Auto-protective redox buffering systems in stimulated macrophages

Background Macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. Using cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-γ), which induce strong endogenous NO production, we examined by which mechanisms a fraction of activated macrophages protect themselves from nitrosative stress and manage to escape destruction? Results We observed that survivors (10–50% depending on the experiments) had acquired a resistant phenotype being capable to survive when further exposed in vitro to an apoptosis inducing dose of the NO donor compound DETA-NO. These cells expressed an increased steady-state levels of Mn SOD, CuZn SOD and catalase mRNA (130–200%), together with an increased activity of the corresponding enzymes. Intracellular concentration of glutathione was also increased (× 3.5 fold at 6 hours, still maintained × 5.2 fold at 48 hours). Neither mRNA for glutathione peroxydase, γ-glutamylcysteine synthase and glutathione reductase, nor thioredoxine and thioredoxine reductase, were significantly modified. Additional experiments in which RAW 264.7 cells were stimulated with LPS and/or IFN-γ in the presence of relatively specific inhibitors of both Mn and Cu/Zn SOD, aminotriazol (ATZ) catalase inhibitor and buthionine sulfoximine (BSO) glutathione inhibitor, showed that inhibiting LPS-induced up-regulation of intracellular redox buffering systems also prevented acquisition of the resistant phenotype. Conclusions Our data suggest a direct causal relationship between survival of a fraction of macrophages and a up-regulation of key sets of auto-protective intracellular redox buffering systems, occurring simultaneously with modulation of expression of apoptotic molecules of the Bcl2-Bcl-XL/Bax-Bad family.


Background
Macrophages, and their circulating form monocytes, are potent defenders of the integrity of our body by mediating crucial physiological and protective functions. Just to mention some : they are central actors in innate immunity and inflammatory reactions; they process and present foreign antigens either by themselves, or through their lineage descendents; they are also dead cells scavengers. Strikingly, their role as front line defense against myriad of potentially pathogenic infectious agents in the outside environment, is essential in many species from insects to humans. Macrophages use various sets of receptor proteins to react to these agents, a central role being plaid by the Toll family molecules [1,2]. Such receptors recognize microbial lipids [3], lipoproteins [4,5], microbial carbohydrates [6] and bacterial DNA specific patterns [7]. Whatever the specific type of Toll like receptors involved, their engagements induce macrophages activation. This results in the production and release of a broad variety of specialized molecules aimed at limiting the multiplication and propagation of the infectious agents (innate immunity). The front line effector molecules produced by activated macrophages are reactive oxygen species (ROS) and reactive nitrogen species (RNS), highly diffusible molecules which have strong cytotoxic activities, including against macrophages themselves [8][9][10]. Such toxic effects are potentially dangerous since extensive macrophage destructions in the body can lead to the development of the fatal complication known as septic shock. Most of the time however, auto-protective redox buffering mechanisms prevent extensive destruction of activated macrophages. The aim of the present work was to elucidate by which mechanisms macrophages stimulated by bacterial products, manage to avoid massive auto-destruction caused by RNS? Experiments were performed with cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-g), two stimuli which induce strong endogenous NO production [11]. Our results directly establish that macrophage resistance is tightly regulated by the expression of definite sets of auto-protective redox buffering molecules.

