Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into listeriocidal activity of oxidative and nitrosative stress

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6)(N[N(O)NO]C3H7) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-gamma (INF-gamma) + tumor necrosis factor-alpha (TNF-alpha or INF-gamma + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Isotope labeling studies on the mechanism of N-N bond formation in denitrification

The mechanism of the denitrification and nitrosation reactions catalyzed by the heme cd-containing nitrite reductase from Pseudomonas stutzeri JM 300 has been studied with whole cell suspensions using H2(18)O, 15NO, and 15NO-2. The extent of H2(18)O exchange with the enzyme-bound nitrosyl intermediate, as determined by the 18O content of product N2O, decreased with increasing nitrite concentration, which is consistent with production of N2O by sequential reaction of two nitrite ions with the enzyme. Reaction of NO with whole cells in H2(18)O gave amounts of 18O in the N2O product consistent with equilibration of nitric oxide with a small pool of free nitrite. Using 15NO and NH2OH, competition between denitrification and nitrosation reactions was demonstrated, as is required if the enzyme-nitrosyl complex is an intermediate in both nitrosation and denitrification reactions. The first evidence for exchange of 18O between H2(18)O and a nitrosation intermediate occurring after the enzyme-nitrosyl complex, presumably an enzyme-bound nitrosamine, has been obtained. The collective results are most consistent with denitrification N2O originating via attack of NO-2 on a coordinated nitrosyl, as proposed earlier (Averill, B. A., and Tiedje, J. M. (1982) FEBS Lett. 138, 8-11).     

Nitric Oxide-Dependent N-Nitrosating Activity of Rat Pleural Mesothelial Cells

Recent studies have demonstrated that nitric oxide (NO)-derived N-nitrosating agents may promote mutagenesis and carcinogenesis from the nitrosative deamination of DNA bases via the formation of nitrosamine intermediates. The objective of this study was to determine if pleural mesothelial cells (PMC) stimulated with proinflammatory cytokines could promote the N-nitrosation of a primary aromatic amine via the L-arginine-dependent formation of NO-derived N-nitrosating agents. N-nitrosating activity was determined by measuring the N-nitrosation of a model amine, 2,3-diaminonapthalene, to yield its fluorescent triazole (1 -naptho-2,3-triazole) derivative. Results show that specific combinations of TNF, IL-1, interferon gamma, and LPS significantly increased N-nitrosating activity. There was a significant positive correlation between nitrite plus nitrate and triazole production. Triazole formation was inhibited by N^G-nitro-L-arginine methyl ester, suggesting that triazole was derived from the L-arginine-dependent formation of NO. These data indicate that PMC have the capacity to promote the N-nitrosation of primary aromatic amines via the formation of NO.     

The chemistry of nitrosative stress induced by nitric oxide and reactive nitrogen oxide species. Putting perspective on stressful biological situations

This review addresses many of the chemical aspects of nitrosative stress mediated by N2O3. From a cellular perspective, N2O3 and the resulting reactive nitrogen oxide species target specific motifs such as thiols, lysine active sites, and zinc fingers and is dependant upon both the rates of production as well as consumption of NO and must be taken into account in order to access the nitrosative environment. Since production and consumption are integral parts of N2O3 generation, we predict that nitrosative stress occurs under specific conditions, such as chronic inflammation. In contrast to conditions of stress, nitrosative chemistry may also provide cellular protection through the regulation of critical signaling pathways. Therefore, a careful evaluation of the chemistry of nitrosation based upon specific experimental conditions may provide a better understanding of how the subtle balance between oxidative and nitrosative stress may be involved in the etiology and control of various disease processes.     

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.     

Distinction between nitrosating mechanisms within human cells and aqueous solution

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.     

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation.

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.     

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes

There is evidence that nitric oxide (NO) formation in adult cardiomyocytes stimulated with lipopolysaccharide (LPS) is not commensurate with iNOS levels. Tetrahydrobiopterin (BH(4)) is a key factor in the stabilization and NO production by iNOS homodimer. Thus we hypothesized that BH(4) is a limiting factor for NO production in adult cardiomyocytes in response to LPS and cytokines (TNF-alpha, IL-1, IFN-gamma alone, or mixed). It was verified that LPS and cytokines induced iNOS expression which did not translate into increased nitrite or [(14)C]citrulline production. This response coincided with defective BH(4) synthesis and low GTP cyclohydrolase activity. Furthermore, supplementation with BH(4) and ascorbate failed to increase iNOS activity. This effect was related to preferential accumulation of BH(2) rather than BH(4) in these cells. Uncoupled iNOS activity in stimulated cells was examined using mitochondrial aconitase activity as an endogenous marker of superoxide anion radical (O(2)(-)) formation, and found not to be significantly inhibited. 2-Hydroxyethidium also was not significantly increased. We conclude that adult cardiomyocytes are an unlikely source of NO and O(2)(-) in inflammatory conditions. This finding adds a new and unexpected layer of complexity to our understanding of the responses of the adult heart to inflammation.     

Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.     

Catalysis of nitrosyl transfer reactions by a dissimilatory nitrite reductase (cytochrome c,d1)

The dissimilatory nitrite reductase (cytochrome c,d1) from Pseudomonas aeruginosa was observed at pH 7.5 to catalyze nitrosyl transfer (nitrosation) between [15N]nitrite and several N-nucleophiles or H2 18O, with rate enhancement of the order of 10(8) relative to analogous chemical reactions. The reducing system (ascorbate, N,N,N',N'-tetramethylphenylenediamine) could reduce nitrite (but not NO) enzymatically and had essentially no direct chemical reactivity toward nitrite or NO. The N-nitrosations showed saturation kinetics with respect to the nucleophile and, while exhibiting Vmax values which varied by about 40-fold, nevertheless showed little or no dependence of Vmax on nucleophile pKa. The N-nitrosations and NO-2/H2O-18O exchange required the reducing system, whereas NO/H2O-18O exchange was inhibited by the reducing system. NO was not detected to serve as a nitrosyl donor to N-nucleophiles. These and other kinetic observations suggest that the enzymatic nitrosyl donor is an enzyme-bound species derived from reduced enzyme and one molecule of nitrite, possibly a heme-nitrosyl compound (E-FeII X NO+) for which there is precedence. Nitrosyl transfer to N-nucleophiles may occur within a ternary complex of enzyme, nitrite, and nucleophile. Catalysis of nitrosyl transfer by nitrite reductase represents a new class of enzymatic reactions and may present another example of electrophilic catalysis by a metal center. The nitrosyl donor trapped by these reactions is believed to represent an intermediate in the reduction of nitrite by cytochrome c,d1.     

Bacterial catalysis of nitrosation: involvement of the nar operon of Escherichia coli.

We have developed a rapid and sensitive fluorimetric method, based on the formation of a fluorescent product from nitrosation of 2,3-diaminonaphthalene, for measuring the ability of bacteria to catalyze nitrosation of amines. We have shown in Escherichia coli that nitrosation can be induced under anaerobic conditions by nitrite and nitrate, that formate is the most efficient electron donor for this reaction, and that nitrosation may be catalyzed by nitrate reductase (EC 1.7.99.4). The narG mutants defective in nitrate reductase do not catalyze nitrosation, and the fnr gene is essential for nitrosation. Induction by nitrite or nitrate of nitrosation, N2O production, and nitrate reductase activity all require the narL gene.     

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma (Ifn-gamma) or Ifn-gamma and lipopolysaccharide in bnl cl.2 murine embryonic liver cells: role of hydrogen peroxide

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils

We investigated the effects of the Th2-like cytokines IL-4 and IL-13 and of IL-10 on the induction of iNOS and NO production in rat eosinophils. Addition of mIL-4 to the eosinophil culture increased iNOS activity and nitrite production but did not improve the stimulatory effect of IFN-gamma and LPS. In contrast to eosinophils, addition of mIL-4 to macrophage cultures inhibited the iNOS expression and nitrite production induced by IFN-gamma plus LPS. Addition of mIL-13 to the eosinophil cultures did not significantly change iNOS activity and nitrite production in cells stimulated or not with IFN-gamma plus LPS. On the other hand, IL-13 inhibited iNOS activity in IFN-gamma plus LPS-stimulated macrophages. In the presence of IL-10, iNOS activity in non-stimulated eosinophil or macrophage cultures was not significantly altered, but the enzyme expression was inhibited in IFN-gamma plus LPS-stimulated eosinophils or macrophages. The production of nitrite by eosinophils stimulated by IFN-gamma plus LPS was inhibited by the presence of IL-10 in the medium. In conclusion, eosinophils might exhibit differential modulation of the L-arginine/iNOS pathway depending on the profile of Th2 cytokines produced during allergic diseases. IL-4 appears to be an important Th2 cytokine involved in the induction of the L-arginine/iNOS pathway in eosinophils.