[Effect of the antioxidants on NO-dependent induction of heme oxigenase 1 gene in U937 monocytes].

Previously it was shown that thiol antioxidants are potent inhibitors of the NO-dependent induction of heme oxygenase 1 (HOX-1) gene. However, the mechanism of HOX-1 gene down-regulation by thiol antioxidants and underlying signaling pathway remain unclear. In this study we have examined, whether the scavenging of reactive oxygen and reactive nitrogen species (ROS and RNS) is the major cause for thiol-mediated suppression of the HOX-1 induction by NO. Further, to identify the ROS family members implicated in the HOX-1 induction, we also exposed cells to various non-thiol antioxidants: dimethyl sulfoxide, dimetylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. A partial inhibition of HOX-1 induction occurred in the presence of non-polar hydroxyl radical scavengers, dimethyl sulfoxide and dimetylthiourea. The other non-thiol antioxidants were ineffective towards HOX-1 expression. Then, in order to determine, whether RNS scavenging is implicated in the HOX-1 down-regulation by thiol antioxidants, we took advantage of the capacity of suboptimal concentrations of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide) to oxidize NO to nitrosating species. We showed that simultaneous cell treatment with NO donor and PTIO significantly enhanced the rate of the HOX-1 gene NO-dependent induction indicating that RNS are mediators of HOX-1 gene transcriptional activation. Thiol antioxidants completely suppressed PTIO stimulatory action. These findings imply that inhibitory action of thiol antioxidants is mediated by RNS scavenging. The study provides an approach for pharmacologycal modulation of cell response to NO and its derivatives through the use of antioxidants.

Effects of hepatocellular iron imbalance on nitric oxide and reactive oxygen intermediates production in a model of sepsis.

BACKGROUND/AIMS: In mammals iron homeostasis is most important, as imbalance of iron such as iron overload may lead to severe diseases. Recently, it has been shown that the iron regulatory protein-1 is partially controlled by nitric oxide and reactive oxygen intermediates, molecules frequently seen in inflammatory events. The aim of the present study was to investigate the effects of impaired iron homeostasis on the interaction of nitric oxide, and reactive oxygen intermediate formation in hepatocytes in a model of acute inflammation. METHODS: Hepatocytes isolated from Corynebacterium parvum (C parvum)-injected rats were used to examine the formation of nitrogen and oxygen intermediates by iron deprivation and iron overload in the presence of lipopolysaccharide. In addition, we investigated the RNA binding and aconitase activity of iron regulatory protein-1. RESULTS: In the present study we show that iron overload in lipopolysaccharide-treated C. parvum-primed hepatocytes downregulated the RNA binding of iron regulatory protein-1 and aconitase activity. Subsequently, we observed a reduced formation of nitrite/nitrate and S-nitrosothiols but an increased production of reactive oxygen species, and hepatocellular damage. Moreover, the addition of iron to cell cultures caused a further increase in cellular damage, a drop in the cellular glutathione pool, and an increase in peroxynitrite and hydroxyl-like radicals. In contrast, addition of deferoxamine (an iron chelator) to lipopolysaccharide-treated C. parvum-primed hepatocytes protected cells by stabilizing the GSH content, maintaining the nitric oxide formation, and by reducing Fenton oxidants. CONCLUSIONS: Our results show that the antioxidative effects of iron chelators prevent the formation of toxic Fenton oxidants in severe inflammatory events, which should be considered in the treatment of disorders characterized by an iron imbalance.

Down-regulation of iron regulatory protein 1 gene expression by nitric oxide.

