Role Of Reactive Nitrogen Intermediates Research Articles

Elucidating the role of reactive nitrogen intermediates in hetero-cyclization during hydrothermal liquefaction of food waste

Understanding the fate of nitrogen in food waste hydrothermal liquefaction.

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells.

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.

Enhancing effect of oxygen radical scavengers on murine macrophage anticryptococcal activity through production of nitric oxide.

We examined the roles of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) in interferon-gamma (IFN-gamma)-induced cryptococcostatic activity of murine peritoneal macrophages using N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of RNI synthesis, and superoxide dismutase (SOD) and catalase, oxygen radical scavengers. IFN-gamma-activated macrophages produced nitric oxide (NO) in a dose-dependent manner, as measured by increased nitrite concentration in the culture supernatant. IFN-gamma also enhanced the suppressive effect on cryptococcal growth in a similar dose-dependent manner. The induction of killing activity and NO production by an optimal dose of IFN-gamma (100 U/ml) was virtually suppressed by 500 microM L-NMMA. These results confirmed the importance of the RNI-mediated effector mechanism in anticryptococcal activity of macrophages. SOD and catalase significantly enhanced the cryptococcostatic activity of macrophages induced by a suboptimal dose of IFN-gamma (20 U/ml). The augmenting effect of these reagents was mediated by NO, since they potentiated the production of NO by macrophages and their effects were totally blocked by L-NMMA. Our results indicate that the IFN-gamma-induced anticryptococcal activity of macrophages is dependent mostly on RNI, and suggest that the ROI system down-regulates the effector mechanism for cryptococcostasis by suppressing the RNI system.

Roles of reactive nitrogen intermediates and transforming growth factor-β produced by immunosuppressive macrophages in the expression of suppressor activity against T cell proliferation induced by TCR stimulation

The suppressor activity of splenic macrophages induced by Mycobacterium intracellulare infection (MI-Mϕs) against T cell concanavalin A (Con A) mitogenesis is mediated by MI-Mϕ's mediators, such as reactive nitrogen intermediates (RNIs), phosphatidylserine, free fatty acids, prostaglandin E 2 and to a minor extent TGF-β. Here, we have compared the roles of RNIs and TGF-β in the expression of MI-Mϕ's suppressor activity against Con A mitogenesis and anti-CD3 monoclonal antibody (mAb)- and anti-CD28 mAb-induced mitogenesis (TCR signal-induced mitogenesis) of the target T cells, and have found the following. First, N G-monomethyl- l-arginine (NMMA) inhibited MI-Mϕ's suppressor activity against TCR signal-induced mitogenesis as well as Con A mitogenesis. Second, anti-TGF-β mAb weakly restored the MI-Mϕ-mediated suppression only in the case of Con A mitogenesis, under limited conditions, such as very low cell densities of MI-Mϕs. Third, the blocking effects of NMMA plus anti-TGF-β mAb were somewhat more prominent in the case of Con A mitogenesis than in the case of TCR signal-induced mitogenesis. Fourth, Con A- or TCR signal-stimulated MI-Mϕs secreted significant amounts of the latent TGF-β but not the active one. These findings indicate that RNIs, but not TGF-β, play important roles in the MI-Mϕ-mediated suppression of TCR signal-induced mitogenesis, as well as Con A mitogenesis, of the target T cells.

Role of reactive nitrogen intermediates and protein nitration during immune response against a rat histiocytoma

The ability of macrophages to produce reactive nitrogen species, particularly nitric oxide (NO) is correlated with an enhanced microbicidal or tumoricidal activity during pathogenic or tumoral invasion, respectively. NO reacts in water with oxygen and its reactive intermediates to yield, among others, nitrite and nitrate, which are relatively, stable anions. In this study, we show the varying concentrations of nitrite and nitrate present in different body fluids during AK-5 tumor growth and regression in Wistar rats. We have followed the tumor progression profile and the corresponding levels of nitrite and nitrate present in three major body compartments: the tumor mass; the serum which is the intermediary site; and the peritoneal compartment which is the priming ground for the macrophages. We are thus able to show that the status of the tumor has a direct correlation with macrophage activation and motility to different sites in the body. We also demonstrate after in vitro coculture, that the levels of nitrite and nitrate secreted by the macrophages correlate with their cytocidal capacity.

