The human peripheral lymph node vascular addressin. An inducible endothelial antigen involved in lymphocyte homing.

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.     

L-selectin and alpha 4 beta 7 integrin homing receptor pathways mediate peripheral lymphocyte traffic to AKR mouse hyperplastic thymus.

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.     

Lymphocyte migration to inflamed lacrimal glands is mediated by vascular cell adhesion molecule-1/alpha(4)beta(1) integrin, peripheral node addressin/l-selectin, and lymphocyte function-associated antigen-1 adhesion pathways

Sjogren's syndrome is an autoimmune disease characterized by inflammation and destruction of lacrimal and salivary glands. The development of the inflammation requires the migration of lymphocytes from the blood into these tissues. This migration involves multistep cascades with binding of lacrimal gland endothelial adhesion molecules to their ligands on circulating lymphocytes. We used nonobese diabetic mice, which develop autoimmune-mediated lacrimal gland inflammation, as an experimental model to define the adhesion molecules that control lymphocyte migration into inflamed lacrimal glands. We found that vascular endothelia in inflamed areas of lacrimal gland expressed vascular cell adhesion molecule (VCAM)-1 and the peripheral node addressin (PNAd), but not mucosal addressin cell adhesion molecule-1. Most lymphocytes in the inflamed glands expressed alpha(4) integrin, L-selectin, and lymphocyte function-associated antigen (LFA)-1. In vivo studies revealed that antibodies against VCAM-1, alpha(4) integrin, PNAd, L-selectin, or LFA-1 almost completely blocked lymphocyte migration from blood into inflamed lacrimal glands. There was no inhibition of migration by antibodies against mucosal addressin cell adhesion molecule-1 or alpha(4)beta(7) integrin. These results indicate that endothelial/lymphocyte adhesion cascades involving VCAM-1/alpha(4)beta(1) integrin, PNAd/L-selectin, and LFA-1 control the migration of lymphocytes into inflamed lacrimal gland. These adhesion molecules offer potential therapeutic targets to block the development of lacrimal gland inflammation and destruction.     

Expression of lymphocyte homing receptors and vascular addressins in low-grade gastric B-cell lymphomas of mucosa-associated lymphoid tissue.

In this study, we examined lymphocyte homing receptor and vascular addressin expression in a case of primary gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) with a secondary intestinal spread. We compared the findings with that observed in B cells of normal MALT and MALT acquired as a consequence of Helicobacter pylori-associated gastritis and other low-grade gastric B-cell MALT lymphomas. The neoplastic B cells in the gastric tumor were alpha 4 beta 7-, CD62L+, whereas the intestinal secondary was alpha 4 beta 7+, CD62L-. Incubation of isolated tumor cells from the stomach by H. pylori generated T-cell-dependent proliferation of neoplastic B cells and induced expression of alpha 4 beta 7 integrin similar to the intestinal tumor. These observations indicate that reversal of homing receptor profile in the gastric tumor by antigen specific stimulation may be responsible for secondary intestinal dissemination. In normal stomach and normal MALT, alpha 4 beta 7 and CD62L expression reflected the differentiation of the B cell. Plasma cells were alpha 4 beta 7+, CD62L-, whereas a subset of memory B cells were alpha 4 beta 7-, CD62L+. Homing receptor expression in MALT lymphoma B cells was heterogeneous, however, in line with their memory B-cell phenotype in the majority of cases, the neoplastic B cells were alpha 4 beta 7-, CD62L+. Neoplastic plasma cells were always alpha 4 beta 7+, CD62L-. The venules in normal gastric mucosa expressed mucosal addressin cell adhesion molecule-1 but not peripheral lymph node addressin. In normal MALT, H. pylori-associated follicular gastritis and MALT lymphomas high endothelial venules coexpressed mucosal addressin cell adhesion molecule-1 and peripheral lymph node addressin. These findings suggest expression of lymphocyte homing receptors by B cells and vascular addressins by mucosal venules are similar in normal MALT and MALT lymphomas, and factors controlling normal mucosal B-cell traffic are also operational in MALT lymphomas.     

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.

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.     

Preferential expression of the mucosal homing receptor integrin alpha 4 beta 7 in gastrointestinal non-Hodgkin's lymphomas.

