Differential regulation of cytokine production by nitric oxide

Nitric oxide (NO) has recently been identified as a potent and pleiotropic intracellular mediator produced by and acting on many cells of the body. Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, there is very little information on the role of this mediator in modulating T-cell-dependent cytokine production. In this study we show that physiological levels of NO (either produced by activated macrophages or by the addition of exogenous NO donors) can selectively down-regulate interleukin-3 (IL-3) production by spleen cells from contact-sensitized mice, while leaving IL-2 activity unaffected. Thus NO may have an important role as an immunomodulatory as well as effector molecule in the immune system.     

Regulation of cytokine production by eicosanoids and nitric oxide

Cytokines are widely regarded as regulatory molecules of inflammatory and immune reactions. Nevertheless, the details of functioning of the complex cytokine network are not yet fully understood. Recent data indicate that eicosanoids, primarily the products of the inducible form of cyclooxygenase (COX-2), are involved in the regulation of cytokine production. We have shown that prostaglandins of E series are no longer only suppressor molecules but they selectively up- or down-regulate the cytokine production. Similarly, nitric oxide (NO) generated in activated immune cells by inducible isoform of nitric oxide synthase (iNOS), is considered to be an immunoregulatory molecule. In this article we present a new concept of interactions between cytokines, eicosanoids (prostaglandins and leukotrienes) and NO. Finally, the impact of these molecules on the regulation of the immune system is discussed.     

Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma

Oxidative stress contributes to airway inflammation and exhaled hydrogen peroxide (H2O2) and nitric oxide (NO) are elevated in asthmatic patients. We determined the concentrations of expired H2O2 and NO in 116 asthmatic (72 stable steroid-naive, 30 stable steroid-treated, and 14 severe steroid-treated unstable patients) and in 35 healthy subjects, and studied the relation between exhaled H2O2, NO, FEV1, airway responsiveness, and eosinophils in induced sputum. Both exhaled H2O2 and NO levels were elevated in steroid-naive asthmatic patients compared with normal subjects (0.72 +/- 0.06 versus 0.27 +/- 0.04 microM and 29 +/- 1.9 versus 6.5 +/- 0. 32 ppb, respectively; p < 0.001) and were reduced in stable steroid-treated patients (0.43 +/- 0.08 microM, p < 0.05, and 9.9 +/- 0.97 ppb, p < 0.001). In unstable steroid-treated asthmatics, however, H2O2 levels were increased, but exhaled NO levels were low (0.78 +/- 0.16 microM and 6.7 +/- 1.0 ppb, respectively). There was a correlation between expired H2O2, sputum eosinophils and airway hyperresponsiveness (methacholine PC20). Exhaled NO also correlated with sputum eosinophils, but not with airway hyperresponsiveness. Our findings indicate that measurement of expired H2O2 and NO in asthmatic patients provides complementary data for monitoring of disease activity.     

Alteration of the cytokine phenotype in an experimental lung granuloma model by inhibiting nitric oxide

Pulmonary granulomatous inflammation modulated by IFN-gamma and IL-12 is also associated with augmented inducible nitric oxide synthase (NOS II). To address the role of increased nitric oxide synthesis in this model, mice received daily i.p. injections of NG-nitro-L-arginine-methyl ester (L-NAME; 8 mg/kg) during both the 2-wk immunization period with purified protein-derivative (PPD) and the subsequent lung challenge with PPD-coated Sepharose beads. Other groups of animals received saline, L-NAME or NG-nitro-D-arginine-methyl ester (D-NAME; 8 mg/kg) during the pulmonary embolization period and not the PPD sensitization period. On day 4 post-PPD bead challenge, PCR analysis of the whole lung revealed that NOS II expression appeared to be similar in both of the L-NAME treatment protocols. L-NAME-treated mice in both dosing protocols had lung lesions that were significantly larger than granuloma lesions measured in mice that received saline or D-NAME. The enlarged lesions from L-NAME-treated mice contained markedly greater numbers of neutrophils and eosinophils. Equivalent numbers of PPD-activated dispersed cells from whole lungs of L-NAME-treated mice produced significantly higher levels of IL-4 and IL-10 and smaller amounts of IL-12 and IFN-gamma compared with similar lung cultures derived from control or D-NAME-treated mice. Levels of C-C chemokines such as monocyte chemoattractant protein-1 (MCP-1), C10, and macrophage inflammatory protein-1alpha (MIP-1alpha) were also significantly elevated in lung cultures from L-NAME-treated mice compared with controls. Thus, nitric oxide regulates the size and cellular composition of the Th1-type lung granuloma, possibly through its effects on the cytokine and chemokine profile associated with this lesion.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H2O2) and leukotriene B4 (LTB4) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated. 2. At a concentration of 100 microM, NAC significantly (P < 0.01) suppressed the accumulation of H2O2 in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml[-1])- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 microM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 microM and above, NAC augmented significantly the level of LTB4 in the supernatants of OZ- and fMLP-stimulated neutrophils (P < 0.01 and P < 0.05, respectively) and OZ-stimulated macrophages (P < 0.05 at 10 microM, P < 0.01 at 100 microM NAC). 3. NAC (100 microM) caused a significant (P < 0.01) reduction in the quantity of measurable H2O2 when incubated with exogenous H2O2 concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB4 when incubated with exogenous LTB4. 4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H2O2 had no significant effect on LTB4 production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H2O2 to H2O and O2, caused a pronounced, statistically significant (P < 0.01) increase in the levels of LTB4 measured in the supernatants of OZ- and fMLP-stimulated neutrophils. 5. H2O2 (12.5 microM and 25 microM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB4 (P = 0.051 and P < 0.05 at 12.5 microM and 25 microM, respectively) that was inhibited by NAC (100 microM) but not by catalase (400 u ml[-1]). 6. In conclusion, the anti-oxidant drug, NAC, increases LTB4 production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H2O2. LTB4 undergoes a H2O2-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB4, suggesting that NAC may increase LTB4 production by blocking the H2O2-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.     

