Active inflammatory conditions in the gastrointestinal tract are characterized by migration of neutrophils across epithelial cells. Previous studies demonstrated that CD47 plays a central role in modulating neutrophil transepithelial migration. This proposal aims to characterize the molecular mechanism(s) by which CD47 modulates neutrophil transepithelial migration. CD47 is a universally expressed cell surface glycoprotein which contains an extracellular domain, three- or five-transmembrane segments and an alternatively spliced intracellular tail. The extracellular domain of CD47 structurally belongs to the immunoglobulin superfamily (IgV type) and is potentially involved in protein homophilic/heterophilic interactions. Function of the CD47 extracellular IgV domain will be investigated by applying site- directed mutagenesis and deletion mutations. The functional inhibitory mAb binding site(s) on this domain will be identified by transfecting mutants in mammalian cells and by surface labeling experiments. Neutrophil transmigration across such transfected epithelial monolayers will be determined. CD47 basolateral sorting signals will be identified in polarized epithelial cells. The topology and tertiary structure of CD47 will be examined. Four CD47 isoforms which differ from each other only at the C-terminal intracellular domain will be individually expressed in epithelial cells and their regulation of neutrophil migration will be investigated. Such studies will provide insight into the molecular basis of neutrophil-epithelial interactions and may lead to new therapeutic intervention in the treatment of acute inflammatory diseases of mucosal surfaces.
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