Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: role of inhibition of NFB activation

^a Children's Hospital Medical Center, Division of Critical Care, Cincinnati, Ohio 45229, USA

	ABSTRACT

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The role of melatonin as an immunomodulator is well established. Recent reports showed that melatonin exerts protective effects in septic and hemorrhagic shock and in inflammation. The expression of the inducible isoform of nitric oxide synthase (iNOS) makes an important contribution to the pathophysiology of shock and inflammation. We studied, in cultured murine macrophages, the role of melatonin in the regulation of the expression of iNOS and defined the mode of melatonin's action. Our results show that melatonin, at 1 µM–1 mM, decreased the production of nitrite/nitrate (the breakdown products of NO) as well as the production of 6-keto-prostaglandin F₁ (the major stable breakdown product of prostacyclin) in macrophages stimulated with bacterial lipopolysaccharide (10 µg/ml). We observed that melatonin reduces iNOS steady-state mRNA levels and iNOS protein expression in the same concentration range (1 µM–1 mM). Melatonin, up to 10 mM, exerted only a slight direct inhibitory effect on iNOS activity. Using iNOS promoter-luciferase constructs, we found that melatonin inhibits iNOS promoter activation. Inhibition of iNOS expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NFB). We conclude that melatonin inhibits NO production in immunostimulated macrophages mainly by inhibiting the expression of iNOS. This is due to inhibition of iNOS transcription, in part through inhibition of NFB activation. Inhibition of iNOS-derived NO production by melatonin may contribute to the anti-inflammatory effects of this pineal secretory product.—Gilad, E., Wong, H. R., Zingarelli, B., Virág, L., O'Connor, M., Salzman, A. L., Szabó, C. Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: role of inhibition of NFB activation. FASEB J. 12, 685–693 (1998)

Key Words: inflammation • antioxidants • scavengers • gene expression • LPS • mesangial cells

	INTRODUCTION

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THE CONVERSION of L-arginine to nitric oxide (NO) by a family of enzymes termed nitric oxide synthases (NOS) plays a major role in regulating cardiovascular, central, and peripheral nervous system functions, immune functions, and other homeostatic mechanisms (1). Increases in NO production have been described in a variety of pathophysiological processes including various forms of circulatory shock and inflammation (2–4). The inducible isoform of NOS (iNOS) is responsible for the overproduction of NO in inflammation (2–4). This isoform can be expressed in many cell types including macrophages, neutrophils, endothelial cells, vascular smooth muscle cells, mesangial cells, and chondrocytes (2–4). Bacterial lipopolysaccharide (LPS), interferon- (IFN-), and a variety of proinflammatory cytokines induce iNOS (1–3). Glucocorticoids, thrombin, transforming growth factor ß, and a variety of anti-inflammatory cytokines suppress the expression of iNOS (2–4). The process of iNOS expression involves multiple signal transduction pathways, including activation of tyrosine kinases and nuclear translocation of the transcription factor nuclear factor kappa B (NFB) (1–4).

Melatonin, the major product of the pineal gland, plays a fundamental role in the neuroimmuno-endocrine system (5–7). Melatonin also functions as a potent antioxidant because it scavenges hydroxyl free radicals and peroxynitrite (a reactive oxidant produced from the reaction of NO and superoxide) (8–10). Melatonin's immunoregulatory effects have been demonstrated in vitro (11, 12) and in vivo (13). Macrophages and T-helper 2 lymphocytes have been proposed as major sites of melatonin's immunomodulatory actions (14–19).

The present study was designed to study whether melatonin inhibits the expression of iNOS in macrophages stimulated with endotoxin in vitro. We attempted to characterize the mechanism by which melatonin inhibits the expression of this enzyme.

