Inflammation is a complex process involving myeloid cell interaction with both endothelial cell and extracellular matrix elements. Integrins are known to play a crucial role in the inflammatory process. Integrins are cell-surface heterodimeric glycoproteins that mediate cell-cell and cell-matrix adhesion. Beta-1 integrins have recently been identified on human neutrophils. Their function remains unclear.
The aim of this grant request is to better delineate the role of beta-1 integrins on human neutrophils, specifically their intracellular signaling mechanisms. This will be evaluated by examining likely second messengers as well as post-translational modification of the receptor. The ultimate aim of this grant request will increase understanding of the mechanisms that mediate beta-1 integrin expression and function and their role in integrin-mediated PMN adhesion. Likely intracellular signaling mechanisms for beta-1 integrin regulation on granulocytes include intracellular calcium, protein kinase C, and tyrosine kinases. The effect of altering intracellular calcium in human PMNs will be studied by adhesion, functionality assays, and flow cytometric analysis. The role of protein kinase C isotypes in integrin expression will be identified in neutrophils, and PKC knockout models designed using transfection of antisense cDNA oligonucleotides. The beta-1 integrins involved with binding to endothelium will be identified and evaluated for changes during migration through an extracellular matrix. An in vitro model of the endothelium/extracellular matrix will be designed and the effect of blocking antibodies to specific beta-1 integrin isoforms studied. Also, the role of alterations in glycosylation of beta-1 integrin expression on migrating neutrophils will be evaluated. Studies include alteration in total number of surface beta-1 integrins, redistribution on the cell surface and its association with cytoskeletal rearrangement, and clustering of beta-1 integrins. Lastly, phenotypic alterations of the beta-1 integrin structure will be evaluated by confocal fluorescent microscopy, flow cytometry, and analysis of actin polymerization. The results of these studies will provide insight into potential mechanisms by which PMNs adhere to and migrate through the extracellular matrix. Understanding the nature of these interactions may suggest strategies to enhance PMN function or to limit PMN-mediated tissue damage.