Modulation of the mitochondrial permeability transition by nitric oxide

The influence of nitric oxide on mitochondrial permeability transition (MPT) phenomenon was studied. NO was generated by photolysis of S-nitroso-N-acetylcysteine, AcCys(NO), with green light (lambda = 550 nm). Two distinct effects of nitric oxide on rat liver mitochondria were identified. First, NO accelerated an onset of swelling in Ca2(+)-loaded mitochondria in a cyclosporin-A-sensitive manner acting as an inducer of permeability transition. This was, apparently, a result of irreversible alteration of mitochondrial function accompanying the inhibition of respiratory chain in the presence of calcium. Formation of ESR-visible iron-sulfur dinitrosyl complexes (g = 2.041) could also contribute to the irreversible changes resulting in MPT induction. Second, NO changed significantly the response of mitochondria to Ca2+/phosphate-induced MPT, acting as a regulator of permeability transition. In this case the action of nitric oxide led to division of the mitochondria into two subpopulations: one which underwent the rapid permeability transition and another in which the MPT was inhibited. The effect of NO on Ca2+/Pi-induced MPT was transient and resulted from reversible inhibition of cytochrome oxidase followed by the changes in transmembrane potential and Ca2+ distribution. The characteristic time of duration of these NO modulated effects depended on nitric oxide as well as on oxygen concentrations. With increasing NO at fixed oxygen concentrations, this time levelled off to reach a maximum value which was inversely related to the oxygen concentration. It is concluded that under physiological condition the duration of reversible NO effects on mitochondrial function could be determined by oxygen concentration.     

Effect of nitric oxide, prostacyclin, and thromboxane on the vasospastic action of hydrogen peroxide on human umbilical artery

During the years 1991-1994, the Norwegian Arthroplasty Register recorded 1,324 primary hip arthroplasties implanted with the Boneloc cement. We have compared the survival until revision due to aseptic loosening for charnley (n 955) and Exeter (n 172) prostheses. The Boneloc cemented hips were also compared with high viscosity cemented hips implanted during the same period. In the Boneloc cemented group, the estimated probability of survival at 4.5 years of a Charnley femoral component was 74% and for an Exeter femoral component 97% (p < 0.0001). Using a Cox regression model with adjustment for age, gender, type of cement, systemic antibiotic and stratified for diagnosis, an 8 times higher risk of revision was found in Boneloc cemented Charnley femoral components than in Exeter femoral components (p < 0.0001). For the acetabular components, the difference between the Charnley and Exeter components with Boneloc cement was not statistically significant. In both the Charnley and the Exeter prostheses, the high viscosity cemented components had significantly better survival than the Boneloc cemented components. The Cox regression model showed that a Boneloc cemented Charnley femoral component had a 14 times higher risk of revision than a high viscosity cemented component (p < 0.0001), and for Exeter femoral components a 7 times higher revision risk was found in the Boneloc cemented components (p = 0.003). Our results confirm the previously reported inferior results of Charnley prostheses implanted with Boneloc cement and inferior results of Boneloc cemented Exeter prostheses as well, but less pronounced than for Charnley prostheses.     

