iNOS Induction In Macrophages Research Articles

Farnesyl thiosalicylic acid prevents iNOS induction triggered by lipopolysaccharide via suppression of iNOS mRNA transcription in murine macrophages

Inducible nitric oxide synthase (iNOS) is a molecule critical for the development of inflammation-associated disorders. Its induction should be tightly controlled in order to maintain cellular homeostasis. Upon lipopolysaccharide (LPS) stimulation, iNOS, in most settings, is induced by the activation of inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) signaling. Farnesyl thiosalicylic acid (FTS), a synthetic small molecule that is considered to detach Ras from the inner cell membrane, has been shown to exhibit numerous anti-inflammatory functions. However, it remains unclear whether and how it affects iNOS induction in macrophages. The present study addressed this issue in cultured macrophages and endotoxemic mice. Results showed that FTS pretreatment significantly prevented LPS-induced increases in iNOS protein and mRNA expression levels in murine cultured macrophages, which were confirmed in organs in vivo from endotoxemic mice, such as the liver and lung. Mechanistic studies revealed that FTS pretreatment did not affect IκB-α degradation and NF-κB activation in LPS-treated macrophages. The nuclear transport of the active NF-κB was also not affected by FTS. But FTS pretreatment reduced the binding of NF-κB to its DNA elements, and reduced NF-κB bindings to iNOS promoter inside LPS-treated macrophages. Finally, our results showed that FST pretreatment increased mouse survival rate compared to LPS alone treatment. Taken together, these results indicate that FTS attenuates iNOS induction in macrophages likely through inhibition of iNOS mRNA transcription, providing further insight into the molecular mechanism of action of FTS in inflammatory disorder therapy.

Fibrin deposition following bile duct injury limits fibrosis through an αMβ2-dependent mechanism

AbstractCoagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMβ2, we tested the hypothesis that disruption of the fibrin(ogen)-αMβ2 interaction in Fibγ390-396A mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)–induced liver fibrosis increased in Fibγ390-396A mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ390-396A mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ390-396A mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ390-396A mice. Fibrin(ogen)-αMβ2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMβ2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)–integrin-αMβ2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.

Smad2 and Smad3 are redundantly essential for the suppression of iNOS synthesis in macrophages by regulating IRF3 and STAT1 pathways

Although transforming growth factor (TGF)-β1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo, compared with wild-type (WT) macrophages. However, TGF-β1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-β1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-β production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-β- and IFN-γ-induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-β-STAT1 pathway.

Eosinophils Preserve Parasitic Nematode Larvae by Regulating Local Immunity

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Induction of iNOS byChlamydophila pneumoniaerequires MyD88-dependent activation of JNK

Innate immune cells produce NO via inducible NO synthase (iNOS) in response to certain infections or upon stimulation with cytokines such as IFN-gamma and TNF. NO plays an important role in host defense against intracellular bacteria including Chlamydophila pneumoniae as a result of its microbicidal activity. In MyD88-deficient mice, which succumb to C. pneumoniae infection, iNOS induction is impaired 6 days postinfection, although pulmonary levels of IFN-gamma and TNF are elevated as in wild-type mice at this time-point. Here, we demonstrate that induction of iNOS in macrophages upon C. pneumoniae infection is controlled by MyD88 via two pathways: NF-kappaB activation and phosphorylation of the MAPK JNK, which leads to the nuclear translocation of c-Jun, one of the two components of the AP-1 complex. In addition, phosphorylation of STAT1 and expression of IFN regulatory factor 1 (IRF-1) were delayed in the absence of MyD88 after C. pneumoniae infection but not after IFN-gamma stimulation. Taken together, our data show that for optimal induction of iNOS during C. pneumoniae infection, the concerted action of the MyD88-dependent transcription factors NF-kappaB and AP-1 and of the MyD88-independent transcription factors phosphorylated STAT1 and IRF-1 is required.