Iron regulatory protein 1 (IRP1) is an RNA binding protein that posttranscriptionally modulates the expression of mRNAs coding for proteins involved in iron metabolism. It has long been held that its RNA binding activity is regulated posttranslationally by the insertion/extrusion of a 4Fe-4S cluster, without changes in IRP1 levels. However, the question of a possible regulation of the expression of this protein has remained open. In the present study we analyzed the modulation of IRP1 expression in murine macrophages. We showed that activation by IFN-gamma and/or lipopolysaccharide, which induces IRP1 RNA binding activity via nitric oxide (NO), results simultaneously in a reduction in IRP1 protein levels, as determined by Western blot analyses. IRP1 expression decreased time-dependently to about 40% of control levels after 16 h. Down-regulation of IRP1 protein levels was correlated with the amount of NO produced and was partially abolished by the NO synthase (NOS) inhibitor N-monomethyl-l-arginine. No changes in IRP1 levels could be detected in stimulated peritoneal macrophages from NOS2 knockout (NOS2(-/-)) mice, unlike wild-type mice. Converse modulation of IRP1 RNA binding activity and IRP1 levels could be reproduced by exogenous NO and also was observed in nonmacrophage cells cocultured with NO-producing macrophages. We also analyzed IRP1 mRNA levels by Northern blotting and found a decrease in IRP1 mRNA expression after stimulation with IFN-gamma plus lipopolysaccharide, which was abrogated in the presence of N-monomethyl-l-arginine. This is evidence that IRP1 is regulated by a physiological stimulus other than posttranslationally.

Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2).

The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)

Intermittent hemophagocytic lymphohistiocytosis is a regular feature of lysinuric protein intolerance.

We describe 4 cases of lysinuric protein intolerance, which all fulfilled the diagnostic criteria for hemophagocytic lymphohistiocytosis. Mature histiocytes and neutrophil precursors participated in hemophagocytosis in the bone marrow. Moreover, serum levels of ferritin and lactate dehydrogenase were elevated, hypercytokinemia was present, and soluble interleukin-2 receptor levels were increased up to 18.6-fold. The diagnosis of lysinuric protein intolerance should therefore be considered in any patient presenting with hemophagocytic lymphohistiocytosis.

Thioredoxin activation of iron regulatory proteins. Redox regulation of RNA binding after exposure to nitric oxide.

Iron regulatory proteins (IRP1 and IRP2) are redox-sensitive RNA-binding proteins that modulate the expression of several genes encoding key proteins of iron metabolism. IRP1 can also exist as an aconitase containing a [4Fe-4S] cluster bound to three cysteines at the active site. We previously showed that biosynthesis of nitric oxide (NO) induces the transition of IRP1 from aconitase to apoprotein able to bind RNA. This switch is also observed when cytosolic extracts are exposed to NO donors. However, the activation of IRP1 under these conditions is far from maximal. In this study we examined the capacity of physiological reducing systems to cooperate with NO in the activation of IRP1. Cytosolic extracts from the macrophage cell line RAW 264.7 or purified IRP1 were incubated with NO donors and subsequently exposed to glutathione or to thioredoxin (Trx), a strong protein disulfide reductase. Trx was the most effective, inducing a 2-6-fold enhancement of the RNA binding activity of NO-treated IRP1. Furthermore, the effect of NO on IRP1 from cytosolic extracts was abolished in the presence of anti-Trx antibodies. We also studied the combined effect of NO and Trx on IRP2, which exhibits constitutive RNA binding activity. We observed an inhibition of IRP2 activity following exposure to NO donors which was restored by Trx. Collectively, these results point to a crucial role of Trx as a modulator of IRP activity in situations of NO production.

Converse modulation of IRP1 and IRP2 by immunological stimuli in murine RAW 264.7 macrophages.

Iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins that control iron metabolism in mammalian cells. Both IRPs bind to specific sequences called iron-responsive elements (IREs) located in the 3' or 5' untranslated regions of several mRNAs, in particular mRNA encoding ferritin and transferrin receptor. In this study, we followed in parallel the in vivo regulation of the two IRPs in physiologically stimulated macrophages. We show that stimulation of mouse RAW 264.7 macrophage-like cells increased IRP1 IRE binding activity 4-fold, whereas IRP2 activity decreased 2-fold 8 h after interferon-gamma/lipopolysaccharide treatment. Decrease in IRP2 was not due to nitric oxide (NO) production and did not require de novo protein synthesis. Our data therefore indicate that the two IRPs can be conversely regulated in response to the same stimulus. In addition, the effect of endogenously produced NO on IRP1 was further characterized in an activated macrophage/target cell system. We show that NO acts as an intercellular signal to increase IRP1 activity in adjacent cells. As the effect was detectable within 1 h and did not require de novo protein synthesis, this result supports a direct action of NO on IRP1.

Inhibitors of ADP-ribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation.

In murine macrophages, inducible NO synthase II (iNOS or NOS-II) is induced at the transcriptional level by IFN-gamma, alone or synergistically with LPS. We investigated the possible role of reactions of ADP-ribosylation in triggering the signaling pathways involved in NOS-II gene expression. Stimulation with IFN-gamma and/or LPS of RAW 264.7 macrophages, transiently transfected with the NOS-II promoter, was inhibited by ADP-ribosylation inhibitors, indicating that they interfered with the signal(s) responsible for NOS-II gene transcription. We therefore explored the effect of these inhibitors on the activity of IRF-1 and NF kappa B transcription factors known to be involved in NOS-II induction by IFN-gamma and LPS. No effect was observed on IRF-1 activation. However, NF kappa B binding to its target sequence diminished and transfection experiments with an NF kappa B-driven reporter plasmid demonstrating that ADP-ribosylation inhibitors suppressed NF kappa B-dependent promoter activity. These results provide evidence that a step involving ADP-ribosylation is required to activate NF kappa B-mediated gene transcription.

Redox modulation of iron regulatory proteins by peroxynitrite.

Expression of several proteins of higher eukaryotes is post-transcriptionally regulated by interaction of iron-responsive elements (IREs) on their mRNAs and iron regulatory proteins (IRP1 and IRP2). IRP1 is a redox-sensitive iron-sulfur protein whose regulatory activity is modulated by iron depletion, synthesis of nitric oxide, or oxidative stress. IRP2 is closely related to IRP1, but it does not possess a [Fe-S] cluster. IRP2 is also regulated by intracellular iron level, but it is assumed that regulation is achieved by accelerated turn-over. In this report, the effect of peroxynitrite, a strong oxidant produced when nitric oxide and O-2 are biosynthesized simultaneously, on the RNA binding activity of IRP1 and IRP2 was investigated in vitro. Macrophage cytosolic extracts were exposed directly to a bolus addition of peroxynitrite or to SIN-1, which releases a continuous flux of peroxynitrite. Under these two experimental conditions, IRP1 lost its aconitase activity but did not gain increased capacity to bind IRE. However, addition of low amounts of the disulfide-reducing agent 2-ME during the binding assay revealed formation of a complex between IRP1 and IRE. Substrates of aconitase, which bind to the cluster of IRP1, prevented this effect, pointing to the [Fe-S] cluster as the target of peroxynitrite. Moreover, single mutation of the redox active Cys437 precluded oxidation of human recombinant IRP1 by SIN-1. Collectively, these results imply that peroxynitrite predisposes IRP1 to bind IREs under a suitable reducing environment. It is assumed that in addition to disrupting the cluster peroxynitrite also promotes disulfide bridge(s) between proximal cysteine residues in the vicinity of the IRE-binding domain, in particular Cys437. When exposed to peroxynitrite, IRP2 lost its spontaneous IRE binding activity, which was restored by further exposure to 2-mercaptoethanol, thus showing that peroxynitrite can also regulate IRP2 by a post-translational event.

Interplay between NO and [Fe-S] clusters: relevance to biological systems.

In mammalian cells, nitric oxide (NO) synthesis results in the inactivation of several mitochondrial iron-sulfur enzymes involved in ATP synthesis that correlates with the appearance of complexes of the [(NO)2Fe(SR)2] type detectable by electron paramagnetic resonance spectroscopy. More specifically, the activity of two [Fe-S] enzymes was followed during the course of NO synthase expression:mitochondrial aconitase, which catalyzes citrate:isocitrate conversion in the Krebs cycle, and cytoplasmic aconitase, or iron regulatory protein (IRP), a trans-regulator that controls expression at the posttranscriptional level of proteins involved in iron metabolism. In response to physiological stimuli, the synthesis of NO leads to inhibition of enzymatic activities of both mitochondrial and cytoplasmic aconitases, whereas the RNA binding activity of IRP is increased. Coordination of the diffusible gas NO with [Fe-S] clusters is thought to result in impairment of metabolic functions. Here it is proposed that the interplay between NO (or some NO-derived molecule) and [Fe-S] clusters at critical catalytic or allosteric sites is crucial in the response to environmental signals within cells.

[Nitric oxide and macrophages]. / Monoxyde d'azote et macrophages.

Nitric oxide (NO) is a gaseous radical enzymatically produced from L-arginine by virtually every cell. This versatile molecule is involved in a variety of biological functions including defense against pathogens. Micro-organisms whose development is inhibited by NO include fungi, bacteria, protozoa, helminths and viruses. In murine macrophages, a high output NO synthase (NOS II) is regulated transcriptionally by cytokines and microbial products. In the past few years, investigators have identified many other cell types expressing NOS II. However, in human monocytes/macrophages, the existence of the L-arginine-NO pathway has long been questioned. Recent findings and new developments in this respect are commented in this short review.

Effects of inhibitors of ADP-ribosylation on macrophage activation.

Nitric oxide (NO) is a potent mediator involved in many biological functions including macrophage cytotoxicity and non-specific immunity against parasites, bacteria and viruses. Murine macrophages possess the capacity to express the inducible NO synthase (iNOS) which is not constitutively expressed but induced at the transcriptional level by interferon gamma (IFN-gamma) alone or synergistically with LPS. We have investigated the possible role of ADP-ribosylation reactions in the signaling pathway involved in NO synthase induction, since ADP-ribosylation has been reported to be involved in the expression of certain IFN-gamma and LPS-inducible genes. We found that inhibitors of ADP-ribosylation inhibited nitrite synthesis in RAW 264.7 macrophages after stimulation by IFN-gamma and LPS. These ADP-ribosylation inhibitors acted by preventing NO synthase mRNA induction, without inhibiting NO synthase enzyme activity. IRF-1, a transcription factor induced and activated by IFN-gamma was recently shown to be involved in iNOS induction. We showed that inhibitors of ADP-ribosylation had no effect on IFN-gamma-mediated mRNA induction of IRF-1 nor on its activation and binding to its target sequence in the iNOS gene. In addition, the inhibitors failed to impair the IFN-gamma-mediated antiviral activity against VSV virus. Since induction by IFN-gamma of IRF-1 and induction of the antiviral state proceed through the JAK/STAT signalling pathway, our results imply that ADP-ribosylation reactions are not involved in triggering this pathway. Although the precise mechanism requires further investigation, our results indicate that ADP-ribosylation is a crucial step restricted to the signalling pathway which leads to iNOS mRNA induction, as well as TNF and MHC class II induction during macrophage activation.

Differential expression of inducible NO synthase in two murine macrophage cell lines.

Although primary macrophages and most murine macrophage cell lines such as RAW 264.7 cells respond to interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) by producing large amounts of nitrite, i.e. the oxidation product of nitric oxide (NO) produced by inducible NO synthase (iNOS), other cell lines like P388.D1 cells do not produce significant amounts. To gain insight into the signalling pathway that leads to the induction of iNOS activity, we compared iNOS expression in RAW 264.7 and P388.D1 cells. We showed that IFN-gamma binds to each cell line with a similar affinity. Furthermore, no differences in iNOS gene structure were detectable by Southern blot analysis. Even though no significant nitrite secretion was found in the supernatant of P388:D1 cells stimulated with IFN-gamma and/or LPS, iNOS mRNA expression was induced. In addition, IFN-gamma induced the interferon regulatory factor-1 (IRF-1) gene and activated the binding of this factor to its target sequence in the iNOS gene. This binding was recently shown to be necessary for iNOS expression. However, in P388.D1 cells, we were unable to detect the corresponding iNOS protein. These results indicate a deficiency in P388.D1 cells which appears to be restricted to the signalling pathway controlling iNOS protein synthesis. This deficiency does not affect the overall IFN-gamma biological response, but rather a convergent post-transcriptional step common to IFN-gamma and LPS.

Modulation by nitric oxide of metalloprotein regulatory activities.

In many cells, a nitric oxide (NO) synthase inducible by immunological stimuli produces a sustained flow of NO that lasts a long time. NO is a short-lived molecule but it is a diffusible ligand believed to be capable of reaching distal target sites. Further, several lines of evidence indicate that cysteine-rich motifs of metal-binding proteins, as well as redox-sensitive metal clusters of metalloproteins, are natural sensors of bioradicals like NO. In metalloregulatory proteins, metals are often conveniently located at binding sites and bound to cysteine residues. Accordingly, disruption of the metal-thiolate polymetallic clusters should trigger significant remodelling of the protein structure involved in regulation. We can therefore postulate that the nitrosation reaction occurring at metal centres or cysteine-rich motifs will preclude correct binding to regulatory sites. Several examples are given of metalloregulatory proteins whose metal is bound to thiols and may then become sensitive to NO. Recent observations indicate that in response to NO synthesis, iron regulatory protein, a eukaryotic bifunctional [Fe-S] protein, switches from acting as aconitase to being an RNA-binding regulator, and we suggest that the interplay between NO or a NO-derived molecule and metal clusters at critical allosteric sites may be a crucial component of the cellular response to environmental stress.

Canine visceral leishmaniasis: successful chemotherapy induces macrophage antileishmanial activity via the L-arginine nitric oxide pathway.

Following successful chemotherapy in canine visceral leishmaniasis, monocyte-derived macrophages can induce antileishmanial activity via a gamma interferon-dependent mechanism in the presence of autologous lymphocytes. The killing of leishmania correlated with the induction of the NO synthase pathway, because it correlated with the generation of nitrogen derivative production and was abrogated in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of the NO synthase pathway. The level of L-citrulline in serum, which was produced after activation of the NO synthase pathway, was markedly enhanced in dogs receiving successful chemotherapy. Taken together, these data indicate that following successful chemotherapy of visceral leishmaniasis, leishmania parasites are killed by macrophages activated by gamma interferon-producing lymphocytes via an NO-dependent mechanism.

Modulation of iron regulatory protein functions. Further insights into the role of nitrogen- and oxygen-derived reactive species.

Iron regulatory protein (IRP) is a cytosolic bifunctional [Fe-S] protein which exhibits aconitase activity or binds iron responsive elements (IREs) in untranslated regions of specific mRNA. The modulators of these activities are the intracellular concentration of iron and, as recently described, NO synthase activity. In this study, we attempted to establish in in vitro experiments whether peroxynitrite (ONOO-, the product of the reaction between NO and O2-.), as well as oxygen-derived radicals (O2-. and H2O2) and various NO donors, allow IRP to bind IREs using cytosol extract of macrophagelike RAW 264.7 cells. Neither the addition of a bolus of ONOO- or H2O2 nor O2-. generation significantly affected IRE binding even though they inhibited its aconitase activity. Moreover, we show that 3-morpholinosydnonimine (SIN-1), a chemical which releases both NO and O2-. enhanced IRE binding activity of IRP only in the presence of superoxide dismutase (SOD). S-Nitrosothiols and the NONOate sper/NO plus gluthathione (GSH) activated IRE binding by IRP whereas oxyhemoglobin prevented enhancement of this binding by SIN-1/SOD and sper/NO plus GSH. cis-Aconitate, substrate, also abolished the effect of SIN-1/SOD on IRE binding by IRP. These results imply that neither O2-. nor ONOO- can convert [4Fe-4S] IRP into IRE-binding protein but rather suggest that an active redox form of NO converts IRP into its IRE binding form by targeting the [Fe-S] cluster.

[NO, ambivalent effector of non-specific immunity and of inflammation]. / NO, effecteur ambivalent de l'immunité non spécifique et de l'inflammation.

Nitric oxide produced from the amino acid L-arginine is a short-lived free radical produced by many types of cells for a variety of biological functions, including defence against a range of pathogens. NO synthesis via NOS-2 is deeply intertwined in the cytokine network. Thus, expression of NOS-2 is induced by IFN-gamma, TNF and IL-1 as well as microbial products whereas IL-4, IL-10 and TGF-beta down-regulate its synthesis. In spite of this tight regulation, excessive production of NO as a result of immunological stimulation via NOS-2 could have potential toxic effects on hosts. Indeed, large amount of nitric oxide (NO) are produced at sites of inflammation through the action of NOS-2. The role of NO in inflammation is unclear and may depend on the balance between NO and O2-. The somewhat paradoxical effects of NO might indeed be explained by its various chemical forms. Besides, understanding the regulation and function of NOS-2 is likely to lead to therapeutic approaches to treat a number of diseases.

Infusion of large quantities of autologous blood monocyte-derived macrophages in two cancer patients did not induce increased concentration of IL-6, TNF-alpha, soluble CD14 and nitrate in blood plasma.

In an attempt to increase the number of macrophages available for reinfusion in immunotherapy trials, GM-CSF was injected in vivo to mobilize circulating blood monocytes in 2 cancer patients. Subsequently mononuclear cells were collected by apheresis, cultured in the presence of GM-CSF and activated with IFN-gamma. This procedure resulted in the harvesting of 1.3 to 3.1 x 10(9) (mean 2 x 10(9)) macrophages per apheresis, product which was very well tolerated at autologous reinfusion. These infusions did not induce increased levels of TNF-alpha, IL-6, soluble CD14 nor nitrates in blood plasma (or urine). The lack of TNF-alpha and IL-6 release in blood plasma could explain the good tolerance of these infusions. No in vivo anti-tumoural activity of these high numbers of infused macrophages could be observed.

Interleukin-4 stimulates cGMP production by IFN-gamma-activated human monocytes. Involvement of the nitric oxide synthase pathway.

Resting human blood monocytes from some donors were found to produce a small amount of 3'-5' guanine cyclic monophosphate (cGMP) in response to interleukin 4 (IL-4). A much higher response was observed when monocytes were preincubated with interferon (IFN-gamma), which alone was ineffective. Preincubation of monocytes with IL-4 led, in contrast, to their subsequent incapacity to generate cGMP in response to IL-4. The accumulation of cGMP induced by IL-4 in IFN-gamma preincubated monocytes was dose-dependent and peaked about 15 min after its addition. It was inhibited in the presence of NG-mono-methyl-L-arginine (L-NMMA), an inhibitor of the nitric oxide synthase pathway. This suppressive effect of L-NMMA was reverted by an excess of L- but not of D-arginine. Accumulation of cGMP was significantly reduced by addition of soluble guanylyl cyclase inhibitors, such as LY83583 [correction of LY83853] and methylene blue, but was not impaired in the presence of EGTA, suggesting that the pathway involved is calcium independent. In addition, IL-4 induced an increased secretion of nitrite by monocytes, that was potentiated by IFN-gamma and inhibited by L-NMMA. Taken together, these results suggest that the sequential exposure of monocytes to IFN-gamma and IL-4 elicits the release of NO from L-arginine, which in turn is capable to stimulate soluble guanylyl cyclase.