AGE-ASSOCIATED ALTERATIONS IN HUMAN NK CELL SYSTEM

人类 NK 细胞系统中与年龄相关的变化

• 批准号: 2049325
• 负责人: RAJABATHER KRISHNARAJ
• 金额: $ 23.26万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-29 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2049325
• 关键词: DNA replication; cell senescence; cell sorting; cellular immunity; complementary DNA; cytokine receptors; gene expression; human subject; interferon gamma; interleukin 2; leukocyte activation /transformation; natural killer cells; polymerase chain reaction; receptor expression; receptor mediated endocytosis; secretion; tissue /cell culture; tumor necrosis factor alpha; DNA replication; cell senescence; cell sorting; cellular immunity; complementary DNA; cytokine receptors; gene expression; human subject; interferon gamma; interleukin 2; leukocyte activation /transformation; natural killer cells; polymerase chain reaction; receptor expression; receptor mediated endocytosis; secretion; tissue /cell culture; tumor necrosis factor alpha

IL2 deficiency and reduced responsiveness to IL2 contribute to the diminished T-cell immunity at advanced age. However, it is not clear if natural killer (NK) cells, the non-specific effectors of anti-tumor and anti-viral immunity are also affected during senescence. By virtue of their ability to secrete cytokines, e.g. interferon gamma (IFN-gamma) rapidly, NK cells may play an important role in the early phases of infection/ malignancy/immunoregulation. We have observed a senescence- related decline in in vitro sensitivity of purified human NK cells to lL2, i.e., release of IFN-gamma or LAK activity. Our long term goal is to investigate the cellular and molecular mechanisms involved in the preferential decline in IL2 inducible cytokine secretory functions of NK cells during senescence. We hypothesize that during senescence, there may be a reduced frequency of IL2 responding, IFN-gamma secreting NK cells and/or a defect in IL2- NK cell interaction at the level of IL2 receptors or the expression of IL2 receptor gamma chain or IFN-gamma genes, resulting in impaired IFN- gamma secretion per cell. To test this hypothesis, the amount of lFN-gamma released by sorted, IL2 activated NK subsets will be measured. The CD56(bright) and CD56(dim), cells represent the two distinct NK subsets. The frequency of IFN-gamma secretors among NK cells from healthy young and elderly will be quantitated by ELISPOT. Quantitative flow cytometric analysis of CD56(bright) ("immature") and CD56(dim) ("mature") NK subsets will be done. To investigate a potentially defective IL2-NK cell interaction, the expression of alpha (CD25) and beta (CD122) lL2 receptor chains (flow cytometry) and their affinity to IL2 or density per NK cell (equilibrium binding studies with (125)I-lL2) will be measured. To rule out a delayed translocation of IL2, the kinetics of internalization of cell surface bound lL2 will be followed. To locate a potential defect in IFN-gamma gene expression, the level of IFN-gamma mRNA in lL2 activated, sorted NK cells will be analyzed by quantitative RT-PCR by using PCR MIMICS. The level of IL2R-gamma mRNA will also be measured by PCR. Since a senescence-related decline in T- & B-cell DNA synthesis was noted by us before, the proliferative potential of IL2 induced NK cells will be assessed. These results will have theoretical implications in malignancy, infection and immune regulation and therapeutic applications in immunopharmacological interventions and cytokine gene therapies, especially in elderly patients.

IL2缺乏症和对IL2的反应性降低有助于 高龄的T细胞免疫降低。但是，尚不清楚是否 天然杀手（NK）细胞，抗肿瘤的非特异性效应子和 在衰老期间，抗病毒免疫也受到影响。依靠 他们分泌细胞因子的能力，例如干扰素伽玛（IFN-GAMMA） 迅速，NK细胞可能在早期的早期阶段起重要作用 感染/恶性/免疫调节。我们已经观察到衰老 纯化的人NK细胞对体外敏感性的相关下降 LL2，即释放IFN-gamma或Lak活性。我们的长期目标是 研究参与的细胞和分子机制 IL2诱导性细胞因子分泌功能的优先下降 NK细胞在衰老过程中。 我们假设在衰老期间，频率可能降低 IL2响应，IFN-gamma分泌NK细胞和/或IL2-中的缺陷 IL2受体水平的NK细胞相互作用或表达 IL2受体伽马链或IFN-GAMMA基因，导致IFN- 每个细胞的伽马分泌。 为了检验这一假设，分类iL2释放的LFN-gamma量 将测量激活的NK子集。 CD56（明亮）和CD56（DIM）， 细胞代表两个不同的NK子集。 IFN-GAMMA的频率 健康的年轻和老年人的NK细胞中的捐赠者将是 由ELISPOT定量。定量流式细胞术分析 CD56（明亮）（“不成熟”）和CD56（DIM）（“成熟”）NK子集将是 完毕。为了研究潜在有缺陷的IL2-NK细胞相互作用， α（CD25）和β（CD122）LL2受体链的表达 （流式细胞仪）及其对IL2或每个NK细胞密度的亲和力 （使用（125）I-LL2的平衡结合研究）将被测量。统治 消除IL2的延迟易位，这是内在化的动力学 将遵循细胞表面结合的LL2。找到潜在的缺陷 在IFN-GAMMA基因表达中，LL2中的IFN-GAMMA mRNA水平 激活的，分类的NK细胞将通过定量RT-PCR分析 使用PCR模拟物。 IL2R-GAMMA mRNA的水平也将通过 pcr。由于T和B细胞DNA合成的衰老相关下降是 以前我们注意到，IL2诱导NK细胞的增殖潜力 将被评估。这些结果将在理论上含义 恶性，感染和免疫调节和治疗应用 在免疫药理学干预措施和细胞因子基因疗法中， 特别是在老年患者中。