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: 3135751
• 负责人: Donald C. Anderson
• 金额: $ 15.96万
• 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/3135751
• 关键词: cell adhesion; cell bank /registry; cell migration; collagen; density gradient ultracentrifugation; glycoproteins; human tissue; immunochemistry; inborn lysosomal enzyme disorder; inborn metabolism disorder; laboratory rat; 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) 功能的了解 识别遗传性异常或缺乏遗传性异常有很大帮助 PMNL 蛋白。 在该提案中，发现了 8 名患者（4 名男性） 和四名女性）缺乏高分子量糖蛋白 家族（LFA-1/Mac-1/p150,95）在细胞表面和细胞内 利用池来了解这些物质的分子机制 蛋白质介导细胞粘附现象。 人内皮细胞 将通过以下方式检查单层细胞的粘附、铺展和渗透 静息条件下正常且 LFA-1/Mac-1/p150-95 缺乏 PMNL 且 用包括 F-Met-Leu-Phe 在内的趋化因子刺激后。 三维 I 型胶原蛋白基质将被研究 正常和有缺陷的 PMNL 在相似条件下的粘附和侵袭 检验 PMNL 依从性对于以下假设很重要的条件： 内皮细胞表面的运动，但不太重要 胶原蛋白基质的渗透。 干扰直接观察 对比显微镜以及扫描和透射电子 显微镜将用于记录细胞的粘附和侵入 PMNL。 LFA-1/Mac-1/p150,95 在 PMNL 依从性调节中的作用 通过测定细胞内的 LFA-1/Mac-1/p150,95 和某些相关中间体的位置 利用单克隆抗体进行独特的定量免疫印迹测定 针对 LFA-1/Mac-1/p150,95 和常见的 Beta 亚基。 亚细胞 分馏将在 Percoll 密度梯度上完成 PMNL 的氮空化。 细胞表面蛋白酶处理， [3H]-半乳糖胺和[3H]-葡萄糖胺和表面代谢标记 NaB3H4 和 [125I] 标记将与 免疫印迹法测定 LFA-1/Mac-1/p150 是否循环，95 发生。 这些研究将通过检测来补充 通过电子显微镜观察包被的凹坑或囊泡中的 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 细胞荧光法）和细胞粘附特性将采用 旨在“上调”或“下调”的趋化或分泌条件 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 粘附反应的作用相关 炎症和宿主防御。