Exposure to lipopolysaccharide (LPS) induces an inflammatory reaction in the lung mediated primarily by blood monocytes and alveolar macrophages, which release an array of inflammatory cytokines including IL-8, TNF, IL-1 and IL-6. Interestingly, the ability of the host to respond to LPS is highly variable. Differences between individuals have been reported in the release and synthesis of cytokines by human monocytes stimulated with LPS. Therefore, elucidating the mechanisms that regulate LPS-mediated gene transcription in alveolar macrophages is crucial for understanding the pathogenesis of lung inflammatory disease and the interactions between lung innate immunity and inflammatory responses. We?ve also demonstrated that LPS stimulated IL-8 expression in alveolar macrophges involves activation of the PI3K, Rho family of small G-proteins, with subsequent activation of NF- B. The primary hypothesis to be tested is that the observed clinical variability in LPS-induced lung inflammation is directly related to differences in LPS-stimulated gene transcription in alveolar macrophages. We further propose that these differences are determined by the signaling cascade linking TLR4 to activation of NF- B. Three broad approaches will be utilized in this proposal. First, we have developed a compelling body of preliminary data suggesting that LPS-induced NF- B activation and IL-8 expression in alveolar macrophages requires Rho GTPase activity. This proposal will therefore elucidate the molecular details of the role of the Rho family of GTPases in LPS-induced NF- B activation and IL-8 expression in alveolar macrophages. Second, our preliminary results showed that inhibitors of PI3K blocked activation of Rho and NF- B induced by LPS and preliminary data also demonstrated that LPS-stimulated MEKK1 activity was blocked by inhibition of RhoA GTPase. We will, therefore, confirm and extend these results by using several complementary approaches to establish the role of PI3K pathway in RhoA activity as well as define their relationship to RhoA and to the possible downstream signaling molecule (MEKK1) that may be responsible for phosphorylation of I B and IL-8 gene expression. Third, A major advance in our understanding of LPS-mediated inflammation was the discovery that the Toll-like receptor 4 gene (TLR4) encodes the LPS receptor and transduces the effect of LPS stimulation. We propose to determine whether mutations in the TLR4 can account for some of differences in LPS-stimulated signaling linked to activation of NF- B.
