TNF-Induced Apoptosis of Lymphocytes in Aged Humans

TNF 诱导的老年人淋巴细胞凋亡

• 批准号: 6475385
• 负责人: SUDHIR GUPTA
• 金额: $ 33.57万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6475385
• 关键词: DNA binding protein; I kappa B beta; T lymphocyte; age difference; aging; apoptosis; biological signal transduction; clinical research; colorimetry; cysteine endopeptidases; cytokine receptors; developmental immunology; enzyme linked immunosorbent assay; flow cytometry; human tissue; immunoprecipitation; immunosenescence; messenger RNA; nuclear factor kappa beta; phosphorylation; protein degradation; protein structure function; receptor expression; terminal nick end labeling; tumor necrosis factor alpha; western blottings

Aging is associated with a progressive decline in T cell immune functions. TNF-a activates T cells via both TNF-RI and TNF-RII. TNF- RI mediates both activation signal (via NF-kB activation) and apoptotic signal (via FADD). FADD is a common conduit for both CD95 and TNF-R-mediated apoptosis. TNF-RII mediates activation signal via NF-kB activation. NF-kB is a repressor of TNF-a-induced apoptosis. Increased TNF-a-induced apoptosis in T cell subsets from aged humans is associated with upregulation of TNF-RI and downregulation of TNF- RII. Therefore, we hypothesized that decreased NF-kB activation (that may be due to decreased TNF-RII expression and signaling), and increased TNF-RI-mediated proapoptotic al (via FADD) play a critical role in increased sensitivity of T cells from aged humans to TNF-a- induced apoptosis. Specific aims of the study are [1 ] To study a role of NF-kB activation and TNF-RII expression in TNF-a-induced apoptosis in T cell subsets from aged and young subjects. [a] examine the expression of TNF-RI and TNF-RII by flow cytometry and Real time PCR, [b] determine the activation of NF-kB by TNF-a using ELISA-based DNA binding assay and cytoplasm to nuclear translocation by Confocal microscopy, [c] determine the expression (by Western blotting) and activation of IKKb (by IKK immune complex kinase assay using GST- IkBa with cytoplasmic extracts of TNR-treated T cells) following treatment with TNF-a, [d] determine the phosphorylation of IkBa following TNF-a treatment by Western blotting, [e] proteosome-mediated degradation of IkBa following treatment with TNF-a, [f] determine the effect of overexpression of TNF-RII on TNF-induced apoptosis in T cell subsets by TUNEL and Annexin V assay, activation of IKKb, phosphorylation of IkBa, and NFk-B activation, [g] determine the effect of overexpression of IKKb on TNF-a-induced apoptosis in T cell subsets, phosphorylation of IkBa, activation of NF-kB, and expression of XIAP, cIAP1 and cIAP2 by Western blotting. [2] To study a role of TNF-RI and FADD in TNF-a-induced apoptosis in T cell subsets from aged and young subjects. [a] examine the expression of FADD at the protein and mRNA level by Western blotting and Real time PCR, [b] examine the effect of dominant negative FADD expression on TNF-a-induced in T cell subsets, recruitment of Caspase 8 to DISK, activation of caspase 8 and caspase 3 by colorimetric assay and flow cytometry,and [c] determine the effect of downregulation of TNF-RI by antisense oligonucleotide on TNF-a-induced apoptosis, Recruitment of FADD to TNF-RI-TRADD complex by immunoprecipitation, and activation of caspase 8 and caspase 3. These studied should mechanism (s) for increased TNF-a-induced apoptosis during aging.

衰老与 T 细胞免疫功能的逐渐下降有关。 TNF-a 通过 TNF-RI 和 TNF-RII 激活 T 细胞。 TNF-RI 介导激活信号（通过 NF-kB 激活）和凋亡信号（通过 FADD）。 FADD 是 CD95 和 TNF-R 介导的细胞凋亡的常见途径。 TNF-RII 通过 NF-kB 激活介导激活信号。 NF-kB 是 TNF-a 诱导的细胞凋亡的抑制因子。老年人 T 细胞亚群中 TNF-α 诱导的细胞凋亡增加与 TNF-RI 的上调和 TNF-RII 的下调有关。因此，我们假设 NF-kB 激活的减少（可能是由于 TNF-RII 表达和信号转导的减少）和 TNF-RI 介导的促凋亡的增加（通过 FADD）在老年人 T 细胞对 TNF-α 诱导的细胞凋亡的敏感性增加中发挥关键作用。该研究的具体目的是 [1] 研究 NF-kB 激活和 TNF-RII 表达在 TNF-a 诱导的老年和年轻受试者 T 细胞亚群凋亡中的作用。 [a] 通过流式细胞术和实时 PCR 检查 TNF-RI 和 TNF-RII 的表达，[b] 使用基于 ELISA 的 DNA 结合测定确定 TNF-a 对 NF-kB 的激活，并通过共聚焦显微镜确定细胞质到核的易位，[c] 确定 IKKb 的表达（通过蛋白质印迹）和激活（通过使用 GST-的 IKK 免疫复合物激酶测定） IkBa 与 TNR 处理的 T 细胞的细胞质提取物）用 TNF-a 处理后，[d] 通过蛋白质印迹确定 TNF-a 处理后 IkBa 的磷酸化，[e] 用 TNF-a 处理后蛋白酶体介导的 IkBa 降解，[f] 确定 TNF-RII 过表达对 TNF 诱导的 T 细胞凋亡的影响 [g] 通过 TUNEL 和膜联蛋白 V 法检测 IKKb 的亚群、IKKb 的激活、IkBa 的磷酸化和 NFk-B 的激活，[g] 通过 Western blotting 确定 IKKb 过表达对 TNF-a 诱导的 T 细胞亚群凋亡、IkBa 的磷酸化、NF-kB 的激活以及 XIAP、cIAP1 和 cIAP2 表达的影响。 [2] 研究 TNF-RI 和 FADD 在 TNF-a 诱导的老年和年轻受试者 T 细胞亚群凋亡中的作用。 [a]通过Western blotting和Real time PCR检测FADD在蛋白和mRNA水平的表达，[b]通过比色法和流式细胞术检测显性失活FADD表达对TNF-α诱导的T细胞亚群、Caspase 8向DISK的募集、Caspase 8和Caspase 3激活的影响，以及[c]通过反义检测下调TNF-RI的影响 寡核苷酸对 TNF-α 诱导的细胞凋亡的影响，通过免疫沉淀将 FADD 招募到 TNF-RI-TRADD 复合物，以及激活 caspase 8 和 caspase 3。这些研究应该是衰老过程中 TNF-a 诱导细胞凋亡增加的机制。