We will determine how regulatory molecules released from resting and stimulated endothelial cells (+/-TNFa, Il- 1, endotoxin) modify neutrophil functions in Arthus and Shwartzman-like models of tissue injury. Using intact human neutrophils, granule-free cytoplasts, cell free systems, liposomes of varying physical structure, and human umbilical vein endothelial cells, we will test four novel hypotheses.
Specific Aim I. To test the hypothesis that the Yin/Yang control of neutrophil function by cAMP/cGMP is exerted at the level of raf phosphorylation in the MAP-kinase cascade. Neutrophils, their cytoplasts and cell-free extracts exposed to the three classes of activators will be studied kinetically (5 sec to 5 min) for delta-psi, [Ca]i phospholipid turnover, lysosomal enzyme release, translocation of 47phox and 67phox components of the NADPH oxidase and ras-related proteins, carboxy methylation, cAMP, cGMP and phosphorylation/ dephosphorylation patterns studied of raf, CD11b/CD 18, MAP kinases +/- inhibitors. (calyculin, genestein, okadaiate, etc.).
Specific Aim II. To test the hypothesis that endothelial cells release autocoids (adenosine, PGI2 and NO) that inhibit neutrophil-mediated endothelial activation (judged by translocation of NFkB, induction of COX II, NO synthases, expression of E-selectin, VCAM-1, ICAM-1). Neutrophils or cytoplasts will be incubated with endothelial cells that do or do not form the autocoid (+/- adenosine deaminase, +/- COX II, NO synthase inhibitors). Endothelial cell activation will be determined by electrophoretic mobility shift, Northern Blot and FACS.
Specific Aim III. To test the hypothesis that lipid metabolites formed in the course of neutrophil/endothelial interactions, e.g. arachidonate (20:4) and phosphatidate (PA), regulate the association of ras-related proteins with their inhibitor/chaperones (GDI's) and with the plasmalemma. In intact cells, cytoplasts and cell-free, oxidase-competent systems we will study membrane/cytosol distribution, GDI association and phospholipid-dependent isoprenyl-directed carboxy methyltransferase (piCMT) activity with respect to O2- and/or aggregation, phosphorylation patterns in response to added 20:4 (+/- other eicosanoids, HODEs) or PA (+/- other phospholipids).
Specific Aim I V. To test the hypothesis that the physical state of phospholipids (lamellar vs Hex(II)) as regulated by phospholipases A, C and D regulates a) the association of prenylated proteins with the plasmalemma and b) the effect of autocoids (PGE1, 14,15 di-HETEs, 13-HODE, 22:6, lipoxins) on the activity of piCMT and the oxidase system in natural and artificial bilayers. Lamellar (phosphatidyl choline, palmitoyl-oleyl ethanolamine) liposomes will be compared with Hex(II) forming liposomes (PA +/- Ca, dioleyl ethanolamine) for presentation to PGI2 receptors and reconstitution of the O2- system.
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