Human activated T cells adhere to synovial fibroblast-like cells in vitro. The present study was conducted to investigate the consequences of T cell-synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence-activated cell sorter (FACS) analysis were used to analyze the induction of VCAM-1 and ICAM-1 expression in T cell-synovial fibroblast cocultures. VCAM-1 and ICAM-1 expression could be induced in synovial fibroblast-like cells by 2 h. PCR amplification showed that both forms of VCAM-1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up-regulation of VCAM-1 and ICAM-1 was confined to synovial fibroblasts; T cells did not express VCAM-1 or increased ICAM-1. In contrast to the T cell-synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up-regulation of ICAM-1 but not VCAM-1, suggesting tissue-specific regulation of VCAM-1. The T cell-synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon-gamma, and interleukin-6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti-TNF could significantly inhibit VCAM-1 and ICAM-1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM-1 and ICAM-1 failed to be up-regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell- and synoviocyte-specific cytokines and suggest a cell contact-mediated and T cell-initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM-1/ICAM-1 by synovial fibroblasts in the rheumatoid synovium.

The interactions of alpha 4 beta 1 integrin with vascular cell adhesion molecule (VCAM) and fibronectin play important roles in many physiological and pathological processes. To understand the mechanism of alpha 4 beta 1 integrin-mediated cell adhesion, we made mutant alpha 4 constructs. Three aspartic acid (Asp) residues in alpha 4, Asp-489, Asp-698, and Asp-811, were replaced with glutamic acids (Glu). The wild-type and mutant alpha 4 constructs were transfected into K562 cells, and stable transfectants with similar levels of alpha 4 surface expression were established. The Asp-->Glu substitutions did not affect alpha 4 beta 1 association or heterodimer formation as demonstrated by immunoprecipitation analysis. However, the glutamate substitutions at Asp-489 and Asp-698 severely impaired cell adhesion to VCAM and fibronectin, whereas the substitution at Asp-811 had no detectable effect on cell adhesion. In contrast to these results, isolated alpha 4 beta 1, containing the D489E or D698E substitution, was able to bind to VCAM, suggesting that these two residues are not critical for ligand recognition. In searching for a mechanism to explain inhibition of adhesion by Asp-489 and Asp-698 mutations, we found that the sequences flanking Asp-698 resemble the DxxxxxD-S-Sx divalent cation/ligand binding motif in beta integrins and the I-domains of alpha integrins. This suggests that Asp-698 in the alpha 4 integrin, which does not possess an I-domain, may also be involved in cation binding and may be part of a sequence functionally similar to that found in the I-domains of other alpha integrins.

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We report here an analysis of the expression and function of the alpha chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the beta chain in these processes. L cells were transfected with human alpha 4 cDNA or alpha 4 and human beta 1 cDNA. Unexpectedly, human alpha 4 cDNA, when transfected alone, could induce de novo surface expression of host beta 7 and increased expression of host beta 1. Induction of mouse beta 7 and beta 1 surface expression was not due to de novo gene activation, but instead represented alpha 4/beta intracellular subunit association and transport to the cell surface. Transfection with human beta 1 prevented surface expression of mouse beta integrins. Whereas human alpha 4 and human beta 1 subunits associated very tightly in anti-alpha 4 immunoprecipitates, human alpha 4 and mouse beta subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for alpha 4 beta 7 and alpha 4 beta 1, was very poor, whereas human alpha 4/human beta 1 receptors bound strongly to VCAM. One alpha 4 transfectant, which exhibited a tight human alpha 4/mouse beta 1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that alpha 4 subunits have specific affinity for beta 7 and beta 1 integrins and require beta subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the alpha 4 and beta subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the beta subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression.