Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.     

Differential susceptibility to nitric oxide-evoked apoptosis in human inflammatory cells

Apoptosis of neutrophils and their subsequent phagocytosis is critical to the successful resolution of inflammation. During inflammation, activated inflammatory cells generate reactive oxygen and nitrogen species, including nitric oxide (NO) and superoxide anion (O₂^??), which rapidly combine to generate peroxynitrite (ONOO^?). NO and ONOO^? are proapoptotic in human neutrophils. This study examines the effects of NO and ONOO^? on caspase activation and mitochondrial permeability in human neutrophils and determines the ability of these species to evoke apoptosis in human monocyte-derived macrophages (MDMs). NO or ONOO^? release from donor compounds was characterized by electrochemistry and electron paramagnetic resonance. Neutrophils and MDMs isolated from the peripheral blood of healthy volunteers were exposed to NO or ONOO^? before analysis of apoptosis by caspase activation, mitochondrial permeability, and annexin V binding. Both NO and ONOO^? induced apoptosis via rapid activation of caspases 2 and 3 in neutrophils. In contrast, only ONOO^? promoted apoptosis in MDMs, whereas a variety of NO donors were ineffective at inducing apoptosis in this cell type. We propose that human macrophages are refractory to NO-stimulated apoptosis in order that they persist long enough within the inflammatory focus to phagocytose apoptotic neutrophils, thereby ensuring successful resolution of inflammation.     

Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling

15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.     

Nitric oxide mediates iron release from ferritin

Nitric oxide (NO) synthesis by cytotoxic activated macrophages has been postulated to result in a progressive loss of iron from tumor target cells as well as inhibition of mitochondrial respiration and DNA synthesis. In the present study, the addition of an NO-generating agent, sodium nitroprusside, to the iron storage protein ferritin resulted in the release of iron from ferritin and the released iron-catalyzed lipid peroxidation. Hemoglobin, which binds NO, and superoxide anion, which reacts with NO, inhibited nitroprusside-dependent iron release from ferritin, thereby providing evidence that NO can mobilize iron from ferritin. These results suggest that NO generation in vivo could lead to the mobilization of iron from ferritin disrupting intracellular iron homeostasis and increasing the level of reactive oxygen species.     

Induction of a Feed Forward Pro-Apoptotic Mechanistic Loop by Nitric Oxide in a Human Breast Cancer Model

We have previously demonstrated that relatively high concentrations of NO [Nitric Oxide] as produced by activated macrophages induced apoptosis in the human breast cancer cell line, MDA-MB-468. More recently, we also demonstrated the importance of endogenous H₂O₂ in the regulation of growth in human breast cancer cells. In the present study we assessed the interplay between exogenously administered NO and the endogenously produced reactive oxygen species [ROS] in human breast cancer cells and evaluated the mechanism[s] in the induction of apoptosis. To this end we identified a novel mechanism by which NO down regulated endogenous hydrogen peroxide [H₂O₂] formation via the down-regulation of superoxide [O₂ ^.−] and the activation of catalase. We further demonstrated the existence of a feed forward mechanistic loop involving protein phosphatase 2A [PP2A] and its downstream substrate FOXO1 in the induction of apoptosis and the synthesis of catalase. We utilized gene silencing of PP2A, FOXO1 and catalase to assess their relative importance and key roles in NO mediated apoptosis. This study provides the potential for a therapeutic approach in treating breast cancer by targeted delivery of NO where NO donors and activators of downstream players could initiate a self sustaining apoptotic cascade in breast cancer cells.     

Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK

The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.     

Signaling and proapoptotic functions of transformed cell-derived reactive oxygen species

Transformed fibroblasts generate extracellular superoxide anions through the recently identified membrane-associated NADPH oxidase. These cell-derived superoxide anions exhibit signaling functions such as regulation of proliferation and maintenance of the transformed state. Their dismutation product hydrogen peroxide regulates the intracellular level of catalase, whose activity has been observed to be upregulated in certain transformed cells. After glutathione depletion, transformed cell-derived reactive oxygen species (ROS) exhibit apoptosis-inducing potential through the metal-catalyzed Haber-Weiss reaction. Moreover, transformed cell-derived ROS represent key elements for selective and efficient apoptosis induction by natural antitumor systems (such as fibroblasts, granulocytes and macrophages). These effector cells release peroxidase, which utilizes target cell-derived hydrogen peroxide for HOCl synthesis. In a second step, HOCl interacts with target cell-derived superoxide anions and forms apoptosis-inducing hydroxyl radicals. In a parallel signaling pathway, effector cell-derived NO interacts with target cell-derived superoxide anions and generates the apoptosis inducer peroxynitrite. Therefore, transformed cell-derived ROS determine transformed cells as selective targets for induction of apoptosis by these effector systems. It is therefore proposed that transformed cell derived ROS interact with associated cells to exhibit directed and specific signaling functions, some of which are beneficial and some of which can become detrimental to transformed cells.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Taurine chloramine produced from taurine under inflammation provides anti-inflammatory and cytoprotective effects

Taurine is one of the most abundant non-essential amino acid in mammals and has many physiological functions in the nervous, cardiovascular, renal, endocrine, and immune systems. Upon inflammation, taurine undergoes halogenation in phagocytes and is converted to taurine chloramine (TauCl) and taurine bromamine. In the activated neutrophils, TauCl is produced by reaction with hypochlorite (HOCl) generated by the halide-dependent myeloperoxidase system. TauCl is released from activated neutrophils following their apoptosis and inhibits the production of inflammatory mediators such as, superoxide anion, nitric oxide, tumor necrosis factor-α, interleukins, and prostaglandins in inflammatory cells at inflammatory tissues. Furthermore, TauCl increases the expressions of antioxidant proteins, such as heme oxygenase 1, peroxiredoxin, thioredoxin, glutathione peroxidase, and catalase in macrophages. Thus, a central role of TauCl produced by activated neutrophils is to trigger the resolution of inflammation and protect macrophages and surrounding tissues from being damaged by cytotoxic reactive oxygen metabolites overproduced during inflammation. This is achieved by attenuating further production of proinflammatory cytokines and reactive oxygen metabolites and also by increasing the levels of antioxidant proteins that are able to scavenge and diminish the production of cytotoxic oxygen metabolites. These findings suggest that TauCl released from activated neutrophils may be involved in the recovery processes of cells affected by inflammatory oxidative stresses and thus TauCl could be used as a potential physiological agent to control pathogenic symptoms of chronic inflammatory diseases.     

Activated macrophages direct apoptosis and suppress mitosis of mesangial cells

During inflammation in the glomerulus, the complement of resident myofibroblast-like mesangial cells is regulated by mitosis and apoptosis, but the cellular mechanisms controlling the size of mesangial cell populations have remained obscure. Prompted by studies of development, we sought evidence that macrophages regulate mesangial cell number. Rat bone marrow-derived macrophages primed with IFN-gamma then further activated in coculture with LPS or TNF-alpha elicited a 10-fold induction of rat mesangial cell apoptosis and complete suppression of mitosis, effects inhibitable by the NO synthase inhibitors L-monomethyl arginine and L-N(6)-(1-iminoethyl) lysine dihydrochloride. Complete dependence upon macrophage-derived NO was observed in comparable experiments employing activated bone marrow macrophages from wild-type and NO synthase 2(-/-) mice. Nevertheless, when mesangial cells were primed with IFN-gamma plus TNF-alpha, increased induction by activated macrophages of mesangial apoptosis exhibited a NO-independent element. The use of gld/gld macrophages excluded a role for Fas ligand in this residual kill, despite increased expression of Fas and increased susceptibility to soluble Fas ligand exhibited by cytokine-primed mesangial cells. Finally, activated macrophages isolated from the glomeruli of rats with nephrotoxic nephritis also induced apoptosis and suppressed mitosis in mesangial cells by an L-monomethyl arginine-inhibitable mechanism. These data demonstrate that activated macrophages, via the release of NO and other mediators, regulate mesangial cell populations in vitro and may therefore control the mesangial cell complement at inflamed sites.     

Nitric oxide production by Leishmania-infected macrophages and modulation by cytokines and prostaglandins

Nitric oxide (NO) produced by an inducible nitric oxide synthase (iNOS or NOS2) plays a major microbicidal role in murine macrophages and its importance is now emerging also in the dog and human models. In dogs we demonstrated that macrophages in vitro infected with Leishmania infantum produced NO, after stimulation with cytokine-enriched peripheral blood mononuclear cell supernatants. In addition, parasite killing was reduced by the NOS inhibitor L-NG monomethylarginine. On the contrary, canine blood monocytes before macrophage differentiation did not release NO, and their leishmanicidal activity was instead correlated with superoxide anion and interferon (IFN)-gamma production. In human macrophage cultures, after infection with Leishmania infantum, we showed both iNOS expression by immunofluorescence and western blotting and NO release by the Griess reaction for nitrites. Various cytokines and prostaglandins can differently modulate NO synthesis. In our experiments, stimulation by recombinant human IFN-gamma and bacterial lipopolysaccharide greatly enhanced iNOS expression and NO production in human macrophages. In addition, the prostaglandin E2 increased NO release in activated, Leishmania-infected human macrophages. These results are interesting in the light of a possible immunological or pharmacological regulation of NO synthesis and microbicidal functions of macrophages.     

Protein tyrosine nitration in cytokine-activated murine macrophages. Involvement of a peroxidase/nitrite pathway rather than peroxynitrite.

