Auto-protective redox buffering systems in stimulated macrophages
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?
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.
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.
KeywordsNitric Oxide Reactive Nitrogen Species Nitrosative Stress Foreign Agent Murine Macrophage Cell Line
inducible isoform of nitric-oxide synthase
- Cu/Zn SOD
Cu/Zn-dependent superoxide dismutase
- Mn SOD
Mn-dependent superoxide dismutase
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 , lipoproteins [4, 5], microbial carbohydrates  and bacterial DNA specific patterns . 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-γ), two stimuli which induce strong endogenous NO production . 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-γ in vitro, stimulate cells of the murine RAW264.7 line along the differentiation pathway of secreting macrophages : induction of TNFα and NO production
Differentiation of RAW 264 7 cells toward secreting-activated macrophages following culture with LPS, IFN-γ or LPS+IFN-γ
nitric oxide metabolism
TNF production (pg/ml)
462 +/ 250
681 +/ 42
Death induction of RAW 264.7 cells stimulated by IFN-γ 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 μg/ml LPS (LPS5), or 50 units/ml of IFN-γ (IFN50), 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.
Nitrosative stress resistance of RAW 264.7 macrophages surviving LPS, or IFN, or LPS+ IFN, stimulation
Upregulation of redox protection/detoxification systems in LPS-differentiated NO resistant RAW 264.7 cells
Pairs of synthetic primers used in RT-PCR to amplify the different molecular systems studied.
5'TGG AAT CCT GTG GCA TCC ATG AAA C 3'
5' TAA AAC GCA GCT CAG TAA CAG TCC G 3'
5' GCA GAT ACC TGT GAA CTG TC 3'
5' GTA GAA TGT CCG CAC CTG AG 3'
5' CCT TCT GGC ACA GCA CGT TG 3'
5' TAA GAC GGC ATC TCG CTC CT 3'
5' CCT CAA GTA CGT CCG ACC TG 3'
5' CAA TGT CGT TGC GGC ACA CC 3'
5' AGC CGC CTG AAC ACC ATC TA 3'
5' CCG TCT GAA TGC CCA CTT TA 3'
5' ACG CTT CAC TTC CAA TGC AAC 3'
5' TGA GGG CTG ACA CAA GGC CTC 3'
5' AAG GCC GTG TGC GTG CTG AA 3'
5' CAG GTC TCC AAC ATG CCT CT 3'
5' GCA CAT TAA CGC GCA GAT CA 3'
5' AGC CTC CAG CAA CTC TCC TT 3'
5' CCC TTC TTC CAT TCC CTC TG 3'
5' AAC TCC CCC ACC TTT TGA CC 3'
5' TCC TCT TTT TCT ACC CAC TG 3'
5' GTA TTC CTT GCT GTC ATC CA 3'
Activities of the redox enzymes catalase, Gpx and SOD, in differentiated NO resistant RAW 264 7 cells
LPS 5 /IFN 50
LPS induced NO resistance of differentiated RAW 264.7 macrophages is abrogated by chemical inhibitors affecting the intra-cellular redox protection/detoxification systems
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 NaDPH oxydase . 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) . 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 . 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-) . All these products, NO and its related nitrosocompounds, mediate toxicity in bacterial assailants. They also mediate growth arrest and apoptosis in normal mammalian cells  (including autocrine and paracrine toxicity for activated macrophages) and tumor cells [21, 22, 23].
Mammalian cells are equiped with intracellular protection systems, « redox buffer », 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-γ), 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 γ-glutamylcysteine synthase controling its synthesis; nor by an increased transcription of the regenerating enzyme glutathione reductase. It might result from the post translational stress-induced allosteric activation of the glutathione generating enzyme γ-glutamylcysteine synthase, or (and) from intra-cellular mobilization of an undefined inactive pool . 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 . 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 .
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.
Materials and Methods
The monoclonal antibody (MoAb) against NOs2 (iNOs) was purchased from Pharmingen (San Diego, CA). The horseradish peroxydase-conjugated goat-anti-rabbit antibody was purchased from BIO-RAD (Bio-Rad laboratories, California). The NO donor compound 2,2'(hydroxynitrosohydrazino)bis-ethanamine (DETA/NO) was purchased from Calbiochem. Purified LPS from Escherichia Coli, Leupeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), EDTA, Hepes, CHAPS, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT), ATZ (aminotriazol), BSO (Buthionine sulfoximine), DETC (Diethylthiocarbamate) and DTT, were from Sigma. ECL chemoluminescence enhancer reagents were from Amersham.
Cells of the mouse macrophage-RAW 264.7 line were obtained from the American Type Culture Collection  and cultured in RPMI 1640 medium supplemented with glutamax 1 (GIBCO/BRL/Life Technologies), 7% (v/v) heat-inactivated foetal calf serum (HyClone), 100 units/ml penicillin and 100 μg/ml streptomycin, at 37°C in a humidified air/CO2 (5%) atmosphere. Standart bulk cultures were with 10 × 106 cells in 150 cm2/50 ml vials (Corning, polystyrene) in culture medium, or culture medium supplemented with either 5 μg/ml Escherichia Coli LPS (serotype O127:B8, from Sigma), or 50 units/ml recombinant mouse IFN-γ (Genzyme), or the combinaison of both (LPS 5 μg/ml plus IFN-γ 50 units/ml).