	MATERIALS AND METHODS

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Cell culture
Murine macrophages (J774 and RAW 264.7) were obtained from the American Type Culture Collection (Rockville, Md.). Cells were cultured in Dulbecco's minimal essential medium (DMEM) supplemented with L-glutamine (3.5 mM) and 10% fetal calf serum. Shortly before the experiments began, the culture medium was replaced with fresh medium. DMEM without fetal calf serum was used for studies of prostaglandin production in order to avoid interference in the radioimmunoassay.

Induction of iNOS in J774 and RAW 264.7 cells
Cells were stimulated with Escherichia coli bacterial LPS (10 µg/ml) in order to induce iNOS. Melatonin dissolved in ethanol (final concentration, 3%) was administered to the cells in different concentrations (1 nM–1 mM) at various times in relation to LPS administration (ranging from 6 h pretreatment to 6 h post-LPS administration). In immunostimulated macrophages, iNOS mRNA induction occurs within 1–6 h after LPS stimulation, but no detectable NO production can be measured during this period. Consequently, agents that inhibit the process of induction of iNOS gradually lose their inhibitory effect on NO production when given at increasing times after LPS stimulation (20–22). Thus, comparison of a pre- vs. posttreatment regimen by melatonin was expected to indicate the mode of the agent's inhibitory action.

In all experiments, treatment with ethanol (3%) alone was used as vehicle control. The supernatant was collected 24 h after LPS administration to measure nitrite/nitrate and 6-keto-prostaglandin F₁, as described below.

Nitrite/nitrate production
The concentration of nitrite and nitrate, breakdown products of NO, were measured as previously described (22). First, nitrate in the culture medium was reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160 µM) at room temperature for 2 h. Nitrite concentration in the samples was then measured by the Griess reaction by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100 µl samples of medium. The optical density at 550 nm (OD₅₅₀) was measured using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, Calif.). Nitrate concentrations were calculated by comparison with OD₅₅₀ of standard solutions of sodium nitrate prepared in culture medium.

In addition to the measurement of nitrite and nitrate in tissue culture supernatants from LPS-stimulated cells in the presence or absence of melatonin, we tested whether melatonin (1 nM–1 mM) interferes with the measurement of nitrite or nitrate or with the Griess reaction, or whether it scavenges NO. For these experiments, melatonin or its vehicle was incubated with sodium nitrate (1–40 µM) or with the NO donor compound S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1–40 µM) for 4 h, followed by nitrite and nitrate measurement, as described above.

Measurement of 6-keto-prostaglandin F
Supernatant samples were diluted 1:5 in a buffer containing 0.1% polyvinylpyrrolidone, 0.9% NaCl, 50 mM Tris base, 1.7 mM MgSO₄, and 0.16 mM CaCl₂ (pH 7.4) before radioimmunoassay. 6-Keto-prostaglandin F₁, the major stable metabolite of prostacyclin, was measured by radioimmunoassay as described (23). Measurements of this prostaglandin metabolite have previously been shown to change in parallel with the expression or activity of the inducible cyclooxygenase (COX-2) in immunostimulated macrophages (23–26). Melatonin did not interfere with the detection of 6-keto-prostaglandin F₁ in this assay.

Cell viability determination
Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan (21, 22, 27). At the end of each experiment, cells in 96-well plates were incubated with MTT (0.2 mg/ml) for 1 h at 37°C. The culture medium was removed and cells were solubilized in dimethylsulfoxide (100 µl). The extent of reduction of MTT to formazan within cells was quantitated by measurement of OD₅₅₀ by the Spectramax reader.

NO synthase activity
J774 macrophages were stimulated with LPS in the presence of various concentrations (1 nM–1 mM) of melatonin or vehicle. Activity of inducible NOS was measured 12 h after LPS stimulation by conversion of [³H]L-arginine to [³H]L-citrulline (21, 27). Briefly, after homogenization, samples were incubated in the presence of [³H]L-arginine (10 µM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 µM), and EGTA (2 mM) for 20 min at 22°C. Reactions were stopped by dilution with 0.5 ml of ice-cold N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic (HEPES) buffer (pH 5.5) containing EGTA (2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W (Na⁺ form) columns and the eluted [³H]L-citrulline activity was measured by a Wallac scintillation counter (Wallac, Gaithersburg, Md.).