LPS and/or IFN-g in vitro, stimulate cells of the murine RAW264.7 line along the differentiation pathway of secreting macrophages : induction of TNFa and NO production
We first made a detailed kinetics analysis of the response of RAW 264.4 cells to in vitro stimulation by LPS, or IFN, or both. We confirmed that these products stimulated the differentiation of the cells toward the phenotype of activated macrophages releasing NO and secreting TNFa. In our experiments, RAW 264.7 cells were exposed in vitro to either 50 units/ml of IFN-g (IFN 50 ), or 5 mg/ml LPS (LPS 5 ), or a mixture of both stimulating products. Cell free culture supernatants were harvested at 0, 6, 24 and 48 H and assessed for the production of TNFa, nitrites/nitrates and citrullin. Titrating TNFa production by ELISA, the strongest stimulation was observed with either LPS or LPS + IFN, leading to abundant secretions of TNFa as early as 6 H after the beginning of the culture, with a subsequent plateau at 24 and 48 H. The quantities of TNFa released by 0.25 ´ 10 6 cells initially seeded were in the range of 1400-1700 pg/ml at 24 and 48 H. Stimulation by IFN alone was less efficient inducing roughly only half of the above TNFa production (table 2). In the same supernatants, the production of NO was evaluated by quantifying citrulline and the total nitrite/nitrate with the Griess reagent. Both products were titrated in quantity (15-30 mM range and 30-70 mM range respectively) in 24 and 48 H culture supernatants of RAW 264.7 cells stimulated by either LPS or LPS+IFN. Smaller quantities were titrated in supernatants of cells stimulated by IFN alone (Table 2). Parallel experiments were performed to monitor NOs2 (iNOs) mRNA induction and NOs2 (iNOs) protein synthesis in RAW 264.7 cells exposed in vitro to either 50 units/ml of IFN-g (IFN 50 ), or 5 mg/ml LPS (LPS 5 ), or a mixture of both stimulating products. Cells were pelleted at 0, 6, 24 H and 48 H. NOs2 (iNOs) mRNA was evaluated by semi-quantitative RT-PCR ( Figure 1B), and NOs2 (iNOs) protein intracellular level by immunoblot ( Figure  1C). NOs2 (iNOs) was undetectable both at the mRNA and protein levels in unstimulated RAW 264.7 cells. It was strongly induced at times 6, 24 and 48 H with the three stimulation protocols ( Figure 1A & 1B). Only slight variations were noted depending of the stimulations used; IFNg alone being as previously, the weakest inducing stimulus compared to LPS and LPS + IFN-g.

Death induction of RAW 264.7 cells stimulated by IFN-g and/or LPS in vitro, with selection of a fraction of surviving activated RAW 264.7
In vitro exposure of cells of the murine macrophage cell line RAW 264.7 to either 5 mg/ml LPS (LPS 5 ), or 50 units/ ml of IFN-g (IFN 50 ), or a mixture of both stimulating products, led to a substantial reduction of the growth rate and number of surviving cells after 24 H or 48 H in culture ( Figure 1A). The strongest deleterious effects were observed culturing the cells with either LPS alone or both IFN and LPS, resulting in the recovery of only approximatively 20% viable cells as estimated by MTT assay (or trypan blue test) at 24 H, and 13% at 48 H. IFN alone induced less cytoxicity with 54% and 50% viable cells recovered at 24 H and 48 H respectively ( Figure 1A). The surviving cells subsequently recovered an in vitro growth rate comparable to untreated parental RAW 264.7 cells in culture (not shown). Additional experiments indicated however that those surviving activated macrophages had acquired peculiar resistance capacity to exogenous oxydative and nitrosative stress. More experiments were performed to characterize this resistance phenotype and the differentiation induced cellular metabolic events responsible for this resistance.

