Analysis of the function of membrane TNF-α

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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.