The inducible nitric oxide synthase locus confers protection against aerogenic challenge of both clinical and laboratory strains of Mycobacterium tuberculosis in mice.

Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosis infection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous, M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2(-/-) mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2(-/-) mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2(-/-) mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.

Nitric oxide and peroxynitrite in ozone-induced lung injury.

One of the hallmarks of the inflammatory response associated with tissue injury is the accumulation of macrophages at sites of damage. These cell types release proinflammatory cytokines and cytotoxic mediators to destroy invading pathogens and initiate wound repair. However, when produced in excessive amounts, these macrophage-derived mediators may actually contribute to tissue injury. This process involves both direct damage to target tissues and amplification of the inflammatory response. One group of macrophage-derived mediators of particular interest are reactive nitrogen intermediates including nitric oxide and peroxynitrite which have been implicated in tissue injury induced by a variety oftoxicants. Our laboratory has been investigating the role of reactive nitrogen intermediates in lung injury induced by oxidants such as ozone. Inhalation of ozone causes epithelial cell damage and Type II cell hyperplasia. This is associated with an accumulation of activated macrophages in the lower lungs which we have demonstrated contribute to toxicity. To analyze the role of macrophage-derived reactive nitrogen intermediates in ozone toxicity, we used transgenic mice lacking the gene for inducible nitric oxide synthase (NOSII). Treatment of wild type control animals with ozone (0.8 ppm) for 3 hr resulted in an increase in bronchoalveolar lavage (BAL) fluid protein reaching a maximum 24-48 hr after exposure. This was correlated with increased expression of NOSII protein and mRNA by alveolar macrophages and increased production of nitric oxide as well as peroxynitrite. Ozone inhalation also resulted in the appearance of nitrotyrosine residues in the lungs, an in vivo marker of peroxynitrite-induced damage. In contrast, in NOSII knockout mice, BAL protein was not increased demonstrating that these mice were protected from ozone-induced epithelial injury. Moreover, alveolar macrophages from the transgenic mice did not produce nitric oxide or peroxynitrite even after ozone inhalation. There was also no evidence for the formation of nitrotyrosine in lung tissue. These data indicate that ozone-induced lung injury is mediated by reactive nitrogen intermediates.

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mphis was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mphi anti-MAC functions. (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect. However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes (Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated. (3) ROI production by murine peritoneal mphis was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1-24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide

The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.

Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro.

This study was carried out to determine the role of reactive nitrogen intermediates in Trypanosoma cruzi infection. In vitro, splenocytes obtained during the acute phase of infection produced elevated amounts of nitric oxide (NO) that were correlated with the resistance or susceptibility of the animals. In vivo, the levels of NO2- plus NO3- in plasma during the later phase of infection were higher in C57BL/6 mice than in BALBL/c mice. The treatment of infected C57BL/6 mice with inhibitors of NO synthase increased parasitemia and mortality. Finally, we found that the NO donor drug S-nitroso-acetyl-penicillamine is able to kill trypomastigotes in vitro in the absence of any other cells, suggesting a direct NO-mediated killing of T. cruzi.

The role of reactive nitrogen intermediates in modulation of gametocyte infectivity of rodent malaria parasites

Direct feeding of Anopheles stephensi mosquitoes on mice infected with Plasmodium vinckei petteri showed that, during the periods of schizogony in the blood, the infectivity of gametocytes was markedly reduced. This could be prevented by prior injection of the L-arginine analogue, Nw-nitro-L-arginine (NwNLA) showing that the altered infectivity was due to reactive nitrogen intermediates (RNI). Similar effects on transmission of P. yoelii nigeriensis were demonstrated in vitro by membrane feeding of the mosquitoes. The in vitro reduction in infectivity could be reversed by injecting the L-arginine analogue either into the infected mouse donor of serum, or into the membrane feeding chamber. Elevated levels of TNF and IL-6 were demonstrated during the course of infection but did not correlate well with nitrogen radical activity. Similarly, direct measurements of NO2- and NO3- did not reflect the nitrogen radical activity revealed by addition of the specific L-arginine analogue.