Figure 1
A/ Growth rate and survival of RAW 264.7 cells cultured in medium, or medium supplemented with LPS or IFN-g or LPS+IFN-g. RAW 264.7 cells were cultured (4 ´ 10 6 cells in 20 ml) in medium (• oe) or medium supplemented with either IFN-g (50 U/ml -.), or LPS (5 mg/ml -) or a mixture of IFN-g (50 U/ml) + LPS (5 mg/ml -). The number of live cells recovered at the indicated times was estimated using trypan blue exclusion. Similar results were obtained estimating the cell survival either by 3H-thymidine incorporation, or by the MTT reduction test. B/ NOS2 (iNOs) mRNA induction in RAW 264.7 cultured with LPS or IFN-g or LPS+IFN-g. RAW 264.7 cells seeded into six-well plates (2.5 ´ 10 6 cells/well) were treated with the different stimuli as indicated above for the indicated times and analyzed by RT-PCR with specific primers for murine NOS2 (iNOs) ( Table 1). HPRT was used as internal control for semi-quantitative estimation. C/ NOS2 (iNOs) protein in RAW 264.7 cultured with LPS or IFN-ã or LPS+IFN-g. RAW 264.7 cells were treated with the different stimuli as indicated above for various times and cell pellets (1 ´ 10 6 cells) were lysed with lysis buffer. Protein concentrations in samples were adjusted and electrophoresed on 15% SDS-polyacrylamide gel, then transferred to nitro-cellulose membrane and Western bloted using polyclonal rabbit anti-murine NOS2 (iNOs), as described in Materials and Methods.   Figure 2A). The susceptibility of LPS, or IFN, or LPS+ IFN activated macrophages to the NO releasing compound DETA-NO, was evaluated in vitro by measuring the number of metabolically active cells (reducing Formazan = MTT assay) or trypan blue negative cells, recovered at the indicated time.
We established previously that the MTT test provides a reproducible quantitative estimate of cell viability giving comparable results with the trypan blue dye exclusion method. As can be seen in Figure 2B, unselected parental RAW 264.7 cells were susceptible to DETA-NO death induction in vitro, with only 55% cells surviving after 24 H, and 45% after 48 hours. By contrast, LPS, or IFN, or LPS+  ND: not detectable RAW 264.7 cells (250 000 cells/well) were cultured with medium or medium supplemented with either IFN-g (50 U/ml), or LPS (5 mg/ml) or a mixture of IFN-g (50 U/ml) + LPS (5 mg/ml). Cell-free culture supernatants were harvested at 0, 6, 24, and 48 hours. The concentrations of TNF-a (measured using an ELISA kit), nitrites/nitrates (measured using the Griess reagent) and citrulline (measured using colorimetric reaction), were evaluated in each culture supernatant as described in Methods. Data represent means +/-SEM of five independent experiments.
IFN activated RAW 264.7 macrophages were less susceptible to DETA-NO death induction in vitro, with variations depending upon the stimulation protocol. (Fig 2B). Activated-macrophages recovered from cultures stimulated with LPS alone were almost completely resistant to the nitrosative stress. Activated-macrophages recovered from IFN, or IFN + LPS stimulated cultures, exhibited an intermediate NO resistance, with about 70% and 60% metabolically active cells, or trypan blue negative cells, recovered after 24 or 48 H in culture with DETA-NO.

Upregulation of redox protection/detoxification systems in LPS-differentiated NO resistant RAW 264.7 cells
LPS (and/or IFN) induced NO resistance of differentiated RAW 264.7 macrophages could result from an up-regulation of their cellular redox protection/detoxification systems. We therefore evaluated the LPS (and/or IFN) induced modulation of a number of redox proteins, critical in the three major systems protecting cells against oxydative and nitrosative stress : the superoxide dismutase/ catalase system (Cu/Zn SOD, Mn SOD and catalase); the glutathione system (glutathione (GSH), g-glutamyl cystein synthase (g-GCS), glutathione peroxidase (Gpx), gluthatione reductase (GR); and the thioredoxin system (thioredoxin (Trx), thioredoxin reductase (TR). The transcriptional modulation of several of these redox proteins was evaluated by semi-quantitative RT-PCR on 24 hours cell extracts, using the specific primers described in table 1. As shown in Figure 3, Cu/Zn SOD, Mn SOD, and catalase, were significantly upregulated (130 to 200 % increased) in NO resistant activated RAW 264.7 macrophages obtained following 24 hours stimulation with LPS (and/or IFN). By contrast, g-glutamyl cystein synthase (g-GCS) and glutathion peroxidase (Gpx) were only marginally increased with IFN, while glutathione reductase (GR), thioredoxin (Trx) and thioredoxin reductase (TR) mRNA were not significantly affected. This gene transcription study was further documented by measuring cellular concentrations of glutathione (GSH) in lysates of activated RAW 264.7 macrophages, recovered 6, 24 or 48 hours after LPS (and/or IFN) stimulations. As can be seen in Figure 4, glutathione level was approximatively 3.5 folds increased at 6 hours in LPS stimulated RAW 264.7 cells, compared to basal level in unstimulated control RAW 264.7 cells. This LPS induced high glutathione level was still detected at 24 and 48 hours (2.6 and 5.2 fold increased respectively). IFN 50 and IFN 50 /LPSs stimulations also increased intra cellular glutathione levels in RAW 264.7 cells, with the same kinetics than LPS alone, although with variable efficiency (Figure 4). Additional experiments were performed to quantify the cellular specific activity of several other redox protection proteins in lysates of RAW 264.7 macrophages, recovered 6, 24 or 48 hours after LPS (and/or IFN) stimulations. As can be seen in table 3, catalase activity was approximately two folds

