Cytoplasmic tyrosine kinases initiate intracellular signaling pathways that regulate myeloid leukocyte responses to inflammatory and infectious stimuli. We have studied the Src-family of tyrosine kinases using a panel of knockout mice that lack one or all Src-family members (Hck, Fgr, and Lyn) normally expressed in myeloid cells. Initial work has revealed that the primary functional role of these kinases is regulating the integrin signaling pathway leading to leukocyte adhesion and activation. Neutrophils lacking Hck and Fgr fail to undergo cell spreading, respiratory burst or granule exocytosis following plating on surfaces which crosslink beta2 or beta3 integrins. Similarly, macrophages from these mice manifest alterations in cytoskeletal structure, cell spreading and migration. This integrin signaling pathway involves the adapter protein c-Cbl and the lipid kinase PI-3 kinase. Using Syk-deficient leukocytes generated in bone marrow chimeras with syk-/- fetal liver cells, we have found that this kinase is also critically involved in leukocyte integrin signaling. Defects in myeloid cell function result in altered immune responses in knockout mice in vivo; hck-/- fgr-/- mutants suffer reduced tissue damage during endotoxemia and blunted development of inflammatory disease when crossed with motheaten mice. To extend these studies, we propose to examine downstream molecules in the integrin signaling pathway using leukocytes from Src- family or Syk-deficient animals. We will focus on the Rho-family GTPases, which regulate actin cytoskeletal dynamics, using both biochemical and genetic approaches. Biochemical approaches will include direct study of the activation state of these GTPases (and their effectors) using binding or enzymatic assays. Genetic approaches will be carried out by transducing macrophages with retroviral vectors encoding signaling molecules that are predicted to rescue the adhesion/migration phenotype of hck-/- fgr-/- lyn-/- or syk-/- cells. Our goal is to compare and contrast the effects of Src-family versus Syk kinase deficiency on downstream signaling responses. We will study the effects of these mutations in vivo using a series of skin inflammation models, all performed in mixed chimeric mice to allow comparison of mutant and wild type leukocytes in the same animal. We will also test the hypothesis that the hematopoietic phenotype seen in lyn-/- mice may be secondary to impaired integrin-mediated adhesion of stem cells. These studies will expand our understanding of the roles of these kinases in hematopoietic cells and will validate whether Src-family or Syk kinases may be potential targets for therapeutics against inflammatory diseases.
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