Measurement of VLA-4/CS-1 and VLA-4/VCAM adhesion inhibition.

Cell adhesion, a critical early step in the inflammatory process, has increasingly become the target of drug discovery efforts. Described in this unit are techniques for measuring inhibitors of VLA-4-mediated adhesion to either VCAM or the connecting segment (CS-1) of fibronectin.

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.

Multiple defects in Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion.

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcepsilonRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcepsilonRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcepsilonRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcepsilonRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.

A small-molecule, tight-binding inhibitor of the integrin alpha(4)beta(1) blocks antigen-induced airway responses and inflammation in experimental asthma in sheep.

The leukocyte integrin very late antigen-4 (alpha(4)beta(1), CD49d/CD29) is an adhesion receptor that plays an important role in allergic inflammation and contributes to antigen-induced late responses (LAR) and airway hyperresponsiveness (AHR). In this study, we show that single doses of a new small-molecule, tight-binding inhibitor of alpha(4), BIO-1211, whether given by aerosol or intravenously, either before or 1.5 h after antigen challenge blocks allergen- induced LAR and post-antigen-induced AHR in allergic sheep. Multiple treatments with doses of BIO-1211 that were ineffective when given singly, were protective. BIO-1211 also provided dose-dependent inhibition of the early airway response (EAR) to antigen. In conjunction with the functional protection against the antigen-induced LAR and AHR, sheep treated with BIO-1211 before challenge showed significantly reduced: (1) numbers of eosinophils in bronchoalveolar lavage (BAL), (2) BAL levels of the inflammatory marker tissue kallikrein, and (3) numbers of inflammatory cells (lymphocytes, eosinophils, metachromatic staining cells, and neutrophils) in bronchial biopsies obtained after challenge when compared with corresponding biopsies after vehicle treatment. More importantly, we show for the first time that an inhibitor of alpha(4) was able to reverse post-antigen-induced AHR, thereby decreasing the time of recovery from the normal period of > 9 d to 3 d. Our results show that effective inhibition of antigen-induced airway responses can be achieved with single doses of a potent small-molecule inhibitor of alpha(4) and that such agents may be used therapeutically, as well as prophylactically, to alleviate allergen- induced inflammatory events. These data provide further support and extend the evidence for the role of alpha(4) integrins in the pathophysiologic events that follow airway antigen challenge.

Modulation of integrin function in hematopoietic progenitor cells by CD43 engagement: possible involvement of protein tyrosine kinase and phospholipase C-gamma.

Attachment of cells to extracellular matrix components is critical for the regulation of hematopoiesis. CD43 is a mucin-like transmembrane sialoglycoprotein expressed on the surface of almost all hematopoietic cells. A highly extended structure of extracellular mucin with negative charge may function as a repulsive barrier to hematopoietic cells. However, some investigators have shown that CD43 has proadhesive properties, and engagement of CD43 has been reported to upregulate integrin-mediated cell adhesion in T cells. We found that cross-linking of CD43 with monoclonal antibodies (MoAbs) enhanced integrin alpha4beta1 (very late antigen [VLA]-4) and alpha5 beta1 (VLA-5)-dependent adhesion of human cord blood CD34(+) cells to fibronectin. CD34(+) CD38(hi), but not CD34(+)CD38(-/low) cells responded significantly to the stimulus, suggesting that committed, but not stem and more immature progenitors are sensitive to CD43-mediated activation of integrin. To elucidate the molecular mechanism leading to integrin activation, we used the growth factor-dependent cell line MO7e. Cross-linking of CD43 induced tyrosine phosphorylation of several intracellular molecules including the protein tyrosine kinase Syk, the proto-oncogene product Cbl, and phospholipase C (PLC)-gamma2 in MO7e cells. Moreover, protein tyrosine kinase inhibitor herbimycin A and PLC inhibitor U73122 both blocked CD43-induced enhancement of adhesion to fibronectin. These results indicate that signals mediated through CD43 may increase integrin affinity to fibronectin via a pathway dependent on protein tyrosine kinase and PLC-gamma activation in hematopoietic progenitors.

Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury.

