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

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.

IL-2缺乏和对IL-2的反应性降低有助于 老年人T细胞免疫力下降然而，目前尚不清楚， 自然杀伤（NK）细胞，抗肿瘤的非特异性效应物， 抗病毒免疫力在衰老期间也受到影响。凭借 它们分泌细胞因子的能力，例如干扰素γ（IFN-γ） 很快，NK细胞可能在早期阶段发挥重要作用， 感染/恶性肿瘤/免疫调节。我们观察到一种衰老- 纯化的人NK细胞体外敏感性的相关下降 IL 2，即，IFN-γ或LAK活性的释放。我们的长期目标是 研究参与的细胞和分子机制， IL-2诱导的细胞因子分泌功能优先下降， 衰老过程中的NK细胞。 我们假设在衰老过程中， IL-2应答性、IFN-γ分泌性NK细胞的缺陷和/或IL-2应答性、IFN-γ分泌性NK细胞的缺陷。 NK细胞在IL 2受体水平的相互作用或 IL 2受体γ链或IFN-γ基因，导致IFN-γ受损。 每个细胞的γ分泌量。 为了检验这一假设，测定了由分选的IL 2释放的IFN-γ的量。 将测量活化的NK亚群。CD 56（亮）和CD 56（暗）， NK细胞代表两个不同的NK亚群。IFN-γ的频率 健康的年轻人和老年人的NK细胞中的分泌物将是 通过ELISPOT定量。流式细胞术定量分析 CD 56（亮）（“未成熟”）和CD 56（暗）（“成熟”）NK亚群将被免疫组化。 完了为了研究潜在缺陷的IL 2-NK细胞相互作用， α（CD 25）和β（CD 122）IL 2受体链的表达 (flow流式细胞术）和它们对IL 2的亲和力或每个NK细胞的密度 （用（125）I-1 L2进行的平衡结合研究）。统治 IL 2的延迟易位， 将跟踪细胞表面结合的IL 2。定位潜在缺陷 在IFN-γ基因表达中，IL 2中IFN-γ mRNA的水平 将通过定量RT-PCR分析活化的、分选的NK细胞， 使用PCR MIMICS。还将通过以下方法测量IL 2 R-γ mRNA的水平 PCR法由于衰老相关的T细胞和B细胞DNA合成下降， 我们以前注意到，IL 2诱导的NK细胞的增殖潜力 将予以评估。这些结果将具有理论意义， 恶性肿瘤、感染和免疫调节以及治疗应用 在免疫药理学干预和细胞因子基因疗法中， 尤其是老年患者。