MECHANISMS OF ALCOHOL RELATED INHIBITION OF NK ACTIVITY

酒精相关的 NK 活性抑制机制

• 批准号: 6168219
• 负责人: GARY G MEADOWS
• 金额: $ 33.94万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6168219
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; athymic mouse; biological signal transduction; calcium; cellular immunity; corticosterone; cytokine; cytotoxicity; enzyme linked immunosorbent assay; flow cytometry; growth inhibitors; humoral immunity; interleukin 2; interleukin 6; laboratory mouse; lymphocyte proliferation; melanoma; metastasis; natural killer cells; neoplastic cell; neoplastic growth; toxicology

It is estimated that alcohol dependency affects over 10 million Americans and that the cost of alcohol related problems totals over 100 billion dollars. Alcohol consumption impairs both humoral and cell-mediated immunity. Natural killer (NK) cells play an important role in immunosurveillance against certain infectious diseases and cancer and are especially active in preventing tumor metastasis. NK cells also control the rate of progression of aids. We showed previously that alcohol consumption specifically results in impaired NK and lymphokine activated killer (LAK) cytolytic activity when mice consume between 25-40% of their calories from ethanol administered in the drinking water. The objective of this proposal is to determine the mechanism(s) underlying this impaired cytolytic activity and to specifically test the hypothesis that alcohol consumption impairs cytolytic activity through disruption of protein kinase C cell signaling, which in turn modulates granule and cytotoxic mediator function. We will examine the effect that alcohol consumption has on NK granule cytotoxicity and the specific function of the cytolytic mediators, perforin, granzyme A, and granzyme B. We will also determine the effect of alcohol consumption on the activity and amount of these proteins in the two primary subpopulations of NK and LAK cells that contribute to inherent NK cell cytolytic activity (NK1.1+LGL1-cells) and to LAK cytolytic activity (NK1.1+LGL-cells). In vitro colorimetric enzymatic assays will characterize granzyme A and granzyme B proteolytic activity. Isolation of enriched NK1.1+ cells and the LGL1 subpopulations will utilize magnetic bead separation technology. Western blot analysis will be used to determine protein expression in granzymes, and reverse transcriptase polymerase chain reaction (RT-PCR) will be used to determine mRNA. Protein kinase C activity will be determined using standard assays. Cytolytic granules will be isolated from NK/LAK cells with nitrogen cavitation.

据估计，酒精依赖影响了超过 1000 万美国人 酒精相关问题造成的损失总计超过 1000 亿 美元。饮酒会损害体液和细胞介导的 免疫。自然杀伤 (NK) 细胞在 针对某些传染病和癌症的免疫监视 特别是在预防肿瘤转移方面具有积极作用。 NK 细胞也控制 艾滋病的进展速度。我们之前表明酒精 消耗特别会导致 NK 和淋巴因子激活受损 当小鼠消耗 25-40% 的杀伤细胞 (LAK) 细胞溶解活性时 来自饮用水中的乙醇的卡路里。的目标 该提案旨在确定造成这种损害的机制 细胞溶解活性并专门检验酒精的假设 消耗通过破坏蛋白质来损害细胞溶解活性 激酶 C 细胞信号转导，进而调节颗粒和细胞毒性 中介功能。我们将研究饮酒的影响 NK颗粒的细胞毒性和溶细胞的具体功能 介质、穿孔素、颗粒酶 A 和颗粒酶 B。我们还将确定 饮酒对这些物质的活性和数量的影响 NK 和 LAK 细胞两个主要亚群中的蛋白质 有助于固有的 NK 细胞溶细胞活性（NK1.1+LGL1 细胞）和 LAK 溶细胞活性（NK1.1+LGL 细胞）。体外比色法 酶测定将表征颗粒酶 A 和颗粒酶 B 的蛋白水解特性 活动。富集 NK1.1+ 细胞和 LGL1 亚群的分离 将利用磁珠分离技术。蛋白质印迹分析 将用于测定颗粒酶中的蛋白质表达，并反向 转录酶聚合酶链反应（RT-PCR）将用于确定 mRNA。蛋白激酶C活性将使用标准测定来确定。 用氮气从 NK/LAK 细胞中分离出溶细胞颗粒 空化。