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 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.

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 Acquired interferon γ responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression

Gut ◽  
1999 ◽  
Vol 44 (5) ◽  
pp. 659-665 ◽  
Author(s):  
A M Chavez ◽  
M J Morin ◽  
N Unno ◽  
M P Fink ◽  
R A Hodin
Keyword(s):  
Nitric Oxide ◽  
Cell Differentiation ◽  
Interferon Γ ◽  
Intestinal Barrier Function ◽  
Inos Mrna ◽  
Pyrrolidine Dithiocarbamate ◽  
No Production ◽  
Mrna Induction ◽  
Ifn Γ ◽  
Inos Induction

BACKGROUNDImpairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells.AIMSTo examine iNOS induction by IFN-γ in vitro as a function of enterocyte differentiation.METHODSPreconfluent and postconfluent Caco-2 cells were treated with IFN-γ in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-γ receptor mRNA and protein levels were determined.RESULTSiNOS mRNA induction by IFN-γ occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-κB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation—that is, in postconfluent Caco-2 cells. This increased IFN-γ responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-γ receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-γ receptor levels as a function of postconfluent growth.CONCLUSIONSWith regard to iNOS mRNA induction, IFN-γ responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-γ receptors.

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 Marylosides A-G, Norcycloartane Glycosides from Leaves of Cymbidium Great Flower ‘Marylaurencin’

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2504
Author(s):  
Tatsuro Yoneyama ◽  
Kanako Iseki ◽  
Masaaki Noji ◽  
Hiroshi Imagawa ◽  
Toshihiro Hashimoto ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Interferon Γ ◽  
Crystallographic Analysis ◽  
Chemical Degradation ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Moderate Activity ◽  
X Ray ◽  
Ifn Γ

Seven novel norcycloartane glycosides, maryloside A–G (1–7), were isolated from the leaves of Cymbidium Great Flower ‘Marylaurencin’, along with a known norcycloartane glycoside, cymbidoside (8). These structures were determined on the basis of mainly NMR experiments as well as chemical degradation and X-ray crystallographic analysis. The isolated compounds (1–6 and 8) were evaluated for the inhibitory activity on lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-stimulated nitric oxide (NO) production in RAW 264.7 cells. Consequently, 1 and 3 exhibited moderate activity.

scholarly journals Fasting-induced intestinal apoptosis is mediated by inducible nitric oxide synthase and interferon-γ in rat

2010 ◽  
Vol 298 (6) ◽  
pp. G916-G926 ◽  
Author(s):  
Junta Ito ◽  
Hiroyuki Uchida ◽  
Takayuki Yokote ◽  
Kazuo Ohtake ◽  
Jun Kobayashi
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Interferon Γ ◽  
No Synthase ◽  
Histological Evaluation ◽  
No Production ◽  
Ros Production ◽  
Nitrite Production ◽  
Ifn Γ

Nitric oxide (NO) is associated with intestinal apoptosis in health and disease. This study aimed to investigate the role of intestinal NO in the regulation of apoptosis during fasting in rats. Male Wistar rats were divided into two groups and subcutaneously injected with saline (SA) or aminoguanidine (AG), followed by fasting for 24, 48, 60, and 72 h. At each time point, the jejunum was subjected to histological evaluation for enterocyte apoptosis by histomorphometric assessment and TUNEL analysis. We performed immunohistochemistry for inducible NO synthase (iNOS) expression in the jejunum and measured tissue nitrite levels using HPLC and 8-hydroxydeoxyguanosine adduct using ELISA, indicative of endogenous NO production and reactive oxygen species (ROS) production, respectively. Jejunal transcriptional levels of iNOS, neuronal NO synthase (nNOS), and interferon-γ (IFN-γ) were also determined by RT-PCR. Fasting caused significant jejunal mucosal atrophy due to attenuated cell proliferation and enhanced apoptosis with increase in iNOS transcription, its protein expression in intestinal epithelial cells (IEC), and jejunal nitrite levels. However, AG treatment histologically reduced apoptosis with inhibition of fasting-induced iNOS transcription, protein expression, and nitrite production. We also observed fasting-induced ROS production and subsequent IFN-γ transcription, which were all inhibited by AG treatment. Furthermore, we observed reduced transcriptional levels of nNOS, known to suppress iNOS activation physiologically. These results suggest that fasting-induced iNOS activation in IEC may induce apoptosis mediators such as IFN-γ via a ROS-mediated mechanism and also a possible role of nNOS in the regulation of iNOS activity in fasting-induced apoptosis.

