Differential Activation of the Nuclear Factor-κB by TNF Muteins Specific for the p60 and p80 TNF Receptors

Human TNF is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors, viz p60 and p80, which transmit their signals independently of each other. Activation of the transcription factor NF-κB is one of the earliest events induced by TNF, but whether it is mediated through one or both forms of the TNF receptor is controversial. In the present studies, we examined the role of each receptor in the activation of NF-κB by using TNFs that has been designed by site-specific mutagenesis to bind either the p60 (R32W; S86T) or the p80 (D143N; A145R) form of the receptor. Human myelogenous leukemic ML-1a cells known to express almost equal amounts of the two receptors were used. The binding of TNF to these cells could be inhibited equally by either TNF(p60) or TNF(p80) mutein. Treatment of these cells with TNF(p60) mutein activated NF-κB within 30 min, whereas TNF(p80) mutein, even at a 1000-fold excess, had no effect, suggesting that the activation of NF-κB is differentially regulated through the p60 receptor. Consistent with these results, treatment with either anti-p80 monoclonal or polyclonal Abs blocked the binding of TNF to the p80 receptor without affecting TNF-mediated activation of NF-κB. TNF(p60) mutein was also effective in cell killing, but the TNF(p80) mutein was totally ineffective. The effect was not cell type-specific, since other p80-expressing cell lines were also unresponsive. Overall, our results clearly demonstrate that the activation of NF-κB and cytotoxicity by TNF is differentially regulated through the p60 receptor.