The inflammatory response is a fundamental component in host defense. The firm adhesion and transmigration of leukocytes across the vascular endothelium during inflammation occurs at the level of post-capillary and collecting venules. Although much attention has been paid to leukocyte- endothelial adhesive interactions, only recently have the molecular mechanisms involved in leukocyte passage through the endothelial cell lateral junctions become a focus of study. Recently, we have shown that neutrophil adhesion triggers disruption of adherens junctions at endothelial lateral borders, prior to the transmigration event (1). Specifically, the VE-cadherin complex, consisting of VE-cadherin, alpha- catenin, beta-catenin, gamma-catenin (also termed plakoglobin), and p120/p100 non-covalently linked, was disrupted within 3 minutes of initial neutrophil binding, and results in loss of this complex from lateral junctions and cleavage of both VE-cadherin and beta-catenin. Washed membranes (protease inhibitor-treated) prepared from neutrophils were able to mediate these changes, suggesting that the stimulus for disruption of the endothelial VE-cadherin complex resides in neutrophil surface and that granule proteolytic components do not appear to be required. The focus of this project is to characterize more fully the endothelial dependent-mechanisms involved in regulation of lateral junctions during leukocyte transmigration using immunological, cell biological and molecular biological strategies in in vitro and in vivo models. The proposed studies will test critically whether the observed changes in the composition of lateral junctions are necessary for leukocyte transmigration.
Specific Aim I will determine the step in cell adhesion that trigger dissociation of the VE-cadherin complex during leukocyte transmigration, using live time immunofluorescence assays to monitor changes in VE-cadherin localization during leukocyte adhesion in a in vitro flow model, and secondly, test the hypothesis that dissociation and cleavage of the VE-cadherin complex is necessary for transmigration.
Specific Aim II will attempt to identify other, as yet uncharacterized, endothelial molecules localized to cell-to-cell junctions that are involved in leukocyte adhesion-transmigration, using a monoclonal antibody screening approach. The experiments proposed in Specific Aim II will determine the intracellular signaling pathways involved in leukocyte- induced VE-cadherin dissociation using pharmacological approaches and the live time immunofluorescence assays developed in Specific Aim I.
Specific Aim I V will utilize in vivo models of acute or chronic inflammation to test the potential pathophysiologic relevance of leukocyte-induced alterations in adherens junctions. The proposed studies are relevant to the overall them of the program and will work in concert with the other project to gain a better understanding of the role of the vascular endothelial lining in inflammation and immune reactions.
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