Repair of the airway epithelium after injury requires that processes such as adhesion and cell migration occur in a defined order. Both of these processes depend on interactions between extracellular matrix (ECM) proteins and appropriate integrins. To study these interactions, we examined monolayer wound repair in a cultured human airway epithelial cell line, 16HBE14o-. Wounds created in confluent monolayers grown on either collagen-IV, laminin-1, or laminin-2 matrix closed quickly in response to 15 ng/ml epidermal growth factor (EGF). Concurrent treatment of cells grown on each matrix protein with EGF and a monoclonal antibody (mAb) to beta1-integrin inhibited wound closure. Treatment with a mAb to alpha2-, alpha3-, and alpha6-integrin blocked wound repair in monolayers grown on collagen-IV but did not do so in monolayers grown either on laminin-1 or laminin-2. Inhibition was not due to cell detachment or apoptosis. These data demonstrate that integrins expressed by airway epithelial cells mediate wound closure on different constitutive ECM proteins. These data suggest that beta1-integrin subunit function is required to permit migration and spreading of epithelial cells, and that alpha-integrin subunits alone do not mediate migration of epithelial cells grown on either laminin-1 or laminin-2. These differences may become important if the matrix protein composition of airway basement membrane changes in disease states such as asthma.

High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow.

The very late Ag-4 (VLA-4) integrin supports both rolling and firm adhesion of leukocytes on VCAM-1 under shear flow. The molecular basis for the unique ability of a single adhesion molecule to mediate these versatile adhesive processes was investigated. VLA-4 occurs in multiple activation states, with different affinities to ligand. In this study we tested how these states regulate VLA-4 adhesiveness under shear flow in Jurkat T cells and PBL. VLA-4 on nonstimulated Jurkat cells supported rolling and spontaneous arrest on VCAM-1, whereas a Jurkat activation mutant with reduced VLA-4 affinity failed to spontaneously arrest after tethering to or during rolling on VCAM-1. The contribution of VLA-4 affinity for ligand to rolling and spontaneous arrests on immobilized VCAM-1 was dissected using soluble VLA-4 ligands, which selectively block high affinity states. VLA-4 saturation with ligand completely blocked spontaneous adhesion strengthening post-tethering to VCAM-1, but did not impair rolling on the endothelial ligand. High affinity VLA-4 was found to comprise a small subset of VLA-4 on resting Jurkat cells and PBL. This subset is essential for firm adhesion but not for tethering or rolling adhesions on VCAM-1. Interestingly, low and high affinity VLA-4 states were found to mediate similar initial tethering to ligand. High affinity VLA-4, constitutively expressed on circulating T cells, may control their early adhesion strengthening on VCAM-1-expressing endothelium before exposure to vascular chemokines and activation of additional integrins.

Cross-species interaction of porcine and human integrins with their respective ligands: implications for xenogeneic tolerance induction.

BACKGROUND: Organ transplantation is limited by the number of available donors. One possible solution would be the use of pigs as organ donors. However, current immunosuppressive protocols cannot prevent rejection of these organs. If donor-specific tolerance toward porcine antigens could be induced in recipients, subsequent implantation of porcine organs would be possible without further immunosuppression. Induction of tolerance can be achieved with a bone marrow transplant if donor antigen-presenting cells successfully differentiate in the recipient thymus to induce deletion of donor-reactive host cells. Migration of porcine progenitor cells to the host marrow and thymus and differentiation into tolerance-inducing antigen-presenting cells is likely to require successful interaction of porcine adhesion molecules with human ligands. In this study, we investigated whether very late antigen (VLA)4 and VLA-6 integrins, which play important roles in homing and differentiation of hematopoietic progenitor cells, function across the pig-to-human species barrier. METHODS: Static cell-to-cell and cell-to-extracellular matrix protein adhesion assays were used to examine the cross-species interaction of porcine adhesion molecules with human ligands. RESULTS: Our studies show that porcine cells adhere to various human endothelial cell monolayers and extracellular matrix proteins and demonstrate that porcine VLA-4 and VLA-6 appear to be fully cross-reactive to the human ligands vascular cell adhesion molecule-1 and laminin, respectively. CONCLUSIONS: It is likely that porcine hematopoietic progenitor cells will be able to successfully employ pVLA-4- and pVLA-6-human ligand interactions in a pig-to-human bone marrow transplantation model in order to induce donor-specific tolerance.

A role for the VLA-4 integrin in the activation of human memory B cells.

