The interaction between leukocytes and endothelium controls immune- inflammatory responses by governing the state of endothelial cell activation and the recruitment of leukocyte subsets at the site of inflammation, in vivo. In addition to cell-to-cell interactions and the release of pleiotropic cytokines, recent studies conducted by the applicant have unraveled a third pathway of cross talk between leukocytes and endothelium. Following chemoattractant stimulation, we have shown that polymorphonuclear leukocytes release a heterogeneous population of membrane MP. In turn, these MP initiate an elaborate pathway of endothelial cell activation with release of chemotactic and inflammatory cytokines IL-6, IL-8, increased expression and function of tissue factor, up-regulation of adhesion receptors ICAM-1 VCAM-1, E- selectin and tyrosine phosphorylation of JNK1 protein. In more recent experiments, we have demonstrated the existence of leukocyte-derived MP in vivo and their up-regulation by inflammatory stimuli. The present proposal is leukocyte-derived MP in vivo and their up-regulation by inflammatory stimuli. The present proposal is designed to elucidate the cellular and molecular requirements of endothelial cell activation by leukocyte MP. In addressing the concerns raised during the first review of the application, this will be accomplished using an experimental design that will broaden the training and experience of the applicant. Experiments will be carried out to analyze the structural organization of leukocyte MP with respect to lipid composition, membrane adhesion receptors and cytoskeletal proteins by chromatography, SDS gel electrophoresis, immunoblotting, and electron microscopy. Secondly, the process of membrane vesiculation will be characterized in various leukocyte subsets, in relationship to its cell activation potential gene induction and upstream and downstream intracellular signaling events. The overall proposal is designed to elucidate an entirely novel aspect of cross talk between leukocytes and endothelial cells, of potential importance for the maintenance of immune surveillance and inflammation, in vivo.
