Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

Inhibition of ethanol-induced changes in rats by Padma 28

In male Wistar rats the protective effect of Padma 28 against changes induced by prolonged treatment with ethyl alcohol was investigated. Exposure of the animals to prolonged ethyl alcohol consumption caused a significant increase in AspAT (by 32%), A1AT (by 50%) and alkaline phosphatase (by 372%) activities as well as an increase in the levels of bilirubin (by 98%) and lipids in the blood serum and lipids in the liver homogenate. Padma 28 administration was found to exert a protective action against these changes: a significant reduction of blood total lipid, triacylglycerol, total cholesterol and bilirubin concentrations as well as in the activity of the liver enzymes was observed.     

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A₂ inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA₄-LTC₄) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB₄, LTC₄ and LTE₄). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.     

Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.     

Inhibition of inducible nitric oxide synthase in persistent pain

The present study was undertaken to investigate the role of inducible nitric oxide synthase in a rat model of persistent pain. The effects of L-N6 (1-iminoethyl) lysine (L-NIL), a relatively potent and relatively selective inhibitor of inducible nitric oxide synthase, were investigated in carrageenan induced hyperalgesia L-NIL (0.1 microMole) injected intraplantar or intrathecal markedly enhanced carrageenan induced hyperalgesia. These effects were reversed during the third hour by co-administration of L-arginine (900 mg/kg i.p.) but not D-arginine. Methylene blue (MB), a soluble guanylate cyclase inhibitor, administered intrathecally (0.1 microg) had no effect on L-NIL potentiation of carrageenan hyperalgesia but abolished antinociception induced by L-arginine. Obtained results suggest that nitric oxide derived from inducible nitric oxide synthase play an inhibitory role in carrageenan produced hyperalgesia in rat.     

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.     

Negative regulation of LPS-stimulated expression of inducible nitric oxide synthase by AP-1 in macrophage cell line J774A.1.

The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-kappaB by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1-like sites (U site, -1126 approximately -1120; L site, -524 approximately -518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS.     

Phosphoinositide 3-kinase in nitric oxide synthesis in macrophage: critical dimerization of inducible nitric-oxide synthase

Phosphoinositide 3-kinase (PI3K) has important functions in various biological systems, including immune response. Although the role of PI3K in signaling by antigen-specific receptors of the adaptive immune system has been extensively studied, less is known about the function of PI3K in innate immunity. In the present study, we demonstrate that macrophages deficient for PI3K (p85alpha regulatory subunit) are impaired in nitric oxide (NO) production upon lipopolysaccharide and interferon-gamma stimulation and thus vulnerable for intracellular bacterial infection such as Chlamydophila pneumoniae. Although expression of inducible nitric-oxide synthase (iNOS) is induced normally in PI3K-deficient macrophages, dimer formation of iNOS protein is significantly impaired. The amount of intracellular tetrahydrobiopterin, a critical stabilizing cofactor for iNOS dimerization, is decreased in the absence of PI3K. In addition, induction of GTP cyclohydrolase 1, a rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, is greatly reduced. Our current results demonstrate a critical role of class IA type PI3K in the bactericidal activity of macrophages by regulating their NO production through GTP cyclohydrolase 1 induction.     

Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs.

The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.).     

Inhibition of lipopolysaccharide-induced inducible nitric oxide synthase expression by endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is induced in infectious and inflammatory conditions, but its role in inflammatory responses still remains elusive. In this study we found tunicamycin (TM) and brefeldin A (BFA), two ER stressors, could attenuate lipopolysaccharide (LPS)-elicited inducible nitric oxide synthase (iNOS) gene expression in murine RAW264.7 macrophages, and this effect was not resulting from the effects on IKK or MAPKs activation. However, ER stressors could block NF-κB binding to the iNOS promoter in late-phase signaling evoked by LPS. Results indicated that inhibition of RelB nuclear translocation and p300 expression are involved in the anti-inflammatory actions of ER stressors. We also found that ER stressors could block LPS- and IFN (α, β, and γ)-mediated STAT1 phosphorylation. Our results suggest that activation of MKP-1 via a Ca/calmodulin/calcineurin pathway accounts for the inhibitory effect of ER stressors on IFN signaling. MKP-1 was downregulated by IFN-γ and is a newly identified protein phosphatase targeting STAT1. Taken together, these results indicate that multiple mechanisms are involved in the inhibition of LPS-induced iNOS gene expression by ER stressors. These include downregulation of RelB and p300, upregulation of MKP-1, and inhibition of the JAK/STAT signaling pathway.     