In additional experiments, the direct effect of melatonin on iNOS activity from immunostimulated macrophages was tested. In these studies, homogenates of immunostimulated macrophages (LPS, 10 µg/ml, 12 h) were treated with increasing concentrations (1 nM–10 mM) of melatonin for 20 min, followed by measurement of the conversion of L-arginine to L-citrulline, as described above.

Western blot analysis
J774 macrophages were stimulated with LPS in the presence and absence of melatonin (1 mM) or vehicle for 4, 8, 12, or 24 h. For Western blot analysis of iNOS (27), cells were first washed in phospate-buffered saline (PBS) and lysed in ice-cold buffer containing 50 mM Tris (pH 8.0), 110 mM NaCl, 5 mM EDTA, 1% Triton X-100, and the protease inhibitor phenylmethylsulfonylfluoride (PMSF: 1 mM). Whole cell lysates were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol) and 50 µg of protein was then loaded onto an 8–16% Tris-glycine gradient gel (Novex, San Diego, Calif.). Proteins were separated electrophoretically and transferred to a nitrocellulose membrane using a Novex Xcell Mini-Gel system. For immunoblotting, the membrane was blocked with 5% non-fat dried milk in Tris-buffered saline (TBS) for 1 h. The primary antibody was a murine monoclonal IgG specific to iNOS (Upstate Biotechnology, Lake Placid, N.Y.) at a 1:1000 dilution for 2–1/2 h. After washing three times in TBS containing 0.1% Tween 20, secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Sigma) was applied at a 1:3000 dilution for 1 h. The blot was then washed in 0.1% Tween 20 in TBS three times over a period of 30 min, incubated in commercial enhanced chemiluminescence reagents (ECL, Amersham, Amersham, U.K.), and exposed to photographic film (27).

Northern blot analysis
J774 macrophages were stimulated with LPS (10 µg/ml) in the presence of various concentrations (1 nM–1 mM) of melatonin or vehicle. Cells were harvested at 180 min post-LPS for total RNA extraction using the Trizol reagent (Gibco, BRL, Grand Island, N.Y.). Total RNA was quantified by spectrophotometry (260 nm) and 15 µg of total RNA per condition underwent electrophoresis on a 1% agarose gel containing 3% formaldehyde. The integrity of the RNA after electrophoresis was confirmed by ethidium bromide staining and UV illumination. RNAs were transferred to nylon membranes (Micron Separation Inc., Westboro Mass.) and were UV auto-crosslinked (UV Stratalinker 1800; Stratagene, La Jolla, Calif.). After a 4 h prehybridization at 42°C, membranes were hybridized overnight at 42°C with an iNOS radiolabeled cDNA probe (27). cDNAs were labeled with [³²P]dCTP (specific activity, 3000 Ci/mM; New England Nuclear Research Products, Boston, Mass.) by random priming (Pharmacia, Piscataway, N.J.). The hybridized filters were serially washed at 53°C using 2x sodium citrate/sodium chloride/0.1% SDS and 25 mM NaHPO₄/1 mM EDTA/0.1% SDS solutions. After washing, exposure was carried out overnight using a Phosphor Imager screen (Molecular Dynamics, Sunnyvale, Calif.). To normalize results for loading differences, membranes were stripped with boiling 5 mM EDTA and rehybridized with an end-labeled ([³²P]dATP) oligonucleotide probe for 18 rRNA. No single blot was stripped more than twice.

