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.
Riches
D W
DW
Division of Basic Science, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA. richesd@njc.org
Chan
E D
ED
Zahradka
E A
EA
Winston
B W
BW
Remigio
L K
LK
Lake
F R
FR
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