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.
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