Abstract

Active inflammatory conditions in the gastrointestinal tract are characterized by migration of neutrophils (PMN) across epithelial mucosal surfaces. Previous studies suggested a central role of CD47 in modulating PMN migration across intestinal epithelium and demonstrated that CD47 on both PMN and epithelial cells acts to enhance PMN transmigration. The goal of this proposal is to further characterize the molecular mechanism(s) by which CD47 regulates PMN transepithelial migration.
The specific aims i n this proposal are 1) to investigate the role of tyrosine phosphorylation in CD47-mediated PMN transmigration; 2) to determine the subcellular localization of CD47 in PMN and analyze its redistribution after chemoattractant stimulation; 3) to investigate the role of epithelial CD47 in PMN transmigration. In this proposal, CD47- mediated tyrosine phosphorylation in PMN will be analyzed by applying specific tyrosine kinase inhibitors in PMN transmigration assays to examine their ability of reversal anti-CD47 antibody inhibition in transmigration. The specific phospho-protein(s) down stream of CD47 during fMLP stimulation will be identified and characterized by 2D-PAGE, phospho-immunoblotting, and proteomics. Subcellular fractionation by sucrose and percoll gradients will be performed to identify the subcellular localization of intracellular pools of CD47 and compared with the localization of SIRPa (CD47 ligand) and CD11bCD18 in PMN before and after fMLP stimulation. Effect of PI3-kinase inhibition in re-distribution of these proteins will also be investigated. Finally, epithelial cell surface CD47 in PMN migration will be studied using T84 epithelial cells and CD47-transfected CaCO2 cells. The potential functionally important residues (or domains) on CD47 extracellular and intracellular domains will be studied by mutagenesis and expression on CaCO2. Such studies will provide insights into the molecular basis of PMN transepithelial chemotaxis which may lead to new therapeutic inventions in the treatment of acute inflammatory diseases of mucosal surfaces.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01DK062894-01
Application #
6559668
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Podskalny, Judith M,
Project Start
2003-03-01
Project End
2005-11-30
Budget Start
2003-03-01
Budget End
2003-11-30
Support Year
1
Fiscal Year
2003
Total Cost
$118,660
Indirect Cost

  Institution

Name
Emory University
Department
Pathology
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322

  Publications

Zen, Ke; Guo, Yalan; Bian, Zhen et al. (2013) Inflammation-induced proteolytic processing of the SIRP? cytoplasmic ITIM in neutrophils propagates a proinflammatory state. Nat Commun 4:2436
Bian, Zhen; Guo, YaLan; Ha, Binh et al. (2012) Regulation of the inflammatory response: enhancing neutrophil infiltration under chronic inflammatory conditions. J Immunol 188:844-53
Zen, Ke; Liu, Yuan (2008) Role of different protein tyrosine kinases in fMLP-induced neutrophil transmigration. Immunobiology 213:13-23
Liu, Yuan; Tong, Qiao; Zhou, Yubin et al. (2007) Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47. J Mol Biol 365:680-93
Zen, Ke; Chen, Celia X-J; Chen, Yi-Tien et al. (2007) Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium. J Immunol 178:2483-90
Zen, Ke; Reaves, Titus A; Soto, Ileana et al. (2006) Response to genistein: assaying the activation status and chemotaxis efficacy of isolated neutrophils. J Immunol Methods 309:86-98
Zen, Ke; Liu, Yuan; McCall, Ingrid C et al. (2005) Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils. Mol Biol Cell 16:2694-703
Liu, Yuan; Soto, Ileana; Tong, Qiao et al. (2005) SIRPbeta1 is expressed as a disulfide-linked homodimer in leukocytes and positively regulates neutrophil transepithelial migration. J Biol Chem 280:36132-40
Liu, Yuan; O'Connor, Miriam B; Mandell, Kenneth J et al. (2004) Peptide-mediated inhibition of neutrophil transmigration by blocking CD47 interactions with signal regulatory protein alpha. J Immunol 172:2578-85
Liu, Yuan; Shaw, Sunil K; Ma, Shuo et al. (2004) Regulation of leukocyte transmigration: cell surface interactions and signaling events. J Immunol 172:7-13

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