Transient transfections, functional promoter analyses, and luciferase assays
Functional analysis of the iNOS promoter was performed as described (27) by transiently transfecting cells with a plasmid containing the reporter gene, firefly luciferase, under the control of the full-length iNOS promoter, a kind gift from Dr. Charles J. Lowenstein, Johns Hopkins University (28, 29). Since the J774 cells were resistant to our transfection attempts (27), these studies were performed in RAW 264.7 cells. Cells were transfected in duplicate, in 6-well plates, at a density of 300,000 cells per well by incubation with cationic liposomes (Lipofectin, Gibco) for 5 h in Optimum (Gibco, BRL). The liposome-to-DNA ratio was 20:3 µg. After transfection, cells were washed once with PBS and allowed to recover overnight. After exposure to LPS (10 µg/ml) in the presence or absence of pretreatment with melatonin (1 mM), cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer's instructions (Promega, Madison, Wis.) using a Berthold AutoLumat LB 953 luminometer. Luciferase activity is reported as light units per milligram of protein.

Preparation of nuclear extracts
RAW 264.7 cells were grown to 80% confluence in 100 mm² dishes. Cells were exposed to LPS (10 µg/ml) with or without pretreatment with melatonin (1 mM). Nuclear protein extracts were prepared as described (30) 60 min after LPS administration. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were washed twice with PBS and harvested by scraping into 1 ml of PBS and pelleted at 6000 rpm for 5 min. The pellet was resuspended in one packed cell volume of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% v/v Nonidet P-40, 1 mM DTT, and 0.1 mM PMSF) and incubated for 5 min with occasional vortexing. After centrifugation at 6000 rpm, one cell pellet volume of extraction buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% v/v glycerol, 1 mM DTT, and 0.5 mM PMSF) was added to the nuclear pellet and incubated on ice for 15 min with occasional vortexing. Nuclear proteins were isolated by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by the Bradford assay. Nuclear extracts were stored at -70°C until used for electromobility shift assay (EMSA).

Electromobility shift assay
The oligonucleotide probe used for EMSA contained the published consensus sequences for NFB (31). The NFB oligonucleotide (5'-GCC TCG AAT GTT CGC GAA GTT TCG-3') was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Oligonucleotide probes were labeled with -[³²P]ATP by using T4 polynucleotide kinase (Gibco, BRL) and purified in Bio Spin chromatography columns (BioRad, Hercules, Calif.). For EMSA analysis, 10 µg of nuclear proteins were preincubated with EMSA buffer (12 mM HEPES, pH 7.9, 4 mM Tris-HCl, pH 7.9, 25 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly[(d-I)(d-C)], 12% glycerol v/v, and 0.2 mM PMSF) on ice for 10 min before addition of the radiolabeled oligonucleotide for another 25 min. Specifications of the binding reactions were tested by incubating duplicate samples with a 100-fold molar excess of the unlabeled oligonucleotide probe. Protein–nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide: bisacrylamide) and run in 0.5x TBE (45 mM Tris-HCl, 45 mM boric aid, 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper, dried under a vacuum at 80°C for 1 h, and exposed to photographic film at -70°C with an intensifying screen. For supershift of the respective samples, samples were incubated for 1 h with p65-NFB antibodies after addition of the radiolabeled probe (Santa Cruz) and loaded onto the gel.

Protein determination
As an indicator of cell density, protein determination was assessed by overnight incubation of cells in the 96-well plates with 1 N NaOH (100 µl/well) at 4°C. The samples were diluted 1:2 and 50 µl of BioRad reagent was added to each well and quantified by measurement of OD₅₉₅ using the Spectramax reader. Bovine serum albumin was used as standard.

	MATERIALS

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Culture media (DMEM and OPTI-MEM), fetal calf serum, Lipofectin, and Trizol reagent were obtained from Gibco. Bacterial LPS (E. coli serotype No. 0127:B8), MTT, and melatonin were obtained from Sigma (St. Louis, Mo.). Lysis buffer for the luciferase reaction was obtained from Promega. Rabbit anti-murine iNOS antibodies were obtained from Upstate Biotechnology (Lake Placid, N.Y.). NFB and anti-p65 NFB monoclonal antibodies were obtained from Santa Cruz. All the other reagents were from Sigma or as indicated in the specified methods.

