The vascular endothelium plays a pivotal role in the initial attachment and diapedesis of peripheral blood leukocytes into the extracellular space in inflammation and vascular diseases. The development of in vitro models to mimic flow conditions likely to exist in the microvascular environment has contributed greatly to our understanding of the cellular and molecular mechanisms involved in polymorphonuclear leukocyte (neutrophil) interactions with the endothelium during acute inflammation. However, most in vitro models have utilized adhesion molecules absorbed to plastic or endothelial cells obtained from large vessel (human umbilical vein), whereas little information is available on microvessel endothelium. In addition, relatively little is known about T lymphocyte - microvascular endothelial cell interactions in non-lymphoid vessels which play an important role in chronic inflammatory diseases, and in immunological reactions such as transplant rejection. Preliminary studies by the applicant have demonstrated that blood monocytes and CD4+ T lymphocytes roll and stably arrest on cytokine-activated endothelial monolayers under defined flow conditions. This application will examine the cellular and molecular mechanisms of T lymphocyte adhesive interactions with human microvascular endothelium under defined flow conditions. The primary goals of this project are to: 1) determine the mechanisms that mediate rolling, stable arrest, and transmigration of CD4+ T lymphocyte subsets to microvascular endothelial cell monolayers using an in vitro flow model which allows direct visualization in live time of lymphocyte-endothelial interactions; 2) to measure live time the temporal patterns and spatial distribution of ICAM-1, VCAM-1, CD31 (PECAM-1) and E-selectin on the surface of activated microvessel endothelium using low- light level immunofluorescence microscopy and image analysis and to examine whether the spatial distribution of these adhesion molecules correlates with the lymphocyte rolling, arrest or spreading normally observed under flow conditions; 3) to examine the effect of soluble and proteoglycan-immobilized chemokine chemoattractants on lymphocyte adhesive interactions with cytokine-activated microvascular endothelium under defined levels of flow and on the activation of T lymphocyte Beta1- and Beta2-integrins.
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