Recent studies have identified the integrin alpha 4 beta 7 as a mucosal homing receptor that mediates lymphocyte migration to the intestinal mucosa by binding to MAdCAM-1, a vascular recognition molecule (addressin) selectively expressed on mucosal endothelium. In the present study, we have assessed the expression of alpha 4 beta 7 on B- and T-cell non-Hodgkin's lymphomas of different primary localization and on related normal lymphocytes. Among B-lineage lymphomas, expression of alpha 4 beta 7 was present in the majority of cases of malignant lymphomatous polyposis of the intestine and low-grade lymphoma of the mucosa-associated lymphoid tissue/monocytoid B-cell lymphoma and in some cases of precursor B-cell lymphoma. CLL/small lymphocytic lymphoma, (nodal) mantle cell lymphoma, follicular center cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma were virtually always alpha 4 beta 7 negative, as was the case when localized in the mucosa-associated lymphoid tissue. The normal B cells of the follicle mantles and part of the B cells of the extrafollicular B-cell compartment of lymphoid tissues expressed moderate levels of alpha 4 beta 7. By contrast, follicular center cells were alpha 4 beta 7 negative. Among T-lineage lymphomas, expression of alpha 4 beta 7 was also strongly related to the primary localization; in mucosal, nodal, and cutaneous T cell lymphomas the percentage of positive cases was 56%, 17%, and 0%, respectively. All cases of precursor T-cell lymphoma were alpha 4 beta 7 negative. High expression of alpha 4 beta 7 was found on a subset of peripheral blood memory T cells as well as on lymphocytes in the intestinal mucosa. We conclude that non-Hodgkin's lymphomas that are related to mucosa-associated B- and T-lymphocyte populations selectively express the mucosal homing receptor alpha 4 beta 7. The presence of this receptor underscores their distinctive character and may play an important role in determining their characteristic mucosal dissemination pattern.     

Developmental regulation of vascular addressin expression: a possible role for site-associated environments

Tissue selective traffic of lymphocytes into different lympholdorgans is mediated by adherence of blood borne lymphocytes tospecialized endothelial cells lining the high endothelial venules(HEV) in lymphoid organs. Lymphocytes discriminate between HEVin peripheral lymph nodes and in mucosal lymphold tissues bymeans of membrane associated lymphocyte homing receptors adheringto their putative HEV ligands, the vascular addressins. Theexpression of particular vascular addressins on HEV is site-or tissue-selective and may be directed by factors unique toa specific location or lymph node environment. In this studywe investigated the impact of regional environments on lymphnode HEV differentiation and function. Experimentally, thisproblem was approached by the transplantation of lymph nodesfrom one region to a second region. The sites selected for receiptof tissues were the mesentery, a mucosal site, and the poplitealfossa, a peripheral site. We found that the phenotype of lymphnode HEV following transplantation was influenced by both donorage and transplantation site. The transplantation site couldinfluence vascular addressin expression, when tissues were obtainedfrom late fetal or early neonatal donors and not when obtainedfrom adult donors. Transplanted adult tissues retained theirpre-transplantation vascular addressin expression phenotyperegardless of transplantation site. Thus the endothellum withinadult lymph nodes may be committed to expression of a particularaddressin or addressins during lymph node development. It isalso possible that regulatory cells or structures present withinlymph nodes at the time of transplantation direct vascular addressinexpression following tissue engraftment. These studies showthat regional environments of the lymph nodes could play a rolein HEV differentiation and function during lymph node development.     

Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue.

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.     

Construction and Adhesive Properties of a Soluble MAdCAM-1-Fc Chimera Expressed in a Baculovirus System: Phylogenetic Conservation of Receptor-Ligand Interaction

