CHARACTERIZATION OF THE LPS RECEPTOR FOR ACUTE PHASE

由革兰氏阴性感染引起的败血症仍然是死亡的主要原因。在全身性感染中最早发生的事件之一是急性期反应，其主要标志之一是血浆蛋白（急性期蛋白，APP）浓度的改变。APP是肝脏中产生的一组功能多样的蛋白质，一般定义为在刺激后血浆浓度（阳性或阴性）变化25%或更多的蛋白质。APP被认为可以增强宿主的防御能力并控制炎症。大量证据表明，细胞因子（TNFalpha， IL-1和IL-6）可以诱导APP的表达。由于脂多糖（LPS，内毒素）是革兰氏阴性菌外膜的一种成分，被认为是革兰氏阴性菌的主要细菌成分，负责诱导一系列导致败血症致死的事件，它既刺激TNFalpha， IL-1和IL-6的产生，也刺激APP的产生，我们有理由认为，LPS诱导APP是巨噬细胞通过CD14-LPS受体刺激巨噬细胞时所分泌的细胞因子的二次效应。为了研究CD14在LPS应答中的作用，我们最近制造了缺乏CD14-LPS受体的小鼠。这些cd14缺陷小鼠对高浓度LPS的反应很少或不产生细胞因子；然而，令人惊讶的是，它们的APP反应正常。这些观察结果表明，小鼠具有LPS的非cd14受体，通过该受体诱导APP的表达。此外，初步研究显示，来自cd14缺陷小鼠的肝细胞直接对LPS产生反应，表明该受体在肝细胞上。因此，我们建议：(1)研究脂多糖和脂质A与肝细胞的结合特性；确定它们的结合是否具有特异性和可饱和性；确定它们的结合常量(2)利用蛋白质纯化和基因克隆的分子方法分离和生化表征参与急性期蛋白诱导的肝细胞非CD14 LPS受体，然后进行功能验证；(2)通过比较LPS- app受体在肝细胞中诱导的基因与LPS通过CD14受体在单核/巨噬细胞上诱导的基因，确定LPS- app受体的分子机制。这些研究不仅将阐明LPS诱导急性期蛋白的机制，而且将增加我们对LPS的多效作用及其在脓毒症中的各种作用的认识。

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.