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Originally published online as doi:10.1189/jlb.1003459 on April 1, 2004

Published online before print April 1, 2004
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(Journal of Leukocyte Biology. 2004;75:1093-1101.)
© 2004 by Society for Leukocyte Biology

A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression

Antonio Vega*,{dagger}, Pedro Chacón*,{dagger}, Javier Monteseirín*,{ddagger}, Rajaa El Bekay{dagger}, Moisés Álvarez{dagger}, Gonzalo Alba{dagger}, José Conde*, José Martín-Nieto§, Francisco J. Bedoya{dagger}, Elizabeth Pintado{dagger} and Francisco Sobrino{dagger},1

{dagger} Departamento de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Spain;
* Servicio de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Sevilla, Spain;
{ddagger} Clínica Sagrado Corazón, Sevilla, Spain; and
§ Departamento de Fisiología, Genética y Microbiología, Facultad de Ciencias, Universidad de Alicante, Spain

1 Correspondence: Departmento Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez Pizjuán 4, 41009-Sevilla, Spain. E-mail: fsobrino{at}us.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This report focuses on the modulatory role of endogenous H2O2 on lipopolysaccharide (LPS)/interferon-{gamma} (IFN-{gamma})-induced inducible nitric oxide synthase (NOS2) gene expression in rat peritoneal macrophages. Exogenously added H2O2 was initially found to inhibit the synthesis of NOS2, which prompted us to assess the effect of the activity of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) as H2O2-forming enzymes on NOS2 gene expression. In the presence of their substrates, tyramine for MAO and benzylamine for SSAO, intracellular synthesis of H2O2 took place with concomitant inhibition of LPS/IFN-{gamma}-induced NOS2 protein synthesis, as detected by Western blotting, flow cytometry, and immunofluorescence microscopy analyses. Pargyline and semicarbazide, specific inhibitors of MAO and SSAO, respectively, canceled this negative effect of MAO substrates on NOS2 expression. In the presence of Fe2+ and Cu2+ ions, inhibition of NOS2 expression was enhanced, suggesting the participation in this regulation of species derived from Fenton chemistry. In addition, the negative effect of H2O2, generated by MAOs, was found to be exerted on NOS2 mRNA levels. These data offer a new insight in the control of NOS2 expression through the intracellular levels of H2O2 and other reactive oxygen species (ROS). The hypothesis can be raised that the inhibition of NOS by H2O2 could constitute a protective mechanism against the cytotoxic consequences of the activation of ROS-generating enzymes, thus providing a new, singular role for the MAO family of proteins.