MKP‐1 inhibits iNOS expression during the innate immune response to LPS

Nitric oxide (NO) is a potent antimicrobial substance and a powerful vasodilator. Inducible NO synthase (iNOS) is responsible for the substantial NO production that occurs with microbial infection. Induction of iNOS in macrophages plays a critical role for the eradication of intracellular pathogens. Moreover, the excessive production of NO by iNOS has also been implicated in the pathogenesis of septic shock syndrome. Previously, we have demonstrated that mice deficient in mitogen‐activated protein kinase phosphatase (Mkp)‐1 exhibit exaggerated inflammatory responses and rapidly succumb to lipopolysaccharides (LPS). In response to LPS, Mkp‐1−/− mice produce greater amounts of inflammatory cytokines and NO than wild type mice, and exhibit severe hypotension. To understand the molecular basis for the increases in NO production, we studied the role of Mkp‐1 in the regulation of iNOS expression. We found that LPS challenge elicited a stronger iNOS induction in Mkp‐1 knockout mice than in wild type mice. Likewise, LPS treatment also resulted in greater iNOS expression in Mkp‐1−/− macrophages than in wild type cells. Both accelerated gene transcription and enhanced mRNA stability contribute to the increases in iNOS expression in LPS‐stimulated Mkp‐1−/− macrophages. We found that two transcription factors known to mediate iNOS induction, Stat1 and IRF1, were more potently activated by LPS in Mkp‐1−/− macrophages than in wild type cells, explaining the accelerated iNOS gene transcription in the Mkp‐1−/− cells. Our results suggest that augmentation of MKP‐1 expression may be a novel treatment for patients suffering from sepsis and septic shock.

Induction of nitric oxide synthase by rotavirus enterotoxin NSP4: implication for rotavirus pathogenicity

Rotavirus non-structural protein (NSP) 4 can induce aqueous secretion in the gastrointestinal tract of neonatal mice through activation of an age- and Ca(2+)-dependent plasma membrane anion permeability. Accumulating evidence suggests that nitric oxide (NO) plays a role in the modulation of aqueous secretion and the barrier function of intestinal cells. This study investigated transcriptional changes in inducible NO synthase (iNOS), an enzyme responsible for NO production, after rotavirus infection in mice and after treatment of intestinal cells with NSP4. Diarrhoea was observed in 5-day-old CD-1 mice from days 1 to 3 after inoculation with 10(7) focus-forming units of different rotavirus strains. Ileal iNOS mRNA expression was induced as early as 6 h post-inoculation, before the onset of clinical diarrhoea in infected mice, and was upregulated during the course of rotavirus-induced diarrhoea. Ex vivo treatment of ilea excised from CD-1 suckling mice with NSP4 resulted in upregulation of ileal iNOS mRNA expression within 4 h. Furthermore, NSP4 was able to induce iNOS expression and NO production in murine peritoneal macrophages and RAW264.7 cells. The specificity of NSP4 inducibility was confirmed by the inhibitory effect of anti-NSP4 serum. Using a series of truncated NSP4s, the domain responsible for iNOS induction in macrophages was mapped to the reported enterotoxin domain, aa 109-135. Thus, rotavirus infection induces ileal iNOS expression in vivo and rotavirus NSP4 also induces iNOS expression in the ileum and macrophages. Together, these findings suggest that NO plays a role in rotavirus-induced diarrhoea.

Molecular and Metabolic Evidence for the Restricted Expression of Inducible Nitric Oxide Synthase in Healing Wounds

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.

Inhibitors of Protein Phosphatase 1 and 2A Differentially Regulate the Expression of Inducible Nitric-oxide Synthase in Rat Astrocytes and Macrophages

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-kappaB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-alpha also depends on the activation of NF-kappaB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-kappaB and production of TNF-alpha in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-kappaB and production of TNF-alpha. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-kappaB activation; however, activation of NF-kappaB is not sufficient for the induction of iNOS in macrophages and that apart from NF-kappaB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-kappaB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.

