MOLECULAR REGULATION OF MACROPHAGE CYTOCIDAL ACTIVITY

巨噬细胞杀细胞活性的分子调控

• 批准号: 3184937
• 负责人: ROBERT DAVID SCHREIBER
• 金额: $ 26.73万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3184937
• 关键词: cell cell interaction; cytokine receptors; electron microscopy; genetic manipulation; glycoproteins; human subject; immunoprecipitation; interferons; leukocyte activation /transformation; macrophage; membrane proteins; molecular cloning; phosphorylation; protein biosynthesis; protein structure function; receptor binding; receptor expression; tissue /cell culture

Activated macrophages play a pivotal role in promoting host defense and pathologic processes. Based on work from several laboratories, including my own, we now know that interferon-gamma (IFN gamma) is one of the major cytokines responsible for effecting macrophage activation both in vitro and in vivo. For the past 7 years, my laboratory has focused its efforts on elucidating IFN gamma's mechanism of action at the molecular level in vitro and in defining the physiologic role of IFN gamma-activated macrophages in vivo. One of our major contributions to this field has been the identification and characterization of the IFN gamma receptor of human and murine mono-nuclear phagocytes. During the past granting period we purified the human IFN gamma receptor, produced receptor-specific monoclonal antibodies, and cloned the human and murine IFN gamma receptor cDNAs. We now wish to use these unique reagents to define the structure of the functionally active human IFN gamma receptor and to elucidate its role in regulating the activity of macrophages and other cells. To achieve this goal, we intend to pursue three specific aims. First, we will continue to study the structure and cell biology of the 90 kDa glycoprotein (gp90) that is the universal IFN gamma binding protein on cell surfaces. Using biosynthetic labelling, immunoprecipitation, and electron microscopic techniques, we will elucidate the receptor's post-translational processing and define its life cycle and intracellular trafficking through cells. Second, we intend to pursue our unique observation that ligand induces the phosphorylation of gp90. We plan to characterize the specificity, kinetics, and reversibility of this reaction at the molecular level and determine its biologic consequences. We will put special emphasis on examining whether receptor phosphorylation accounts for the well documented but poorly understood desensitization of macrophages effected by ligand, immune complexes, and other biologic particles. Third, we will use state of the art molecular biology techniques to obtain high level expression of human gp90 in murine cells and investigate whether these cells become responsive to human IFN gamma. The latter experiments will also examine whether accessory polypeptides are needed to form a functionally active receptor. These studies should help to better define the molecular basis of macrophage activation and provide new insights into the roles of IFN gamma and activated macrophages in promoting physiologically important immune responses.

活化的巨噬细胞在促进宿主防御和 病理过程。基于几个实验室的工作，包括 我自己，我们现在知道干扰素-伽马(干扰素-伽马)是主要的 影响体外和体外巨噬细胞活化的细胞因子 在活体内。在过去的7年里，我的实验室一直致力于 从分子水平阐明干扰素-γ的体外作用机制 以及在确定干扰素-γ激活的巨噬细胞在 活着。我们对这一领域的主要贡献之一是 人和人干扰素-γ受体的鉴定与鉴定 小鼠单核吞噬细胞。在过去的授权期内，我们 提纯人干扰素-γ受体，产生受体特异性 并克隆了人和小鼠干扰素-γ受体 CDNA。我们现在希望使用这些独特的试剂来定义 功能性人干扰素-γ受体及其作用机制的研究 调节巨噬细胞和其他细胞的活动。要做到这一点 为了实现这一目标，我们打算追求三个具体目标。第一，我们将继续 90 kDa糖蛋白(Gp90)的结构和细胞生物学研究 是细胞表面普遍存在的干扰素-γ结合蛋白。vbl.使用 生物合成标记、免疫沉淀和电子显微镜 技术，我们将阐明受体的翻译后处理 并定义其生命周期和通过细胞的细胞内运输。 其次，我们打算继续我们独特的观察，即配体诱导 Gp90的磷酸化。我们计划描述这一特性， 该反应在分子水平上的动力学和可逆性 确定其生物学后果。我们将特别强调 检查受体磷酸化是否解释了已有文献记载的 但对配体对巨噬细胞的脱敏作用知之甚少， 免疫复合体和其他生物颗粒。第三，我们将使用状态 最先进的分子生物学技术获得高水平表达 人gp90在小鼠细胞中的表达，并研究这些细胞是否会 对人干扰素-伽马有反应。后一项实验也将检验 是否需要辅助性多肽来形成功能活性 受体。这些研究应该有助于更好地定义分子基础 巨噬细胞活化，并为干扰素的作用提供了新的见解 γ-和活化巨噬细胞在促进生理学上的重要作用 免疫反应。