A leading cause of morbidity and mortality in critically ill patients is septic shock. The major factor causing septic shock is bacterial lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Preexposure to low concentrations of LPS leads to acquired resistance to LPS-induced lethaIity in vivo and cellular inflammatory responses in vitro. This phenomenon is known as LPS tolerance. LPS tolerance produces cross-tolerance to a variety of noxious stimuli and may constitute a form of heterologous desensitization. Altered macrophage function is a hallmark of LPS tolerance, but molecular mechanisms mediating cellular changes remain unknown. LPS tolerance is associated with decreases in guanine nucleotide regulatory (G) protein content and function in peritoneal macrophages from rats. These changes are accompanied by altered signal transduction pathways and a reorientation of LPS-stimulated macrophage inflammatory mediator production. The major hypothesis is that down- regulation of G protein content and function are essential cellular mechanisms of acquired LPS tolerance.
Specific aims proposed to test this hypothesis will: 1) determine the changes in G protein content and function in rat peritoneal macrophages and hepatic Kupffer cells during induction of and recovery from LPS tolerance; 2) examine molecular mechanisms for down-regulation of G proteins in tolerance through potential posttranslational modifications of G proteins, and/or alterations in G protein expression; 3) determine which macrophage second messenger systems are affected or unaffected by altered G protein function during tolerance; and 4) characterize and develop an in vitro model of LPS tolerance in the human monocytic cell line THP- 1. A final specific aim will assess the importance of down-regulation of specific G protein function on induction of LPS tolerance. The latter studies will employ inhibitors of specific G proteins to distinguish essential macrophage signal transduction pathways required for induction of LPS tolerance from pathways associated with macrophage activation. The goal is to identify molecular mechanisms by which LPS alters cellular signal transduction pathways to produce resistance to lethal LPS shock. These studies may provide new strategies and effective therapies for the prevention or treatment of sepsis.
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