The role of a phosphatidylcholine-specific phospholipase C in the production of diacylglycerol for nitric oxide synthesis in macrophages activated by IFN-gamma and LPS

Murine macrophages activated by interferon (IFN)-gamma and bacterial lipopolysaccharide (LPS) produce large amounts of nitric oxide (NO), which is a critical mediator for a variety of biological functions. The expression of this inducible NO synthase (iNOS) involves a protein kinase C (PKC)-dependent pathway, but the mechanism for the PKC activation in this system is unclear. Through analysis of diacylglycerol (DAG) synthesis and choline metabolism in activated macrophages, direct evidence is provided that NO synthesis involves the activation of an unusual phosphatidylcholine-specific phospholipase C (PC-PLC) and not a phosphatidylinositol-specific phospholipase C (PI-PLC) or phospholipase D (PLD).     

Aflatoxin B1-induced suppression of nitric oxide production in murine peritoneal macrophages

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and non-specific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 microM reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 microM decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 microg/ml LPS and about 12% and 24%, respectively, after stimulation with 10 microg/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.     

Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma

The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.     

Mechanism of suppression of macrophage nitric oxide release by IL-13: influence of the macrophage population

IL-13 is a cytokine produced by T lymphocytes, mast cells, basophils, and certain B cell lines that up-regulates or inhibits various macrophage functions. In the present study we analyzed the mechanisms of suppression of nitric oxide (NO) release by IL-13 in the macrophage cell line J774A.1 and in thioglycolate-elicited mouse peritoneal macrophages. In both cell types efficient reduction (>80%) of NO production required treatment of the macrophages with IL-13 for at least 7 h before stimulation with IFN-gamma and LPS. In J774A.1 cells, increasing concentrations of IFN-gamma partially antagonized the suppression mediated by IL-13, whereas in peritoneal macrophages, the inhibitory effect of IL-13 was largely independent of the concentrations of IFN-gamma and LPS. In J774A.1 cells, IL-13 strongly reduced both the mRNA and protein levels of inducible nitric oxide synthase (iNOS, NOS-2), as determined by Northern blot analysis and immunoprecipitation. In peritoneal macrophages, in contrast, IL-13 decreased iNOS protein and enzyme activities after 8 to 48 h of stimulation, without altering the expression of iNOS mRNA. Pulse labeling with [35S]methionine revealed that IL-13 caused a 4.7-fold reduction of the de novo synthesis of iNOS protein in these cells. These data demonstrate for the first time that IL-13 is capable of regulating iNOS at both the mRNA and translational levels and underline the important influence of the macrophage population when studying mechanisms of cytokine functions.     

Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells

We have examined whether modulation of the polyamine biosynthetic pathway, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate limiting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J774 macrophages. DFMO potentiated LPS-stimulated nitrite production in both a concentration- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM. This effect was observed in cells pre-treated with DFMO for 24 h prior to stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate LPS-induced nitrite production. Supplementation of the culture medium with horse serum (10%) in place of foetal calf serum (10%) caused no significant change in either LPS-induced nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-induced NO synthesis. Metabolism of L-[3H]arginine to L-[3H]citrulline by partially purified inducible nitric oxide synthase (iNOS) was not significantly altered by either DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) or spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but was markedly attenuated (70+/-0.07%) by L-NMMA (100 microM). Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with LPS resulted in enhanced (approximately 2 fold) iNOS protein expression. These results show that DFMO potentiates LPS-induced nitrite production in the murine macrophage cell line J774. Since the only known mechanism of action of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we conclude that expression of iNOS can be critically regulated by endogenous polyamines.