Effects of macrophage inducible nitric oxide synthase in murine septic lung injury

Inducible nitric oxide synthase (iNOS) contributes importantly to septic pulmonary protein leak in mice with septic acute lung injury (ALI). However, the role of alveolar macrophage (AM) iNOS in septic ALI is not known. Thus we assessed the specific effects of AM iNOS in murine septic ALI through selective AM depletion (via intratracheal instillation of clodronate liposomes) and subsequent AM reconstitution (via intratracheal instillation of donor iNOS+/+ or iNOS-/- AM). Sepsis was induced by cecal ligation and perforation, and ALI was assessed at 4 h: protein leak by the Evans blue (EB) dye method, neutrophil infiltration via myeloperoxidase (MPO) activity, and pulmonary iNOS mRNA expression via RT-PCR. In iNOS+/+ mice, AM depletion attenuated the sepsis-induced increases in pulmonary microvascular protein leak (0.3 +/- 0.1 vs. 1.4 +/- 0.1 microg EB.g lung(-1).min(-1); P < 0.05) and MPO activity (37 +/- 4 vs. 67 +/- 8 U/g lung; P < 0.05) compared with that shown in non-AM-depleted mice. In AM-depleted iNOS+/+ mice, septic pulmonary protein leak was restored by AM reconstitution with iNOS+/+ AM (0.9 +/- 0.3 microg EB.g lung(-1).min(-1)) but not with iNOS-/- donor AM. In iNOS-/- mice, sepsis did not induce pulmonary protein leak or iNOS mRNA expression, despite increased pulmonary MPO activity. However, AM depletion in iNOS-/- mice and subsequent reconstitution with iNOS+/+ donor AM resulted in significant sepsis-induced pulmonary protein leak and iNOS expression. Septic pulmonary MPO levels were similar in all AM-reconstituted groups. Thus septic pulmonary protein leak is absolutely dependent on the presence of functional AM and specifically on iNOS in AM. AM iNOS-dependent pulmonary protein leak was not mediated through changes in pulmonary neutrophil influx.     

Inhibition of inducible nitric oxide synthase lowers the cochlear damage by lipopolysaccharide in guinea pigs

Endotoxin-treated cochleas of the guinea pig were examined electrophysiologically and immunohistochemically concerning the expression of inducible nitric oxide synthase (iNOS/NOS II). One mg of bacterial lipopolysaccharide (LPS, 5 mg/ml) or mixed solution of 1 mg of LPS plus 1 mg of N^G-nitro-L-arginine methyl ester (L-NAME, 5 mg/ml) (L-NAME/LPS) was injected into the middle ear of guinea pigs transtympanically. The electrocochleograms were measured prior to, immediately and 48 h after the injection. Immunohistological studies for iNOS followed after fixation, embedding and sectioning of the temporal bones.

The threshold and amplitude of the compound action potential (CAP) became significantly worse in the LPS treated group. In contrast, the changes of the threshold and amplitude of CAP were decreased in the L-NAME/LPS group. iNOS was expressed in the stria vascularis, the spiral ligament, the organ of Corti and the spiral ganglion in the LPS group. These immunore-activities in the L-NAME/LPS group were less intense than that in the LPS group. These results indicate that LPS has an ototoxic effect on the cochlea and that this effect could be mediated by iNOS produced high nitric oxide under inflammatory conditions.     

Inhibition of inducible nitric oxide synthase lowers the cochlear damage by lipopolysaccharide in guinea pigs

Endotoxin-treated cochleas of the guinea pig were examined electrophysiologically and immunohistochemically concerning the expression of inducible nitric oxide synthase (iNOS/NOS II). One mg of bacterial lipopolysaccharide (LPS, 5 mg/ml) or mixed solution of 1 mg of LPS plus 1 mg of N^G-nitro-L-arginine methyl ester (L-NAME, 5 mg/ml) (L-NAME/LPS) was injected into the middle ear of guinea pigs transtympanically. The electrocochleograms were measured prior to, immediately and 48 h after the injection. Immunohistological studies for iNOS followed after fixation, embedding and sectioning of the temporal bones.

The threshold and amplitude of the compound action potential (CAP) became significantly worse in the LPS treated group. In contrast, the changes of the threshold and amplitude of CAP were decreased in the L-NAME/LPS group. iNOS was expressed in the stria vascularis, the spiral ligament, the organ of Corti and the spiral ganglion in the LPS group. These immunore-activities in the L-NAME/LPS group were less intense than that in the LPS group. These results indicate that LPS has an ototoxic effect on the cochlea and that this effect could be mediated by iNOS produced high nitric oxide under inflammatory conditions.     

Cutting edge: urease release by Helicobacter pylori stimulates macrophage inducible nitric oxide synthase

Inducible NO synthase (iNOS) expression and production of NO are both up-regulated with Helicobacter pylori infection in vivo and in vitro. We determined whether major pathogenicity proteins released by H. pylori activate iNOS by coculturing macrophages with wild-type or mutant strains deficient in VacA, CagA, picB product, or urease (ureA(-)). When filters were used to separate H. pylori from macrophages, there was a selective and significant decrease in stimulated iNOS mRNA, protein, and NO(2)(-) production with the ureA(-) strain compared with wild-type and other mutants. Similarly, macrophage NO(2)(-) generation was increased by H. pylori protein water extracts of all strains except ureA(-). Recombinant urease stimulated significant increases in macrophage iNOS expression and NO(2)(-) production. Taken together, these findings indicate a new role for the essential H. pylori survival factor, urease, implicating it in NO-dependent mucosal damage and carcinogenesis.