It is generally recognized that activation through membrane effector molecules such as CD40 or the B cell receptor (BCR) is mandatory to allow B cells to proliferate and differentiate into antibody (Ab)-secreting cells in response to cytokines. We show here that purified tonsillar B cells can be stimulated directly by a cytokine combination to proliferate and secrete immunoglobulins when cultures are performed at high cell density. The contact-mediated activation of B cells in this experimental system is strongly inhibited both by anti-very late antigen (VLA)-4 monoclonal Ab and by a peptide containing the LDV sequence specifically recognized by the alpha 4 integrin binding site. These reagents also significantly suppressed the B cell responses elicited by engagement of the BCR or CD40. Our data reveal that memory B cells but not virgin or germinal center B cells are sensitive to the direct stimulatory effect of cytokines in high-density cultures. Finally, we found that the dual expression of the alpha and beta chains of VLA-4 is a distinctive feature of the memory B cell population. Collectively, our findings support the notion that VLA-4-dependent homotypic B cell interactions can mediate a co-stimulatory signal to human memory B cells and might participate in the B cell activation triggered through the BCR and CD40.

Mesenchymal cells stimulate human intestinal intraepithelial lymphocytes.

BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) from human intestinal mucosa proliferate minimally to T-cell stimuli. Optimal growth may depend on factors that are missing in vitro, such as accessory cells. The aim of this study was to determine whether mesenchymal cells costimulate IELs. METHODS: IELs were isolated from human jejunum and cultured with fibroblasts or smooth muscle cells (mesenchymal cell models for mucosal myofibroblasts) and various T-cell stimuli. Proliferation was determined by [3H]thymidine incorporation, and interleukin 2 (IL-2) production was measured by enzyme-linked immunosorbent assay. Surface molecules were detected by immunofluorescence and flow cytometry. RESULTS: The proliferative responses of IELs to mitogen (phytohemagglutinin), superantigen (staphylococcal enterotoxin B), or anti-CD3 antibody were increased greatly by coculture with mesenchymal cells, while only slightly by peripheral-blood monocytes, the classical antigen-presenting cells. IL-2 production and receptor expression also increased. Mesenchymal cell costimulation of IEL growth required direct contact between the two cell types and was partly dependent on the integrin alpha4beta1 (very late activation 4[VLA-4]) and major histocompatibility complex (MHC) class I, as their respective antibodies blocked the effect. The surface molecules B7 (CD80), CD2, and MHC class II were not involved. CONCLUSIONS: Optimal IEL growth depends on their contact with mesenchymal cells, an interaction that is mediated by VLA-4 and MHC class I. In mucosal immunity, basement membrane myofibroblasts likely serve this role.

Granulocyte-macrophage colony-stimulating factor regulates the functional adhesive state of very late antigen-4 expressed by eosinophils.

As very late antigen-4 (VLA-4) can exist in different functional states, we have sought to determine whether a cytokine expressed by inflamed endothelium (i.e., granulocyte-macrophage CSF (GM-CSF)) could regulate the functional state of VLA-4 expressed by eosinophils. Using a micropipette single cell adhesion assay able to measure the strength of adhesion forces, eosinophils exhibited low levels of basal adhesion to unstimulated endothelium (separation force, 0.022 +/- 0.003 mdynes). In contrast, individual eosinophils bound to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes), TNF-stimulated endothelium (0.62 +/- 0.05 mdynes), or IL-4-stimulated endothelium (0.11 +/- 0.01 mdynes) with increased avidity as assessed by separation force. Eosinophil binding to IL-4-stimulated endothelium was significantly inhibited by neutralizing Abs to either vascular cell adhesion molecule (VCAM) or VLA-4. The strength of eosinophil adhesion to VCAM (0.31 +/- 0.02 mdynes) or to connecting segment-1 (CS-1) (0.18 mdynes) was greater than the strength of eosinophil adhesion to unstimulated endothelium (0.02 mdynes), but was less than the strength of eosinophil adhesion to IL-1beta-stimulated endothelium (0.49 +/- 0.02 mdynes). After incubating eosinophils for 30 min with GM-CSF, the mean adhesion strength of eosinophils to CS-1 and VCAM increased significantly by 84 and 54%, respectively, compared with that of controls. This increased binding of eosinophils to VCAM or CS-1 was not due to alterations in VLA-4 receptor number (assessed by FACS analysis) or alterations in VLA-4 receptor distribution (assessed by confocal microscopy). These studies suggest that endothelial-derived cytokines such as GM-CSF have the potential to alter the functional state of eosinophil-expressed VLA-4 from a low affinity state to a high affinity state.

