Exogenous nitric oxide regulates IFN-gamma plus lipopolysaccharide-induced nitric oxide synthase expression in mouse macrophages.

This study was performed to determine the effects of nitric oxide (NO) on the expression of inducible NO synthase (iNOS) in mouse macrophages. We used the NO donor diethylamine dinitric oxide (DEA/NO) and the mouse macrophage cell line ANA-1 in these experiments. ANA-1 macrophages did not express iNOS mRNA either constitutively or following exposure to 100 U/ml IFN-gamma alone, to 10 ng/ml LPS alone, or to 200 microM DEA/NO alone. Similarly, ANA-1 macrophages did not express detectable levels of iNOS mRNA following treatment with 100 U/ml IFN-gamma plus 200 microM DEA/NO. However, IFN-gamma (100 U/ml) plus LPS (10 ng/ml) induced high levels of iNOS mRNA in ANA-1 macrophages after 6 h. Low concentrations of DEA/NO (approximately 1 to 12 microM) caused up to a 2.5-fold augmentation of IFN-gamma plus LPS-induced iNOS mRNA expression. In contrast, 200 microM DEA/NO suppressed IFN-gamma plus LPS-induced iNOS mRNA expression (60% decrease). The effects of DEA/NO were gene-specific because DEA/NO did not affect the IFN-gamma plus LPS-induced expression of TNF-alpha mRNA. Moreover, the biphasic effects of DEA/NO were specifically due to released NO. Diethylamine and nitrite were unable to regulate IFN-gamma plus LPS-induced gene expression in ANA-1 macrophages. Time-response experiments suggested that the effects of NO were short-lived and occurred early during the induction of iNOS gene expression. The effects of NO were not limited to iNOS mRNA expression but were apparent at the level of iNOS protein expression and enzymatic activity. Overall, these results suggest that NO has immunoregulatory effects and may control the extent and duration of cytokine- and/or endotoxin-induced iNOS expression in macrophages.

Characterization of nitric oxide-stimulated ADP-ribosylation of various proteins from the mouse macrophage cell line ANA-1 using sodium nitroprusside and the novel nitric oxide-donating compound diethylamine dinitric oxide.

We examined the ability of nitric oxide (NO) to stimulate the ADP-ribosylation of proteins from the mouse macrophage cell line ANA-1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (M(r) = 28,000, 33,000 and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (EC50 = 80 microM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-gamma plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weight of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.

Macrophage-stimulating protein inhibits induction of nitric oxide production by endotoxin- or cytokine-stimulated mouse macrophages.

Human serum macrophage-stimulating protein (MSP) is a disulfide-linked heterodimer that induces motile and phagocytic activity of mouse resident peritoneal macrophages. In this work, we found that MSP blocked the increase in macrophage nitric oxide synthase mRNA, as well as the associated increase in nitric oxide production, that occurred in response to several stimuli. These included bacterial products and mammalian cytokines: endotoxin, and interferon-gamma plus endotoxin, interleukin-2, or tumor necrosis factor-alpha. The inhibition by MSP of induction of nitric oxide synthase mRNA and nitric oxide secretion was concentration-dependent. The concentration of MSP that caused maximal inhibition of nitric oxide production was comparable with the optimum for stimulation of macrophage motile and phagocytic activity. Time course studies showed that nitrite was first detected in culture fluid about 8 h after endotoxin stimulation, and it accumulated at a linear rate during the ensuing 16 h. Inhibition by MSP occurred during the 8-h lipopolysaccharide (LPS) induction period; inhibition was maximal when MSP and LPS were added together and decreased progressively to no inhibition as the interval between LPS and MSP addition increased to 11 h.

Regulation of nitric-oxide synthase mRNA expression by interferon-gamma and picolinic acid.

Picolinic acid, a catabolite of L-tryptophan, is a potent co-stimulatory agent for the induction of tumoricidal activity and the production of L-arginine-dependent reactive nitrogen intermediates (RNI) in murine macrophages. We studied whether picolinic acid could affect nitric-oxide synthase (NOS) expression at the gene level in the macrophage cell line ANA-1. NOS mRNA was neither constitutively expressed nor induced by treatment with picolinic acid alone. However, low levels of NOS mRNA were induced by interferon (IFN)-gamma alone. In contrast, a major increase of NOS mRNA expression was observed after treatment with IFN-gamma plus picolinic acid. The synergism was already detectable after 5-6 h and increased up to 20 h of treatment. The ability of picolinic acid to augment IFN-gamma-dependent NOS mRNA expression was associated with a parallel increase in transcription, as demonstrated by nuclear run-on experiments. Protein synthesis was required for the induction of NOS mRNA because addition of cycloheximide dramatically reduced IFN-gamma plus picolonic acid-induced NOS mRNA expression. Finally, interleukin-4 significantly decreased IFN-gamma plus picolinic acid-induced NOS mRNA expression and NOS transcription. These data provide evidence of a molecular event connecting arginine and tryptophan metabolic pathways in the generation of RNI, and they indicate that picolinic acid can induce transcriptional activation of gene expression.