Nitric oxide production by peritoneal macrophages of cirrhotic rats: A host response against bacterial peritonitis

BACKGROUND & AIMS: Patients and rats with cirrhosis and ascites are prone to develop peritonitis. The aim of this study was to assess whether peritoneal macrophages of cirrhotic rats without peritoneal infection produce nitric oxide and express inducible NO synthase (iNOS). METHODS: NO2- accumulation produced by macrophages from control rats and cirrhotic rats with ascites was determined. iNOS messenger RNA and protein expression were analyzed by Northern and Western blot and immunocytochemical analysis. The in vivo effects of inhibiting iNOS were investigated by giving the specific iNOS inhibitor L-N-(1- iminoethyl)-lysine (L-NIL) or sterile saline to 9 and 7 cirrhotic rats with ascites, respectively. RESULTS: Cirrhotic macrophages produced NO2- that was around fourfold greater than that of control macrophages after 30 hours in culture. Northern and Western blot and immunocytochemical analysis showed the presence of iNOS messenger RNA and protein in macrophages of cirrhotic rats. Ascites cultures were positive in all rats administered L-NIL and negative in those administered saline. CONCLUSIONS: Macrophages of cirrhotic rats produce NO and express iNOS messenger RNA and protein, and these changes are not a consequence of overt bacterial infection. Because iNOS inhibition results in peritoneal infection, these results suggest that iNOS induction in macrophages of cirrhotic rats is a host defense response to prevent bacterial peritonitis. (Gastroenterology 1997 Jun;112(6):2056-64)

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.

Serine and Cysteine Proteinase Inhibitors Prevent Nitric Oxide Production by Activated Macrophages by Interfering with Transcription of the Inducible NO Synthase Gene

The objective of this study was to ascertain the mechanism by which serine and cysteine proteinase inhibitors interfere with production of NO by LPS-activated rat alveolar macrophages. Macrophages were incubated in the presence of LPS + test agent for 24 hr. Culture media were analyzed for NO − X accumulation, harvested cells were assayed for iNOS activity, and cellular RNA was extracted for determination of iNOS mRNA by Northern blot analysis. TPCK, TLCK, calpain inhibitor 1 (CPI-1) and calpain inhibitor 2 (CPI-2) each inhibited NO − X production and inducible iNOS expression in a concentration-dependent manner at 1-100 μM. TPCK and CPI-1 were about 10-fold more potent than TLCK and CPI-2, respectively. These data suggested that a chymotrypsin-like serine or cysteine proteinase is required for the LPS-inducible expression of the iNOS gene, perhaps by mechanisms involving activation of transcription factor NF-κB. Accordingly, a potent inhibitor of NF-κB activation whose action is attributed to inhibition of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) was tested. Z-IE(O- t-Bu)A-Leucinal abolished NO − X production and inducible iNOS expression at 1 μM and showed over 50% inhibition at 10 nM. These observations indicate that inhibitors of MPC interfere with iNOS induction and provide strong evidence that MPC functions importantly in iNOS induction in macrophages.

NF-κB and transcriptional control of renal epithelial-inducible nitric oxide synthase

Expression of the inducible isoform of nitric oxide synthase (iNOS) is subject to strict tissue specific transcriptional control. Recently, the NF-kappa B/Rel family of transcription factors, and particularly c-rel, was shown to mediate bacterial lipopolysaccharide (LPS) induction of iNOS in macrophages. Since LPS is only a weak inducer of iNOS in most nonimmune cells, we investigated the role of NF-kappa B in the regulation of iNOS expression in mouse renal epithelial cells. We report that LPS activates NF-kappa B in renal epithelium, but that this is not sufficient for induction of iNOS activity. The NF-kappa B complexes activated by LPS in renal epithelium differ from those in macrophages in that they lack c-rel, which may explain the absence of iNOS induction in renal epithelium. Conversely, LPS and interferon-gamma (IFN) synergize to induce renal epithelial iNOS. Functional iNOS promoter analysis indicate that this synergistic induction requires NF-kappa B. We conclude that NF-kappa B is necessary but not sufficient for the induction of renal epithelial iNOS expression, and that in contrast to macrophages, c-rel does not appear to play a major role in the regulation of renal epithelial iNOS.