MAdCAM-1 is a high endothelial venule adhesion molecule composed of immunoglobulin and mucin-like domains which binds the leucocyte integrin LPAM-1 (α4β7), and is largely responsible for the selective homing of lymphocytes to mucosal tissues. A novel soluble form of mouse MAdCAM-1 which is normally membrane bound has been produced by joining the extracellular region of the receptor to the Fc domain of human IgGl. The MAdCAM-1-Fc cDNA was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda insect cells infected with the recombinant virus produced MAdCAM-1-Fc as a disulfide-linked homodimer of 82kDa polypeptides, which was secreted into the culture medium at > 1 μg/ml. The product purified by Protein G-Sepharose was identified as authentic MAdCAM-1-Fc by the anti-MAdCAM-l monoclonal antibody (MoAb) MECA-367 using Western blot and ELISA analysis. When immobilized on glass it was fully functional in supporting the binding of mouse α4β1⁺α4β7⁺ mesenteric lymph node lymphocytes, and the α4β1^?αβ7⁺ TK1 T cell lymphoma. Binding was enhanced by Mn⁺⁺ -induced integrin activation, and specifically blocked by anti-integrin α4 subunit and anti-MAdCAM-1 MoAbs. Binding was blocked by pretreatment of cells with sodium azide, and EDTA, indicating that binding is an energy-dependent process which requires divalent cations. Thus the mouse MAdCAM-1-Fc chimera produced in insect cells retains certain functional properties that typify the native receptor, and should be valuable in analysing the role of MAdCAM-1 in lymphocyte recirculation and emigration. However it was not sialylated despite being post-translational modified with N- and O-linked carbohydrate moieties, suggesting that the ability of MAdCAM-1 to support cell adhesion under static conditions is sialylation-independent. A rabbit polyclonal antibody raised against the entire cytopiasmic domain of the human integrin β7 subunit recognized LPAM-1-like molecules in human, rat, and mouse cells, suggesting a high degree of conservation of the MAdCAM-1 receptor across species. In agreement with this notion MAdCAM-1-Fc immobilized on glass was fully functional in supporting the cation-dependent binding of peripheral blood or spleen cells from a range of other species including human, rat, and guinea pig; and for human myeloid HL60 cells, binding was mediated by o4 integrins.     

Complexity and differential expression of carbohydrate epitopes associated with L-selectin recognition of high endothelial venules.

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.     

A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules leads to rolling and is critical for recruitment of lymphocytes into peripheral lymph nodes. Peripheral node addressin represents a class of L-selectin ligands recognized by the function-blocking monoclonal antibody MECA-79. Its epitope overlaps with sialyl 6-sulfo Lewis X, an L-selectin recognition determinant. Here, mice lacking two N-acetylglucosamine-6-O-sulfotransferases (GlcNAc6ST-1 and GlcNAc6ST-2) demonstrated elimination of both peripheral node addressin and sialyl 6-sulfo Lewis X in high endothelial venules, considerably reduced lymphocyte homing to peripheral lymph nodes and reduced sticking of lymphocytes along high endothelial venules. Our results establish an essential function for the sulfotransferases in L-selectin ligand synthesis and may have relevance for therapy of inflammatory diseases.     

Lymphocyte ‘homing’ and chronic inflammation

Chronic inflammation is a response to prolonged exposure to injurious stimuli that harm and destroy tissues and promote lymphocyte infiltration into inflamed sites. Following progressive accumulation of lymphocytes, the histology of inflamed tissue begins to resemble that of peripheral lymphoid organs, which can be referred to as lymphoid neogenesis or formation of tertiary lymphoid tissues. Lymphocyte recruitment to inflamed tissues is also reminiscent of lymphocyte homing to peripheral lymphoid organs. In the latter, under physiological conditions, homing receptors expressed on lymphocytes adhere to vascular addressin expressed on high endothelial venules (HEVs), initiating a lymphocyte migration process composed of sequential adhesive interactions. Intriguingly, in chronic inflammation, HEV‐like vessels are induced de novo, despite the fact that the inflamed site is not originally lymphoid tissue, and these vessels contribute to lymphocyte recruitment in a manner similar to physiological lymphocyte homing. In this review, we first describe physiological lymphocyte homing mechanisms focusing on vascular addressins. We then describe HEV‐like vessel‐mediated pathogenesis seen in various chronic inflammatory disorders such as Helicobacter pylori gastritis, inflammatory bowel disease (IBD), autoimmune pancreatitis and sclerosing sialadenitis, as well as chronic inflammatory cell neoplasm MALT lymphoma, with reference to our work and that of others.     

De novo expression of MECA-79 glycoprotein-determinant on developing B lymphocytes in gut-associated lymphoid tissues

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.     

Mucosal vaccination increases endothelial expression of mucosal addressin cell adhesion molecule 1 in the human gastrointestinal tract

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-alpha) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-alpha. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.     

The influence of dietary fat on the interaction of lymphocytes with high endothelial venules.