Immunoregulatory roles of adhesive interactions between lymphocytes and gingival fibroblasts.

Chronic adult periodontitis is usually characterized by inflammatory cell accumulation in the extravascular periodontal connective tissue. In order to reveal how the lymphocyte migration and retention in periodontal lesions is regulated, we have focused on the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). In this study, we investigated the involvement of cell adhesion molecules in adhesive interactions between lymphocytes and HGF. We found that activated lymphocytes bound strongly to HGF and VLA integrins, extracellular matrix receptors, play crucial roles in the binding. Interestingly, we first revealed that CD44 molecules (hyaluronate receptor) on lymphocytes also participated in lymphocyte-HGF interactions and that hyaluronate anchored on the surface of HGF functioned as the ligand for CD44. In addition, when HGF were stimulated with inflammatory cytokines such as IL-1, TNF alpha and IFN gamma, the binding avidity between lymphocytes and HGF was significantly increased and the adhesion was mainly mediated by LFA-1/ICAM-1 pathway. We then examined the possibility whether lymphocyte-HGF interaction may cause activation of HGF. When HGF directly interacted with lymphocytes for 3 h, IL-1 beta mRNA expression was clearly increased in HGF. These findings suggested that the adhesive interactions between lymphocytes and HGF was mediated at least by VLA integrins, LFA-1/ICAM-1 and CD44/hyaluronate and that the heterotypic cell-cell interactions could mutually cause intracellular signal transduction.

Correlation of very late activation integrin and CD44 expression with extrarenal invasion and metastasis of renal cell carcinomas.

Cell adhesion molecules mediate cell-cell and cell-matrix interactions, and they are thought to play an important role in tumor invasion and metastasis. Altered expression of integrins and CD44 in renal cell carcinoma has been recently demonstrated, but an association with invasive or metastatic behavior has not been reported. We examined very late activation (VLA) integrin and CD44 expression in 37 renal cell carcinomas and correlated adhesion molecule expression with multiple histological and clinical parameters. Most tumors exhibited positive staining for VLA3 (81%). Approximately one third of the tumors stained positively for VLA6 and CD44, and fewer (27%) were positive for VLA2. Only a few tumors were positive for VLA4 (8%) and VLA5 (14%). Most of the tumors exhibiting positive staining showed a combination of membranous and cytoplasmic staining patterns. Low-grade tumors positive for VLA6 showed a tendency for basilar staining of the tumor cells, whereas high-grade tumors exhibited diffuse cytoplasmic staining. All tumors exhibiting weak or strong positive staining for VLA4 or VLA5 showed extrarenal invasion or were known to have developed metastases at the time of nephrectomy. All tumors strongly positive for VLA2 or CD44 showed invasion beyond the renal capsule or metastases. In contrast to a previous study, no association was observed between positive staining and tumor grade. Nor were tumor size, architectural pattern, cell type, or DNA ploidy found to be associated with particular staining patterns. Although many of the invasive tumors showed no difference in VLA integrin or CD44 expression compared with tumors confined to the kidney, increased expression in some of them suggests that these cell adhesion molecules may contribute to the invasive or metastatic phenotype.

Role of VLA-integrin receptor in invasion and metastasis of human fibrosarcoma cells.

Anti-invasive and anti-metastatic effects of anti-integrin antibodies (against VLA-alpha 2, alpha 4, beta 1) were examined on human fibrosarcoma cells using in vitro invasion assay in a reconstituted basement membrane (Matrigel) and experimental metastatic assay in a chick embryo. The effects of anti-integrin antibodies were compared with those of RGD-containing peptides (GRGDS), which have been known as effective inhibitors of tumor cell metastasis. Although slight differences in effective concentration among antibodies were observed, invasion and metastasis were significantly inhibited by anti-integrin antibodies. The results also showed partial inhibitory effect of GRGDS on the invasion and metastasis of human fibrosarcoma cells. These results indicate that integrin receptors mediating cell-cell/cell-extracellular matrix components interactions play a key role in the invasion and metastasis of human fibrosarcoma cells.

