scholarly journals Antimicrobial Actions of the Nadph Phagocyte Oxidase and Inducible Nitric Oxide Synthase in Experimental Salmonellosis. I. Effects on Microbial Killing by Activated Peritoneal Macrophages in Vitro

2000 ◽  
Vol 192 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Andrés Vazquez-Torres ◽  
Jessica Jones-Carson ◽  
Pietro Mastroeni ◽  
Harry Ischiropoulos ◽  
Ferric C. Fang
Keyword(s):  
Nitric Oxide ◽  
Antibacterial Activity ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Independent Effect ◽  
No Production ◽  
Bacterial Killing ◽  
Phagocyte Oxidase ◽  
Inducible Nitric Oxide

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox−/−, iNOS−/−, and doubly immunodeficient phox−/−iNOS−/− mice. The respiratory burst and NO radical (NO·) made distinct contributions to the anti-Salmonella activity of macrophages. NADPH oxidase–dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S. typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O2·−), hydrogen peroxide (H2O2), and peroxynitrite (ONOO−) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S. typhimurium. Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon γ appears to augment antibacterial activity predominantly by enhancing NO· production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 µg chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P < 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P < 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P < 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P < 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

scholarly journals Effect ofTityus serrulatusvenom on cytokine production and the activity of murine macrophages

2002 ◽  
Vol 11 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Vera L. Petricevich
Keyword(s):  
Cytokine Production ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
Dependent Manner ◽  
Immune Functions ◽  
Macrophage Activity ◽  
Ifn Γ

The purpose of this study was to investigate the effects ofTityus serrulatusvenom (TSV) on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2) and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed including anin vitromodel for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6) and interferon-γ (IFN-γ) were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-γ. Incubation of macrophages with TSV increased production of IL-6 and IFN-γ in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-γ. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functionsin vitro.

scholarly journals Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

2008 ◽  
Vol 295 (1) ◽  
pp. L96-L103 ◽  
Author(s):  
Viktor Brovkovych ◽  
Xiao-Pei Gao ◽  
Evan Ong ◽  
Svitlana Brovkovych ◽  
Marie-Luise Brennan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Bacterial Colonization ◽  
Inos Expression ◽  
No Production ◽  
Bacterial Killing ◽  
E Coli ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO−/−mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO−/−mice unexpectedly had improved survival compared with wild-type (WT) mice within 5–12 h after intraperitoneal E. coli challenge. Lungs of MPO−/−mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO−/−mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO−/−mice. Inhibition of iNOS in MPO−/−mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO−/−mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

scholarly journals Cholecystokinin Inhibits Inducible Nitric Oxide Synthase Expression by Lipopolysaccharide-Stimulated Peritoneal Macrophages

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Rafael Simone Saia ◽  
Fabíola Leslie Mestriner ◽  
Giuliana Bertozi ◽  
Fernando Queiróz Cunha ◽  
Evelin Capellari Cárnio
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Peritoneal Macrophages ◽  
Inos Expression ◽  
No Production ◽  
Camp Content ◽  
Systemic Complications ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Cholecystokinin (CCK) was first described as a gastrointestinal hormone. However, apart from its gastrointestinal effects, studies have described that CCK also plays immunoregulatory roles. Taking in account the involvement of inducible nitric oxide synthase- (iNOS-) derived NO in the sepsis context, the present study was undertaken to investigate the role of CCK on iNOS expression in LPS-activated peritoneal macrophages. Our results revealed that CCK reduces NO production and attenuates the iNOS mRNA expression and protein formation. Furthermore, CCK inhibited the nuclear factor- (NF-)κB pathway reducing IκBαdegradation and minor p65-dependent translocation to the nucleus. Moreover, CCK restored the intracellular cAMP content activating the protein kinase A (PKA) pathway, which resulted in a negative modulatory role on iNOS expression. In peritoneal macrophages, the CCK-1R expression, but not CCK-2R, was predominant and upregulated by LPS. The pharmacological studies confirmed that CCK-1R subtype is the major receptor responsible for the biological effects of CCK. These data suggest an anti-inflammatory role for the peptide CCK in modulating iNOS-derived NO synthesis, possibly controlling the macrophage activation through NF-κB, cAMP-PKA, and CCK-1R pathways. Based on these findings, CCK could be used as an adjuvant agent to modulate the inflammatory response and prevent systemic complications commonly found during sepsis.