Nitric Oxide Production in Peritoneal Macrophages from Peritoneal Dialysis Patients with Bacterial Peritonitis

1999 ◽  
Vol 19 (2_suppl) ◽  
pp. 378-383 ◽  
Author(s):  
Jörg Plum ◽  
Maryam Mirza Tabatabaei ◽  
Mohammad Reza Lordnejad ◽  
Olga Pipinika ◽  
Peter Razeghi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Peritoneal Macrophages ◽  
Cell Protein ◽  
Inos Mrna ◽  
No Production ◽  
Blood Monocytes ◽  
Rt Pcr ◽  
Bacterial Peritonitis ◽  
Protein Versus

Nitric oxide (NO) is produced by various cell types, and it is an important mediator in many biological processes, including macrophage-mediated cellular host defense. The relevance and amount of NO production in peritonitis during peritoneal dialysis (PD) treatment is still not clear. We studied whether human peritoneal macrophages (PMΦ) isolated from healthy PD patients or PD patients with peritonitis showed different spontaneous or lipo-polysaccharide (LPS)linterferon gamma (IFN-y) -induced NO production (LPS, 1 nglmL 10 μglmL; IFN-y, 101000 UlmL; incubation between 6 -48 hours; measured by Griess reagent). Results were compared with human blood monocytes (HBM) isolated from buffy coats. Inducible nitric oxide synthetase (iNOS) mRNA expression was looked for in PMΦ by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, plasma (P) and peritoneal dialysate effluent (D) nitrite concentrations were measured in vivo. The dialysate-to-plasma ratio (DIP) of nitrite concentration was inverse in the case of peritonitis compared to infection-free patients (peritonitis DIP = 1.3, non peritonitis DIP = 0.4; p < 0.01). PMΦ from peritonitis patients produced higher amounts of NO than did those from infection-free patients (0.040 ± 0.044 nmol per microgram cell protein versus 0.018 ± 0.015 nmol per microgram cell protein, p < 0.05). NO release could not be further enhanced by stimulation with LPS plus IFN-y (1 ng/mL, 250 UlmL, respectively). However, NO production in PMΦ from infection-free patients increased during in vitro stimulation (0.044 ± 0.031 nmol per microgram cell protein versus 0.018± 0.015 nmol per microgram cell protein, p < 0.01). An increase of iNOS mRNA expression could be demonstrated by RT-PCR. Blood monocytes from healthy donors also increased NO release during cytokine stimulation (0.032± 0.015 nmol per microgram cell protein versus 0.019 ± 0.009 nmol per microgram cell protein, p < 0.05). Our results indicate that significant amounts of NO are released intraperitoneally in the case of bacterial peritonitis. PMΦ represent a site of NO production, though the absolute amounts released in vitro are only moderate. NO production can be induced in PMΦ and HBM by LPSIIFN-y stimulation in vitro.

Microbial epidemiology in cirrhotic patients with spontaneous bacterial peritonitis in Vienna

2013 ◽  
Vol 51 (05) ◽  
Author(s):  
P Schwabl ◽  
K Soucek ◽  
T Bucsics ◽  
M Mandorfer ◽  
A Blacky ◽  
...  
Keyword(s):  
Spontaneous Bacterial Peritonitis ◽  
Bacterial Peritonitis ◽  
Cirrhotic Patients
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