REGULATION OF IMMUNE RESPONSES IN HUMANS AND NON-HUMAN PRIMATES

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L-selectin (LAM-1/LECAM-1) the selectin expressed on leukocytes mediates a number of leukocyte-endothelial interactions, including the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes and Peyer's patches, the binding of neutrophil to endothelial cells and the binding of all leukocytes to inflamed venules. Recently we showed that L-selectin ligation influences the T cell activation process: immobilized Leu-8 mAb influences the anti-CD3-mediated proliferation of T cells and co-immunoprecipitates molecules in the TCR/CD3 complex. In the present study, we provide further evidence of the involvement of L- selectin in signal transduction by showing that ligation of L-selectin augments phosphoinositol (PI) hydrolysis induced by anti-CD-3 and accelerates and enhances tyrosine phosphorylation induced by anti-CD3. In further studies, we examine the signaling function using immunoprecipitation techniques aimed at identifying molecules associated with L-selectin under various conditions. First, we showed that in resting cells--metabolically labeled with 35-S methionine and 35-S cysteine and then exposed to non-dissociating detergent lysing agents, anti-L-selectin coimmunoprecipitates both a 94 kD and a 210 kD molecule identified on SDS-PAGE. These molecules are likely to be intracytoplasmic (non-surface) molecules, since they are not detected in lysates derived from surface-labeled cells. Second, we showed that in metabolically labeled and anti-L-selectin clusterred cells, anti-L- selectin coimmunoprecipitates a 210 kD molecule which, in an in vitro kinase assay, takes up 32-P. Furthermore, in the same cells activated with Con A, anti-L-selectin coimmunoprecipitates a 150 kD molecule as well as the 210 kD molecule that take up 32-P. These data establish that L-selectin is a signal transduction molecule that associates with several other molecules, at least some of which have tyrosine kinase activity. We hypothesize that the latter are critical for the ability of L-selectin to signal cells directly or via TCR/CD3. In additional studies, we sought to determine if L-selectin-mediated cell signaling has functional consequences other than an effect on proliferation. In particular, we investigated the role of L-selectin in T cell cytokine production. We demonstrated utilizing quantitative RT-PCR, that L-selectin crosslinking of anti-CD3 activated CD4+ T cells leads to an approximately 10-fold higher steady state IFN-gamma mRNA levels at an early time point (6 hours), but not at later time points (12 and 24 hours) and this effect was seen when L-selectin and TCR/CD3 ligations were applied simultaneously or sequentially. L-selectin ligation alone, in the absence of anti-CD3 signalling did not induce an INF-gamma mRNA increase. The above results are relevant to T cell-endothelial interaction occurring in the vasculature at sites of inflammation, as well as to immune response at particular sites.