Cooperative Signaling by Tumor Necrosis Factor Receptors CD120a (p55) and CD120b (p75) in the Expression of Nitric Oxide and Inducible Nitric Oxide Synthase by Mouse Macrophages

Tumor necrosis factor-alpha (TNF alpha) is recognized by the cell-surface receptors CD120a (p55) and CD120b (p75). In the present study, we have investigated the role of these receptors in the expression of NO2- a stable metabolite of nitric oxide, and inducible nitric oxide synthase (iNOS) by mouse macrophages. Specific antibody-mediated aggregation of CD120a (p55) induced NO2- accumulation in culture supernatants and iNOS mRNA expression in macrophage lysates, whereas cross-linking of CD120b (p75) had a minimal effect. In contrast, simultaneous cross-linking of both receptors led to a marked augmentation in NO2- and iNOS mRNA expression. Antibody-mediated blockade of CD120a (p55) completely inhibited NO2- expression in response to TNF alpha, whereas blockade of CD120b (p75) reduced NO2- accumulation by similar to 50%. Specific Ligation of CD120a (p55) with either (i) human TNF alpha or (ii) by incubation with mouse TNF alpha following pretreatment of macrophages with blocking concentrations of anti-CD120b (p75) antibody resulted in a similar reduction in NO2- production in response to TNF alpha. Quantification of iNOS mRNA, protein, and NO2- expression during independent and coligation of CD120a (p55) and CD120b (p75) indicated that iNOS mRNA and protein expression was transient in nature when CD120a (p55) was cross-linked alone but was prolonged when both receptors were simultaneously cross-linked. In addition, cross-linking both receptors also led to a potentiation of NO2- accumulation in culture supernatants that was more pronounced at later time points. These findings suggest that while cross-linking of CD120a (p55) is necessary and sufficient for iNOS mRNA and NO(2)(-)expression, CD120b (p75) participates by (i) increasing the sensitivity of the cells to TNF alpha, probably by "passing" ligand to CD120a (p55), and (ii) initiating a signaling event that results in a more sustained induction of iNOS mRNA and protein and thereby augments the production of nitric oxide.