Highly sensitive host responses to endotoxin (E) are needed for optimal defense against many species of invading Gram-negative bacteria (GNB) but can also lead to significant host pathology when exposure to endotoxin is not adequately controlled. Maximum sensitivity of E signaling requires the ordered interaction of E with several extracellular and cell surface host proteins, including: lipopolysaccharide (LPS)-binding protein (LBP), CD14, MD-2 and Toll-Like Receptor (TLR) 4. Recent observations, including our own, indicate an essential role for MD-2, linking E recognition initiated by LBP and CD14 to activation of TLR4 and determining, at least in certain settings (e.g. airway epithelium), cellular responsiveness to E. ? The long-term goals of this project are to better understand the molecular basis of innate immune recognition of E and of the coupling of E recognition to induction of pro-inflammatory responses. Our immediate focus will be the structure, function and expression of MD-2, including: 1) further characterization of the structural determinants of E-MD-2 interactions; 2) identification of molecular properties of E:MD-2 complexes required either for potent agonist or antagonist function; and 3) characterization of mechanism(s) regulating MD-2 expression in human airway epithelial cells. ? We will use metabolically labeled and chromatographically purified E and recombinant E-binding proteins (LBP, sCD14 and MD-2) to analyze protein-E interactions at low, patho-physiologically relevant E concentrations and to preparatively purify protein:E complexes for further structural and functional analyses. MD-2 expression will be monitored by mRNA and protein analyses of fresh tissue and primary cultures of human bronchial epithelium to identify mediators and mechanisms of regulation of MD-2 expression. ? These studies should yield novel insights concerning the structural determinants of host-E interactions important in innate immune recognition and response to invading GNB. A better understanding of the structure, function and expression of MD-2 should improve understanding of the regulation of host responsiveness to E and could pave the way for development of compounds that, by modulating cellular responses to E, may have prophylactic and/or therapeutic applications. ? ? ?
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