Understanding of polymorphonuclear leukocyte (PMNL) function has been greatly aided by the recognition of heritable abnormalities or absence of PMNL proteins. In this proposal, the discovery of eight patients (4 males and four females) with an absence of a high molecular weight glycoprotein family (LFA-1/Mac-1/p150,95) on both the cell surface and an intracellular pool are utilized to understand the molecular mechanism by which these proteins mediate cell adherence phenomena. Human endothelial cell monolayers will be examined for adherence, spreading, and penetration by normal and LFA-1/Mac-1/p150-95 deficient PMNL under resting conditions and after stimulation with chemotactic factors including F-Met-Leu-Phe. Three-dimensional type I collagen matrices will be investigated for adherence and invasion by normal and deficient PMNL under similar conditions to test the hypothesis that PMNL adherence is important for movement on endothelial cell surfaces, but is less important for penetration of collagen matrices. Direct observation with interference contrast microscopy as well as scanning and transmission electron microscopy will be used to document the adherence and invasion of the PMNL. The role of LFA-1/Mac-1/p150,95 in the modulation of PMNL adherence dependent function will be studied by determining the intracellular locations of LFA-1/Mac-1/p150,95 and certain related intermediates by a unique, quantitative immunoblotting assay utilizing monoclonal antibodies against LFA-1/Mac-1/p150,95 and the common Beta subunit. Subcellular fractionation will be accomplished on Percoll density gradients after nitrogen cavitation of the PMNL. Cell surface protease treatment, metabolic labeling with [3H]-galactosamine and [3H]-glucosamine and surface labeling with NaB3H4 and [125I] will be employed concurrently with the immunoblotting assay to determine whether cycling of LFA-1/Mac-1/p150,95 takes place. These studies will be complemented by detection of LFA-1/Mac-1/p150,95 in coated pits or vesicles by electron microscopy experiments utilizing colloidal gold or ferritin labeling experiments. Comparative assessments of surface Mac-1 subunit expression (flow cytofluoragraphy) and cell adherence properties will be performed employing chemotactic or secretory conditions designed to """"""""up regulate"""""""" or """"""""down regulate"""""""" Mac-1 proteins. These studies should provide new information relevant to the role of Mac-1 dependent PMNL adherence reactions in inflammation and host defense.
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