Key Words: NOS2 • oxidative stress • H2O2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are pivotal phagocytic cells, which upon activation, kill microorganisms as well as damaged and tumor cells during inflammation processes. Macrophage activation involves the participation of two separate oxidative pathways generating reactive oxygen species (ROS) [1 ] and nitric oxide (NO) [2 ], which is a short-lived messenger molecule involved in neurotransmission, regulation of blood pressure, and cytotoxicity, whose synthesis is catalyzed by NO synthases (NOS) [3 ]. Inducible NOS, designed as NOS2 or iNOS, is a NOS isoform whose expression becomes induced in macrophages by the presence in the extracellular medium of cytokines and microbial products, such as interferon-{gamma} (IFN-{gamma}) and bacterial lypopolysaccaride (LPS) [4 ]. It has been described that in the absence of a previous macrophage activation, the amount of NO produced is very low. The activation of macrophages is thought to take place in two stages, known as priming and triggering [5 ]. IFN-{gamma} is considered to be the predominant lymphocyte-derived cytokine involved in this process [6 ], and one of the genes whose expression becomes induced by IFN-{gamma} is NOS2 [4 ]. As for ROS generation, monoamine oxidases (MAOs) catalyze the oxidative deamination of primary amines, following the reaction R-H2-NH2 + O2 + H2O -> R-CHO + H2O2 + NH3 [7 ]. There are two groups of MAOs: flavin adenine dinucleotide-containing enzymes (MAO-A and -B) and a novel group designed as semicarbazide-sensitive amine oxidases (SSAOs). They differ in their substrates, cofactors, specific inhibitors, and subcellular distribution [8 ]. MAOs are mitochondrial enzymes with roles in the metabolism of neurotransmitters and other biogenic amines (e.g., tyramine) [9 ]. SSAOs are mostly soluble or expressed on the cell surface, are insensitive to classical MAO inhibitors (e.g., pargyline), and mediate the catabolism of biogenic amines [10 ], regulation of glucose uptake [11 ], and leukocyte-endothelial cell interactions [12 ]. Recently, the identity of murine SSAO as the vascular adhesion protein-1 (VAP-1) has been reported [13 14 15 ]. In the context of the present study, it is noteworthy that both oxidase types generate H2O2 through the oxidative deamination of monoamines and that H2O2 is recognized to constitute a signal-transducing molecule [16 ]. As several inflammatory, pathological states are associated with pro-oxidant conditions, in which intracellular levels of H2O2 are increased, the present study was undertaken to analyze whether oxidative stress could modulate NOS2 expression. We now report for the first time that exogenously added H2O2 acutely inhibits NO production and NOS2 expression. Further research was undertaken to analyze whether endogenous synthesis of H2O2, by reactions coupled to MAOs, could mimic such an inhibitory effect. Present findings reveal a novel function of MAO and SSAO enzymes, i.e., an involvement in the negative control of NOS2 gene expression at protein and mRNA levels, exerted by H2O2, derived from their catalytic activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of rat peritoneal macrophages
Macrophages were isolated from the peritoneal cavity of male Wistar rats (200–230 g), as described previously [17 ], but omitting previous casein treatment of the animals. Briefly, the cells were recovered by centrifugation at 200 g for 10 min and were washed twice with 10 ml ice-cold phosphate-buffered saline (PBS). Macrophages were seeded at 1 x 106 cells/cm2 in 24-well tissue-culture plates containing phenol red-free RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 50 mg/ml each gentamicin, penicillin, and streptomycin. The possibility of active bovine serum amine oxidase being present in FCS [18 ] was eliminated by its heat treatment at 68°C for 5 h before use. Additionally, this protein was not detectably found by Western blotting analysis of concentrated FCS (performed using anti-SSAO polyclonal antibodies as described below). After incubation for 1 h at 37°C in a 5% CO2 atmosphere, nonadherent cells were removed by extensive washing with PBS. Experiments were performed in the same medium above. Substrates, such as tyramine and benzylamine, and inhibitors, such as 3-amino-1,2,4-triazole, sodium ortovanadate, pargyline, and semicarbazide (Sigma Chemical Co., Alcobendas, Spain), were added 1 h before stimulation with 1 µg Escherichia coli LPS (Sigma Chemical Co.) plus 100 U recombinant rat IFN-{gamma} (Peprotech EC Ltd., London, UK) per ml medium. None of these reagents affected the viability of the cells at the concentrations used, as confirmed by the trypan blue dye-exclusion test.

Analysis of NO production
NO release was determined spectrophotometrically on the basis of accumulation of nitrite in the medium. Nitrite levels were determined using the Griess reagent as indicated previously [17 ].

Measurement of H2O2 levels
2,7-Dichlorohydrofluorescein diacetate (DCFDA) was used as an indicator of intracellular H2O2 levels [19 ], as described previously [20 ]. Briefly, 2 x 106 macrophages were incubated at 37°C for 1 h in the dark in the presence of 2.5 µM DCFDA (dissolved in ethanol). The cells were then rinsed twice with phenol red-free RPMI medium, and fluorescence intensity and distribution from 1 x 104 cells were analyzed on an Epics Elite flow cytometer (Coulter Electronics Inc., Hialeah, FL), equipped with a Coulter Elite software workstation. Results are expressed as the percentage of fluorescence-positive cells.

Cell lysis
For the analysis of NOS2 and SSAO expression by Western blotting, cells (5x106) were pelleted, lysed in 40 µl ice-cold lysis buffer A [20 mM Hepes, pH 7.9, 5 mM KCl, 0.1% Nonidet P-40 (NP-40), 1 mM EDTA, 1 mM dithiothreitol, 10 mM NaF, and the following protease-inhibitor mixture: 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml N-tosyl-L-phenylalanyl-chloromethyl ketone, 10 µg/ml captopril, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 mM iodoacetamide], and vortexed for 10 s following sonication for 10 s, and insoluble material was removed by centrifugation at 14,000 g for 2 min. For SSAO Western blotting analysis, a similar procedure was followed, except that the cells were disrupted in ice-cold lysis buffer B, with the following composition: 50 mM Tris-HCl, pH 7.9, 10 mM EDTA, 50 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), and the protease-inhibitor mixture indicated above. The supernatants were used for protein measurement by the Bradford procedure [21 ].

