Inflammation plays a major role in the pathogenesis of many diseases including atherosclerosis, immune complex-mediated responses and the long term sequelae of many infections. Leukocyte migration across the vascular endothelium is a critical step in an inflammatory response. While much is known about leukocyte adhesion to EC, the mechanism by which leukocytes open the junctions between EC is unknown. Stimulated PMN signal an increase in EC cytosolic free calcium concentrations. Such increases in EC (Ca++ (i) regulate transendothelial migration indicated that simulated PMN produce an extracellular substance. Monocytes and a human monocytic cell line, THP-1, also initiate increases in EC *(Ca+++(i) which regulates transendothelial monocyte migration. These findings form the basis for the hypothesis that leukocytes signal the opening of junctions between EC in order to migrate across EC barrier. The following aspects of the hypothesis will be studied: 1). identify/characterize the substance (s) by stimulated PMN s that increase in EC ((Ca++(i), phosphorylation of EC MLC, and the permeability of EC MLC, and the permeability of EC megalocytes establish the biological properties of this product 3). determine the role of EC protein kinases, such as MLC kinase, in medicating leukocyte migration across EC monolayers. An in vitro system of culturing EC on human amniotic membranes and methods for measuring EC ((Ca++(i), EC monocyte permeability to ions, EC MLC phosphorylation and leukocyte migration across EC monolayers will be used to perform these studies. The studies proposed in this application should contribute to an understanding of the mechanisms by which leukocytes migrate across EC barriers and by which EC regulate vascular permeability. An understanding of these processes well provide a basis for developing rational strategies for athe prevention/treatment of diseases characterized by inflammation.
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