ROLE OF LFA-1/MAC-1/P150,95 IN PMN LEUKOCYTE ADHERENCE

LFA-1/MAC-1/P150,95 在 PMN 白细胞粘附中的作用

• 批准号: 3135749
• 负责人: Donald C. Anderson
• 金额: $ 14.1万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3135749
• 关键词: cell adhesion; cell bank /registry; cell migration; density gradient ultracentrifugation; human tissue; immunochemistry; inborn lysosomal enzyme disorder; inborn metabolism disorder; membrane activity; molecular weight; monoclonal antibody; neutrophil; phase contrast microscopy; radiotracer; scanning electron microscopy; vascular endothelium

Understanding of polymorphonuclear leukocyte (PMNL) function has been greatly aided by the recognition of heritable abnormalities or absence of PMNL proteins. In this proposal, the discovery of eight patients (4 males and four females) with an absence of a high molecular weight glycoprotein family (LFA-1/Mac-1/p150,95) on both the cell surface and an intracellular pool are utilized to understand the molecular mechanism by which these proteins mediate cell adherence phenomena. Human endothelial cell monolayers will be examined for adherence, spreading, and penetration by normal and LFA-1/Mac-1/p150-95 deficient PMNL under resting conditions and after stimulation with chemotactic factors including F-Met-Leu-Phe. Three-dimensional type I collagen matrices will be investigated for adherence and invasion by normal and deficient PMNL under similar conditions to test the hypothesis that PMNL adherence is important for movement on endothelial cell surfaces, but is less important for penetration of collagen matrices. Direct observation with interference contrast microscopy as well as scanning and transmission electron microscopy will be used to document the adherence and invasion of the PMNL. The role of LFA-1/Mac-1/p150,95 in the modulation of PMNL adherence dependent function will be studied by determining the intracellular locations of LFA-1/Mac-1/p150,95 and certain related intermediates by a unique, quantitative immunoblotting assay utilizing monoclonal antibodies against LFA-1/Mac-1/p150,95 and the common Beta subunit. Subcellular fractionation will be accomplished on Percoll density gradients after nitrogen cavitation of the PMNL. Cell surface protease treatment, metabolic labeling with [3H]-galactosamine and [3H]-glucosamine and surface labeling with NaB3H4 and [125I] will be employed concurrently with the immunoblotting assay to determine whether cycling of LFA-1/Mac-1/p150,95 takes place. These studies will be complemented by detection of LFA-1/Mac-1/p150,95 in coated pits or vesicles by electron microscopy experiments utilizing colloidal gold or ferritin labeling experiments. Comparative assessments of surface Mac-1 subunit expression (flow cytofluoragraphy) and cell adherence properties will be performed employing chemotactic or secretory conditions designed to "up regulate" or "down regulate" Mac-1 proteins. These studies should provide new information relevant to the role of Mac-1 dependent PMNL adherence reactions in inflammation and host defense.

对多形核白细胞（PMNL）功能的理解一直是 这在很大程度上得益于对遗传性异常或缺乏 PMNL蛋白。 在这项提案中，发现了8名患者（4名男性 和4只雌性），缺乏高分子量糖蛋白 LFA-1/Mac-1/p150，95）在细胞表面和细胞内表达。 池被用来了解这些分子机制， 蛋白质介导细胞粘附现象。 人内皮细胞 将通过以下方法检查单层细胞的粘附、铺展和渗透性： 静息状态下正常和LFA-1/Mac-1/p150-95缺陷型PMNL， 用包括F-Met-Leu-Phe的趋化因子刺激后。 将研究三维I型胶原基质， 正常和缺陷性PMNL在类似条件下的粘附和侵袭 条件来检验PMNL依从性对以下假设的重要性： 在内皮细胞表面的运动，但不太重要， 胶原基质的渗透。 干扰直接观测 对比显微镜以及扫描和透射电子显微镜 显微镜将用于记录粘附和侵入的 PMNL。 LFA-1/Mac-1/p150，95在PMNL粘附调节中的作用 依赖性功能将通过测定细胞内 LFA-1/Mac-1/p150，95和某些相关中间体的位置 利用单克隆抗体的独特的定量免疫印迹分析 针对LFA-1/Mac-1/p150，95和共同的β亚基。 亚细胞 分馏将在Percoll密度梯度上完成， PMNL的氮气空化。 细胞表面蛋白酶处理， 用[3 H]-半乳糖胺和[3 H]-葡糖胺和表面进行代谢标记 用NaB 3 H4和[125 I]标记将与 免疫印迹分析以确定LFA-1/Mac-1/p150、95 会发生 这些研究将通过检测 通过电子显微镜观察包被小凹或囊泡中的LFA-1/Mac-1/p150，95 利用胶体金或铁蛋白标记实验的实验。 表面Mac-1亚基表达的比较评估（流动性） 细胞荧光照相术）和细胞粘附特性将采用 旨在“上调”或“下调”的趋化性或分泌条件 调节”Mac-1蛋白。 这些研究应该提供新的信息 与Mac-1依赖性PMNL粘附反应在 炎症和宿主防御。