Inflammation involves extensive granulocyte extravasation from the blood into tissues at a site of infection or immunologic challenge. Adhesion molecules and complementary ligands on granulocytes and on the blood vessel wall endothelium control the extent and identity of granulocytes entering the tissue at a site of insult. E-selectin, expressed de novo on activated endothelial cells, is a selectin adhesion protein which mediates cell-cell adhesion early in this immune response by binding to leukocyte surface glycoconjugates. The identification of normal human granulocyte glycoconjugates responsible for E-selectin-mediated cell adhesion will be important to the understanding of inflammation in its many forms, and may offer novel avenues for inflammation control. Although several glycoconjugates which bind to selectins have been identified, the natural ligands for E-selectin on human granulocytes have yet to be fully elucidated. The applicants and others have obtained strong published and preliminary data indicating that glycosphingolipids on normal human neutrophils and eosinophils, not glycoproteins, are the most important legends for E-selectin, and that these glycosphingolipids may differ from those previously identified in cell lines such as HL-60. This proposal combines expertise from three laboratories in granulocyte cell adhesion under both static and physiologic flow conditions with glycosphingolipid glycobiology to evaluate the roles of normal human granulocyte glycosphingolipids in E-selectin binding and granulocyte function.
Aim 1 tests the hypothesis that glycosphingolipids constitute major ligands for E-selectin on intact normal human neutrophils and eosinophils using cell adhesion assays and highly specific enzymes, antibodies, and glycoconjugate biosynthesis inhibitors.
Aim 2 uses methods for glycosphingolipid extraction, resolution and functional analysis to isolate and identify glycosphingolipids from normal peripheral blood neutrophils and eosinophils capable of specific binding to E-selectin. TLC analysis, antibodies, specific enzymes, and matrix assisted laser desorption ionization mass spectroscopy will be used to elucidate and characterize the structures of these glycosphingolipids.
Aim 3 will test glycosphingolipids on intact cells, and glycosphingolipids identified from Aim 2, for their roles in granulocyte cell adhesion under static conditions and physiological flow, and will allow us to test whether these glycosphingolipids are the natural ligands for E-selectin on normal human granulocytes. Knowledge gained from these studies will advance the design of improved anti-inflammatory drugs, and our understanding of the physiological roles of glycosphingolipids in leukocyte adhesion.
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