Western blotting analysis of NOS2 and SSAO protein expression
Proteins (40 µg/lane) were separated on 7.5% polyacrylamide-SDS gels under reducing conditions and electrophoretically transferred to polyvinylidene difluoride membranes using a semidry device (Bio-Rad, Richmond, CA). The membranes were probed without need of prior blocking [22 ] with specific mouse anti-NOS2 monoclonal antibodies (mAb; Transduction Laboratories, Lexington, KY) or else with rabbit polyclonal anti-SSAO antiserum (provided by Mercedes Unzeta, Universidad de Barcelona, Spain) or anti-ß-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 2 h at a 1:2000 dilution in PBS containing 1% bovine serum albumin (BSA) and 0.2% Tween-20. Next, the membranes were washed with PBS and incubated at room temperature for 30 min with horse anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG secondary antibodies (Innogenetics Diagnóstica y Terapéutica, Barcelona, Spain) linked to horseradish peroxidase at a 1/5000 dilution. After washing with PBS, immunoreactive bands were visualized by means of enhanced chemiluminescence, as described previously [23 ]. Different exposure times were used to ensure that the intensities of the bands in the films were proportional to the amount of blotted proteins in all cases. Reprobing blots with rabbit polyclonal antibodies against ß-actin (Santa Cruz Biotechnology) was performed to verify even protein-loading throughout lanes.

Flow cytometry analysis of NOS2 protein expression
Expression of NOS2 was analyzed by flow cytometry as described previously [24 ] with minor modifications. Cells were treated with the Fix and Perm cell permeabilization kit (Caltag Laboratories, Burlingame, CA), following the manufacturer’s indications. After washing, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-NOS2 mAb (Transduction Laboratories) or with its isotypic control FITC–IgG2a (Nion-Izasa, Barcelona, Spain), and the fluorescence intensity and distribution from 1 x 104 cells were analyzed on an Epics Elite flow cytometer (Coulter Electronics Inc.), equipped with a Coulter Elite software workstation. The results are expressed as the percentage of fluorescence-positive cells.

Immunofluorescence microscopy analysis of NOS2 and SSAO protein expression
The presence of NOS2 and SSAO/VAP-1 proteins was also assessed by immunofluorescence cell staining, as described previously [25 ] with the following modifications: After stimulation, macrophages (2x106 cells) were harvested, washed with PBS, and smeared onto poly-L-lysine-coated glass slides. The cells were fixed at room temperature with 2% paraformaldehyde for 10 min. After washing with PBS, unspecific binding was blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 15 min, and the cells were then incubated with mouse anti-NOS2 (Transduction Laboratories) or anti-SSAO/VAP-1 mAb (Alexis, Madrid, Spain) [12 , 14 , 26 27 28 29 30 31 ] at a 1:100 dilution for 1 h, washed extensively, and then stained with FITC-conjugated anti-mouse IgG at a 1:100 dilution for 30 min. After final washing, coverslips were mounted on the slides using 90% glycerol in PBS. Immunostained cells were observed and photographed using a Nikon EFD-3 microscope (Nikon-Izasa).

Northern blotting analysis of NOS2 mRNA expression
Total RNA extraction and Northern blotting analysis were performed as described previously [32 , 33 ]. Briefly, macrophages stimulated for 5 h were harvested by scraping, washed twice with PBS, and stored as cell pellets at –80°C until isolation of total RNA by the acid guanidinium thiocyanate-phenol-chloroform extraction was performed [33 ]. Total RNA (10 µg) was separated on 1% agarose/2.2 M formaldehyde gels and then transferred to nylon membranes. After ultraviolet-A cross-linking and prehybridization for 6–8 h at 65°C, the blots were hybridized overnight at 65°C with 106 cpm/ml [{alpha}-P32]deoxy-cytidine 5'-triphosphate-radiolabeled NOS2 cDNA probe (Oxford Biomedical Research, Oxford, MI) or with a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) cDNA probe as an internal control of loaded RNA amounts. The hybridization solution contained 3x saline sodium citrate (SSC), 0.1% SDS, 0.1% sodium pyrophosphate, 10% dextran sulfate, 10x Denhardt’s solution (0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, and 0.2% BSA), and 1 mg/ml denaturated salmon sperm DNA. The blots were washed subsequently at 65°C with 2x SSC/0.5% SDS twice for 30 min and then with 0.1x SSC/0.5% SDS twice for 30 min. Finally, they were exposed for up to 2 days to XRP-5 films (X-OMAT RP, Kodak, Liverpool, UK) with intensifying screens at –80°C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
H2O2 and vanadate act synergistically to inhibit LPS/IFN-{gamma}-induced NOS2 expression
The stimulatory effect exerted by LPS/IFN-{gamma} on NOS2 induction is well established [4 ]. Figure 1A illustrates that LPS/IFN-{gamma}-induced NOS2 protein expression was inhibited significantly in a dose-dependent manner by exogenously added H2O2 and that its negative effect was potentiated by aminotriazole, an inhibitor of the activity of the intracellular H2O2-removing enzyme, catalase. NOS2 synthesis was notably sensitive to H2O2, as a clear inhibition was already detectable at a dose as low as 10 µM H2O2. The combination of vanadate and H2O2 was necessary to obtain a nearly complete inhibition of NOS2 expression at 10 µM H2O2 (Fig. 1B) . Further analysis, performed using flow cytometry (Fig. 1C) and immunofluorescence staining (Fig. 1D) yielded results similar to those illustrated in Figure 1A on LPS/IFN-{gamma}-induced NOS2 expression. Besides, in separate experiments, we observed a positive correlation between the concentration of H2O2 added to the cultures and the degree of inhibition of LPS/IFN-{gamma}-promoted NO2 release obtained (Fig. 2 ).



