Selectin-dependent leukocyte adhesion to vascular endothelium is an essential precursor to leukocyte emigration in inflammation. Antagonism of selectin-dependent leukocyte adhesion in vivo can diminish the pathological consequences of selectin-dependent inflammation. This Program Project brings together an interactive group of individuals with expertise in oligosaccharide synthesis, cell adhesion, cell and molecular biology, and the physiology of inflammation, for the purpose of further evaluating the roles of oligosaccharide-based selectin molecules in inflammation. This group will generate and evaluate a diverse set of oligosaccharide-based selectin antagonists in in vitro assays of selectin-dependent cell adhesion, and in animal models of selectin-dependent acute and chronic inflammation. Four categories of oligosaccharide-based selectin antagonists will be prepared and studied; (i) multivalent sialyl Lewis x- bearing N-linked oligosaccharides (Project V), (ii) chemically synthesized selectin ligand analogues composed of low molecular weight sialylated, sulfated, fucosylated oligosaccharides (Project I), (iii) sulfatides (Project II), and (iv) soluble recombinant glycoproteins bearing sialyl Lewis x-type oligosaccharides (Projects I, III, IV). These molecules will be evaluated for selectin blocking activity in vitro, using static and flow based assays of selectin-dependent leukocyte adhesion (Projects III and IV). Their in vitro potencies will be compared to their anti-inflammatory efficacy in vivo, in acute models of selectin-dependent inflammation in the rat (Project II). Complementary studies will define the biodistribution of N-linked selectin antagonists in the rat (Project V). A rat model of T- cell/monocyte-dependent arthritis will define the role of selectins in a chronic inflammatory disorder (Project II). This model, and a rabbit arthritis model (Project IV), will be available for evaluation of the selectin uantagonists. Related studies will focus on an analysis of mice genetically deficient in alpha(1,3)fucosyltransferase genes (Fuc-TIV and Fuc-TVII: Project I) responsible for selectin ligand synthesis. Biochemical analyses will define the acceptor substrate specificity of these two enzymes (Project I). The Fuc-TVII and Fuc-TTV """"""""knock out"""""""" mice, and mice deficient in ICAM-1, P-selectin, NADPH oxidase, will be evaluated in models of acute inflammatory lung injury (Project II). Biochemical studies will define the structures of L-selectin ligands on rabbit endothelial cells (Project IV), and of P- and E-selectin ligands on human T-lymphocytes (Project III) and mouse leukocytes (Project I). Together, these interrelated studies will add substantially to our understanding of (i) the roles of selectins in acute and chronic inflammation, (ii) the relationships between the structures of oligosaccharide-based selectin antagonists, their ability to block selectin-dependent leukocyte adhesion in vitro, and their in vivo anti-inflammatory efficacy.
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