Inflammation is marked by extensive granulocyte extravasation from the blood into tissues at a site of infection or immunologic challenge. Adhesion molecules and their 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 injury or insult. E- selectin, which is expressed on activated endothelial cells, is a member of the selectin adhesion protein family which mediates cell-cell adhesion events early in this immune response by binding to cell surface glycoconjugates. The identification of normal human granulocyte glycoconjugates responsible for E-selectin-mediated cell adhesion will be important to our understanding of inflammation in its many guises, and may offer novel avenues for inflammation control. Although carbohydrate structures which bind to selectins have been identified, the natural ligands for E-selectin on normal human granulocytes have yet to be fully elucidated. The principal investigators have obtained data indicating that one or more glycosphingolipids on normal human neutrophils and eosinophils act as E-selectin ligands. The current application proposes to combine proven expertise in granulocyte cell adhesion, flow cytometry, and glycosphingolipid glycobiology to identify and characterize normal human granulocyte glycosphingolipid ligands for E-selectin, and to evaluate their role(s) in granulocyte function.
Aim 1 will directly test the hypothesis that glycosphingolipids constitute major ligands for E-selectin on intact human neutrophils and eosinophils using in vitro cell adhesion assays in conjunction with highly specific enzymes, antibodies, and glycoconjugate biosynthesis inhibitors.
Aim 2 will use techniques for glycosphingolipid isolation, resolution and functional analysis to identify and isolate glycosphingolipid(s) from normal human neutrophils and eosinophils capable of specific binding to E- selectin.
Aim 3 will use microanalytical techniques, antibodies, and specific enzymes, to determine the structure of glycosphingolipid ligand(s) identified and isolated in Aim 2.
Aim 4 will test the glycosphingolipids on intact cells and individual glycosphingolipids purified and characterized in Aims 2 and 3 for their role(s) in granulocyte cell adhesion under static conditions and physiological flow. The knowledge gained from these studies may be applicable to the design of improved anti-inflammatory drugs, to understanding certain immune dysfunctions, and may provide insight into the physiological roles of glycosphingolipids.
