Durng infection of the urogenital tract with Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), bacteria encounter macrophages in the endometrium and other tissues. Although previous efforts have focused on defining immunologic responses to protein antigens, recent studies strongly suggest that when phagocytes encounter Chlamydia and the gonococcus, the subsequent cytokine response is due to the interactions of bacterial lipopolysaccharides (LPS) with macrophage receptors. The goal of this proposal is to analyze both the nature of these unique lipopolysaccharides and the mammalian receptors likely to be activated in response to bacterial invasion. The lipid A structures of CT and GC will be definitively determined. In addition, the structure of several lipid A species from serum resistant vs. serum sensitive phenotypes of N. gonorrhoeae will be analyzed to determine if distinct structures correlate with bacteremic phenotype. The biological characteristics of CT and GC LPS and lipid A will be analyzed to characterize the immunostimulatory effects of these endotoxins, and to determine if CT LPS or lipid A might actually inhibit cellular activation by GC. Efforts to characterize mammalian receptors for these endotoxins will focus on the leukocyte (beta2) integrins, which we have demonstrated to be LPS-signaling receptors. While most investigators of LPS receptors have focused upon CD14, which requires the presence of serum components for efficient binding to LPS, the mucosal environment of the urogenital tract is likely to be relatively serum-free. We propose to begin by defining the milieu of the endometrium by immunohistochemical approaches. Unlike CD14, heterologous expression of CD11c in Chinese hamster ovary fibroblasts imparts LPS-responsiveness in the complete absence of serum proteins. The characteristics of CD11c/CD18 mediated signaling will be determined, including the relationship of CD11c-mediated cellular activation and tyrosine kinase activity. Monoclonal and polyclonal Abs will be assessed for their ability to influence CD11c-signaling. The identification of the appropriate reagents will lead to studies designed to determine if CD11/CD18 function in native cells as LPS receptors. We will engineer several new transfected cell lines which express mutant CD11/18 receptors and wild-type CD11a/CD18 and CD11b/CD18 to determine the importance of the cytoplasmic domains in LPS signaling and whether other leukocyte integrins function as LPS receptors, respectively. A soluble form of recombinant CD11c/CD18 will be produced to determine if, like soluble CD14, this molecule enables LPS responses in endothelia. Finally, we will engineer a cell line in which CD11c and CD14 are co-expressed, to determine the influence of each LPS-binding protein on the other's function. These studies will help define the role of LPS in the pathogenesis of these two common sexually transmitted diseases.
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