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  		Figure 1. H2O2 inhibits LPS/IFN-{gamma}-induced NOS2 expression. Additive effect of aminotriazole and vanadate. Macrophages were cultured in the presence of H2O2 with or without 25 mM aminotriazole (AMT; A) and/or vanadate (VAN; B) at the indicated doses for 1 h before the addition of 1 µg LPS plus 100 U IFN-{gamma} per ml. The cells were lysed after 18 h of incubation, and NOS2 expression was assessed by Western blotting. Blot-reprobing with anti-ß-actin is shown as a control of loaded protein amounts. (C) The cells were left untreated (1) or treated for 18 h with 1 µg/100 U LPS/IFN-{gamma} per ml in the absence (2) or presence (3) of 50 µM H2O2. Results are expressed as the percentage of intracellular NOS2 fluorescence-positive cells obtained by flow cytometry analysis using FITC-conjugated anti-NOS2 mAb. (D) The cells were left untreated (1 and 2) or treated with 1 µg/100 U LPS/IFN-{gamma} per ml for 18 h in the absence (3 and 4) or presence (5 and 6) of 50 µM H2O2 and were subjected to immunostaining using anti-NOS2 antibodies. The same representative fields were photographed under visible (1, 3, and 5) and fluorescent (2, 4, and 6) light conditions.

 


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  		Figure 2. Exogenous-added H2O2 inhibits NO2– production by peritoneal macrophages. Macrophages were cultured in the absence or presence of 25 mM aminotriazole (AMT) and/or H2O2 at the indicated doses for 1 h before the addition of 1 µg/100 U LPS/IFN-{gamma} per ml. Then, nitrite was measured in the culture medium as indicated in Materials and Methods. All data are expressed as the mean ± SD from three separate experiments.

 
Effect of MAO substrates on H2O2 production and on NOS2 expression
MAOs catalyze the conversion of primary amines into aldehydes, ammonia, and H2O2 [7 ]. We tested the possibility that tyramine (an endogenous substrate of MAO) and benzylamine (a synthetic sustrate of SSAO) could influence LPS/IFN-{gamma}-induced NOS2 expression. With this purpose, macrophages were exposed to LPS/IFN-{gamma} and MAO substrates for 18 h. We found that 2 mM tyramine (Fig. 3A ) or 500 µM benzylamine (Fig. 4A ) alone promoted a drastic inhibition of LPS/IFN-{gamma}-induced NOS2 protein expression. These experiments were also performed in the presence of vanadate and aminotriazole, as previous studies have shown that the effects of MAO sustrates on glucose transporters could be mediated by the formation of peroxovanadium compounds [34 ] and that they were potentiated by the presence of the catalase inhibitor. As shown, a greater inhibitory capacity of tyramine (Fig. 3B) and benzylamine (Fig. 4B) on NOS2 expression was observed in the presence of vanadate and aminotriazole, as compared with their absence. These results are in line with previous work on the glucose transporter-4 [34 ] and suggest that peroxovanadate, generated from the reaction of H2O2 with vanadate, could be one of the reactive species negatively modulating NOS2 expression. In any case, it is noteworthy that the substrates of MAO and SSAO are able to promote by themselves inhibition of LPS/IFN-{gamma}-induced NOS2 synthesis. Further analyses by immunofluorescence staining (Fig. 5A ) and flow cytometry (Fig. 5B) yielded similar results regarding inducible expression of NOS2. By contrast, neither p-formaldehyde nor ammonia altered the synthesis of this protein induced by LPS/IFN-{gamma}, as assessed by Western blotting analysis (data not shown).



