Aggarwal S, Gollapudi S, Gupta S
Department of Medicine, University of California, Irvine 92697, USA.
J Immunol. 1999 Feb 15;162(4):2154-61.
Aging is characterized by increased T cell lymphopenia, T cell dysfunction, and increased serum TNF levels. In this study, we have examined the role of TNF-induced apoptosis in T cell deficiency in lymphocytes from aged humans. The constitutive expression of TNF receptors (TNFRI and TNFRII) and the adapter molecules, including TNFR-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF-2), and receptor interacting protein (RIP), were analyzed both at the protein level by flow cytometry or Western blotting, and at the mRNA level using quantitative PCR or Northern blotting in lymphocytes from aged and young subjects. The susceptibility of T cells to undergo TNF-induced apoptosis was analyzed using terminal deoxynucleotidyltransferase-mediated UTP-end-labeling (TUNEL) and DNA ladder assays. Caspase (caspase-8 and caspase-3) activation was compared between aged and young subjects using Western blotting and colorimetric assays. In lymphocytes from aged humans, there was an increased susceptibility of CD4+ and CD8+ T cells to undergo TNF-alpha-induced apoptosis, as observed by TUNEL assay and DNA fragmentation ladder assay. Increased TNF-alpha-induced apoptosis was also observed in both CD45RA+ and CD45RO+ T cells from aging subjects. An increased constitutive expression of TNFRI and TRADD and decreased expression of TNFRII and TRAF-2 were observed in lymphocytes from aged as compared with young controls. In addition, there was an early and increased activation of caspases (caspase-8 and caspase-3) involved in TNFR/TNF signaling pathway, as evident by early cleavage of caspase-8, poly(ADP-ribose) polymerase (PARP), and caspase-3 substrate DEVD-p-nitroamilide NA. These data suggest that an increased TNF-alpha-induced apoptosis may play a role in T cell deficiency associated with human aging.
衰老的特征是T细胞淋巴细胞减少、T细胞功能障碍以及血清TNF水平升高。在本研究中，我们检测了TNF诱导的细胞凋亡在老年人类淋巴细胞T细胞缺乏中的作用。通过流式细胞术或蛋白质印迹在蛋白质水平分析了TNF受体（TNFR I和TNFR II）以及衔接分子（包括TNFR相关死亡结构域蛋白（TRADD）、TNFR相关因子2（TRAF-2）和受体相互作用蛋白（RIP））的组成性表达，并使用定量PCR或Northern印迹在老年和年轻受试者的淋巴细胞中在mRNA水平进行了分析。使用末端脱氧核苷酸转移酶介导的UTP末端标记（TUNEL）和DNA梯状分析检测T细胞对TNF诱导的细胞凋亡的敏感性。使用蛋白质印迹和比色分析比较老年和年轻受试者之间的半胱天冬酶（半胱天冬酶-8和半胱天冬酶-3）激活情况。通过TUNEL分析和DNA片段化梯状分析观察到，在老年人类的淋巴细胞中，CD4+和CD8+ T细胞对TNF-α诱导的细胞凋亡的敏感性增加。在衰老受试者的CD45RA+和CD45RO+ T细胞中也观察到TNF-α诱导的细胞凋亡增加。与年轻对照相比，在老年受试者的淋巴细胞中观察到TNFR I和TRADD的组成性表达增加，而TNFR II和TRAF-2的表达降低。此外，参与TNFR/TNF信号通路的半胱天冬酶（半胱天冬酶-8和半胱天冬酶-3）有早期且增加的激活，这可通过半胱天冬酶-8、聚（ADP-核糖）聚合酶（PARP）和半胱天冬酶-3底物DEVD-p-硝基酰胺NA的早期裂解来证明。这些数据表明，TNF-α诱导的细胞凋亡增加可能在与人类衰老相关的T细胞缺乏中起作用。
