Inflammation is resolved, in part, by cell surface signaling molecules on leukocytes that have extracellular ligand binding domains linked to intracellular immune inhibitory domains. Among these are members of the siglec family that have ITIM and ITSM inhibitory sequences on their short intracellular domains that are actuated when they bind, with high specificity, to extracellular sialoglycans (siglec ligands) expressed on inflammatory target tissues. Project 3 is based on the hypothesis that endogenous human siglec ligands are multivalent sialoglycans produced by inflammatory target tissues including airway, skin and intestine. In terms of allergic inflammation, Siglec-8 is expressed on human eosinophils, whereas CD33 (Siglec-3), Siglec-6 and Siglec-8 are expressed by mast cells. When siglecs on these granulocytes encounter their sialoglycan ligands, inflammation is halted; engagement of Siglec-8 on eosinophils results in their apoptosis whereas engagement of siglecs on mast cells inhibits immune mediator release. We propose that identification of immune inhibitory sialoglycan structures and knowledge of how their expression is regulated in health and disease will provide new insights into the molecular basis of immune regulation and perhaps new targets for therapeutic intervention in human allergic inflammation.
Three aims are proposed to accomplish these goals.
Aim 1 is based on our finding that Siglec-8 ligands on human airway are robustly expressed in submucosal glands and ducts, and that these ligands are secreted onto the airway mucus layer.
This aim will compare Siglec-8 sialoglycan ligands in mucus secretions from inflamed and non-inflamed human airways, revealing quantitative as well as qualitative (protein carrier, glycan structure) information. Our studies on airway Siglec-8 ligands will be extended to other human tissues subject to allergic inflammation, using healthy and inflamed human skin and GI tract. Tissue distributions and molecular characteristics of Siglec-8 ligands will be compared among these tissues.
Aim 2 will explore the glycan structures required for optimal Siglec-8 engagement on eosinophils and mast cells. Based on our finding that specifically sulfated and sialylated keratan sulfate chains are responsible for Siglec-8 binding on human airways, we will use chemoenzymatic methods to generate modified glycan structures to test for binding requirements. We will create multivalent forms of these ligands to functionally engage Siglec-8 on human eosinophils and mast cells. The knowledge gained will be used to identify glycan biosynthetic enzymes that, when transfected into human submucosal gland cell lines, result in expression of Siglec-8 ligands. Concurrently, in Aim 3, we will apply the technologies we established to identify Siglec-8 ligands to the search for endogenous human ligands for CD33 and Siglec-6. Through these aims we anticipate significantly enhancing our understanding of glycans involved in regulating allergic inflammation and their expression in health and disease.
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