TNF-Induced Apoptosis of Lymphocytes in Aged Humans

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通道。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-a诱导的凋亡增加与TNF- ri上调和TNF- RII下调有关。因此，我们假设NF-kB激活的减少（可能是由于TNF-RII表达和信号传导的减少）和tnf - ri介导的促凋亡的增加（通过FADD）在老年人T细胞对TNF-a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研究NF-kB活化和TNF-RII表达在tnf -a诱导的老年和青年T细胞亚群凋亡中的作用。[a]通过流式细胞术和Real - time PCR检测TNF-RI和TNF-RII的表达，[b]使用基于elisa的DNA结合试验和共聚焦显微镜检测细胞内核易位对TNF-a的激活，[c]检测TNF-a处理后IKKb的表达（通过Western blotting）和激活（使用GST- IkBa与tnr处理的T细胞的细胞质提取物进行IKK免疫复合物激酶试验）。[d]通过Western blotting测定TNF-a处理后IkBa的磷酸化情况，[e] TNF-a处理后蛋白体介导的IkBa降解情况，[f]通过TUNEL和Annexin V试验测定过表达的TNF-RII对tnf诱导的T细胞亚群凋亡的影响，IKKb的活化，IkBa的磷酸化和NFk-B的活化情况，[g]测定过表达的IKKb对TNF-a诱导的T细胞亚群凋亡的影响，IkBa的磷酸化，Western blotting检测NF-kB的激活，以及XIAP、cIAP1和cIAP2的表达。研究TNF-RI和FADD在tnf -a诱导的老年和青年T细胞亚群凋亡中的作用。[a]通过Western blotting和Real time PCR检测FADD蛋白和mRNA水平的表达，[b]通过比色法和流式细胞术检测FADD显性阴性表达对tnf -a诱导的T细胞亚群、Caspase 8向DISK募集、Caspase 8和Caspase 3的激活的影响，[c]通过反义寡核苷酸下调TNF-RI对tnf -a诱导的凋亡的影响，免疫沉淀法检测FADD向TNF-RI- tradd复合物募集的影响。以及caspase 8和caspase 3的激活。这些研究应该在衰老过程中增加tnf -a诱导的细胞凋亡机制。

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.