Statistical analysis
All values shown in the figures are expressed as means ± standard error of the mean (SEM) of n determinations, obtained on at least three independent experimental days. The blots presented are representative of blots performed on at least three independent experimental days. The results were examined by one- and two-way ANOVA; individual group means were then compared with the Bonferroni test. A P value of less than 0.05 was considered significant.

	RESULTS

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Melatonin inhibits nitrite/nitrate and prostaglandin production
Since melatonin has been shown to act as a scavenger of oxyradicals and peroxynitrite (see opening paragraphs), we tested whether melatonin interferes with detection of nitrite or nitrate by the Griess reaction. Melatonin, up to 1 mM, failed to affect nitrite/nitrate measured from the NO donor compound SNAP and did not affect the nitrate standard curve ( Fig. 1a). We conclude, therefore, that melatonin does not scavenge NO or interfere with the detection of nitrite or nitrate by the Griess reaction.

View larger version (19K): [in this window] [in a new window]   Figure 1. a) Lack of effect of melatonin (1 µM–1 mM) on nitrate concentrations generated from sodium nitrate standard curve (1–40 µM) or from the NO donor SNAP (1–40 µM), as measured by the Griess reaction. b, c) Effect of melatonin (10 nM–1 mM) on nitrite/nitrate (b) and 6-keto-prostaglandin F1 (c) production in immunostimulated J774 cells. d) Effect of melatonin on mitochondrial respiration in the presence of LPS. Nitrite/nitrate and 6-keto-prostaglandin F1 are expressed as percent of the LPS-stimulated values in the absence of melatonin; mitochondrial respiration is expressed as a percent of the values measured in unstimulated cells. Nitrite/nitrate increased from 2.4 ± 0.7 (baseline) to 44 ± 5 µM in response to LPS stimulation; 6-keto-prostaglandin F1 concentrations increased from 0.4 ± 0.1 ng/ml (baseline) to 4.2 ± 0.5 ng/ml in response to LPS stimulation. *P < 0.05 and **P < 0.01 indicate significant inhibition of nitrite/nitrate or 6-keto-prostaglandin F1 production or enhancement of the mitochondrial respiration by melatonin in the presence of LPS (n=9–12).

Stimulation of J774.2 macrophages with LPS caused a marked increase in nitrite/nitrate production, as measured 24 h after stimulation. Melatonin, at 1 µM–1 mM, dose-dependently inhibited NO production ( Fig. 1b). The inhibitory effect of melatonin was most pronounced when administered 6 h before LPS and diminished gradually when it was added along with LPS or at increasing intervals (up to 6 h) after LPS stimulation ( Fig. 2). Only in the experiments involving 1 mM melatonin was there a small residual inhibitory activity when the agent was given 6 h post-LPS: the inhibitory effect of 300 or 100 µM melatonin was completely abolished when the agent was given at 4–6 h post-LPS stimulation ( Fig. 2). Experiments in RAW 264.7 macrophages produced results identical to those in the J774 cells (not shown). When cells were stimulated with a combination of LPS and IFN-, melatonin exhibited a more modest inhibition of NO production. For example, melatonin at 1 mM inhibited nitrite/nitrate production by 11 ± 4% (n=9; P<0.05).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect in J774 cells of melatonin at 100 µM, 300 µM, and 1 mM on nitrite/nitrate production when given 6 h before LPS (10 µg/ml) administration (-6 h), together with LPS (0), or 2, 4, and 6 h after LPS. The most pronounced inhibition was found when the cells were pretreated with melatonin for 6 h before LPS stimulation, and there was a gradual loss of the inhibition with posttreatment. *P < 0.05 and **P < 0.01 indicate significant suppression of nitrite/nitrate production (n=8).