Effect of Peganum Harmala Seeds Extract on Nitric Oxide in U937 Monocytes and Macrophages

2020 ◽  
Author(s):  
Nima Rahmati ◽  
Fatemeh Hajighasemi
Keyword(s):  
Nitric Oxide ◽  
Aqueous Extract ◽  
Calf Serum ◽  
Peritoneal Macrophages ◽  
No Production ◽  
Peganum Harmala ◽  
Logarithmic Growth Phase ◽  
Anti Inflammatory

Background and Aims: Nitric oxide (NO) has an essential role in inflammation and has been related to pathogenesis and the progress of numerous inflammatory-based diseases, including some cancers. Peganum harmala (P. harmala) is a medicinal plant used for the treatment of numerous diseases such as several infections. Also, anti-inflammatory effects of P. harmala extracts and its derivatives (harmaline and harmine) by suppressing myeloperoxidase, NO, and other mediators have been demonstrated in vivo. In this study, the effect of P. harmala seeds aqueous extract on NO production in U937 monocytic cells and peritoneal macrophages has been evaluated in vitro. Materials and Methods: U937 and mice peritoneal macrophages were cultured in Roswell Park Memorial institute-1640 with 10% fetal calf serum. Then, the cells at the logarithmic growth phase were incubated with different concentrations of aqueous extract of P. harmala seeds (0.1-1 mg/ml) for 24 hours. Next, NO production was assessed by the Griess method in the culture medium. Results: P. harmala seeds aqueous extract did not significantly affect lipopolysaccharide-induced NO production in U937 cells and peritoneal macrophages after 24 hours incubation time compared with untreated control cells. Conclusion: These results suggest that the anti-inflammatory effects of P. harmala may be mediated through NO-independent mechanism(s). However, further studies are warranted to define the P. harmala aqueous extract impact on NO expression in other related normal and cancerous cells.

Up-regulation of nitric oxide production by interferon-γ in cultured peritoneal macrophages from patients with cirrhosis

1999 ◽  
Vol 97 (4) ◽  
pp. 399-406 ◽  
Author(s):  
P. N. BORIES ◽  
B. CAMPILLO ◽  
E. SCHERMAN
Keyword(s):  
Nitric Oxide ◽  
Western Blot ◽  
Spontaneous Bacterial Peritonitis ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
No Production ◽  
Inos Protein ◽  
Bacterial Peritonitis ◽  
Cirrhotic Patients ◽  
Ifn Γ

We previously described a long-lasting overproduction of nitric oxide (NO) in cirrhotic patients with spontaneous bacterial peritonitis. The aim of the present study was to investigate the presence of the inducible NO pathway in peritoneal macrophages. Ascitic fluids were collected from 29 patients with cirrhosis, aged between 35 and 82 years. Peritoneal macrophages were isolated and cultured in the presence or absence of 1 μg/ml lipopolysaccharide and/or 500 units/ml interferon-γ (IFN-γ) for 6 days. NO production was measured as nitrate+nitrite (NOx), inducible NO synthase (iNOS) protein expression was analysed by immunocytochemistry and Western blot analysis using a specific anti-(human iNOS) antibody, and the catalytic activity of NOS was revealed by cytochemical staining for NADPH-dependent diaphorase. Cultured macrophages spontaneously released small amounts of NOx [median (10–90th percentile) of 18 separate experiments: 3.3 (0–8) μmol/l]. Addition of lipopolysaccharide alone or in combination with IFN-γ to the culture medium did not change the levels of NOx, while IFN-γ alone dramatically increased NO production [13.4 (3.5–28.3) μmol/l; P< 0.001]. Macrophages were stimulated by IFN-γ to a greater extent in patients with recent spontaneous bacterial peritonitis (n = 13) than in those in a stable clinical condition (n = 18) [19.8 (10.5–30.1) and 10.0 (3.2–14.5) μmol/l respectively; P< 0.001]. Macrophages freshly isolated or stimulated with IFN-γ expressed iNOS protein, as shown by Western blot and immunocytochemical analysis, and stained for NADPH diaphorase. Our findings demonstrate the presence of iNOS protein in peritoneal macrophages from cirrhotic patients. The role of IFN-γ appears to be a determinant for the up-regulation of NO production, particularly under conditions of infection. Therefore peritoneal macrophages producing large amounts of NO at the site of infection may contribute to maintaining splanchnic vasodilation in these patients.

scholarly journals Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1184-1195 ◽  
Author(s):  
JB Weinberg ◽  
MA Misukonis ◽  
PJ Shami ◽  
SN Mason ◽  
DL Sauls ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Peritoneal Macrophages ◽  
Mononuclear Phagocytes ◽  
Inos Mrna ◽  
No Production ◽  
Human Monocytes ◽  
Low Levels ◽  
Nitrite Nitrate ◽  
Oxide Synthase