Peroxynitrite, formed in a rapid reaction of nitric oxide (NO) and superoxide anion radical (O(2)), is thought to mediate protein tyrosine nitration in various inflammatory and infectious diseases. However, a recent in vitro study indicated that peroxynitrite exhibits poor nitrating efficiency at biologically relevant steady-state concentrations (Pfeiffer, S., Schmidt, K., and Mayer, B. (2000) J. Biol. Chem. 275, 6346-6352). To investigate the molecular mechanism of protein tyrosine nitration in intact cells, murine RAW 264.7 macrophages were activated with immunological stimuli, causing inducible NO synthase expression (interferon-gamma in combination with either lipopolysaccharide or zymosan A), followed by the determination of protein-bound 3-nitrotyrosine levels and release of potential triggers of nitration (NO, O(2)*, H(2)O(2), peroxynitrite, and nitrite). Levels of 3-nitrotyrosine started to increase at 16-18 h and exhibited a maximum at 20-24 h post-stimulation. Formation of O(2) was maximal at 1-5 h and decreased to base line 5 h after stimulation. Release of NO peaked at approximately 6 and approximately 9 h after stimulation with interferon-gamma/lipopolysaccharide and interferon-gamma/zymosan A, respectively, followed by a rapid decline to base line within the next 4 h. NO formation resulted in accumulation of nitrite, which leveled off at about 50 microm 15 h post-stimulation. Significant release of peroxynitrite was detectable only upon treatment of cytokine-activated cells with phorbol 12-myristate-13-acetate, which led to a 2.2-fold increase in dihydrorhodamine oxidation without significantly increasing the levels of 3-nitrotyrosine. Tyrosine nitration was inhibited by azide and catalase and mimicked by incubation of unstimulated cells with nitrite. Together with the striking discrepancy in the time course of NO/O(2) release versus 3-nitrotyrosine formation, these results suggest that protein tyrosine nitration in activated macrophages is caused by a nitrite-dependent peroxidase reaction rather than peroxynitrite.     

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Sustained nitric oxide delivery delays nitric oxide-dependent apoptosis in macrophages: contribution to the physiological function of activated macrophages

Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN-gamma accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.     

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.     

Protection by nitric oxide against liver inflammatory injury in animals carrying a nitric oxide synthase-2 transgene.

The effect of pre-existent hepatic NO synthesis on liver injury induced by lipopolysaccharide was studied in animals carrying a nitric oxide synthase-2 (NOS-2) transgene under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. These animals expressed NOS-2 in liver cells under fasting conditions. Lipopolysaccharide-induced liver injury in D-galactosamine-conditioned mice, which enhanced notably the effect of the endotoxin on the liver, was impaired in animals expressing NOS-2. This protection against inflammatory liver damage was dependent on NO synthesis and was caused by an inhibition of nuclear factor kB (NF-kB) activity and an impairment of the synthesis of the proinflammatory cytokines tumor necrosis factor a and interleukin 1b. These data indicate that intrahepatic synthesis of NO protects liver by inhibiting the release of cascades of proinflammatory mediators and suggest a beneficial role for local delivery of NO in the control of liver injury.     

Decreased exhaled nitric oxide as a marker of postinsult immune paralysis.

Nitric oxide (NO) regulates neutrophil migration and alveolar macrophage functions such as cytokine synthesis and bacterial killing, both of which are impaired in immune paralysis associated with critical illness. The aim of this study was to determine whether NO is involved in immune paralysis and whether exhaled NO measurement could help to monitor pulmonary defenses. NO production (protein expression, enzyme activity, end products, and exhaled NO measurements) was assessed in rats after cecal ligation and puncture to induce a mild peritonitis (leading to approximately 20% mortality rate). An early and sustained decrease in exhaled NO was found after peritonitis (from 1 to 72 h) compared with healthy rats [median (25th-75th percentile), 1.5 parts per billion (ppb) (1.2-1.7) vs. 4.0 ppb (3.6-4.3), P < 0.05], despite increased NO synthase-2 and unchanged NO synthase-3 protein expression in lung tissue. NO synthase-2 activity was decreased in lung tissue. Nitrites and nitrates in supernatants of isolated alveolar macrophages decreased after peritonitis compared with healthy rats, and an inhibitory experiment suggested arginase overactivity in alveolar macrophages bypassing the NO substrate. Administration of the NO synthase-2 inhibitor aminoguanidine to healthy animals reproduced the decreased neutrophil migration toward alveolar spaces that was observed after peritonitis, but L-arginine administration after peritonitis failed to correct the defect of neutrophil emigration despite increasing exhaled NO compared with D-arginine administration [4.8 (3.9-5.7) vs. 1.6 (1.3-1.7) ppb, respectively, P < 0.05]. In conclusion, the decrease in exhaled NO observed after mild peritonitis could serve as a marker for lung immunodepression.