CHARACTERIZATION OF THE LPS RECEPTOR FOR ACUTE PHASE

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Sepsis due to Gram-negative infection remains a major cause of mortality. One of the earliest events occurring in a systemic infection is the acute phase response which has, as one of its major hallmarks, alteration of the concentration of plasma proteins (acute phase proteins, APP). APP are a set of functionally diverse proteins produced in the liver and defined in general as those proteins which show changes in plasma concentration (positive or negative) of 25 percent or more following the stimulus. APP are thought to increase host defenses as well as to control inflammation. There is a large body of evidence showing that cytokines (TNFalpha, IL-1 and IL-6) can induce the expression of APP. Since lipopolysaccharide (LPS, endotoxin), a component of the outer membrane of Gram-negative bacteria which is thought to be the major bacterial component of Gram-negative bacteria responsible for inducing the cascade of events leading to lethality in sepsis, stimulates both the production of TNFalpha, IL-1 and IL-6 as well as the production of APP, it has been reasonable to assume that the LPS induction of APP results from a secondary effect of cytokines secreted by macrophages when LPS stimulates them through the CD14-LPS receptor. To study the role of CD14 in the response to LPS, we have recently produced mice which lack the CD14-LPS receptor. These CD14-deficient mice produce little or no cytokines in response to very high concentrations of LPS; surprisingly however, they have a normal APP response. These observations indicate that mice have a non-CD14 receptor for LPS through which expression of APP is induced. Furthermore, as shown in Preliminary Studies, hepatocytes from CD14-deficient mice respond directly to LPS, indicating that this receptor is on hepatocytes. Accordingly, we propose to (1) study the binding characteristics of LPS and Lipid A to hepatocytes; determine whether their binding is specific and saturable; determine their binding constants (2) isolate and biochemically characterize the hepatocyte non-CD14 LPS receptor involved in the induction of the acute phase proteins using molecular methods of protein purification and gene cloning followed by functional verification and (2) determine the molecular mechanism(s) by which the LPS-APP receptor functions by comparing the genes induced via this receptor in hepatocytes to those induced by LPS via the CD14 receptor on monocytes/macrophages. These studies will not only clarify our understanding of the mechanisms involved in the induction of acute phase proteins by LPS, but will also increase our understanding of the pleiotropic effects of LPS and its various roles in sepsis.