P-selectin is a cell adhesion molecule that resides in the storage granules of platelets and endothelial cells. Upon cell stimulation, the protein is translocated to the plasma membrane where it functions as a leukocyte receptor for PSGL-1 on neutrophils and monocytes. The current application represents a continuation of studies of the biology of P-selectin and PSGL-1. Since PSGL-1 has been shown to bind to all selectins, the kinetic and equilibrium binding of soluble PSGL-1 to soluble P-selectin, E-selectin and L-selectin will be analyzed by fluorescence spectroscopy. During the past grant period, we have prepared a PSGL-1 deficient mouse by homologous recombination and have completed the initial characterization. Cells from this mouse will be used to establish the physiologic role of PSGL-1 in selectin function by comparing the interaction of PSGL-1 (-/-), (+/-) and (+/+) leukocytes with P-selectin, E- selectin and L-selectin under rolling conditions in a parallel plate ex vivo assay using neutrophils and T lymphocytes. The PSGL-1 deficient mouse and double knockout mice including PSGL-1 null/P-selectin null mice, PSGL-1 null/E-selectin null mice and PSGL-1 null/L-selectin null mice will be employed in model systems to determine the physiologic function of PSGL-1. Pathologic processes to be studied include models of non- immune mediated and T-cell mediated skin inflammation, leukocyte rolling following trauma and TNF, experimental glomerulonephritis, chemical peritonitis, bacterial pneumonitis, thrombosis, atherosclerosis, wound healing and platelet rolling. To understand the molecular basis of signal transduction and effector function induced by platelet activation or P-selectin binding to the P-selectin ligand on leukocytes, the induction of Ca2+ flux in platelets by PSGL-1 via P-selectin will be analyzed. Furthermore, binding of cytoplasmic tails of P-selectin and PSGL-1 to cytoplasmic signalling proteins in platelets and monocytes respectively will be examined using dimer constructs of cytoplasmic tails. If these studies indicate that PSGL-1 and ESL-1 are not physiologically critical counterreceptors for E-selectin or that there is evidence for another P- selectin ligand, we propose to expression clone a novel E-selectin ligand from a leukocyte library prepared from WEHI cells and a novel P-selectin ligand from a library prepared from neutrophils isolated from the PSGL-1 null mouse. The putative ligands will undergo characterization of their full length cDNAs and comparison of their predicted amino acid sequences with that of the PSGL-1, ESL-1 and GlyCAM-1. These studies will contribute to our understanding of the physiologically relevant receptors and counterreceptors that define cell-cell interaction during inflammation.
Showing the most recent 10 out of 18 publications
