Identification of cell surface molecules responsible for LPS recognition, signal transduction, and cellular activation is important to understanding the pathophysiology of septic shock. CD14 is a cell surface lipopolysaccharide (LPS) binding glycoprotein involved in LPS mediated signal transduction; it also exists as a soluble plasma protein which potentiates cellular responses to LPS. The primary hypothesis of this proposal is that CD14 is of central importance in determining the responses of myeloid and nonmyeloid (e.g., epithelial) cells to LPS. The specific objective is to identify the tissues, cells, and signals that regulate CD14 expression and antigen processing in vivo. Mice will be treated with LPS or other agonists and their effect on CD14 mRNA and protein production evaluated. To measure CD14 mRNA expression, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays will be developed; these will be complemented by in situ hybridization to identify responsible cell specific phenotypes. A combination of ELISA, SDS PAGE, Western blot, immunochemistry, and Triton X 114 phase separation techniques will be developed for analysis of membrane bound and soluble CD14 protein in plasma and tissues. In separate studies, neutralization of TNFa, IL 1, and IL 6, and experiments involving the LPS hyporesponsive mouse strain C3H/HeJ will be performed to determine the role of these mediators in cell specific induction of CD14 by LPS. SDS PAGE and phase separation studies will characterize the proportion of membrane bound versus soluble CD14 in tissues under various conditions. These analyses of the biosynthesis and processing of CD14 should elucidate the origin of plasma CD14.
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