Selective homing of human leukemic B-cell precursors to specific lymphohematopoietic microenvironments in SCID mice: a role for the beta 1 integrin family surface adhesion molecules VLA-4 and VLA-5.

We used a SCID mouse xenograft model to study the in vivo growth patterns of primary leukemic cells from six patients with newly diagnosed B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), including two patients with t(1;19) ALL, two patients with t(4;11) ALL, and two patients with t(9;22) ALL. Leukemic cells from these six patients caused overt leukemia in SCID mice with extensive multiple organ involvement. Leukemic BCP from SCID mice xenografted with leukemic cells from two t(9;22) ALL patients expressed very high levels of both VLA-4 and VLA-5 regardless of the tissue of origin. By comparison, in SCID mice xenografted with leukemic cells from the two patients with t(1;19) ALL and two patients with t(4;11) ALL, leukemic BCP from the bone marrow samples expressed high levels of VLA-4 as well as VLA-5, whereas the vast majority of leukemic BCP in the liver or spleen samples expressed neither of these adhesion molecules at significant levels. These results suggest that the expression of VLA-4 and VLA-5 on t(1;19) or t(4;11) leukemia cells likely determines their binding capacity to bone marrow stroma and may affect their migration to extramedullary tissues. Our findings are in accord with and extend previous studies which demonstrated that extracellular matrix and integrins influence development, compartmentalization, and migration of BCP during B-cell ontogeny. The described SCID mouse model system provides a unique opportunity to study the adhesion receptors which regulate the selective homing of human leukemic BCP to specific SCID mouse organs.

Signal transduction through the beta1 integrin family surface adhesion molecules VLA-4 and VLA-5 of human B-cell precursors activates CD19 receptor-associated protein-tyrosine kinases.

We demonstrate that the CD19 receptor associates with the beta1 family integrin receptors on human B-cell precursors as well as mature B-lymphocytes, and engagement of the beta1 family integrin receptors with monoclonal antibody homoconjugates leads to rapid activation of the CD19-associated protein-tyrosine kinases (PTK) and results in hyperphosphorylation of CD19 on tyrosine residues. Our findings prompt the hypothesis that homoconjugate-induced integrin clustering may effect the approximation and, by intermolecular cross-phosphorylation, activation of the CD19-associated PTK and subsequent tyrosine phosphorylation of the CD19 receptor. The ability of the beta1 family integrin receptors to transmit a biochemical signal triggering the CD19-linked multifunctional PTK pathway provides a possible explanation for the pleiotropic biologic responses generated though adhesive VLA-4- and VLA-5-mediated contacts.

Chemokines regulate T cell adherence to recombinant adhesion molecules and extracellular matrix proteins.

Chemokines are a family of structurally related, low m.w. proteins that regulate leukocyte migration both in vitro and in vivo. By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation during the genesis of an immune response. Chemokines have been shown to induce leukocyte adhesion to endothelium, to facilitate trans-endothelial passage, and to direct cell migration along a protein gradient (chemotaxis). The chemokines (macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, RANTES, and IFN-inducible protein-10) have recently been reported to be chemotactic for T cells. We have investigated the potential activity of these proteins in regulation of T cell adhesion. These chemokines induce T cell adhesion to purified, recombinant human adhesion molecules (rhICAM-1, rhVCAM-1) and to ECM proteins: fibronectin, collagen, and laminin. The chemokine-induced adhesion process occurs rapidly, is dose-dependent, and appears to be mediated via beta 1 and beta 2 integrins. The enhanced T cell adhesion is not associated with an increased surface expression of adhesion proteins, suggesting that chemokines stimulate the development of a high affinity state in the integrin molecules. Our findings provide in vitro evidence of a critical role for chemokines in T cell adhesion to endothelial adhesion molecules and ECM proteins, thereby promoting haptotactic migration of T cells to sites of inflammation in vivo.

Expressions of very late antigen-6 and vitronectin receptor, and their interactions to laminin and vitronectin during tonsillar B-cell activation.

