Bosco M. C. Chan

The integrin VLA-2 binds echovirus 1 and extracellular matrix ligands by different mechanisms.

The integrin VLA-2 mediates cell adhesion to collagen and laminin and also functions as a virus receptor, mediating cell surface attachment and infection by a human pathogen, echovirus 1. To determine whether extracellular matrix proteins and virus interact with VLA-2 in the same manner, we carried out a detailed comparison of these two functions and found that they differed markedly in six different respects. In contrast to the ECM/VLA-2 interaction, echovirus 1 binding did not discriminate between functional forms of VLA-2, showed a different pattern of inhibition by anti-beta1 and -alpha 2 antibodies, was not stimulated by phorbol esters, was not activated by beta 1 antibodies that stimulate ECM binding, was not inhibited by any particular divalent cation, and most notably was not inhibited by EDTA. These striking differences were found both with intact cells expressing VLA-2 and with solubilized VLA-2, suggesting that VLA-2 interacts with these different ligands by markedly different mechanisms, and probably at different functional sites. In addition, alterations in the alpha 2 cytoplasmic domain that had marked effects on cellular responses to collagen and laminin had no effect on virus internalization and cell killing. Thus VLA-2-mediated events that occur after receptor occupancy by extracellular matrix proteins also appear to be distinct from those that occur after receptor interaction with virus.

The Mouse VLA-2 Homologue Supports Collagen and Laminin Adhesion but Not Virus Binding

Human VLA-2 (alpha 2 beta 1) mediates cellular adhesion to collagen and laminin and cell attachment by the human pathogen echovirus 1. We report here the cloning, sequencing and functional expression of the mouse VLA-2 alpha subunit homologue. This integrin subunit is closely related to its human counterpart, with 84% amino acid identity between the human and murine proteins. Conserved structural features include an identical number of amino acids, the presence of an I domain, and identity in the number and position of N-linked glycosylation sites and putative divalent cation binding regions. Murine and human alpha 2 show 30% amino acid divergence within the cytoplasmic tail, a difference that can be detected with antisera directed against the C-terminal peptides. Functionally, mouse alpha 2 was capable of mediating cell attachment to collagen and laminin, and responded to both intra- and extracellular signals with changes in its ligand affinity. In contrast, unlike its human homologue, mouse alpha 2 did not promote binding of echovirus 1. Comparison of the primary structure of the homologues leads us to predict that echovirus 1 may bind in the region of the first two thirds of the human alpha 2 I domain, where the sequences are most divergent, whereas more conserved flanking regions, and the conserved terminal one third of the I domain, may be involved in adhesion to collagen and laminin.

Monoclonal antibody against beta7 integrins, but not beta7 deficiency, attenuates intestinal allograft rejection in mice.

Background. &bgr;7 integrins mediate homing and retention of lymphocytes to the normal and inflamed small bowel in a tissue-selective fashion. In the present study, we investigated the expression of &bgr;7 integrins after small bowel transplantation (SBT) and tested the effects of blocking &bgr;7-mediated pathways by using monoclonal antibody (mAb) or knockout mice. Methods. Heterotopic SBT from BALB/c to C57BL/6 (B6) was used as a surgical model. Expression of &bgr;7 integrins was measured on recipient lymphocytes (CD4+ and CD8+) in spleen, blood, and mesenteric lymph nodes (MLN) using flow cytometry. To analyze the effects of blocking &bgr;7 integrins on graft survival, either &bgr;7-deficient B6 or wild-type B6 mice that were treated with mAb against &bgr;7 were studied. Results. After allogeneic SBT, there were markedly increased levels of &agr;4&bgr;7high recipient CD8+ lymphocytes in MLN, blood, and spleen as early as 3 days postoperatively. In contrast, &agr;4&bgr;7 integrin levels in isograft recipients were similar to those of normal mice. Mean survival time of intestinal allografts was not affected by &bgr;7 deficiency (7.3±1 days) compared with wild-type mice (7.5±0.8 days). However, mAb against &bgr;7 integrins significantly prolonged graft survival (12.8±1 days) compared with treatment with control mAb (7.3±1 days, P <0.001). Histologic changes of SBT rejection were significantly attenuated when mice were given mAb against &bgr;7. Conclusion. As indicated by the increased levels of &agr;4&bgr;7high CD8+ lymphocytes, activation of this integrin contributes to the immune response in SBT rejection. Furthermore, blocking &bgr;7 integrins with mAb provides a suitable target for immunosuppressive therapy. The discrepancy in survival data obtained by mAb and &bgr;7 deficiency may be a result of the more rapid activation of compensatory mechanisms in the knockout mice.

