Neutrophil extravasation and fluid leak into the alveolar space is a hallmark of the acute lung injury that occurs in adult respiratory distress syndrome (ARDS) and other human disease conditions. The long- term objective of this work is to treat acute lung injury in humans by interrupting the initial adhesion of leukocytes to the blood vessel wall. E-, P-, and L-selectin are endothelial, platelet, and leukocyte glycoproteins that support adhesion through the recognition of carbohydrate ligands. Antibody-, peptide-, and carbohydrate-based approaches will be used to elucidate the molecular interactions of selectins and their ligands in vitro and in vivo.
Four specific aims are proposed: I. Determine the expression of selectins and their carbohydrate ligands in acute lung injury. Specific antibodies, selectin-Ig fusion proteins, and cDNA probes will be used to localize and quantitate antigen and mRNA expression in lung tissue of rodents given bacterial endotoxin and/or inflammatory cytokines by intratracheal and intravenous routes. Selectin and carbohydrate ligand expression will be determined in blood and bronchoalveolar lavage samples from patients undergoing surgical removal of pulmonary arterial thromboemboli. II. Develop specific selectin-blocking reagents based on antibodies and peptides. Monoclonal antibodies generated against recombinant murine selectins and peptides representing sequences found in their extracellular domains will be evaluated as blockers of adhesion and direct molecular binding in vitro. III. Identify oligosaccharide ligands of murine selectins. Cell adhesion assays and direct molecular binding studies, including a competitive ELISA, titration microcalorimetry and fluorescence spectroscopy, will be used to study the carbohydrate specificities of murine selectins. Comparisons will be made to ongoing investigations of human selectin-carbohydrate interactions. IV. Inhibit selectin-carbohydrate interactions in acute lung injury utilizing antibodies, peptides, and oligosaccharides. Potent blockers of selectin-carbohydrate interactions in vitro will be evaluated for efficacy in the treatment of acute lung injury in rodent models. Alternative routes of administration will be tested. We anticipate that our studies will provide new insights into the role of selectin-carbohydrate interactions in acute lung injury, and directly point to avenues for therapeutic intervention. In addition, the reagents generated in the course of these studies may prove useful in the study of selectin-carbohydrate interactions in other human disease conditions.
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