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  		Figure 3. Activation of MAOs (MAO-A/-B) inhibits LPS/IFN-{gamma}-induced NOS2 expression though H2O2 production. (A) Macrophages were cultured in the presence of 2 mM tyramine (TYR), a substrate of MAO-A and -B, at the indicated doses for 1 h before the addition of 1 µg/100 U LPS/IFN-{gamma} per ml. (B) Macrophages were cultured with 2 mM TYR, 10 µM vanadate (VAN), and/or 25 mM aminotriazole (AMT), as indicated, for 1 h before the addition of 1 µg/100 U LPS/IFN-{gamma} per ml. In both panels, the cells were lysed after 18 h of incubation, and NOS2 expression was assessed by Western blotting. (C) Intracellular production of H2O2 was analyzed by the DCFDA assay in macrophages, which were left untreated (1) or treated for 10 min with 2 mM TYR in the absence (2) or presence (3) of 500 µM pargyline (PARG), a specific MAO-A/-B inhibitor. Intracellular levels of H2O2 were measured by flow cytometry, and the results obtained are expressed as the percentage of fluorescence DCFDA-positive cells.

 


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  		Figure 4. Activation of SSAO inhibits LPS/IFN-{gamma}-induced NOS2 expression through H2O2 production. (A) Macrophages were cultured with benzylamine (BENZ), a substrate of SSAO, at the indicated doses for 1 h before the addition of 1 µg/100 U LPS/IFN-{gamma} per ml. (B) Macrophages were cultured with BENZ at the indicated doses, 10 µM vanadate (VAN), and/or 25 mM aminotriazole (AMT), as indicated, for 1 h before the addition of 1 µg/100 U LPS/IFN-{gamma} per ml. In both panels, the cells were lysed after 18 h of incubation, and NOS2 expression was assessed by Western blotting. (C) Intracellular production of H2O2 was assessed by the DCFDA assay in macrophages, which were left untreated (1) or treated for 10 min with 3 mM BENZ in the absence (2) or presence (3) of 500 µM semicarbazide (SZ), a specific SSAO inhibitor. Results are expressed as the percentage of fluorescence DCFDA-positive cells after flow cytometry analysis. (D) The presence of SSAO/VAP-1 protein in untreated macrophages was assessed by Western blotting using a specific polyclonal antiserum. (E) The presence of SSAO/VAP-1 protein in untreated macrophages was further assessed by immunofluorescence staining using a different mAb (1 and 2). Isotypic controls of the FITC-conjugated anti-mouse IgG antibodies are also shown (3 and 4). Representative fields were photographed under visible (2 and 4) and fluorescent (1 and 3) light conditions.

 


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  		Figure 5. Effect of MAO substrates on NOS2 expression assessed by flow immunocytochemistry and flow cytometry analyses. (A) Macrophages were left untreated (1 and 2) or treated for 18 h with 1 µg/100 U LPS/IFN-{gamma} per ml in the absence (3 and 4) or presence of 3 mM benzylamine (BENZ; 5 and 6) or 3 mM tyramine (TYR; 7 and 8). The same representative fields were photographed under visible (1, 3, and 5) and fluorescent (2, 4, and 6) light conditions. (B) Macrophages were treated as in A, and NOS2 expression was assesed by flow cytometry analysis. Results are expressed as the percentage of NOS2 fluorescence-positive cells.

 
Subsequent experiments were addressed to quantify directly by fluorescence, using DCFDA as indicator, the intracellular levels of H2O2 generated by MAO-A/-B and SSAO catalytic activities. Figure 3C shows that H2O2 production was promoted by treatment of macrophages with tyramine and that this process was abolished by pargyline, a MAO-A/-B-specific inhibitor [35 ]. Figure 4C shows the H2O2 synthesis taking place after treatment of macrophages with benzylamine and the complete inhibition of H2O2 production elicited by semicarbazide, a SSAO-specific inhibitor [36 ]. By contrast, when pargyline or semicarbazide was added alone to the cells, the rate of H2O2 synthesis was not altered (data not shown). These results suggested that the effect of tyramine and benzylamine on LPS/IFN-{gamma}-induced NOS is mediated, at least in part, by H2O2 generated by MAOs. Consequently, to augment the intracellular concentration of H2O2 and its inhibitory capacity on NOS2 expression, subsequent experiments were performed in the presence of aminotriazole and vanadate. As expression of MAO has been reported in macrophages previously [37 ], we investigated whether SSAO was also present in these cells. Western blotting analysis using specific anti-SSAO polyclonal antibodies illustrated this to be the case—this protein being detectable in macrophage lysates as a band corresponding to a molecular mass of ca. 100 kDa (Fig. 4D) . Immunofluorescence microscopy analysis using a different mAb further confirmed the presence of the SSAO/VAP-1 protein in peritoneal rat macrophages (Fig. 4E) .