Abstract Nitric oxide (NO) is produced by numerous different cell types, and it is an important regulator and mediator of many processes including smooth muscle relaxation, neurotransmission, and murine macrophage- mediated cytotoxicity for microbes and tumor cells. Although murine macrophages produce NO readily after activation, human monocytes and tissue macrophages have been reported to produce only low levels of NO in vitro. The purpose of this study was to determine if stimulated human mononuclear phagocytes produce inducible nitric oxide synthase (iNOS) mRNA, protein, and enzymatic activity. By reverse transcriptase- polymerase chain reaction (RT-PCR) analysis, we show that human monocytes can be induced to express iNOS mRNA after treatment with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By immunofluorescence and immunoblot analyses, we show monocytes and peritoneal macrophages contain detectable levels of iNOS antigen after stimulations with cytokines in vitro. Control monocytes or those cultured with LPS and/or various cytokines have low levels of NOS functional activity as measured by the ability of cell extracts to convert L-arginine to L-citrulline, and they produce low levels of the NO catabolites nitrite and nitrate. Peritoneal macrophages have significantly enhanced nitrite/nitrate production and NOS activity after treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate production and NOS activity are not altered by the treatments. Monocytes cultured with various live or heat-killed bacteria, fungi, or human immunodeficiency virus (HIV)-1 do not produce high levels of nitrite/nitrate. Antibodies against transforming growth factor-beta (TGF-beta), a factor known to inhibit iNOS expression and NO production in mouse macrophages, do not enhance NO production in human monocytes or macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity, is undetectable in freshly isolated or cultured human monocytes and peritoneal macrophages. However, replenishment of intracellular levels of tetrahydrobiopterin by culture with the cell- permeable, nontoxic precursor sepiapterin does not enhance the abilities of the human mononuclear phagocytes to produce NO in vitro. Mixing experiments show no evidence of a functional NOS inhibitor in human mononuclear phagocytes. Thus, we demonstrate that human mononuclear phagocytes can produce iNOS mRNA and protein, and (despite this) their abilities to generate NO are very low.

scholarly journals Kinetic modelling of the nitric oxide gradient generated in vitro by adherent cells expressing inducible nitric oxide synthase

1996 ◽  
Vol 314 (1) ◽  
pp. 109-113 ◽  
Author(s):  
Michel LAURENT ◽  
Michel LEPOIVRE ◽  
Jean-Pierre TENU
Keyword(s):  
Nitric Oxide ◽  
Kinetic Modelling ◽  
No Synthase ◽  
Target Cells ◽  
No Production ◽  
Adherent Cells ◽  
True Rate ◽  
Neutral Radical ◽  
Inducible Nitric Oxide

Inducible nitric oxide (NO) synthase produces a long-lasting NO flux which can exert cytotoxic effects on target cells. A prerequisite for the understanding of the molecular basis of NO action is quantitative data on the availability of this small neutral radical molecule at both the spatial and temporal levels. The limits of NO availability depend on the respective rates of NO production, diffusion and autoxidation by molecular oxygen. Kinetic modelling of these processes has been performed for a widely used experimental system consisting of a monolayer of adherent cells cultured in vitro for hours in unstirred culture medium. It appears that: (i) the maximal NO concentration in the culture is in the immediate vicinity of the monolayer, where target cells will sediment; (ii) the steady-state NO concentration in this area is lower than 4 to 5 μM; and (iii) measurements of nitrite/nitrate or citrulline accumulation in the bulk cell medium culture during a given time period significantly underestimate (by a factor of up to 3 to 4) the true rate of NO synthesis at the level of the producer cell. This rate can be, nevertheless, easily estimated from the rate of production of the stable NO synthase products.

scholarly journals Role of cytokines and nitric oxide in the induction of tuberculostatic macrophage functions

2000 ◽  
Vol 9 (6) ◽  
pp. 261-269 ◽  
Author(s):  
Vera L. Petricevich ◽  
Rosely C. B. Alves
Keyword(s):  
Nitric Oxide ◽  
Culture Media ◽  
Peritoneal Macrophages ◽  
Interferon Γ ◽  
Enzyme Linked Immunosorbent Assay ◽  
No Production ◽  
Phenotypic Differences ◽  
Ifn Γ ◽  
Necrosis Factor

The aim of this study was to determine phenotypic differences when BCG invades macrophages. Bacilli prepared from the same BCG primary seed, but produced in different culture media, were analysed with respect to the ability to stimulate macrophages and the susceptibility to treatment with cytokines and nitric oxide (NO). Tumour necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, interleukin-6 (IL-6) and interferon γ (IFN-γ) were assayed by enzyme-linked immunosorbent assay (ELISA), whereas NO levels were detected by Griess colorimetric reactions in the culture supernatant of macrophages incubated with IFN-γ , TNF or NO and subsequently exposed to either BCG-I or BCG-S. We found that BCG-I and BCGS bacilli showed different ability to simulate peritoneal macrophages. Similar levels of IL-6 were detected in stimulated macrophages with lysate from two BCG samples. The highest levels of TNF and IFN-γ were observed in macrophages treated with BCG-S and BCG-I, respectively. The highest levels of NO were observed in cultures stimulated for 48h with BCG-S. We also found a different susceptibility of the bacilli to ex ogenous treatm ent w ith IFN-γ and TNF which were capable of killing 60 and 70% of both bacilli, whereas NO was capable of killing about 98 and 47% of BCG-I and BCG-S, respectively. The amount of bacilli proportionally decreased with IFN-γ and TNF, suggesting a cytokine-related cytotox ic effect. Moreover, NO also decreased the viable number of bacilli. Interestingly, NO levels of peritoneal macrophages were significantly increased after cytokine treatment. This indicates that the treatment of macrophages with cytokines markedly reduced bacilli number and presented effects on NO production. The results obtained here emphasize the importance of adequate stimulation for guaranteeing efficient killing of bacilli. In this particular case, the IFN-γ and TNF were involved in the activation of macrophage bactericidal activity.

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