LPS induced NO resistance of differentiated RAW 264.7 macrophages is abrogated by chemical inhibitors affecting the intra-cellular redox protection/detoxification systems
The causal relationship between the acquisition of NO resistance and the upregulation of a selective set of redox protection/detoxification systems in LPS differentiated RAW 264.7 macrophages was established using chemical inhibitors targeting those redox systems. The compounds used were relatively specific inhibitors affecting the SOD/ catalase system and the glutathione system, the two redox protection/detoxification biochemical sets significantly up-regulated in LPS-differentiated macrophages. DETC is an inhibitor of both Mn SOD and Cu/Zn SOD, ATZ is a catalase inhibitor, and BSO reduces the intra-cellular level of glutathione by inhibiting the g-glutamyl cysteine synthase. In our experiments, RAW 264.7 cells were first differentiated in vitro, as previously, by stimulation with an optimal dose of LPS (5 mg/ml) for 48 h. Selected-surviving-LPS-differentiated RAW 264.7 macrophages (and con-trol unstimulated RAW 264.7 cells) were washed and incubated for an additional 2 hours period in the presence of the redox inhibitors (or not). Subsequently, nitrosative stress resistance of the treated cells was evaluated by culturing them in the presence of 1 mM DETA-NO and assessing cell viability after 24 hours (number of metabolically active cells reducing formazan = MTT test).
The results are presented in Figure 5. LPS differentiated macrophages were 90-100% resistant to the toxic effects of NO released by 1 mM DETA-NO in vitro, while unstimulated cells were susceptible to NO, with 45% cells only surviving to the same in vitro treatment. Noteworthy, incubation of LPS differentiated cells in the presence of the redox inhibitors abrogated their NO resistance and restored their susceptibility to the toxic effects of DETA-NO. The SOD inhibitor DETC was the most effective compound. It completely reversed the NO resistance status of LPS-differentiated macrophages : only 10% of DETCtreated LPS-differentiated macrophages were recovered at 48 hours following exposure to DETA-NO; compared to 90% recovery of LPS-differentiated macrophages cultured without DETC ( Figure 5C). The two others redox inhibitors BSO (g-GCS inhibitor) and ATZ (catalase inhibitor), also very significantly abrogated NO resistance in LPS-differentiated macrophages (with only 35% and 30% viable cells recovery following DETA-NO exposure of LPS-differentiated macrophages treated with these two compounds, respectively) ( Figure 5A & 5B). In addition we noted that the SOD inhibitor DETC substantially sensitized unstimulated RAW 264.7 cells to the toxic effects of DETA-NO : only 10% DETC treated unstimulated RAW 264.7 cells survived NO exposure, compared to 40% control unstimulated RAW 264.7 not incubated with DETC ( Figure 5C).

