TNF-Induced Apoptosis of Lymphocytes in Aged Humans

衰老与T细胞免疫功能的进行性下降有关。肿瘤坏死因子-α通过肿瘤坏死因子受体I和肿瘤坏死因子受体II激活T细胞。肿瘤坏死因子-RI既可介导激活信号(通过核因子-kB激活)，又可介导凋亡信号(通过FADD)。FADD是CD95和肿瘤坏死因子受体介导的细胞凋亡的共同途径。肿瘤坏死因子-RII通过激活核因子-kB介导激活信号。核因子-kB是肿瘤坏死因子-α诱导的细胞凋亡的抑制因子。肿瘤坏死因子-α诱导的老年人T细胞亚群凋亡率的增加与肿瘤坏死因子-RI上调和肿瘤坏死因子-受体II下调有关。因此，我们推测，降低核因子-kB的活性(可能是由于减少了肿瘤坏死因子-RII的表达和信号)，以及增加了肿瘤坏死因子-RI介导的促凋亡活性(通过FADD)，在提高老年人T细胞对肿瘤坏死因子-α诱导的凋亡的敏感性方面发挥了关键作用。本研究的具体目的是[1]研究核因子-kB的激活和肿瘤坏死因子-Ⅱ的表达在肿瘤坏死因子-α诱导的老年人和年轻人T细胞亚群凋亡中的作用。[a]用流式细胞仪和实时定量聚合酶链式反应检测肿瘤坏死因子-RI和肿瘤坏死因子-受体II的表达；[b]用双抗体夹心法和共聚焦显微镜检测肿瘤坏死因子-α对核转录因子-kB的激活作用；[c]用Western blotting法检测细胞内IKKB的表达和活化(用GST-IkBA和经TNR处理的T细胞胞浆提取液进行IKK免疫复合体检测)；[d]用Western blotting检测经TNF-a处理后IkBA的磷酸化；[F]通过原位末端标记法和Annexin V法检测肿瘤坏死因子-受体II过表达对肿瘤坏死因子诱导的T细胞亚群凋亡的影响，用免疫印迹法检测IKKB过表达对肿瘤坏死因子-α诱导的T细胞亚群凋亡的影响，通过Western blotting检测IkB的磷酸化、核因子-kB的激活以及XIAP、cIAP1和cIAP2的表达。[2]探讨肿瘤坏死因子-RI和FADD在肿瘤坏死因子-α诱导的老年人和年轻人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]检测显性负性FADD表达对T细胞亚群诱导的肿瘤坏死因子-α的影响，通过比色法和流式细胞术检测Caspase8和caspase3的激活，[c]确定反义寡核苷酸下调肿瘤坏死因子-RI对肿瘤坏死因子-α诱导的细胞凋亡的影响，免疫沉淀法向肿瘤坏死因子-RI-Tradd复合体募集FADD，以及激活caspase8和caspase3。这些都是S研究的肿瘤坏死因子-α诱导的细胞凋亡增加的机制(S)。

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.