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 (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.
Division of Basic Science, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA. richesd@njc.org
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Riches
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Chan
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Zahradka
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Winston
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Remigio
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Lake
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015703:Antigens, CD / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D018124:Receptors, Tumor Necrosis Factor / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000906:Antibodies; D015703:Antigens, CD; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse