The goal of this project is to define the molecular mechanisms that regulate the adhesion of polymorphonuclear leukocytes (PMNs, neutrophils, granulocytes) to endothelial cells (EC) and the biologic consequences of this interaction, such as activation (and subsequent polarization, migration, degranulation, etc) of the PMNs. The regulation of PMN adhesion to the Ec that form the intima of blood vessels is a central issue in vascular biology because of its requisite role in physiologic inflammation and host defense. Furthermore, unregulated adhesion of PMNs to EC, followed by release of granular enzymes, oxygen radicals, and other mediators by the activated leukocytes, contributes to vascular damage in ischemia-reperfusion injury, myocardial infarction, acute pulmonary vascular injury, nad other syndromes in human disease. We have described a novel mechanism of EC-dependent adhesion of PMNs. The adhesive interaction occurs rapidly (within minutes) when cultured human EC are activated by an agonist and is mediated, at least in part, by the rapid, time-dependent, synthesis and surface expression by the EC of platelet-activating factor (""""""""PAF""""""""; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a unique glycero- phospholipid that interacts with PMNs and other cell types by binding to a surface receptor. This rapid EC-dependent mechanism of PMN adhesion is clearly different from that induced by cytokines, which occurs over hours, and likely is involved in the rapid localization of PMNs to specific areas of the intima that occurs in physiologic and pathologic inflammation. The current project has 4 specific objectives: 1) We will define the requirement for an activation response by the PMNs for the adhesive interaction and the mechanisms that are involved. This also the first step in determining how adhesion of PMNs to activated EC influences subsequent events (such as polarization, release of enzymes and lipid mediators, etc by the PMNs). 2) We will define the role of the PMN CD11/CD18 adhesive glycoproteins, with the hypothesis that they play an amplifying rather than an obligate role. 3) We will characterize the role of other EC molecules in rapid EC- dependent adhesion, focusing initially on granule membrane protein 140 (GMP-140), a novel glycoprotein that is localized in secretory granules of the EC and that can be rapidly translocated to the plasma membrane. 4) We will define the mechanism(s) of PMN adhesion to EC activated by oxidants, a model relevant to inflammatory vascular injury in human disease. Based on the results of this line of investigation, we will be able to explore the mechanisms that lead to unregulated PMN adhesion and activation in other models of vascular injury and potentially, in vivo. The experiments will employ cultured EC, isolated native, fixed, and radiolabeled PMNs, neutrophil cytoplasts, myeloid cell lines, studies of signal transduction mechanisms in PMNs activated by agonists presented in the EC plasma membrane, in model membranes, or in the fluid phase, studies of surface expression of lipid (PAF) and glycoprotein (GMP-140) mediators by Ec, metabolic labeling of PAF and other products of activated EC and, potentially, isolation and characterization of new mediators expressed by EC or PMNs.
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