The goal of this proposal is to understand the molecular mechanisms of leukocyte recruitment to areas of inflammation. The trafficking of leukocytes to inflamed tissue is a multi-step process starting with the tethering and rolling on the blood vessel wall. Two endothelial cell molecules, P-selectin and E-selectin, have overlapping roles in this initial process. The molecules expressed on leukocytes, that interact with the selectins have been difficult to elucidate. While PSGL-1 appears to be the major physiologic ligand for P-selectin, and several candidate molecules for the E-selectin ligand(s) have been proposed, the major physiologic E-selectin ligands, remain undetermined. In order to elucidate further the role of the two endothelial selectins, the precise spatial and temporal expression pattern of P-selectin and E-selectin after stimulation of the endothelial cells will be determined in vivo using intravital microscopy. The role of the interacting ligands will also be addressed in vivo. The function of PSGL-1 and ESL- 1, two E-selectin ligand candidates, will be examined by eliminating their function in vivo using a mouse deficient in both proteins: Leukocyte tethering, rolling, and firm adhesion will be characterized in the mouse cremaster muscle vasculature. An experimental model of mouse peritonitis will be used to examine neutrophil accumulation during inflammation. Finally, a novel blot rolling assay using a parallel-plate flow chamber will be used to characterize and identify other E-selectin ligands. Novel ligands will be isolated, a portion of its amino acid sequence obtained, and the coding sequence cloned. This research will not only help further the understanding of the normal inflammatory response, but it will also contribute to the understanding of the pathophysiologic; responses in inflammatory disorders and in the vascular inflammation associated with thrombosis and atherosclerosis.
