Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

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.     

Interactions between endothelial cells and leukocytes

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.     

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor

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.     

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.     

Dynamic control of lymphocyte trafficking by fever-range thermal stress

Migration of blood-borne lymphocytes into tissues involves a tightly orchestrated sequence of adhesion events. Adhesion molecules and chemokine receptors on the surface of circulating lymphocytes initiate contact with specialized endothelial cells under hemodynamic shear prior to extravasation across the vascular barrier into tissues. Lymphocyte–endothelial adhesion occurs preferentially in high endothelial venules (HEV) of peripheral lymphoid organs. The continuous recirculation of naïve and central memory lymphocytes across lymph node and Peyer’s patch HEV underlies immune surveillance and immune homeostasis. Lymphocyte–endothelial interactions are markedly enhanced in HEV-like vessels of extralymphoid organs during physiological responses associated with acute and chronic inflammation. Similar adhesive mechanisms must be invoked for efficient trafficking of immune effector cells to tumor sites in order for the immune system to have an impact on tumor progression. Here we discuss recent evidence for the role of fever-range thermal stress in promoting lymphocyte–endothelial adhesion and trafficking across HEV in peripheral lymphoid organs. Findings are also presented that support the hypothesis that lymphocyte–endothelial interactions are limited within tumor microenvironments. Further understanding of the molecular mechanisms that dynamically promote lymphocyte trafficking in HEV may provide the basis for novel approaches to improve recruitment of immune effector cells to tumor sites.     

Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion and transmigration. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with sulphated ligands expressed by the specialized endothelial cells of high endothelial venules (HEVs). The sulphate-dependent monoclonal antibody MECA79 stains HEVs in peripheral lymph nodes and recognizes the complex of HEV ligands for L-selectin termed peripheral node addressin. High endothelial cell GlcNAc-6-sulphotransferase/L-selectin ligand sulphotransferase is a HEV-expressed sulphotransferase that contributes to the formation of the MECA79 epitope and L-selectin ligands on lymph node HEVs. MECA79-reactive vessels are also common at sites of chronic inflammation, suggesting mechanistic parallels between lymphocyte homing and inflammatory trafficking.     

Evolutionary conservation of tissue-specific lymphocyte-endothelial cell recognition mechanisms involved in lymphocyte homing

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.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

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)     

Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes

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.     

Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies

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.     

Demonstration that a lectin-like receptor (gp90MEL) directly mediates adhesion of lymphocytes to high endothelial venules of lymph nodes

Lymphocyte migration from the blood into most secondary lymphoid organs is initiated by a highly selective adhesive interaction with the endothelium of specialized blood vessels known as high endothelial venules (HEV). The propensity of lymphocytes to migrate to particular lymphoid organs is known as lymphocyte homing, and the receptors on lymphocytes that dictate interactions with HEV at particular anatomical sites are designated "homing receptors". Based upon antibody blockade experiments and cell-type distribution studies, a prominent candidate for the peripheral lymph node homing receptor in mouse is the approximately 90-kD cell surface glycoprotein (gp90MEL) recognized by the monoclonal antibody MEL-14. Previous work, including sequencing of a cDNA encoding for this molecule, supports the possibility that gp90MEL is a calcium-dependent lectin-like receptor. Here, we show that immunoaffinity-purified gp90MEL interacts in a sugar-inhibitable manner with sites on peripheral lymph node HEV and prevents attachment of lymphocytes. Lymphocyte attachment to HEV in Peyer's patches, a gut-associated lymphoid organ, is not affected by gp90MEL. The results demonstrate that gp90MEL, as a lectin-like receptor, directly bridges lymphocytes to the endothelium.     

Lymphocyte recirculation and homing: roles of adhesion molecules and chemoattractants

Lymphocyte migration from high endothelial venules into lymphoid organs is mediated by a sequence of interactions between cell adhesion molecules on lymphocytes and those on the vascular endothelial cells that line the vessels. recent studies suggest that the so-called lymphocyte homing receptors and vascular addressins regulate the first stages of this process, that of binding of lymphocytes from flowing blood. The subsequent crawling of lymphocytes over the endothelial cell surface and migration across the vessel wall (diapedesis) are regulated independently of initial binding. These latter stages are thought to be mediated by functional activation of integrins on the lymphocyte by chemoattractants located in the vessel wall.     