L-arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric oxide synthase-dependent mechanism

The hip abductor muscles are considered important for gait and biomechanics of the hip joint; however, their specific function has not been defined precisely. The intensity of magnetic resonance imaging signals in skeletal muscle has been reported to increase immediately after exercise. Making use of this phenomenon, we evaluated the hip abductor muscles. Magnetic resonance imaging was performed after isometric exercise of the hip abductor in three positions (20 degrees of abduction, neutral, and 20 degrees of adduction). The abduction force of the hip was measured with a dynamometer, and electromyographic measurements were made simultaneously for the same hip positions. Additionally, magnetic resonance imaging was performed after one-legged stance. As the hip was more adducted, the signal intensity increased on the scans. The values for muscle force, as evaluated with the dynamometer and integrated electromyography, also supported the results. The increase in signal intensity of the gluteus minimus at 20 degrees of abduction and after one-legged stance was significantly greater than that of the gluteus medius (p < 0.0001 and p < 0.0001, respectively). The results of this study indicate that the gluteus minimus muscle, along with the gluteus medius, plays an important role in hip abduction, gait, and stabilization of the pelvis.     

Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Nitric oxide synthase inhibition by haem oxygenase decreases macrophage nitric-oxide-dependent cytotoxicity: a negative feedback mechanism for the regulation of nitric oxide production

Nitric oxide (NO) production in macrophages by inducible nitric oxide synthase (NOS2) has multiple tissue damaging effects and is involved in the pathogenesis of inflammation and graft rejection. Haem oxygenase (HmOx) is the enzyme which degrades haem. Its inducible isoform, HmOx1, was recently shown to increase cellular resistance against oxidative stress and to decrease inflammation and graft rejection. Since haem is an essential cofactor for NOS2 activity, we investigated the effects of HmOx1-induction upon NO secretion in macrophages. We induced HmOx1 in BALB/c bone-marrow-derived macrophages by short-term exposure to haemin (20 micromol/l, 30 min); then we incubated them for 24 h to allow maximal expression of HmOx1 activity. Next, we activated the macrophages with lipopolysaccharide (LPS) and measured their NO production and their NO-dependent cytotoxicity against P815 cells. We found that HmOx induction 24 h before LPS activation in mouse macrophages suppresses their production of NO, while HmOx inhibition (with zinc protoporphyrin) increases NO secretion. NOS2 inhibition is reflected by the decrease of macrophage NO-dependent cytotoxicity against the P815 targets. We therefore propose that HmOx1 is a physiological inhibitor of NOS2 in activated macrophages because it decreases haem availability for NOS2 synthesis. NOS2 inhibition may explain the antinflammatory effects of HmOx induction which could also be used therapeutically in situations when NO hyperproduction leads to cytotoxic effects such as inflammation or transplant rejection.     

Modulation of ion channels in rod photoreceptors by nitric oxide

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Up-regulation of inducible nitric oxide synthase and production of nitric oxide by the Swarm rat and human chondrosarcoma

Phospholipids are the major constituents of cell membranes, and have numerous structural and functional roles in the nervous system. Although the metabolic pathways responsible for the syntheses of the phosphatides phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), and phosphatidylserine (PtdSer) are well understood, the mechanisms controlling these pathways in neural tissue have not been fully characterized. Recent studies have suggested that the main factors controlling PtdCho and PtdEtn synthesis by the Kennedy cycle tend to be the intracellular levels of key substrates for the biosynthetic enzymes, or changes in the activities of the rate-limiting enzymes. Moreover, different control mechanisms may operate, depending upon the functional state of the tissue.     

Modulation of human T cell responses by nitric oxide and its derivative, S-nitrosoglutathione

OBJECTIVE: To examine the effects of nitric oxide (NO) and its more stable derivative, S-nitrosoglutathione (SNO-GSH), on the response of activated T lymphocytes. METHODS: The effects of NO and SNO-GSH on DNA synthesis, interleukin-2 (IL-2) production, IL-2 receptor expression, and cGMP accumulation were determined in phytohemagglutinin-activated peripheral blood mononuclear cells (PBMC) and spleen T cells. RESULTS: Nitric oxide (half-life [T1/2] < 15 seconds) did not inhibit T cell proliferation. However, the derivative SNO-GSH (25 microM) (T1/2 > 2 hours) inhibited DNA synthesis by a mean +/- SD of 65 +/- 19.6% (P < 0.001) in PBMC and 75 +/- 15% (P < 0.001) in spleen cells. Macrophage depletion of PBMC did not abrogate the inhibition. SNO-GSH had no effect on IL-2 production or IL-2 receptor expression. NO (25 microM) increased the cGMP content of PBMC (0.65 +/- 0.15 pmoles/10(6) cells; P < 0.04), as did SNO-GSH (25 microM) in both PBMC (3.8 +/- 1; P < 0.001) and spleen T cells (5.2 +/- 1.2; P < 0.001). Methylene blue and hemoglobin, which are NO inhibitors, inhibited SNO-GSH-induced cGMP accumulation (P < 0.001). CONCLUSION: SNO-GSH inhibits T cell DNA synthesis independently of IL-2 production and in association with cGMP accumulation via a NO-dependent mechanism. We suggest that NO and its S-nitrosothiol derivatives may act as endogenous inhibitors of T cell-mediated inflammation.     

Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.