Analysis of the function of membrane TNF-α

TNF-□拮抗剂是。因其在活动性慢性炎症性疾病中具有显著的临床疗效而备受关注。在类风湿性关节炎中，英夫利昔单抗（嵌合抗TNF-□抗体）和依那西普（p75 TNF-□受体融合蛋白）都是非常有效的，而在克罗恩病中，只有英夫利昔单抗可以诱导临床缓解。由于不同的临床疗效可能是由生物效应引起的，而不仅仅是可溶性TNF-□的中和，我们在这里研究了这些药物通过跨膜TNF-□mTNF)诱导的反向信号传导。英夫利昔单抗和依那西普均能诱导转染mTNF的人Jurkat T细胞稳定表达e -选择素，但只有英夫利昔单抗能诱导IL-10产生、细胞凋亡、ROS积累和G1细胞周期阻滞。接下来，我们研究了英夫利昔单抗诱导的这些生物学效应所必需的mTNF元素。mTNF细胞质中2、5和27位的丝氨酸残基通过定点诱变被依次替换为丙氨酸。通过替换所有这三种细胞质丝氨酸残基，英夫利昔单抗诱导的细胞凋亡和细胞周期阻滞被完全消除。在这项研究中，我们发现英夫利昔单抗和依那西普诱导的新的生物学效应是由mTNF的反向信号介导的，这可能解释了这些抗tnf -□药物的临床疗效差异。我们还澄清了细胞质丝氨酸残基对mTNF的信号转导至关重要。

TNF-□ antagonists are the. center of attention for their dramatic clinical efficacy in active chronic inflammatory diseases. In rheumatoid arthritis, both infliximab (chimeric anti-TNF-□ antibody) and etanercept(p75 TNF-□ receptor fusion protein) are highly effective while in Crohn's disease, only infliximab can induce clinical remission. As the differential clinical efficacy was likely to be caused by biological effects other than mere neutralization of soluble TNF-□, we here investigated reverse signaling through transmembrane TNF-□mTNF) induced by these drugs. Both infliximab and etanercept induced E-selectin expression on human Jurkat T cells stably transfected with mTNF, however only infliximab was able to induce IL-10 production, apoptosis, ROS accumulation and G1 cell cycle arrest. We next examined the element of the mTNF essential for these biological effects induced by infliximab. Cytoplasmic serine residues at positions 2,5 and 27 of mTNF were sequentially substituted to alanine by site-directed mutagenesis. Infliximab-induced apoptosis and cell cycle arrest were completely abrogated by substitution of all of these three cytoplasmic serine residues. In this study, we revealed that novel biological effects induced by infliximab and etanercept were mediated by reverse signaling of mTNF, which might explain the differential clinical efficacy of these anti-TNF-□ agents. We also clarified that cytoplasmic serine residues were critical for the signal transduction through mTNF.