POSTTRAUMATIC SEPSIS--REGULATION OF LPS-BINDING PROTEIN

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Gram (-) sepsis remains a major cause of morbidity and mortality following trauma. We hypothesize that gram (-)sepsis is initiated when lipopolysaccharide (LPS) is bound by a protein synthesized by hepatocytes known as LPS binding protein (LBP). We also propose that the resultant LBP-LPS complex binds to receptors on macrophages which then, depending on the receptor type, the complex either activates the macrophage to secrete cytokines (CD14 receptor) or facilitates the uptake and degradation of LPS (acetylated-LDL receptor). Since the liver Kupffer cells are already known to be the principal site of circulating LPS uptake, we propose that LBP released by hepatocytes promote the uptake of LPS by the adjacent Kupffer cell. It is likely that cytokines released by the Kupffer cells then further upregulates hepatocyte LBP synthesis creating an important feedback loop and function of LBP in the liver. AIM I: TO DETERMINE THE EXTRACELLULAR AND INTRACELLULAR REGULATION OF LPS BINDING PROTEIN IN HEPATOCYTES. We will begin by defining the external signals (e.g., cytokines, glucocorticoids, etc.) which regulate hepatocyte LBP synthesis in vitro. We predict that LBP will respond in a similar manner to several well-described acute phase reactants. Next, we will determine the role of Kupffer cells in providing these signals both in vitro and in vivo. In addition to our studies on the extracellular signals, we will study the intracellular mechanisms of LBP regulation. We will determine if the synthesis of LBP is transcriptionally regulated. If so, we will isolate the LBP gene promoter, which will allow us to study and compare, in detail, LBP regulation with other secreted hepatocyte proteins that are upregulated in sepsis (acute phase reactants). AIM II: TO DETERMINE THE FUNCTIONAL ROLE OF LPS BINDING PROTEIN IN THE INTERACTION OF LPS WITH CD14 AND THE ACETYLATED-LDL RECEPTOR ON LIVER CELLS. To study the role of LBP in the processing of LPS by the liver, we will first need to examine the expression of the receptors for LPS-LBP complexes. Therefore, we will define the expression of CD14 and acetylated-LDL receptors on liver cells both in the resting state and under septic and inflammatory conditions. If receptor expression is regulated in vivo, we will study the role of LBP and other hepatocyte-derived factors in this regulation. Finally, we will determine the role of LBP in the uptake of LPS by these receptors in isolated Kupffer cells and the whole organ. We will determine the functional consequence (activation vs no activation) of LBP-LPS binding to Kupffer cells. At the completion of our studies we will have characterized the regulation of LBP synthesis and the functional role of LBP in LPS clearance within the liver. This information will provide important insights into the mechanisms of host-LPS interaction and the induction of the septic response.