Uncontrolled neutrophil adhesion and activation contributes to significantly to tissue damage in inflammatory diseases such as rheumatoid arthritis and vasculitis. Integrins are the major adhesive receptors on neutrophils that regulate cellular activation. Investigating the basic mechanisms of neutrophil integrin signaling is therefore critical to our understanding of the pathogenesis of inflammatory disease. This application is focused on the """"""""outside-in"""""""" integrin signaling pathway that induces neutrophil and macrophage activation. In previous work, we have demonstrated that outside-in integrin signaling is mediated by Src-family and Syk tyrosine kinases. In turn, these enzymes signal to SLP-76 leading to downstream activation of MAP kinases. The sequential involvement of Src-family kinases, Syk and SLP-76 is reminiscent of classical immunoreceptor signaling, such as B or T-cell receptor pathways, which depends on ITAM-containing adapter proteins. In preliminary data, we have found that neutrophils derived from DAP-12-/- FcRgamma-/- mice, which lack the two major ITAM adapters present in myeloid cells, are also completely defective in neutrophil integrin signaling. Based on these observations we hypothesize that leukocyte outside-in integrin signaling mimics classical immunoreceptor signaling pathways. We will test this hypothesis is a series of genetic and biochemical experiments. Using retroviral-mediated fetal liver hematopoietic stem cell transduction, we will introduce a series of mutant versions of Syk and DAP-12 into syk-/- or DAP-12-/- FcRgamma-/- mice that will test whether ITAM-Syk interactions are required for integrin signaling in primary neutrophils and macrophages. We will also test whether beta2 integrins form a complex with ITAM-containing DAP-12 and Syk, using biochemical methods and FRET-based microscopy techniques. If Syk and DAP12/FcRgamma are critical in outside-in integrin signaling, then deficiency of these molecules should result in equivalent defects in inflammatory processes in vivo. We will test this using two disease models that are known to be integrin dependent. If our hypothesis that integrin outside-in signaling mimics immunoreceptor pathways by use of ITAM molecules is validated, this will significantly change the view of integrin function in neutrophil-dependent inflammation. Therapeutic targeting of the integrin outside-in signaling pathway may provide novel classes of anti-inflammatory drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI065495-03
Application #
7196544
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Palker, Thomas J
Project Start
2005-07-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
3
Fiscal Year
2007
Total Cost
$359,123
Indirect Cost
Name
University of California San Francisco
Department
Pathology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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