The influence of afferent lymphatic vessel interruption on vascular addressin expression

Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation. Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins. After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall. Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing. In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side. In addition, an HEV-specific differentiation marker, defined by mAb MECA-325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed: subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.     

Pathways to chronic inflammation in rheumatoid synovitis

Postcapillary venules resembling the high endothelial venules (HEVs) of lymphoid tissues have often been observed at sites of chronic inflammation. We have therefore postulated that such venules may be an important site of lymphocyte migration into rheumatoid synovial membrane and that inflammatory cell products may act on endothelial cells (ECs) to increase lymphocyte emigration. Electron microscopic examination of rheumatoid synovial membranes showed that a strong correlation existed between the proportion of lymphocytes in perivascular tissue and the height/base ratio of the ECs in those areas. In addition, binding experiments showed that peripheral blood mononuclear cells preferentially bound to ECs in sections of rheumatoid synovial membrane that had the morphological appearance of HEVs. In vitro binding experiments, in which lymphocyte adhesion to human umbilical vein EC monolayers was measured, showed that adhesion was enhanced by preincubation of the ECs with interferon-gamma or interleukin 1 (IL 1). The central role of IL 1 in increasing lymphocyte migration into the rheumatoid synovial membrane was also supported by the findings that IL 1 is chemotactic for lymphocytes, ECs can secrete IL 1, and IL 1 activity is readily detectable in synovial fluids of rheumatoid arthritis patients.     

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

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.     

Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation

We are investigating the hypothesis that carbohydrate-binding molecules on the cell surface are involved in the recirculation of lymphocytes from the bloodstream into lymphoid organs. This phenomenon requires the specific attachment of circulating lymphocytes to the endothelial cells of postcapillary venules. Using an in vitro assay to measure the adhesive interaction between lymphocytes and postcapillary venules, we have found that L-fucose, D mannose, and the L-fucose-rich, sulfated polysaccharide fucoidin specifically inhibit this binding interaction. L-fucose shows stereo-selective inhibitory activity at concentrations greater than 18 mM while fucoidin produces 50% inhibition at approximately 1-5 X 10(-8) M. Fucoidin appears to interact with the lymphocyte, and not the postcapillary venule, to inhibit binding. These data suggest that cell surface carbohydrates (fucoselike) and carbohydrate-binding molecules (cell surface lectins) may contribute to the specific attachment of lymphocytes to postcapillary venules.     

Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction: MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptor

The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-l) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-l transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1. Scanning electron microscopy revealed that MCAM-l induced microvilli formation and extension. In contrast, MCAM short or mock transfection had no effect on adhesion of NKL1 cells and microvilli formation. As shown by mutagenesis, serine 32 of the MCAM-l cytoplasmic tail, belonging to a putative protein kinase C phosphorylation site, was necessary for MCAM-l-actin cytoskeleton interaction and microvilli induction. Accordingly, chelerythrine chloride, a protein kinase C inhibitor, abolished MCAM-l-induced microvilli and rolling of MCAM-l-transfected NKL1 cells. Inhibition of adhesion under shear stress by anti-MCAM Abs suggested that both lymphoid MCAM-l and endothelial MCAM were also directly involved in lymphocyte endothelium interaction. MCAM-l-transfected NKL1 and activated CD4 T cells adhered to rMCAM under shear stress whereas anti-MCAM Ab treatment inhibited this process. Taken together, these data establish that MCAM is involved in the initial steps of lymphocyte endothelium interaction. By promoting the rolling on the inflammation marker VCAM-1 via microvilli induction and displaying adhesion receptor activity involving possible homophilic MCAM-l-MCAM-l interactions, MCAM might be involved in the recruitment of activated T cells to inflammation sites.     

Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin.

