Uncontrolled development of neutrophil adhesion and activation contributes to tissue damage in inflammatory diseases, such as occurs in the lung during TRALI reactions. Integrins are the major receptors on neutrophils that regulate adhesion and cellular activation. Investigating the basic mechanisms of neutrophil integrin signaling is therefore critical to our understanding of the pathogenesis of TRALI. This application is focused on the """"""""outside-in"""""""" integrin signaling that induces neutrophil and macrophage activation, hi previous work, we have demonstrated that outside-in integrin signaling is mediated by Srcfamily 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, 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-/-FcR-/- mice, which lack the two major IT AM 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-/- orDAP-12-/-FcRy-/- mice that will test whether ITAM-Syk interactions are required for integrin signaling in primary neutrophils and macrophages. We will also test whether (52 integrins form a complex with ITAM-containing DAP-12 and Syk, using biochemical methods and FRET-based microscopy techniques. We will also test whether activation of integrin-signaling pathways is a major feature of murine or human TRALI using intracellular staining/flow cytometry methods. These later experiments will involve collaborations with other projects in this SCCOR application. If our hypothesis that integrin outside-in signaling mimics immunoreceptor pathways by use of IT AM molecules is validated, this will significantly change the view of integrin function in neutrophildependent inflammation. Therapeutic targeting of the integrin outside-in signaling pathway may provide novel classes of anti-inflammatory drugs.
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