The specific adherence of lymphocytes to high endothelial venules (HEV) represents the first step in the lymphocyte emigration from blood into most lymphoid tissues. The interaction of lymphocytes with HEV exhibits a remarkable organ specificity, which appears to be mediated by complementary receptors on both recirculating lymphocytes (homing receptors) and tissue-specific HEV (vascular addressins). The expression of homing receptors varies and depends on factors such as lymphocyte subtype, stage of activation and maturation. As these receptors are glycoproteins, which are anchored in the cell membrane, it can be envisaged that their position and function are determined by the overall composition of the cell membrane itself. In this study we investigated the significance of dietary fat concentration and saturation for the interaction between lymphocytes and HEV. In addition to these functional studies, the expression of homing receptors in combination with T and B cell markers were analyzed. Using immunohistochemistry the effect on the presence and characteristics of lymphocytes and HEV in situ was studied. Changes in the dietary fatty acid composition resulted in an altered ability of T and B cells to adhere to HEV, without affecting their binding preference. The changed adhesion patterns seemed to be associated with alterations in the expression of adhesion molecules, that are essential for the lymphocyte migration. The latter observation might in turn be explained by the observed modifications in the fatty acid composition of the lymphocytes. These results suggest a role for the fatty acid composition of the nutrition in the process of lymphocyte recirculation.     

Homing to central nervous system vasculature by antigen-specific lymphocytes. II. Lymphocyte/endothelial cell adhesion during the initial stages of autoimmune demyelination.

A previous study from this laboratory on adoptively transferred experimental allergic encephalomyelitis (EAE) induced by myelin basic protein-responsive (MBP+) 11C-labeled lymphocytes showed that MBP+ cells entered the central nervous system (CNS) before signs, migrated through the endothelium, and remained within the perivascular space. The majority of cells effecting CNS damage were nonradiolabeled and appeared to be host-derived and non-CNS antigen specific. The present study defined the immunocytochemical and initial structural events occurring between lymphocytes and endothelial cells (EC) on CNS blood vessels during EAE induced with MBP+ lymph node cells or T-cell lines. Monoclonal antibodies against lymphocyte function-associated molecule LFA-1 and its ligand, intercellular adhesion molecule-1 (ICAM-1), and the addressin MECA-325, a marker of mouse lymph node high endothelial venules, were tested on frozen sections in combination with the avidin-biotin-complex technique. The attachment and infiltration of lymphocytes correlated with the onset of signs and the appearance in the CNS of MECA-325 and ICAM-1 on vessels with cellular infiltrates and sometimes with plump EC. The cellular infiltrates were composed largely of LFA-1+ lymphocytes. Ultrastructurally, pseudopodia from lymphocytes were seen to attach to and penetrate EC in the CNS and form small gap junction-like contacts. On the EC surface, some processes from lymphocytes made larger synapse-like contacts while others were associated with coated pits suggestive of receptor mediation. The results are in accord with specific homing and attachment of lymphocytes to the CNS vasculature being early features of the disease process in EAE and with some CNS vessels acquiring properties of lymph node elements. Understanding of the mechanisms underlying these lymphocyte/EC interactions has therapeutic import for multiple sclerosis, for which EAE is the prime model.     

Functional capacities of high endothelial venules appear not to be controlled by recirculating lymphocytes.

The influence of recirculating lymphocytes on the function and morphology of high endothelial venules (HEV) has been studied. Mice were depleted of lymphocytes by lethal (1200 cGy) total body irradiation; subsequently, the HEV in mesenteric and cervical lymph nodes were studied up to 7 days after irradiation for: 1) capacity to bind lymphocytes by using the in vitro HEV-binding assay, 2) for morphological aspects such as ultrastructure and endothelial height, 3) for presence of RNA (pyroninophylia) and MECA-325 expression. Although, commencing 3 days after irradiation, lymphocyte depletion was intense and no extravasation of lymphocytes was observed; HEV were capable of binding lymphocytes at normal levels. Also the ratio of B/T cell binding to HEV was comparable to normal. MECA-325 expression, pyroninophilia, and ultrastructure of high endothelial cells were not affected by lymphocyte depletion. However, the average height of endothelial cells, which is a measurement related to cell volume, declined during lymphocyte depletion, stabilizing at about 70% of normal levels from day 4. After intravenous injection of viable lymph node cells, endothelial cell height rapidly increased within a few hours in conjunction with lymphocyte extravasation and homing into the nodes. Restoration of endothelial cell height was not observed after infusion of thymocytes, lethally irradiated lymph node cells or supernatants rich in cytokines. We conclude that recirculating lymphocytes in blood and lymphoid tissues are not involved in controlling high endothelial cell activity including the specific function in lymphocyte extravasation. However, recirculating/extravasating lymphocytes contribute to the development of endothelial cell height. The significance of non-lymphoid (radioresistant) cells in the control of characteristic high endothelial function is suggested.     

