Carbohydrates on the surfaces of T cells, neutrophils and monocytes are reactive with a family of adhesive receptors (termed selectins) on endothelial cells. This program is designed to determine if carbohydrates, glycoconjugates and glycoproteins can be used to block adhesive interactions between leukocytes and endothelial cells in a manner that effectively blocks immunologically-triggered inflammatory reactions. In Section 1 (Lowe), studies will be undertaken to isolate and chemically characterize naturally occurring glycoconjugates reactive with E- and P- selectins. the sialyl Lex tetrasaccharide will be synthesized using recombinant glycosyltransferases. Recombinant glycoproteins expressing sialyl Lex (L-selectin, lamp-1, and CD43/leukosialin) will also be constructed and characterized. These preparations will be used in Sections 2 and 4 for blocking of inflammatory reactions. In Section 2 (Ward), the ability of 14C-sialyl Lex preparations and other irrelevant preparations to bind in vitro and in vivo to TNF-alpha-stimulated endothelial cells will be assessed, as will the ability of anti-ELAM-1 to block this binding. In companion studies we will assess the ability of these carbohydrate preparations to block the in vitro adhesion of neutrophils to endothelial cells as well as the ability of these carbohydrate components to block the ability of activated neutrophils to kill TNFa stimulated endothelial cells. The in vivo experiments will employ the ELAM-1 dependent model of immune complex-induced acute lung injury and assess the ability of carbohydrate compounds that bind to E-selectin and P-selectins (Sections 1, 3) to block immunologically triggered lung injury. Section 3 (Stoolman), will focus on selectin-dependent monocyte attachment to inflamed microvasculature in human rheumatoid arthritis. The monocytic structures containing the binding sites for P- and E-selectin will be characterized. The endothelial binding site for monocytic L-selectin will be defined and the impact of glycosylation on L-selectin function determined. Finally, the specificity and potency of carbohydrate-based inhibitors of selectin function (Section 1) will be assessed in humans using a battery of in vitro assays. These assays will quantitate the activity of compounds against all three selectins under physiologically relevant conditions and thus complement the in vivo studies (Sections 2, 4). In Section 4 (Marks), antibodies to rat and rabbit, E-, P- and L-selectins as well as glycoconjugates (Sections 1, 3) will be obtained and used to define the contributions in vitro of these selectins to neutrophil adhesion to endothelial cells, the expression of selectins in human rheumatoid arthritis and in animal models of arthritis and the ability of these compounds to protect in vivo against chronic immune arthritis. In summary, this program should permit definition of the role of carbohydrate moieties in immunologically triggered tissue injury and should facilitate the development of novel anti-inflammatory carbohydrate-based molecules that have the ability to block the emigration from the vasculature of tissue-damaging leukocytes.
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