Evaluation of nitrite production by human monocyte-derived macrophages

Reactive nitrogen intermediates are important in the anti-tumor and anti-microbial activities of rodent macrophages, but it is not known whether this is the case for human macrophages. In the present study, nitrite concentrations in vitro were used as an indicator of reactive nitrogen intermediate production by mouse, rat, and human macrophages. Human macrophages derived by culturing peripheral blood monocytes did not consistently produce detectable nitrite levels in response to any stimulus examined. Human macrophages were viable and metabolically active as indicated by the MTT assay, and their respiratory burst response to phorbol myristate acetate was increased following incubation with Interferon-γ, as expected for typical macrophages. In contrast, rat or mouse peritoneal macrophages produced nitrite concentrations of ∼20–100 μM in response to lipopolysaccharide, Interferon-γ, or both. These results demonstrate substantial differences in the production of nitrites by rodent and human macrophages. Because of the heterogeneity among macrophage populations, these findings may not be applicable to all human macrophage populations, but they suggest a need for caution in extrapolating from rodent studies regarding the role of reactive nitrogen intermediates in anti-tumor or anti-microbial functions of human macrophages.

Preliminary communication: possible role of reactive nitrogen intermediates in leucocyte-mediated sperm dysfunction.

This preliminary study was designed to examine whether nitric oxide, a reactive nitrogen intermediate produced by leukocyte metabolism of L-arginine, could reduce sperm motility. Increasing doses (10(-6)-10(-4) M) of the nitric oxide-generating drug, sodium nitroprusside (SNP) were added to motile sperm preparations and incubated over 20 hr. Quantitative sperm motility measurements were made using a computer-assisted motility analyzer in each treated sample and controls over this time period. The percentage of motile sperm, progressive motility, and concentration of motile cells were all significantly reduced with all doses of SNP. This effect was not observed in preparations treated with oxyhaemoglobin (HbO2). Mean path velocity was unaltered. Sperm viability in SNP treated sperm did not differ significantly from that of control sperm over the same time period. We conclude that further experiments are required to determine whether the production of nitric oxide/reactive nitrogen intermediates in vivo by activated leukocytes could be a contributory factor in the development of immunologic infertility.

Mechanisms involved in mycobacterial growth inhibition by gamma interferon-activated bone marrow macrophages: role of reactive nitrogen intermediates

Murine bone marrow-derived macrophages are able to inhibit the growth of Mycobacterium bovis after stimulation with recombinant gamma interferon. This antimycobacterial activity was inhibited by NG-monomethyl-L-arginine, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Furthermore, there was a complete lack of mycobacterial growth inhibition in a medium deficient in L-arginine. Nitrite is generated by gamma interferon-activated bone marrow-derived macrophages after infection with M. bovis, and a correlation between mycobacterial growth inhibition and nitrite production was observed. These results indicate that reactive nitrogen intermediates derived from L-arginine are crucially involved in macrophage antimycobacterial activity.

Mechanisms of macrophage‐mediated tumor cell killing: A comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor

The roles of tumor necrosis factor (TNF alpha) and reactive nitrogen intermediates (RNI) as effectors of macrophage-mediated tumor cell killing were investigated in a variety of tumor cell lines. Three TNF alpha-sensitive tumor targets were also susceptible to resting bone-marrow-derived mononuclear phagocytes (BMMP). This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha, indicating that TNF alpha is the major effector of macrophage-mediated killing of these targets. The other 21 tumor cell lines examined were resistant to TNF alpha but, in their large majority, were more or less susceptible to killing by interferon gamma (IFN gamma)- and Corynebacterium parvum (CP)-activated BMMP. Among the various analogues of L-arginine used to assess the role of L-arginine-derived RNI as mediators of macrophage tumoricidal activity, NG-monomethyl-L-arginine (NMMA) was most efficient in suppressing RNI secretion by activated macrophages. In some macrophage tumor-cell combinations, NMMA inhibited both the generation of RNI and the expression of tumoricidal activity in a dose-dependent manner, suggesting a central role for RNI as effectors. In other combinations, NMMA in concentrations that abolished secretion of RNI either affected tumor-cell killing only after its induction by IFN gamma, or not at all. The findings not only support the thesis that macrophages posses various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophage activation and the properties of the tumor-cell type.