Neutrophil localization to the lung to the pulmonary capillary and migration in the lung parenchyma are central events in the genesis of the adult respiratory distress syndrome (ARDS). In the previous grant period, we have promulgated the hypothesis that the viscoelastic properties of neutrophils in response to lipopolysaccharide (LPS) and other stimuli circulating in patients with ARDS accounts, at least in part, for the neutrophil localization in the pulmonary capillary. We now hypothesize that LPS and LPS-binding protein (LBP) interact with CD14, a glycosyl-phosphatidyl inositol linked surface glycoprotein results in activation of a src-family cytoplasmic tyrosine kinase, which results in the activation of MAP kinase and cytoskeletal assembly through involvement of the rho family of small molecular weigh GTP-binding proteins. In the endothelial cell, we postulate a similar sequence of events (although the transduction) mechanisms more clearly involve protein kinase C as an important pathway) which leads to the induction of IL-8 gene expression through the involvement of the NF-kB class of transcriptional regulators. These hypotheses will be tested in human neutrophils and human endothelial cells in vitro. The tyrosine kinases involved in neutrophil activation will be sought through a co- immunoprecipitation approach as will the identity of a putative transmembrane transducer which is hypothesized to interact with src-type tyrosine kinases. The potential role of other phosphorylated products will be linked to activation of low molecular weight G proteins by expression of rho A and other members of this family in Sf9 cells and their addition or microinjection into neutrophils and neutrophil-like cell lines. The transduction mechanisms involving activation both tyrosine kinases and PKC will be characterized in endothelial cells as will the presence of the putative transmembrane transducer. The IL-8 promoter region will be cloned and reporter gene studies carried out. Finally, DNAse I hypersensitivity will be used to confirm the precise sites of protein-DNA interaction in induction of IL-8 gene expression. These studies will elucidate mechanisms by which LPS induces cytoskeletal assembly in neutrophils and new gene expression in endothelial cells, which we suggest contribute to lung injury induced by sepsis in humans.
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