Loss of expression of alpha4beta7 integrin and L-selectin is associated with high-grade progression of low-grade MALT lymphoma.

Expression of adhesion molecule in low-grade B-cell mucosa-associated lymphoid tissue (MALT) lymphoma of the gastrointestinal tract has been reported in recent years, but these reports have primarily focused on low-grade gastrointestinal MALT lymphoma. In this study, we examined the lymphocytic homing receptor alpha4beta7 integrin, L-selectin, and VLA-4 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in low-grade lymphoma of the gastrointestinal tract and other organs such as the ocular adnexa and thyroid. We also observed changes in the expression pattern associated with high-grade transformation. Neoplastic cells in the gastrointestinal low-grade lymphoma and the low-grade component of high-grade MALT lymphoma were found to be alpha4beta7 integrin(+), L-selectin(+), whereas the gastrointestinal high-grade component and diffuse large B-cell lymphoma were found to be alpha4beta7 integrin(-), L-selectin(-). High endothelial venules in the gastric MALT lymphomas expressed MAdCAM-1. In the ocular adnexa low-grade MALT lymphoma, most cases were alpha4beta7 integrin(-), L-selectin(+); and in the thyroid, most cases of both low- and high-grade MALT lymphoma were alpha4beta7 integrin(-), L-selectin(-). These findings show that alpha4beta7 integrin and L-selectin may play an important role in the lymphocyte homing of gastrointestinal low-grade MALT lymphoma and in the loss of alpha4beta7 integrin expression throughout the course of high-grade progression.     

Culture characterization of differentiated high endothelial venule cells from human tonsils

High endothelial venules (HEV) are specialized vessels that support abundant lymphocyte emigration from peripheral blood into secondary lymphoid organs. HEV endothelial cells (HEVEC) exhibit particular structural and functional features, including secretion of the HEV-specific extracellular matrix protein hevin and an array of uniquely glycosylated counter-receptors for L-selectin expressed on lymphocytes. These ligands are collectively called the peripheral lymph node addressin (PNAd), originally defined by the monoclonal antibody MECA-79. PNAd expression was used to purify HEVEC by positive immunoselection from enzyme-digested human tonsils after negative immunoselection for other cells. Purified HEVEC maintained secretion of hevin and homogenous expression of intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), and CD31, at high levels following 8 days in culture. Expression of functional PNAd was maintained during the first 4 to 5 days of culture but decreased gradually and disappeared on day 8, while the expression of CD34 remained strong. However, the CD34 glycoform shifted toward the in situ phenotype of flat-walled vessels, suggesting that the observed loss of L-selectin binding determinants and MECA-79 antigen was due to down-regulation of the glycosyl- and sulfo-transferases essential for their expression. Our rapid and reproducible method to establish HEVEC cultures provides a useful mechanistic tool for identification of the factors that induce and maintain the HEV phenotype, as well as a source for isolation of HEV-specific genes.     

Random entry of circulating lymphocyte subsets into peripheral lymph nodes and Peyer's patches: no evidence in vivo of a tissue-specific migration of B and T lymphocytes at the level of high endothelial venules.

Lymphocytes continuously migrate through the body and thus immune competent cells are constantly delivered to most tissues. They interact with high endothelial venules (HEV) via specific homing receptors and vascular addressins, and these molecules seem to be the reason for a preferential homing of B lymphocytes into Peyer's patches and of T lymphocytes into peripheral lymph nodes. When lymphocytes derived from lymph node cell suspensions were applied in the in vitro lymphocyte/endothelium binding assay, the well-known preference of mouse lymph node B lymphocytes for Peyer's patch HEV compared to peripheral lymph node HEV was confirmed in the rat (2.8 times). When in the same in vitro assay thoracic duct lymphocytes (TDL) were used this preference was far less obvious (1.4 times). However, by injecting rat TDL intravenously and by tracing them directly in HEV, B, T, CD4+ and CD8+ lymphocytes are seen to enter Peyer's patches and peripheral lymph nodes in vivo without preference. Thus, in contrast to lymphocytes from lymph node cell suspensions, no evidence was found of a tissue-specific migration of thoracic duct B, T, CD4+ and CD8+ lymphocytes at the HEV level. This finding demonstrates the importance of considering both experimental conditions and the cell source used when investigating lymphocyte traffic.