Effect of Fe2+/Cu2+ ions on H2O2 levels and MAO substrate-dependent inhibition of NOS2 expression
After passive diffusion through the plasma membrane, H2O2 can be converted into other ROS, such as O2.– and OH·. The most likely mode of intracellular OH· radical production is via Fenton reactions, involving the reduction of H2O2 by Fe2+ and Cu2+ [38 ]. Therefore, we analyzed the possible involvement of OH· ions in the H2O2/MAO substrate-dependent NOS2 inhibition. Figure 6 illustrates that the presence of Fe2+/Cu2+ strongly enhanced the inhibition of NOS2 expression, promoted by H2O2 (Fig. 6A) , benzylamine (Fig. 6B) , or tyramine (Fig. 6C) . By contrast, the pretreatment of cells with 1,10-phenanthroline, a chelator of Fe2+ and Cu2+ ions, partially canceled the inhibitory effect of H2O2 and MAO substrates (Fig. 6D) . The addition of aminotriazole plus vanadate to the cells did not modify the LPS/IFN-{gamma}-dependent NOS2 expression (data not shown). These results suggested that the intracellular reduction of H2O2 to yield other ROS, such as OH·, constitutes an efficient mechanism for the control of NOS2 expression.



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  		Figure 6. MAO substrates inhibit NOS2 expression via increased ROS synthesis from Fenton reactions. Macrophages were preincubated for 1 h with FeSO4 (Fe2+) plus CuSO4 (Cu2+; 10 µM each; A–C) or 20 µM 1,10-phenanthroline (1, 10-PHEN; D). During this preincubation, the cells were treated at the indicated doses of H2O2 in the presence or absence of 25 mM aminotriazole (AMT; A), the cells were treated with the indicated doses of benzylamine (BENZ) and tyramine (TYR), respectively, with the addition of 25 mM AMT plus 10 µM vanadate (VAN) where indicated (B and C), and the cells were treated with 25 mM AMT and 10 µM VAN in the presence or absence of 200 µM BENZ or 2 mM TYR or 10 µM H2O2 where indicated (D). Thereafter, the cells were incubated in the presence of 1 µg/100 U LPS/IFN-{gamma} per ml for 18 h, and NOS2 expression was assessed in cell lysates by Western blotting.

 
To examine the specificity of MAO-negative effects on NOS2 synthesis, the effect of the SSAO and MAO inhibitors, semicarbazide and pargyline, respectively, was tested on NOS2 expression. Pargyline was found to completely cancel the inhibitory effect of MAO on NOS2 expression obtained upon enzyme activation with tyramine (Fig. 7A ), and reciprocally, semicarbazide prevented the negative effect of SSAO observed upon its activation with benzylamine (Fig. 7D) . The effect of both inhibitors was specific, as pargyline did not alter SSAO-inhibitory action on NOS2 expression (Fig. 7B) , and conversely, semicarbazide failed to affect the MAO-negative effect (Fig. 7C) .



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  		Figure 7. Inhibition of MAOs prevents inhibition of NOS2 expression. Macrophages were preincubated for 1 h with the MAO inhibitor pargyline (PARG; A and B) or the SSAO inhibitor semicarbazide (SZ; C and D) in the presence of the respective MAO and SSAO substrates, tyramine (TYR; 2 mM; A and C) or benzylamine (BENZ; 200 µM; B and D) and of 25 mM aminotriazole (AMT) plus 10 µM vanadate (VAN). Thereafter, the cells were incubated in the presence of 1 µg/100 U LPS/IFN-{gamma} per ml for 18 h, and NOS2 expression was assessed in cell lysates by Western blotting.

 
MAO substrates and H2O2 inhibit NOS2 mRNA expression
To analyze whether H2O2 and MAO substrates also acted negatively on NOS2 mRNA synthesis, Northern blotting analysis was performed after 5 h of LPS/IFN-{gamma} stimulation in the presence and absence of these compounds. Figure 8 illustrates that the incubation of cells with H2O2 or the MAO substrates benzylamine or tyramine completely canceled LPS/IFN-{gamma}-promoted induction of NOS2 mRNA expression in cultured rat peritoneal macrophages.