Treatment of cardiac allografts with established leukocyte infiltration by modulation of alpha4 (CD49d) and leukocyte function-associated antigen-1 (CD11a/CD18) integrin function.

BACKGROUND Leukocyte infiltration is a landmark feature of organ rejection. The present study was undertaken to determine whether monoclonal antibodies (mAb) against alpha4 (CD49d) and/or leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) would reverse ongoing rejection in a mouse C57BL/6-to-BALB/c heart transplant model. METHODS Control animals had rejection on postoperative day (POD) 8. Treatment with mAb started on POD 4 when leukocyte infiltration was well established. The recipients were treated with (1) mAb LFA-1, (2) mAb alpha4, and (3) mAbs LFA-1 + alpha4 at a dose of 6 mg/kg/day i.v. on PODs 4, 5, and 7. Untreated and rat IgG-treated animals were used as controls. RESULTS Control animals experienced rejection on POD 8. Treatment with mAb against LFA-1 or alpha4 alone prolonged allograft survival to 17.0+/-3.2 and 24.3+/-4.6 days, respectively (P < 0.01 vs. controls). Combination therapy with both mAb increased allograft survival to 28.2+/-3.7 days (P < 0.01 vs. controls). Sequential pathological studies showed the mAb to alpha4, but not LFA-1, markedly reduced the degree of lymphocytic infiltration in cardiac allografts. In contrast, a different pattern was observed using in vitro studies: mAb to LFA-1, not alpha4, significantly reduced proliferative responses in mixed lymphocyte culture and interleukin-2 production from recipient splenocytes on POD 8. CONCLUSION These data indicate that integrins play an important role in rejection. Although the effect of mAb against alpha4 and LFA-1 may involve different mechanisms, treatment with mAbs to integrins may be valuable in future clinical transplantation by averting ongoing rejection and prolonging graft survival.

Integrin α2β1 on rat myeloma cells modulates interaction of α4β1 integrin with vascular cell adhesion molecule-1 but not fibronectin

It is well established that α2β1 integrin functions as a receptor for collagen and laminin; whereas α4β1 integrin binds fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In the present study, we showed that rat myeloma YB2/0 cells constitutively expressed α4β1 but not α2β1 integrin. Transfection of cDNA of mouse α2 integrin subunit resulted in the expression of heterologous α2β1 integrin on YB2/0 cells (YBmα2). The expression of α2β1 conferred YBmα2 cells the ability to interact with collagen and laminin. In comparison with mock transfected YB2/0 cells (YBpF), YBmα2 cells exhibited increases in the binding and migration on VCAM-1; in contrast, both YBpF and YBmα2 were similar in their interactions with fibronectin or fibronectin fragment FN-40 that contains the binding site for α4β1 integrin. The interaction of α4β1 with VCAM-1 was further stimulated upon ligation with α2β1-specific mAb. The use of specific inhibitory mAb demonstrated the role of α4β1 in mediating the observed interactions with fibronectin and VCAM-1. Therefore, results show that expression of α2β1 differentially regulated α4β1 integrin function by stimulating its interactions with VCAM-1 but not fibronectin. The in vivo significance of α2β1 integrin expression was demonstrated by intravital videomicroscopy showing that ligation of α2β1 enhanced α4β1-mediated extravasation of YBmα2 cells in the liver.

Extracellular and Intracellular Functions of vla Proteins

An extensive assortment of cell adhesion processes are mediated by receptors called integrins, a family composed of at least 20 distinct α-β heterodimers. Ligands bound by integrins include a variety of extracellular matrix molecules, cell surface Ig-like proteins, and some serum and complement proteins (Hemler, 1990; Hynes, 1992; Larson and Springer, 1990). Adhesion mediated by integrins is an essential feature of diverse processes such as tumor cell growth and metastasis, development, wound healing, and immunolocalization of leukocytes.