Stimulation of J774.2 cells with LPS resulted in a significant increase in 6-keto-prostaglandin F₁ production. Melatonin exhibited an inhibitory effect on 6-keto-prostaglandin F₁ production in the concentration range of 1 µM–1 mM ( Fig. 1c). In the concentration range used, melatonin did not affect mitochondrial respiration in the LPS-stimulated macrophages except at 1 mM, when it improved respiration ( Fig. 1d).

Effects of melatonin on iNOS gene expression
We then studied whether melatonin affects transcription of the iNOS gene. Figure 3 shows the results of a Northern blot analysis. At 1 nM, melatonin had already tended to decrease iNOS steady-state mRNA levels. Most of the inhibitory effect on iNOS steady-state mRNA occurred in the concentration range of 1 µM–1 mM, consistent with the effect of melatonin on NO production.

View larger version (35K): [in this window] [in a new window]   Figure 3. Northern blot analysis demonstrating iNOS mRNA expression in J774 cells treated with melatonin (1 nM, 1 µM, or 1 mM) or vehicle before stimulation with LPS (10 µg/ml). Cells were harvested 180 min after LPS administration (10 µg/ml). The results were normalized with an end-labeled ([32P]dATP) oligonucleotide probe for 18s rRNA. Top panel: individual blots are shown. Bottom panel: normalized results are shown. Melatonin decreased transcription of the steady-state iNOS mRNA compared to LPS or vehicle (*P<0.05; **P<0.01; n=6).

Direct measurements of iNOS activity (by measurements of the conversion of L-arginine to L-citrulline) were also performed. In J774.2 cells stimulated with 10 µg/ml LPS for 12 h, pretreatment with 1 µM–1 mM melatonin caused a dose-dependent inhibition of iNOS activity ( Fig. 4a). In the presence of a 6 h pretreatment with 1 mM melatonin, a decrease in iNOS activity by 40 ± 3% was measured (n=8; P<0.01). Figure 4b shows a Western blot analysis, confirming that melatonin reduces the amounts of iNOS protein in immunostimulated J774 macrophages. The appearance of iNOS protein occurred between 4 and 8 h after LPS stimulation ( Fig. 4b).

View larger version (40K): [in this window] [in a new window]   Figure 4. A) Effect of melatonin (1 µM–1 mM, given 6 h before LPS) on iNOS activity measured 12 h after LPS (10 µg/ml) stimulation in J774 cells. B) Western blot analysis confirming the inhibitory effect of melatonin on iNOS expression. iNOS expression was measured at 4, 8, 12, and 24 h post-LPS in the presence or absence of 1 mM melatonin applied 6 h before LPS (10 µg/ml). C) Direct inhibitory effect of melatonin on iNOS activity from immunostimulated macrophages. LPS (10 µg/ml for 12 h) was used to induce iNOS. Cell homogenates were treated with increasing concentrations (10 µM–10 mM) of melatonin in vitro for 20 min. A, C) *P < 0.05, **P < 0.01 indicate significant suppression of iNOS activity; n = 6. In LPS-stimulated cells, iNOS activity amounted to 2.7 ± 0.4 pmol/(mg·min). In the absence of LPS stimulation, no detectable iNOS activity or iNOS protein immunoreactivity was detected.

We also tested the direct effect of melatonin on the iNOS activity of immunostimulated macrophages. In homogenates of immunostimulated macrophages, melatonin caused only a slight inhibition of iNOS activity ( Fig. 4c). Even at the highest concentration of melatonin tested (10 mM), the activity of iNOS was reduced by only 10 ± 4% (n=6, P<0.05). From these studies, we conclude that melatonin mainly exerts its effects on iNOS mRNA and protein expression in immunostimulated macrophages; its direct inhibitory effect on the activity of iNOS is modest.