The lymphocyte-high endothelial venule (HEV) cell interaction is an essential element of the immune system, as it controls lymphocyte recirculation between blood and lymphoid organs in the body. This interaction involves an 85-95-kD class of lymphocyte surface glycoprotein(s), CD44. A subset of lymphocyte CD44 molecules is modified by covalent linkage to chondroitin sulfate (Jalkanen, S., M. Jalkanen, R. Bargatze, M. Tammi, and E. C. Butcher. 1988. J. Immunol. 141:1615-1623). In this work, we show that removal of chondroitin sulfate by chondroitinase treatment of lymphocytes or incubation of HEV with chondroitin sulfate does not significantly inhibit lymphocyte binding to HEV, suggesting that chondroitin sulfate is not involved in endothelial cell recognition of lymphocytes. Affinity-purified CD44 antigen was, on the other hand, observed to bind native Type I collagen fibrils, laminin, and fibronectin, but not gelatin. Binding to fibronectin was studied more closely, and it was found to be mediated through the chondroitin sulfate-containing form of the molecule. The binding site on fibronectin was the COOH-terminal heparin binding domain, because (a) the COOH-terminal heparin-binding fragment of fibronectin-bound isolated CD44 antigen; (b) chondroitin sulfate inhibited this binding; and (c) finally, the ectodomain of another cell surface proteoglycan, syndecan, which is known to bind the COOH-terminal heparin binding domain of fibronectin (Saunders, S., and M. Bernfield. 1988. J. Cell Biol. 106: 423-430), inhibited binding of CD44 both to intact fibronectin and to its heparin binding domain. Moreover, inhibition studies showed that binding of a lymphoblastoid cell line, KCA, to heparin binding peptides from COOH-terminal heparin binding fragment of fibronectin was mediated via CD44. These findings suggest that recirculating lymphocytes use the CD44 class of molecules not only for binding to HEV at the site of lymphocyte entry to lymphoid organs as reported earlier but also within the lymphatic tissue where CD44, especially the subset modified by chondroitin sulfate, is used for interaction with extracellular matrix molecules such as fibronectin.     

A family of cell-surface glycoproteins defined by a putative anti-endothelial cell receptor antibody in man

An efficient immune response depends directly on the ability of lymphocytes and other blood borne leukocytes to leave the blood and enter lymphoid organs and other sites of immune response or inflammation. A new murine mAb termed 515 recognizes a 85- to 90-kDa structure involved in lymphocyte binding to specialized endothelium in postcapillary venules of lymphoid organs (high endothelial venules). In crossed immunoprecipitations followed by SDS-PAGE analysis, 515 appears to be identical to the antilymphocyte homing receptor mAb, Hermes-1. We show here that 515 recognizes peripheral blood leukocytes of all classes examined, but, like Hermes-1, fails to recognize germinal center cells or nearly all cortical (but not medullary) thymocytes. In addition, we demonstrate that 515 recognizes both fibroblasts and epidermal keratinocytes, both in situ and in cultured cell lines. Immunoprecipitation and SDS-PAGE analysis reveals that 515-defined Ag from T lymphocytes, neutrophils, and the epidermal keratinocyte cell line HaCaT are clearly distinct, both before and after treatment with peptide N-glycosidase F, which removes all asparagine-linked oligosaccharides. In contrast, the Mr of 515-defined Ag from T cells and fibroblasts appear indistinguishable. Thus, the 515 mAb recognizes a broadly distributed family of glycoproteins which may be involved in a variety of adhesive functions.     

Ligand-specificity of the selectins

The selectins are carbohydrate-binding cell adhesion molecules acting in the vascular system. They mediate the docking of leukocytes to the blood vessel wall and the rolling of these cells along the endothelial cell surface. These adhesion phenomena initiate the entry of leukocytes into sites of inflammation as well as the migration of recirculating lymphocytes into secondary lymphoid tissues. Blocking selectin function with antibodies or oligosaccharides has proven to be beneficial in various animal models of inflammation and models of ischemia/reperfusion damage. This has raised much interest in the identification of the physiological ligands of the selectins. Several glycoprotein ligands have been identified, some of which can even be selectively isolated from cellular detergent extracts using a selectin as an affinity probe. Four of these “high affinity” ligands have been cloned. The structural requirements of their interaction with the selectins is discussed. © 1996 Wiley-Liss, Inc.