TNF-Induced Apoptosis of Lymphocytes in Aged Humans

TNF 诱导的老年人淋巴细胞凋亡

• 批准号: 6624500
• 负责人: SUDHIR GUPTA
• 金额: $ 34.74万
• 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/6624500
• 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细胞免疫功能的进行性下降有关。TNF-α通过TNF-RI和TNF-RII激活T细胞。TNF-RI介导活化信号（通过NF-κ B活化）和凋亡信号（通过FADD）。FADD是CD 95和TNF-R介导的细胞凋亡的共同通道。TNF-RII通过NF-kB活化介导活化信号。NF-kB是TNF-α诱导的细胞凋亡的阻遏物。老年人T细胞亚群中TNF-α诱导的凋亡增加与TNF-RI的上调和TNF- RII的下调有关。因此，我们假设降低的NF-kB活化（这可能是由于降低的TNF-RII表达和信号传导）和增加的TNF-RI介导的促凋亡α 1（通过FADD）在来自老年人的T细胞对TNF-α诱导的凋亡的敏感性增加中起关键作用。本研究的具体目的是[1 ]研究NF-kB活化和TNF-RII表达在老年和年轻受试者的T细胞亚群中TNF-α诱导的细胞凋亡中的作用。[a]通过流式细胞术和真实的时间PCR检测TNF-RI和TNF-RII的表达，[B]使用基于ELISA的DNA结合分析和通过共聚焦显微镜确定TNF-α对NF-κ B B的激活，[c]确定表达式（通过蛋白质印迹法）和IKKb活化（通过IKK免疫复合物激酶测定，使用GST-IkBa和TNR处理的T细胞的细胞质提取物）在用TNF-α处理后，[d]通过蛋白质印迹法测定TNF-α处理后IkBa的磷酸化，[e]用TNF-α处理后蛋白体介导的IkBa降解，[f]通过TUNEL和膜联蛋白V测定法测定TNF-RII过表达对T细胞亚群中TNF诱导的细胞凋亡的影响，IKKb的活化，通过Western印迹法测定IKKb过表达对T细胞亚群中TNF-α诱导的细胞凋亡、IkBa的磷酸化、NF-kB的活化以及XIAP、cIAP 1和cIAP 2表达的影响。[2]研究TNF-α诱导的老年人和青年人T细胞亚群凋亡中TNF-RI和FADD的作用。[a]通过蛋白质印迹和真实的时间PCR检测FADD在蛋白质和mRNA水平的表达，[B]通过比色测定和流式细胞术检测显性负性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的活化的影响，和[c]确定通过反义寡核苷酸下调TNF-RI对TNF-α诱导的细胞凋亡的影响，通过免疫沉淀将FADD募集至TNF-RI-TRADD复合物，并激活半胱天冬酶8和半胱天冬酶3。这些研究应该是衰老过程中TNF-α诱导的细胞凋亡增加的机制。