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  		Figure 8. Exogenous and endogenous H2O2 cancels NOS2 expression at the mRNA level. Macrophages were cultured for 1 h in the absence or presence of 3 mM benzylamine (BENZ), 3 mM tyramine (TYR), or 50 µM H2O2. Thereafter, the cells were incubated in the presence of 1 µg/100 U LPS/IFN-{gamma} per ml for 5 h, and NOS2 mRNA levels were analyzed by Northern blotting using a NOS2 cDNA probe, as described in Materials and Methods. As a control of loaded RNA amounts, rehybridization of the same blot with a GAPDH cDNA probe was subsequently performed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The intracellular processes that become triggered upon oxidative stress constitute nowadays a focus of extensive research. The present work demonstrates for the first time that LPS/IFN-{gamma}-induced NOS2 expression in peritoneal macrophages is inhibited acutely by H2O2. The fine sensitivity of NOS2 expression toward H2O2 is noteworthy, as at doses as low as 5–10 µM, a clear inhibition was already detected. In the search for biological processes implicated in the genesis of intracellular H2O2, we here report a singular role for the MAOs, MAO and SSAO, in mediating the inhibition of NOS2 expression in response to primary amines. Several lines of experimental evidence support the idea: Tyramine and benzylamine, substrates of MAO and SSAO, respectively, elicited a dose-dependent decrease of LPS/INF-{gamma}-induced NOS2 expression (Figs. 3 and 4) . Pargyline and semicarbazide, specific inhibitors of MAO and SSAO, respectively, antagonized the negative effect of these substrates on NOS2 expression (Fig. 7) . Evidence here presented that in addition to MAO [37 ], SSAO is expressed in macrophages (Fig. 4D) and is a functionally active enzyme, as its specific substrate, benzylamine, elicited intracellular H2O2 production (Fig. 4C) . Finally, H2O2, tyramine, and benzylamine were separately able to cancel net NOS2 mRNA synthesis induced by LPS/INF-{gamma} (Fig. 8) , likely by exerting a negative, modulatory action on transcription and/or NOS2 mRNA stability. The experimental results presented here strengthen the view that H2O2 production mediates MAO- and SSAO-dependent inhibition of NOS2 expression, as neither p-formaldehyde nor ammonia exerted any negative effect on this process. Such inhibition is reinforced by the exposure of cells to aminotriazole, which augments the intracellular concentration of H2O2 upon inhibition of catalase activity. The fact that H2O2-negative effects become enhanced by vanadate suggests that the actual inhibitory species acting on NOS2 expression is peroxovanadium. Experimental evidence is also presented that the ROS responsible for the observed inhibition on NOS2 expression are likely generated through Fenton reactions.

In examining the events leading to H2O2 release, it is accepted that the responsiveness of macrophages to stimulation is markedly enhanced by a previous stage-reaction process—priming. Two classical agents known to induce the priming effect on macrophages are IFN-{gamma} and LPS, although through different mechanisms. In this light, the priming-stage induction by treatment with the lymphokine is dependent on protein synthesis [39 ], whereas LPS-induced priming correlates with the potentiation of protein kinase C-dependent responses, such as enhanced arachidonic acid release [40 ]. However, the complexity of the interaction between IFN-{gamma} and LPS is evidenced by the fact that the pre-exposure of resident mouse peritoneal macrophages for 1 h to traces of LPS rendered the cells refractory to subsequent activation by IFN-{gamma}, as evaluated by the release of H2O2 upon stimulation with phorbol 12-myristate 13-acetate, and that such an inhibited state persisted for at least 4 days [41 ]. Therefore, it has been suggested that an elevated, intracellular cyclic adenosine monophosphate concentration in response to LPS-induced prostaglandin synthesis might mediate the observed inhibited state. In this light, it has also been observed that LPS-induced interleukin-8 gene expression is negatively modulated by IFN-{gamma} treatment in human granulocytes [42 ].