Titration of macrophage-derived pro-inflammatory products
TNF-α 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 μl of culture supernatants collected at 6, 24 and 48 hours were mixed with 150 μl 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 . Briefly, 30 μl of urease (25 U/ml) was added to 300 μl of supernatant, and incubated for 1 hour at 37°C. After addition of 37.5 μl trichloroacetic acid (TCA, 59% vol/vol), the precipitated proteins were pelleted by 5 minutes centrifugation at 12 000 g. 250 μl of supernatant was harvested and mixed with 300 μl of a 1:1 (vol/vol) mixture of 240 mmol diacetylmonoxime and a solution of phenazone (3 g in 104 ml H2O reacted with 12 mg FeSO4 and 21 ml H2SO4 36N, and incubated 15 minutes at 90°C in dark. 200 μl of the reaction mixture was transferred to a microtitration plate for measurement of the optical density at 492 nm using Dynex Revelation F 3.21 microplate reader. Citrulline concentrations were determined by extrapolation from a citrulline standart curve.
RNA isolation and RT-PCR analysis
Following in vitro stimulation for the indicated times with the indicated stimulus, total cellular RNAs were extracted from 2.5 × 106 RAW 264.7 cells using TRI reagent (Euromedex, Souffelweyersheim, France) according to manufacturer's instructions. cDNAs were synthesised in 40 μl reaction mixtures containing 2 μg of RNA, 8 μl of 5X buffer (250 mM TRIS-HCL, 250 mM KCL, 50 mM MgCl2, 50 mM DTT and 2.5 mM spermidine), 2 μl of each dNTP 25 mM, 0.4 μl of 50 mM oligo-dT primers, 0.4 μl of RNAsin at 22 000 U/ml and 0.6 μl of Avian Myeloblastosis Virus-reverse transcriptase (Appligene, Strasbourg, France) at 42°C for 2 hours. PCR was performed using 1/20 of the cDNA reaction mixture in a 100 μl reaction volume containing 10 μl of 10X buffer (10 mM TRIS-HCL, 50 mM KCL, 1.5 mM MgCl2, 0.1% Triton X 100 and 0.2 mg/ml gelatine), 0.5 μl of each dNTP 25 mM, 0.4 μl of Taq Polymerase 5 000 U/ml (Thermus aquaticus, Appligene) and 1 μl of each primer 20 μM. The RT-PCR products were subjected to electrophoresis on agarose gel, stained with ethidium bromide, scanned and quantified by densitometry. The sequences of sense and anti-sense PCR-primers used for amplification and the predicted size of products are described on Table 1. HPRT mRNA was used as internal standard. Modulation of mRNA expression in RAW 264.7 cells either undifferentiated, or differentiated, was standardized as follow. Bands were scanned and quantified by densitometry. The ratios : redox protein mRNA band intensity / HPRT mRNA band intensity were calculated for each stimulation conditions. The ratio values were then standardized taking the relevant mRNA band intensity ratio in undifferentiated RAW 264.7 cells as reference value 1.
Pellets of 1 × 106 cells were lysed in lysis buffer (25 mM HEPES, 0.5% Nonidet P40, 0.1% SDS, 0.5 M NaCl, 5 mM EDTA, 0.1 mM sodium deoxycholate, 1 mM PMSF, and 0.1 mg/ml leupeptin/pepstatin, pH 7.8) at 4°C for 30 min. Nuclei and membranes were removed by centrifugation at 12 000 g for 15 min. The amount of protein in each lysate was measured using the BSA microbiuret assay from Pierce. Loading buffer (42 mM tris-HCl, pH 6.8, 10% glycerol, 2.3% SDS, 5% 2-mercaptoethanol, and 0.002% bromophenol blue) was added to each lysate, which was subsequently boiled for 3 min and electrophoresed on an 15% SDS-polyacrylamide gel. Proteins were transferred to nitro-cellulose membrane. The membrane was saturated with TBS (150 mM NaCl, 10 mM tris HCl, pH 7.5) containing 10% skim milk overnight, washed twice 15 min with TTBS 0.1% (150 mM NaCl, 10 mM tris HCl, 0.1% Tween 20, pH 7.5), and incubated with a polyclonal rabbit-anti-murine NOS2 (iNOs) (1/1000 dilution), in TTBS 0.1% containing 3% skimmed milk for 1 h. The membrane was subsequently washed twice 15 min with TTBS 0.1%, and incubated for 45 min with the second antibody, peroxidase conjugated goat-anti-rabbit (1/5000). Bound antibodies were visualised by chemiluminescence using an ECL Western Immunoblotting Kit (Amersham).
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 (4.104 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) 2,5-diphenyltetrazolium 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 μl of MTT (5 mg/ml in PBS 1X) was added in each wells and incubated at 37°C for 4 hours. Then, 150 μl of medium was removed from each well, and 100 μl of 0,5% HCl/propanol-1 was added to dissolve cristals. The net absorbance ratio (ratio A550-A630) 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 freezing-thawing 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 H2O2 for 1 min at 37°C. The assay mixture consisted of 2.98 ml of H2O2 solution from a stock solution of 0,1 ml of 30% (w/v) H2O2 diluted in 50 ml of sodium phosphate buffer, pH 7, and 20 μl 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 . 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.
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.
This work was supported by the Institut National de la Sante et de la Recherche Medicale, the Association pour la Recherche contre le Cancer (Grants n° 9974 and n° 5483), the Ministère de l'Education Nationale de la Recherche et de la Technologie. PJF has received a research grant from the Association Française pour la Recherche Thérapeutique.
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