This proposal targets endothelial cell (EC) biology in leukocyte trafficking and inflammation, focusing on the role of vascular adhesion receptors and activating factors in the control of lymphocyte homing. I. The biology of the mucosal vascular addressin, MAdCAM-1 will be explored through: 1) In situ videomicroscopic analyses of the contribution of MAdCAM-1 to L-selectin-mediated lymphocyte rolling in Peyer's patch (PP)-high endothelial venules (HEV); 2) determination of the ability of MAdCAM-1 to bind and present lymphocyte activating chemoattractants of the chemokine family; 3) immunohistologic assessment of the physiologic distribution of MAdCAM-1 variant forms; 4) morphologic and functional analyses of MAdCAM-1 knockout mice; and 5) extension of these studies to man. II. The molecular and functional biology of the peripheral lymph node addressin (PNAd)--HEV ligands for the L-selectin--will be studied through: 1) structural analyses of L- selectin-binding sulfated glycotopes of HEV, taking advantage of a novel panel of anti-PNAd glycotype MAbs: 2) identification of dominant PNAd glycoproteins (gp's) responsible for glycotope presentation by HEV: PNAd gp's with demonstrable physiologic relevance in in vivo homing studies will be targeted for molecular cloning; 3) identification of genes encoding enzymes involved in PNAd glycotope synthesis, or other HEV differentiation antigens, by mammalian expression cloning with anti-PNAd glycotope MAbs; and through isolation of HEV-specific cDNA's by hybridization selection. III. HEV- or EC-associated factor(s) responsible for rapid G-protein-mediated activation of lymphocytes during homing will be identified through immunohistologic and in vivo antibody inhibition studies of chemokines on HEV; and if indicated through production of MAbs capable of inhibiting the activation step in lymphocyte interactions with EC in in vitro artificial vessel models. IV. In vitro migration and/or chemotaxis assays will determine whether MAdCAM-1 can serve as a selective substrate for alpha4Beta7+ lymphocyte crawling, acting coordinately with local chemoattractants in regulating the chemotaxis or haptotaxis of lymphocyte subsets. V. The developmental regulation of vascular addressins and of other EC adhesion receptors will be studied immunohistologically; and the role of known or novel cytokines in addressin regulation will be explored through in vitro studies of cultured EC lines, including a novel transformed HEV cell line; and also in analyses of cytokine knockout mice. VI. The physiologic role(s), functions, and if indicated, the molecular biology of newly identified EC antigens involved in leukocyte trafficking will be explored: These include the MECA-32 antigen (antibodies to which inhibit lymphocyte homing); the PA 67 antigen (involved in T cell adhesion to activated mouse EC); and 92-ELAM (involved in adhesion of monocytes). VII. Finally, the role of vascular selectins in lymphocyte trafficking to inflamed joints will be explored. The proposed studies should expand our understanding of the critical role of the vascular endothelium in regulating lymphocyte trafficking during normal and pathologic immune responses.
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