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Abstract
Tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha, whereas blockade of CD120b (p75) reduced NO2- accumulation by approximately 50%. Specific ligation of CD120a (p55) with either (i) human TNFalpha or (ii) by incubation with mouse TNFalpha following pretreatment of macrophages with blocking concentrations of anti-CD120b (p75) antibody resulted in a similar reduction in NO2- production in response to TNFalpha. Quantification of iNOS mRNA, protein, and NO2- expression during independent and co-ligation 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 NO2- expression, CD120b (p75) participates by (i) increasing the sensitivity of the cells to TNFalpha, 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.
Highlights
Expression in macrophage lysates, whereas cross-linking of CD120b (p75) had a minimal effect
In this study we have investigated the roles of CD120a (p55) and CD120b (p75) in the induction of NO2Ϫ and inducible nitric oxide synthase (iNOS) mRNA expression by mouse macrophages and the mechanisms underlying their involvement
Agonistic Effects of Anti-TNF Receptor Antibodies on NO2Ϫ Production—The independent roles of CD120a (p55) and CD120b (p75) in NO2Ϫ production were initially addressed by incubating macrophage monolayers with increasing concentrations of polyclonal antibodies directed against each receptor type in the presence and absence of IFN␥
Summary
NO1⁄7, nitric oxide; iNOS, inducible nitric oxide synthase; TNF␣, tumor necrosis factor-␣; IFN␥, interferon-␥; JNK, c-Jun NH2-terminal kinase; TRAF, TNF receptor-associated factor; PAGE, polyacrylamide gel electrophoresis. Cross-linking of CD120b (p75) may generate a signal that may or may not synergize with signals produced during ligation of CD120a (p55). Since both concepts are not mutually exclusive, cross-linking of CD120b (p75) may contribute to the functions elicited by ligation of CD120a (p55) may not necessarily, by itself, stimulate a cellular response. Our findings show that whereas cross-linking of CD120a (p55) is necessary and sufficient for iNOS and NO2Ϫexpression, additional cross-linking of CD120b (p75) and signaling by this receptor are required for the optimal and prolonged induction of iNOS and NO2Ϫ
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