Discussion
Macrophages mediate crucial functions protecting the organism against pathogenic infectious agents. Upon encounter with micro-organisms or stimulation by cytokines, they produce and release various sets of effector molecules aimed at destroying the foreign agents. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are their front line effector molecules [13,14]. These highly diffusible products exert strong cytotoxic activities against micro-organisms and many cells, including against macrophages themselves. However, a fraction of activated macrophages exposed to redox stress manage to escape destruction. Our results establish that nitrosative resistance results from the adapatative tuning of the redox buffers in these cells.
Reactive oxygen species and nitrogen monoxide are the earliest cytotoxic defense molecules produced by activated murine macrophages after contact with the infectious agents. Elevated oxygen production in macrophages (and polymorpho nuclear cells) relies upon the enzyme

Figure 4
Glutathione concentrations in RAW 264.7 either undifferentiated, or differentiated cells. Cells were cultured for the indicated times in medium, or medium supplemented with 5 mg/ml LPS, 50 U/ml IFN-g or a mixture of IFNg (50 U/ml) + LPS (5 mg/ml). Total glutathione was quantified using "Glutathione Cellular Assay" kit (specific evaluation of glutathione-SH groups). Results are expressed as means +/-SEM of five independent experiments.
NaDPH oxydase [15]. Elevated and sustained production of nitric oxide (NO), a short-lived radical molecule generated from the guanido nitrogen group of L-arginine, is controled by an inducible NO synthase isoform (iNOS) [15]. This iNOS enzyme is tightly regulated in macrophages at variance with the two additional constitutive isoforms of NO synthase (cNOS) which are expressed in different tissues and function in basal physiological conditions. These later enzymes produce low levels of NO involved respectively in neurotransmission, and vascular relaxation [16][17][18][19]. Elevated production of NO in activated macrophages by the inducible NOS isoform following cytokines and/or bacterial lipopolysaccharide (LPS) challenge has been recognized as an essential anti-bacterial defense mechanism in rodent [14]. NO is not a strong oxidant by itself. However, NO can react with transition metals and reactive oxygen species (ROS) leading to the secondary generation of very toxic and reactive products like peroxynitrite (ONOO-) [20]. All these products, NO and its related nitrosocompounds, mediate toxicity in bacterial assailants. They also mediate growth arrest and apoptosis in normal mammalian cells [13] (including autocrine and paracrine toxicity for activated macrophages) and tumor cells [21][22][23].
Mammalian cells are equiped with intracellular protection systems, « redox buffer t, which protect them from endogen oxygen radicals produced by the respiration in mitochondria [24,25]. Three biochemical sytems are the most important redox buffers. A predominant protection is by the Cu/Zn-dependent or Mn-dependent superoxide dismutase enzymes (SOD) which convert superoxide ions to hydrogen peroxide, then detoxified by catalase [26,27].
A second protection is by gluthatione (GSH), an intracellular peptidic thiol molecule with both oxidant scavenger and redox regulating capacities [28][29][30]. Thiols on gluthatione combine with NO and form the less reactive product S-nitrosoglutathione (GSNO). A third protection is performed by thioredoxine (Trx) which can buffer ROS and RNS through oxidation of its intra chain disulfide bridge, reduced thioredoxine being then regenerated by the enzyme thioredoxine reductase (TR) [31][32][33]. The respective roles of these intracellular antioxidant autoprotective systems against ROS and/or RNS-mediated auto-injury in activated macrophages have not been defined in details.
Using a murine macrophage cell line (RAW 264.7), abundantly producing NO upon stimulation with lipopolysaccharide (LPS) and/or interferon (IFN-g), we examined changes in those intracellular redox homeostatic systems and, using relatively specific inhibitors, identified the most important redox protection systems.
