CHARACTERIZATION OF THE LPS RECEPTOR FOR ACUTE PHASE

急性期 LPS 受体的特征

• 批准号: 6194383
• 负责人: Sanna M Goyert
• 金额: $ 18.16万
• 依托单位: NORTH SHORE UNIVERSITY HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6194383
• 关键词: CD14 molecule; acute phase protein; antibody formation; antigen receptors; cytokine; gene induction /repression; genetically modified animals; hamsters; laboratory mouse; laboratory rabbit; laboratory rat; lipopolysaccharides; liver cells; macrophage; monoclonal antibody; monocyte; receptor binding

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.

革兰氏阴性菌感染引起的败血症仍然是死亡的主要原因。 全身感染中发生的最早期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变化（阳性或阴性）为25%或更多的蛋白质。APP被认为可以增加宿主的防御能力以及控制炎症。 有大量证据表明，细胞因子TNF α、IL-1和IL-6可诱导APP的表达。内毒素（LPS，内毒素）是革兰氏阴性菌外膜的组分，被认为是革兰氏阴性菌的主要细菌组分，负责诱导导致败血症致死性的级联反应，它刺激TNF α的产生，IL-1和IL-6以及APP的产生，已经合理地假设当LPS通过CD 14-LPS受体刺激巨噬细胞时，LPS诱导APP是由巨噬细胞分泌的细胞因子的次级效应引起的。 为了研究CD 14在LPS应答中的作用，我们最近制备了缺乏CD 14-LPS受体的小鼠。这些CD 14缺陷型小鼠对非常高浓度的LPS产生很少或不产生细胞因子;然而，令人惊讶的是，它们具有正常的APP反应。 这些观察结果表明，小鼠具有LPS的非CD 14受体，通过该受体诱导APP的表达。 此外，如初步研究所示，CD 14缺陷小鼠的肝细胞直接对LPS产生反应，表明该受体位于肝细胞上。因此，我们建议（1）研究LPS和Lipid A与肝细胞的结合特征;确定它们的结合是否具有特异性和饱和性;确定它们的结合常数（2）使用蛋白纯化和基因克隆的分子方法分离并生化表征参与诱导急性期蛋白的肝细胞非CD 14 LPS受体，随后进行功能验证，和（2）通过比较肝细胞中通过LPS-APP受体诱导的基因与LPS通过单核细胞/巨噬细胞上的CD 14受体诱导的基因，确定LPS-APP受体发挥功能的分子机制。 这些研究不仅将阐明我们对LPS诱导急性时相蛋白的机制的理解，而且还将增加我们对LPS的多效性效应及其在脓毒症中的各种作用的理解。