To confirm that melatonin inhibits iNOS promoter activation, experiments were performed in RAW 264.7 cells transfected with the full-length iNOS-promoter/luciferase constructs. Melatonin significantly inhibited iNOS promoter activation (P<0.02) ( Fig. 5).

View larger version (32K): [in this window] [in a new window]   Figure 5. Effect of melatonin on the induction of luciferase activity in RAW cells transfected with full-length iNOS-promoter/luciferase constructs. Cells treated with melatonin (1 mM) and LPS (10 µg/ml) showed reduced luciferase activity when compared to LPS alone. **P < 0.01; n = 6.

Effects of melatonin on NFB activation
A variety of signal transduction processes precede the induction of iNOS and production of other inflammatory mediators in response to LPS stimulation in macrophages. Activation of the NFB pathway (32, 33), tyrosine kinase activation (34), and the mitogen-activated protein (MAP) kinase (35) have all been implicated in the process of iNOS expression. Since the activation of NFB is a redox-sensitive process and melatonin is known to affect cellular redox processes (31–37), we tested the potential effect of this pineal secretory hormone on the activation of NFB. The results demonstrate that melatonin exerts an inhibitory effect on the LPS-induced activation of NFB ( Fig. 6).

View larger version (87K): [in this window] [in a new window]   Figure 6. Effect of melatonin of NFB activation, as assessed by EMSA analysis. J774 cells were exposed to LPS (10 mg/ml) in the presence of vehicle or 1 mM melatonin. Cells were harvested 60 min after LPS, followed by extraction of nuclei and EMSA analysis. Melatonin caused a decrease in LPS-induced NFB activation.

	DISCUSSION

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In recent years, melatonin, the major product of the pineal gland, has been found to affect various neuroimmuno-endocrine functions (6, 7, 11, 12). To our knowledge, this report is the first to show that melatonin inhibits NO production from iNOS. This inhibitory effect is most pronounced when melatonin is given before LPS and is markedly attenuated when melatonin is administered after stimulation with LPS.

NO production can be regulated in several ways, affecting 1) cofactor or substrate binding, 2) enzyme stability, or 3) gene expression (at the level of either mRNA expression or mRNA stability) (4). Based on the current results, we propose that melatonin acts via suppression of iNOS promoter activation and iNOS mRNA transcription. This conclusion is based on the following findings: 1) melatonin dose-dependently reduces iNOS promoter activation; 2) melatonin dose-dependently reduces iNOS steady-state mRNA levels, and 3) melatonin dose-dependently reduces iNOS protein expression. Moreover, the time-dependent decrease in the degree of inhibition of NO production with increasing times of posttreatment also supports the above proposition.

Melatonin exerted its inhibitory actions in the high micromolar concentration range. This is the same range where melatonin can exert its antioxidant effects in vitro (31–34). Since 1) the activation of NFB is involved in the process of iNOS expression (4) and 2) NFB activation is a redox-sensitive process that can be attenuated by antioxidants (35), we tested whether the inhibitory effect by melatonin of iNOS expression may be mediated, at least in part, by inhibition of NFB activation. The inhibition by melatonin of NFB activation in LPS-stimulated macrophages, as demonstrated by our study, indicates that inhibition of this signal transduction pathway may be involved in melatonin's actions. In line with our observation, it has recently been reported that melatonin inhibits NFB activation by a variety of stimuli (tumor necrosis factor-, phorbol ester, irradiation) (36). Furthermore, melatonin can exert an inhibitory effect on NFB-DNA binding (37). Melatonin may also interact with additional signal transduction pathways of iNOS expression, such as the one involving MAP kinase (38, 39).

6-Keto-prostaglandin F₁ production in immunostimulated J774 macrophages is due to the expression of COX-2 (23–26). Regulation of this enzyme shares similarities with the regulation of iNOS and is also under the regulation of NFB and MAP kinase activation (26, 40). Suppression of NFB activation and COX-2 expression by melatonin may explain, at least in part, the reduction in prostaglandin production by melatonin observed in our experiments.