Our experiments indicate that macrophage stimulation induce an up-regulation of the two major intracellular redox buffering systems : the enzymatic SOD-catalase system and the RNS acceptor-neutralizer molecule glutathione. Such differentiated macrophages are resistant to exogenous NO This activation-induced new redox status is stable since the resistant phenotype of RAW 264.7 cells persisted after induction for at least one week in culture (data not shown). The regulation is both at the transcriptional and protein levels for SOD and catalase. The increased glutathione content in cells following stimulation is not explained by an increased transcription of the enzyme g-glutamylcysteine synthase controling its synthesis; nor by an increased transcription of the regenerating   Figure 2A). The % viability of cell exposed to exogenous NO was assessed after 48 hours as the percent of metabolically active cells reducing formazan (MTT test) compared to control (number of RAW 264.7 cells undifferentiated, not exposed to exogeneous NO, surviving at the end of the culture period = 100% cell viability). enzyme glutathione reductase. It might result from the post translational stress-induced allosteric activation of the glutathione generating enzyme g-glutamylcysteine synthase, or (and) from intra-cellular mobilization of an undefined inactive pool [34]. We did not observed any modulation of the third redox buffering system thioredoxine-thioredoxine reductase. This is at variance with our own previous results with THP1 monocytic human cells which heavily relied upon the thioredoxine system to maintain their redox homeostasis [33]. Such variation might represent inter species difference. The present data with RAW 264.7 cells are in good agreement with previous work from Brockaus & Brune group who reported that stably transfected RAW 264.7 cells overexpressing CuZn SOD were more resistant to endogenous or exogenous NO [35].
Using relatively specific inhibitors, we were able to establish a causal relationship between the upregulation of the intra cellular redox buffering systems mentioned above and the acquisition of their nitrosative stress resistant phenotype. The SOD inhibitor DETC almost completely reversed the NO resistance status of LPS-differentiated macrophages. It also substantially sensitized unstimulated RAW 264.7 cells to the toxic effects of DETA-NO. The two others inhibitors BSO (g-GCS inhibitor) and ATZ (catalase inhibitor), also very significantly abrogated NO resistance in LPS-differentiated macrophages. Indeed the activation-induced resistance of macrophages has been studied by many groups (also designated as endotoxintolerized macrophages). Their results linked the resistance of macrophages to several, non exclusive, mechanisms such as : complex impaired expression and/or function of common signaling pathways [36] increased expression of Bcl-xL anti-apototic proteins [37], increased expression of Bfl-1 anti-apototic gene and simultaneous down regulation of caspase-8 mRNA [38], alteration of the Fas-Fas ligand transduction pathway [39], over-expression of CD14 receptor [40], autocrine cytokine regulatory network between IL-12 and IL-10 [41,42]. The multiplicity and complexity of the macrophage responses was recently documented using the gene array technology which detected a broad spectrum of genes overexpressed in response to S. typhimurium and S. typhimurium LPS in RAW 264.7 cells [43]. We also observed in our experiments an increased expression of Bcl 2 and Bcl-XL and decreased expression of Bax and Bad in NO-resistant activated macrophages (not shown). Nevertheless, our results clearly demonstrated the crucial auto-protective importance of the up-regulation of the two major redox buffering systems SOD-catalase and glutathione, for nitrosative stress resistance of activated macrophage. A schematic representation of the many factors regulating survival of activated macrophages is presented in Figure 6.

Conclusion
Activated macrophages exposed to endogenous NO are submited to a redox nitrosative stress which acts as a strong selection process allowing the survival of the fraction of cells up-regulating key sets of autoprotective redox buffering molecules. What are the cascade of events and the mechanisms leading to the transcriptional activation of SOD genes and increased glutathione availability in activated macrophages, remain to be determined. Such findings might help define new strategies aimed at improving the induction of nitrosative stress resistant macrophages which could be the best efficient defenders of the organisms against infection and possibly cancer.