Most studies have been devoted to analyze the conditions under which release of H2O2 to the extracellular medium takes place by primed or activated macrophages, and much is known about the mechanisms by which this release takes place when phagocytic cells respond to soluble stimuli [38 ]. However, the knowledge on endogenous H2O2-generating systems, other than the classical O2.–-scavenging enzyme superoxide dismutase, and the potential mechanisms regulating such systems are limited by the inaccessibility of subcellular granules or membranes to standard ROS detectors and scavengers [43 ]. Using cytoplasts from neutrophils, which are devoid of granules but bear an intact, ligand receptor-coupling mechanism, it has been reported that formyl-methionyl-leucyl-phenylalanine, which acts through cell-membrane receptors, promotes the release of H2O2, whereas ionomycin, which bypasses cell-surface receptors, stimulates the generation of H2O2, which is retained inside the cells [44 ]. Thus, the existence of two differently regulated pools of H2O2 in macrophages is conceivable. However, and it is interesting that for the present study, the simultaneous presence of LPS and IFN-{gamma}, at variance with ROS release, has been shown to induce the synthesis of NOS2 in macrophages in a synergistic manner with generation of high levels of NO [45 , 46 ]. From these studies, it was concluded that the pathways leading to extracellular secretion of H2O2 and NO are independent. On this basis, present observations argue in favor of the hypothesis that the regulation of NOS2 synthesis in macrophages is modulated mainly according to intracellular H2O2 pool(s). However, it must be recognized that the role of oxidants in the regulation of NO release and NOS2 expression by macrophages and other cells remains a controversial issue. Thus, several authors have described that different antioxidants, when acting on the murine RAW 264.7 [47 ] or J774.1 macrophage cell lines [48 ] as well as on neuronal cells [49 ], markedly attenuate the induction of NOS2 expression elicited by LPS/IFN-{gamma}. Also, a direct, stimulating effect of H2O2 on NOS2 synthesis by RAW 264.7 and peritoneal murine macrophages has been reported recently [50 ]. At present, we lack a clear explanation for these conclusions, apparently opposed to our results, using H2O2 as a pro-oxidative reagent, although several methodological differences can be brought in regarding the work by Han et al. [50 ]. First, they used mainly the RAW 264.7 macrophages cell line, and in the experiments performed on peritoneal macrophages, the BALB/c mice were primed previously by injection with thioglycollate, whereas in our case only resident peritoneal macrophages were used. Second, these authors did not exclude contaminating cells after peritoneal lavage. Finally, the animal models used (rats in our studies and mice in theirs) were different. In an opposite manner and in line with present data, it has been described previously that under pro-oxidative conditions, such as reduced glutathione depletion in macrophages and hepatocytes [51 ], by using L-3,4-dihydroxyphenylalanine as an oxidant reagent in resident glial cells [52 ] or upon direct addition of H2O2 to macrophages [53 ], strong decreases of NOS2 expression and NO production are obtained. In conclusion, it appears that the use of different experimental conditions and cell lines may lead to obtain a wide and sometimes contradictory spectrum of NOS2 expression in response to intracellular redox imbalance.

Although the presence of MAO in macrophages was known, no data on the expression of SSAO in these cells were hitherto available, which we hereby report. In addition to its function as scavenger of amines [8 ], other physiological roles of SSAO are now beginning to be unraveled. Recently, it has been observed that SSAO/VAP-1 works as a molecular brake early in the cell-adhesion cascade and as a regulator of granulocyte extravasion during acute granulocyte-dependent peritoneal inflammation [26 ]. There exist various inflammatory disorders in which MAOs appear to play a role. For instance, SSAO is intensely expressed in high endothelial venules in inflammed synovial membranes [54 ]. It is interesting that several drugs in clinical use (e.g., the antimicrobial isoniazide and the antihypertensive hidralazine) are also known to inhibit SSAO activity [55 ]. In most cases, however, the contribution of SSAO inhibition to the biological effects of these drugs remains undefined. The synthesis of monoamines in the brain during carrageenan-induced acute paw inflammation in rats has been shown previously [56 ]. There is evidence that in diabetes, soluble SSAO activity may cause atherogenic lesions typical of this disorder by extensively yielding aldehydes and ROS from soluble substrates [57 ]. It has also been shown that intact cells produce ROS in response to incubation with tyramine and that inhibition of H2O2 production takes place by specific inhibitors of SSAO [58 ]. Taken together, these results strongly suggest that inflammatory processes are associated with an increase in the production of endogenous, biogenic amines and the subsequent activation of MAOs.

The potential, physiological significance of the interplay described here between the oxidative stress promoted by MAO/SSAO and NO synthesis is unknown. It is well documented that in the inflammatory response, many of the cell types that generate ROS, such as macrophages, also express NOS2 and that in some cases, the interaction between nitrosants and oxidants may yield products that are more toxic than either reactant alone [59 ]. In this context, it has been suggested that the balance between NO and O2.– generation is a critical determinant in the etiology of many inflammatory diseases [60 ]. As indicated previously, MAOs appear to be involved in a number of inflammatory diseases. In this scenario, it may be proposed that the inhibition of NO synthesis by H2O2 and/or other ROS could constitute a cytoprotective mechanism against the negative consequences for the cell of the activation of MAOs, which provides a novel clue to extend the physiological roles of this enzyme family.


   ACKNOWLEDGEMENTS
 
This work was financed by grants from the Ministerio de Ciencia y Tecnología (MCyT; Spain) SAF/2000-117, awarded to F. S., and SAF/2000-161, given to F. J. B., and by grants from the Fundación SEIAC, Spain, and Bial-Arístegui and Hycor Biomedical Inc., USA, awarded to J. M. A. V. and P. C. were supported by fellowships from the MCyT. A. V. and P. C. contributed equally to this work. We thank Dr. M. Unzeta, Universidad Autónoma de Barcelona, for her kind gift of anti-SSAO antiserum and for her helpful discussions and F. A. Prada, Instituto de Biología del Desarrollo, Universidad de Sevilla, for facilities and help on fluorescence and light microscopy.

Received October 7, 2003; revised February 2, 2004; accepted February 9, 2004.


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