatively spared. Cellular fluorescence is fairly stable at 4 and 25 degrees C, decays rapidly at 37 degrees C, but is nonetheless visible for days even at this temperature. In the case of lymphocytes, intense fluorescence is obtained without affecting cell viability, and without alteration of the ability to mount a graft versus host response.

  View details for Web of Science ID A1980KM67800001

  View details for PubMedID 7003013

• THYMUS-CELL MIGRATION QUANTITATIVE ASPECTS OF CELLULAR TRAFFIC FROM THE THYMUS TO THE PERIPHERY IN MICE EUROPEAN JOURNAL OF IMMUNOLOGY SCOLLAY, R. G., BUTCHER, E. C., Weissman, I. L. 1980; 10 (3): 210-218

  Abstract

  We have used intrathymic injection of fluorescein isothiocyanate to label thymocytes in situ. The method gives random labeling of the thymocyte population and so can be used to quantitate the extent of migration of cells from the thymus to the periphery. Migrant cells can be visualized in frozen sections or cell suspensions of peripheral organs by their fluorescence. Our data show that in young adults, about 1% of thymocytes leave the thymus per day. Since the bulk of thymocytes turn over every 5 to 7 days, this indicates that the vast majority (95%) of thymocytes die within the thymus. Cells that do leave the thymus, go mainly to the T areas of lymph nodes, spleen and Peyer's patches. Migrants are extremely rare in bone marrow, gut and liver. Migration is about the same in neonates as in adults relative to the size of the thymus, but is considerably lower in older animals where it is only about 0.1% of thymocytes per day at the age of six months.

  View details for Web of Science ID A1980JQ94600009

  View details for PubMedID 7379836

• DIRECT FLUORESCENT LABELING OF CELLS WITH FLUORESCEIN OR RHODAMINE ISOTHIOCYANATE .2. POTENTIAL APPLICATION TO STUDIES OF LYMPHOCYTE MIGRATION AND MATURATION JOURNAL OF IMMUNOLOGICAL METHODS BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1980; 37 (2): 109-121

  Abstract

  The effect of direct cell labeling with fluorescein or tetramethyl rhodamine isothiocyanate on lymphocyte migration is examined. In vitro conditions of labeling are defined which (1) do not significantly affect immediate or long term viability of lymphocytes (up to 2 weeks after transfer in vivo), (2) do not alter normal lymphocyte migration, (3) do not affect expression or detectability of surface antigens, and (4) permit direct visualization and counter-staining with fluorescent antibody reagents for days after intravenous injection. The potential application of this method to studies of lymphocyte migration and maturation is discussed.

  View details for Web of Science ID A1980KM67800002

  View details for PubMedID 6777427

• LYMPHOCYTE ADHERENCE TO HIGH ENDOTHELIAL VENULES - CHARACTERIZATION OF A MODIFIED INVITRO ASSAY, AND EXAMINATION OF THE BINDING OF SYNGENEIC AND ALLOGENEIC LYMPHOCYTE POPULATIONS JOURNAL OF IMMUNOLOGY BUTCHER, E. C., SCOLLAY, R. G., Weissman, I. L. 1979; 123 (5): 1996-2003

  View details for Web of Science ID A1979HT92000015

  View details for PubMedID 314954

• Lymphocyte-high endothelial venule interactions: examination of species specificity. Advances in experimental medicine and biology Butcher, E., Scollay, R., Weissman, I. 1979; 114: 65-72

  View details for PubMedID 313685

• EVIDENCE OF CONTINUOUS EVOLUTIONARY CHANGE IN STRUCTURES MEDIATING ADHERENCE OF LYMPHOCYTES TO SPECIALIZED VENULES NATURE Butcher, E., Scollay, R., Weissman, I. 1979; 280 (5722): 496-498

  View details for Web of Science ID A1979HF85100050

  View details for PubMedID 460429

• PATHOLOGY AND HOMING OF A TRANSPLANTABLE MURINE B CELL LEUKEMIA (BCL1) JOURNAL OF IMMUNOLOGY Warnke, R. A., Slavin, S., Coffman, R. L., BUTCHER, E. C., Knapp, M. R., Strober, S., Weissman, I. L. 1979; 123 (3): 1181-1188

  Abstract

  The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.

  View details for Web of Science ID A1979HH67000037

  View details for PubMedID 381519

• LYMPHOID SYSTEM - ITS NORMAL ARCHITECTURE AND POTENTIAL FOR UNDERSTANDING SYSTEM THROUGH STUDY OF LYMPHOPROLIFERATIVE DISEASES HUMAN PATHOLOGY Weissman, I. L., Warnke, R., BUTCHER, E. C., Rouse, R., Levy, R. 1978; 9 (1): 25-45

  Abstract

  This article presents a view of lymphoid tissue architecture as defined by the traffic of defined lymphoid cell classes. The compartmentalization of lymphocytes is discussed in reference to specific cell-cell interactions that occur in antigen-driven immune responses. Finally, the distribution of normal and neoplastic lymphocytes in humans is defined and compared with animal model systems.

  View details for Web of Science ID A1978EK34700004

  View details for PubMedID 344190

• LYT MARKERS ON THYMUS-CELL MIGRANTS NATURE Scollay, R., Kochen, M., Butcher, E., Weissman, I. 1978; 276 (5683): 79-80

  View details for Web of Science ID A1978FU83100046

  View details for PubMedID 310964

• LYMPHOCYTE INTERACTION WITH HIGH ENDOTHELIAL VENULES - LACK OF SPECIES SPECIFICITY Butcher, E., Scollay, R., Weissman, I. GUSTAV FISCHER VERLAG. 1978: 304–

  View details for Web of Science ID A1978FD02400011

• MEASUREMENT OF NANOGRAM QUANTITIES OF PROTEIN BY HYDROLYSIS FOLLOWED BY REACTION WITH ORTHOPHTHALALDEHYDE OR DETERMINATION OF GLUTAMATE ANALYTICAL BIOCHEMISTRY BUTCHER, E. C., Lowry, O. H. 1976; 76 (2): 502-523

  View details for Web of Science ID A1976CN80400011

  View details for PubMedID 11709