CHARACTERIZATION OF THE LPS RECEPTOR FOR ACUTE PHASE

革兰氏阴性菌感染引起的脓毒症仍然是死亡的主要原因。全身性感染最早发生的事件之一是急性时相反应，其主要特征之一是血浆蛋白(急性时相蛋白，APP)浓度的变化。APP是一组在肝脏中产生的功能不同的蛋白质，通常被定义为那些在刺激后血浆浓度(正或负)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(TNFα、IL-1和IL-6)可诱导APP的表达。内毒素是革兰氏阴性菌外膜的一种成分，被认为是导致脓毒症致死性事件级联反应的主要细菌成分，它既刺激肿瘤坏死因子α、IL-1和IL-6的产生，又刺激APP的产生，因此有理由认为，内毒素诱导APP是由于巨噬细胞通过CD14-脂多糖受体刺激巨噬细胞分泌的细胞因子的次要作用。为了研究CD14在内毒素应答中的作用，我们最近建立了缺乏CD14-内毒素受体的小鼠。这些CD14缺陷小鼠很少或根本不会产生细胞因子，以应对非常高浓度的内毒素；然而，令人惊讶的是，它们有正常的APP反应。这些观察表明，小鼠有一种非CD14的内毒素受体，通过这种受体可以诱导APP的表达。此外，初步研究表明，CD14缺陷小鼠的肝细胞直接对内毒素产生反应，表明该受体位于肝细胞上。因此，我们建议(1)研究内毒素和脂蛋白A与肝细胞的结合特性；确定它们的结合是否特异和饱和；确定它们的结合常数；(2)利用蛋白质纯化和基因克隆等分子方法分离参与急性时相蛋白诱导的肝细胞非CD14LPS受体并进行生化鉴定，然后进行功能验证；(2)通过比较肝细胞通过该受体诱导的基因与内毒素通过单核细胞/巨噬细胞上的CD14受体诱导的基因，确定内毒素-APP受体发挥作用的分子机制(S)。这些研究不仅将澄清我们对内毒素诱导急性时相蛋白的机制的理解，而且也将加深我们对内毒素的多效性及其在脓毒症中的各种作用的理解。

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.