Exposure to lipopolysaccharide (LPS) induces an inflammatory reaction in the lung mediated by alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF, IL-1 and IL-6. Asthmatic subjects are hyperresponsive to the inflammatory and bronchospastic effects of inhaled LPS, and this hyperresponsiveness is further heightened following allergen exposure. The inflammatory response to LPS involves de novo gene expression. Therefore, elucidating the mechanisms that regulate LPS-mediated gene transcription in alveolar macrophages is crucial for understanding the clinical consequences of environmental exposure to LPS in asthma. A major advance in our understanding of LPS-mediated inflammation was the discovery by us and others that the TLR4 gene encodes the LPS receptor and transduces the effect of LPS stimulation. We have also shown that LPS stimulated IL-8 expression in alveolar macrophages involves activation of the small GTPase RhoA with subsequent activation of the transcription factor NF-kappaB. The primary hypothesis to be tested is that LPS leads to activation of pro-inflammatory gene expression in alveolar macrophages through signaling cascades that critically require TLR4 and the small GTPase RhoA. Three broad approaches will be utilized in this project. First, we have developed a compelling body of preliminary data suggesting that LPS-induced NF-kappaB activation and IL-8 expression in alveolar macrophages requires Rho GTPase activity. This project will therefore elucidate the molecular details of the role of the Rho family of GTPases in LPS-induced NF-kappaB activation and IL-8 expression in alveolar macrophages. Second, our preliminary results suggest that the signaling pathways initiated by LPS may require activation of Rho-kinase. We will, therefore, confirm and extend these results by using several complementary approaches to establish the role of Rho-kinase pathways as well as define their relationship to RhoA and to the possible downstream signaling molecule (MEKK1) that may be responsible for phosphorylation of IkappaB and IL-8 gene expression. Third, published data have shown that LPS causes airway inflammation at a significantly lower dose in asthmatic subjects than in normal controls. The molecular mechanisms that mediate the different responses to LPS are unknown. We will, therefore, analyze the potential role of TLR4 polymorphisms in mediating the variability of responses to LPS seen among normal subjects and between asthmatic and normal subjects.
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