The goal of the proposed project is to characterize the adhesive interactions between T lymphocytes and vascular endothelial cells. This will involve quantifying the contributions of the various adhesion pathways in T cell rolling, capture, and long-term binding, and correlating levels of receptor and ligand expression with the adhesive function of the cells. The project will accomplish these goals by using a variety of independent yet complementary techniques. The binding of T cells with human umbilical vein endothelial cells (HUVECs) will be quantified using the parallel plate flow chamber to examine the detachment of cells that have been pre-incubated on the monolayer and have attained stable adhesion. The flow chamber will also be used to mimic in vivo conditions of cell capture by flowing T cell suspensions past an endothelial monolayer at various physiological shear stresses. This will yield critical threshold stresses for adhesion as well as binding efficiencies over a range of shear stresses. The molecules involved and their relative contributions to cell capture, rolling and firm binding will be studied through antibody blocking of the known adhesion pathways. The kinetics of expression of the relevant adhesion molecules will be quantified using immuno-staining, flow cytometric analysis and fluorescent microscopy. In this way, the expressions of specific adhesion receptors can be related to their ability to mediate adhesion under various activation protocols. In addition, the possible contribution of erythrocytes in increasing the rate of cell capture to the endothelium will be investigated. The information gained will greatly enhance our understanding of lymphocyte trafficking and may allow us to define conditions under which the delivery of cells to specific sites is optimized.
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