Also of interest was the finding that the degree of inhibition of NO production decreases when IFN- and LPS (rather than LPS alone) are used as a stimulus of iNOS induction. This effect, which has been observed with several other inhibitors of iNOS expression (1–4, 27), may be due to the ability of an IFN- induced transcription factor to bypass the inhibition of iNOS expression by melatonin or other inhibitors of iNOS expression. Melatonin still exerted a significant, although relatively minor, inhibitory effect on NO production even when administered at 6 h post-LPS. One possibility is that, in addition to inhibition of the process of iNOS expression, melatonin at higher concentrations also directly inhibits the catalytic activity of iNOS. In fact, Pozo and colleagues (41, 42) have described inhibition of the constitutive brain isoform of NOS by melatonin. However, in our experiments we have not seen a marked direct inhibitory effects of melatonin on iNOS activity, even at concentrations as high as 10 mM. This is probably because the inhibitory effect of melatonin on iNOS is due to an interaction with calmodulin (42) and iNOS is associated with calmodulin in a tightly bound form (4). We cannot exclude the possibility that, in the immunostimulated macrophages, a prolonged exposure to melatonin during the induction of iNOS and its association with calmodulin increases its inhibitory effect on iNOS activity.

Improved survival has been reported in septic mice treated with melatonin (1–10 mg/kg) (43). Melatonin also decreased plasma nitrite/nitrate concentrations at 18 and 24 h after LPS administration (43). Attenuation by melatonin of the hypotensive effect and lethality after hemorrhage or interleukin-2 therapy have also been reported (44–46). Furthermore, melatonin exerts potent antiinflammatory effects and reduces NO production in murine models of carrageenan-induced inflammation (47). Considering the crucial role of iNOS expression and NO overproduction in rodent models of septic and hemorrhagic shock and inflammation (2, 48–50), we propose that some of the protection provided by melatonin in shock and inflammation is due to inhibition of iNOS expression.

In conclusion, we have demonstrated that melatonin inhibits NO production by iNOS by suppressing its gene transcription. The concentrations of melatonin needed for this inhibitory action are in the range of its antioxidant effects (29–34) and are higher than the physiological concentrations of this pineal secretory product. Therefore, it is unlikely that endogenously produced melatonin may be present in sufficient concentrations to suppress the process of iNOS induction. However, the current data may offer a novel mechanism for melatonin's anti-shock and anti-inflammatory effects (24, 43–47). Melatonin's regulatory effect on gene expression appears to be selective in the sense that melatonin can inhibit the expression of some genes and at the same time enhance the expression of others. For instance, in a study investigating neuronal damage caused by porphyrins, melatonin showed a simultaneous capability to decrease porphyrin synthesis and aminolevulinate synthase mRNA and to increase mRNA levels for antioxidant enzymes (51). Additional studies are needed to establish the utility of high-dose melatonin as a novel anti-inflammatory therapy.

	ACKNOWLEDGMENTS

 
The assistance of Ms. Vivien Xue with the tissue culture studies and the help of Ms. Marnie Ryan with Western and Northern blots are appreciated. This work was supported, in part, by a Grant-In-Aid from the American Heart Association to C.S.

	FOOTNOTES

 
¹ Correspondence: Children's Hospital Medical Center, Division of Critical Care, 3333 Burnet Ave., Cincinnati, OH 45229, USA.

² Abbreviations: COX-2, inducible isoform of cyclooxygenase; EMSA, electromobility shift assay; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NO, nitric oxide; NFB, nuclear factor kappa B; MAP, mitogen-activated protein; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltet-razolium bromide; OD, optical density; SNAP, S-nitroso-N-acetyl-DL-penicillamine; IFN, interferon; DMEM, Dulbecco's minimal essential medium; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.

Received for publication August 15, 1997. Accepted for publication January 19, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS MATERIALS RESULTS DISCUSSION REFERENCES

 

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