This study examined the expressions of a-subunits of very late antigen-6 (VLA-6; alpha 6) and vitronectin receptor (VNR; alpha V) on tonsillar B cells and interactions between those integrins and their respective ligands, laminin (LM) and vitronectin (VN). alpha 6 and alpha V were expressed on about 30 to 40% of tonsillar B cells. When purified tonsillar B cells were separated by a discontinuous Percoll gradient, the number of alpha 6- and alpha V-positive cells decreased as the cell density went down, while the number of activated cells went up. After in vitro activation of tonsillar B cells by Staphylococcus aureus Cowan I strain (SAC), the expressions of alpha 6 and alpha V and their adhesiveness to LM or VN decreased significantly. Increased proliferation of B cells was observed when tonsillar B cells were cultured with immobilized LM or VN. The results of immunohistological staining showed VLA-6, VNR, LM and VN in the follicular area. These results suggest that the expressions of VLA-6 and VNR on tonsillar B cells may be decreased during B cell activation, and the interaction between VLA-6, VNR, and LM, VN may give a costimulatory effect on B cell activation in the follicular area of the tonsil.

Mononuclear leukocytes invade rabbit arterial intima during thickening formation via CD18-and VLA-4-dependent mechanisms and stimulate smooth muscle migration.

The role of mononuclear leukocytes for the migration of smooth muscle cells (SMCs) during intimal thickening was investigated in the rabbit model of electrically stimulated carotid artery. The approach was to inhibit leukocyte entry into the arterial intima with antibodies against the adhesion molecules very late activation antigen-4 (VLA-4) and CD11/CD18. In electrically stimulated control rabbits treated either with saline or a nonspecific antibody, all types of granulocytes, monocytes, and lymphocytes migrated across an intact endothelium into the acellular subendothelial space, followed by the movement of SMCs from the media into the intima within 36 hours of applying electrical current. Treatment of the rabbits with monoclonal antibody (mAb) HP1/2 directed toward the alpha 4 subunit (CD49d) of VLA-4 inhibited mononuclear leukocyte invasion (consisting of monocytes and lymphocytes) by approximately 70% compared with the IgG-treated control rabbits and completely abolished the minimal influx of basophils and eosinophils after 36 hours. Neutrophil infiltration, however, remained unaffected by anti-VLA-alpha 4 treatment. Under these conditions, SMC migration across the internal elastic lamina was reduced by 50%. The use of mAb HP1/2 together with mAb 60.3 (directed to the beta 2 chain of CD11/CD18) completely abolished the influx of monocytes, lymphocytes, and all types of granulocytes into the arterial intima. This complete blockade of leukocyte infiltration resulted in a 70% reduction of intimal SMC accumulation. Together with our previous findings excluding neutrophils as stimulators of SMC migration, the present results indicate that mononuclear leukocyte promote lesion development by stimulating SMC migration.

Administration of monoclonal antibodies against vascular cell adhesion molecule-1/very late antigen-4 abrogates predisposing autoimmune diabetes in NOD mice.

The interaction of vascular cell adhesion molecule-1 and very late antigen-4 (alpha 4 beta 1-integrin) has been recently known to be profoundly involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues. To elucidate the role of these molecules in the development of autoimmune diabetes, the expression of these adhesion molecules on inflamed islets and the effects of administration of monoclonal antibodies to these molecules on insulitis and overt diabetes were evaluated in nonobese diabetic (NOD) mice. Immunohistochemical study revealed the overexpression of vascular cell adhesion molecule-1 on vascular endothelium near or within inflamed islets and alpha 4-integrin on islet-infiltrating mononuclear cells. Either anti-vascular cell adhesion molecule-1 or anti-alpha 4-integrin monoclonal antibody prevented the transfer of diabetes in irradiated NOD mice which received spleen cells from acutely diabetic NOD mice. When both monoclonal antibodies were administrated to NOD mice during 2-30 weeks of age, neither lymphocytic infiltration to islets nor overt diabetes was observed. Furthermore, administration of these antibodies even from 10 weeks of age could inhibit the development of insulitis and diabetes, whereas administration during 2-5 weeks of age could not. Splenocytes obtained from these treated mice showed no significant change of cytokine production and preserved the ability to transfer diabetes into NOD scid/scid mice. This suggests that treatment with antibodies against these adhesion molecules can inhibit insulitis and diabetes without affecting the Th1/Th2 balance or effector T cells. The blockade of vascular cell adhesion molecule-1/very late antigen-4 interaction would be suitable for therapeutical treatment of the predisposing and latent type I (insulin-dependent) diabetic subjects.