Titration of macrophage-derived pro-inflammatory products
TNF-a were measured in undiluted supernatants using ELISA kit purchased from Medgenix Diagnostics (Rungis, France) according to manufacter's instructions, in triplicates for each sample. The total amount of nitrite/nitrate, the stable products of NO, was determined in supernatants using the Griess reagent. Briefly, 50 ml of culture supernatants collected at 6, 24 and 48 hours were mixed with 150 ml of Griess reagent (1% sulfanilamide/0,1% naphtylethylenediamine-dihydrochloride) at RT for 30 s. The absorbance at 543 nm was immediately determined on Dynex Revelation F 3.21 microplate reader. Nitrite concentrations in each sample were determined by extrapolation from a sodium nitrite standart curve.
L-citrulline levels were determined by the colorimetric reaction of carbamido groups with diacetyl monoxime in acid solution [10]. Briefly, 30 ml of urease (25 U/ml) was added to 300 ml of supernatant, and incubated for 1 hour at 37°C. After addition of 37.5 ml trichloroacetic acid (TCA, 59% vol/vol), the precipitated proteins were pelleted by 5 minutes centrifugation at 12 000 g. 250 ml of supernatant was harvested and mixed with 300 ml of a 1:

Susceptibility-resistance of RAW 264.7 cells to NO-mediated cell injury
The susceptibility-resistance of RAW 264.7 cells exposed in vitro to exogenous NO was evaluated on cells distributed in 96 wells plates (4.10 4 per well). Diethylene triamine-nitric oxide (DETA-NO) 1 mM was added to each well at t0 and incubation processed for the indicated times. This 1 mM dose induced substantial cytotoxicity of undifferentiated RAW 264.7 cells after 24 h (Figure 2A). After various incubation times, cell viability was assessed using 3 methods: trypan blue dye exclusion test, 3H thymidine incorporation and MTT assay (mitochondria-dependant reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (MTT) to formazan. The 3 tests give highly concordant results in every experiments, with or without NO-donor molecules. Results with the MTT assay only are presented. It was performed as follow : 20 ml of MTT (5 mg/ml in PBS 1X) was added in each wells and incubated at 37°C for 4 hours. Then, 150 ml of medium was removed from each well, and 100 ml of 0,5% HCl/propanol-1 was added to dissolve cristals. The net absorbance ratio (ratio A 550 -A 630 ) in each well was immediately recorded using an ELISA microplate reader. The net absorbance in wells containing cells cultured in control medium was taken as the 100% viability value. The percent of viable cells exposed to DETA-NO was calculated by comparison.

Determination of cellular content of glutathion
The cellular concentration of glutathion was evaluated using the reagent kit Bioxytech GSH-400 (Bioproducts, Gagny, France) according to manufacturer instructions', a glutathione colorimetric quantification test based upon specific evaluation of glutathione-SH groups.

Activities of redox enzymes
In vitro cultures of RAW 264.7 cells, in medium or medium supplemented with the differentiating agents, were interruped at the indicated times. Cells were pelleted, washed three times and lysed by three successive freezingthawing cycles. The enzymatic activities were evaluated on cell lysates recovered after centrifugation at 12 000 g for 15 mn. The catalase activity was assayed by monitoring the decreased absorbance at 240 nm resulting from the decomposition of H 2 O 2 for 1 min at 37°C. The assay mixture consisted of 2.98 ml of H 2 O 2 solution from a stock solution of 0,1 ml of 30% (w/v) H 2 O 2 diluted in 50 ml of sodium phosphate buffer, pH 7, and 20 ml of the incubation solution (0.025 mg of enzyme/ml of buffer). All assays were performed in triplicate. Activity is expressed in comparision with control activitis determined for each incubation time set as 100% value [12]. SOD and Gpx activities were determined using the reagent kits SOD-525 and Gpx-340 kits (from Alexi and Calbiochem respectively) following manufacturer's instructions.

Statistical analysis
Results are reported as mean ± SD except where indicated, and differences between groups are calculated by means of the nonpaired Student's t-test. Experiments were performed in triplicate.