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(Circulation Research. 2004;95:1174.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Peroxisome Proliferator–Activated Receptor Induces NADPH Oxidase Activity in Macrophages, Leading to the Generation of LDL with PPAR- Activation Properties

Elisabeth Teissier, Atsushi Nohara, Giulia Chinetti, Réjane Paumelle, Bertrand Cariou, Jean-Charles Fruchart, Ralf P. Brandes, Ajay Shah, Bart Staels

From the UR 545 INSERM-Institut Pasteur de Lille and Faculté de Pharmacie (E.T., A.N., G.C., R.P., B.C., J.-C.F., B.S.), Université de Lille II, Lille, France; Klinikum Goethe-Universität (R.B.), Frankfurt-am-Main, Germany; and the Department of Cardiology (A.S.), GKT School of Medicine, King’s College, London, UK.

Correspondence to Bart Staels, UR545 INSERM, Département d’Athérosclérose, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP245, Lille 59019, France. E-mail Bart.Staels{at}pasteur-lille.fr

	Abstract

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Peroxisome proliferator–activated receptors (PPARs) are nuclear receptors controlling lipid and glucose metabolism as well as inflammation. PPARs are expressed in macrophages, cells that also generate reactive oxygen species (ROS). In this study, we investigated whether PPARs regulate ROS production in macrophages. Different PPAR-, but not PPAR- agonists, increased the production of ROS (H₂O₂ and ) in human and murine macrophages. PPAR- activation did not induce cellular toxicity, but significantly decreased intracellular glutathione levels. The increase in ROS production was not attributable to inherent prooxidant effects of the PPAR- agonists tested, but was mediated by PPAR-, because the effects were lost in bone marrow–derived macrophages from PPAR-^–/– mice. The PPAR-–induced increase in ROS was attributable to the induction of NADPH oxidase, because (1) preincubation with the NADPH oxidase inhibitor diphenyleneiodinium prevented the increase in ROS production; (2) PPAR- agonists increased production measured by superoxide dismutase–inhibitable cytochrome c reduction; (3) PPAR- agonists induced mRNA levels of the NADPH oxidase subunits p47^phox, p67^phox, and gp91^phox and membrane p47^phox protein levels; and (4) induction of ROS production was abolished in p47^phox–/– and gp91^phox–/– macrophages. Finally, induction of NADPH oxidase by PPAR- agonists resulted in the formation of oxidized LDL metabolites that exert PPAR-–independent proinflammatory and PPAR-–dependent decrease of lipopolysaccharide-induced inducible nitric oxide synthase expression in macrophages. These data identify a novel mechanism of autogeneration of endogenous PPAR- ligands via stimulation of NADPH oxidase activity.

Key Words: macrophages • nuclear receptors • NADPH oxidase • reactive oxygen species • inflammation

	Introduction

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Peroxisome proliferator-activated receptor (PPAR-) is a transcription factor belonging to the superfamily of ligand-activated nuclear receptors that heterodimerizes with the retinoid X receptor. In rodents, but not in humans, PPAR- activation causes hepatomegaly attributable to parenchymal peroxisome proliferation, resulting in a marked increase in oxidative stress.¹ In humans, PPAR- agonists are used for the treatment of dyslipidemia. PPAR- regulates the expression of genes controlling lipid and lipoprotein metabolism and cholesterol and glucose homeostasis. PPAR- activation results in decreased plasma triglyceride and small dense LDL levels and increased HDL.² PPAR- also exerts antiinflammatory activities by transrepressing inflammatory signaling pathways.²

PPAR- is also expressed in vascular wall cells, including monocyte-derived macrophages, where it modulates cholesterol homeostasis. In macrophages, PPAR- regulates the expression of the HDL receptor CLA-1/SR-B1 and the cholesterol/phospholipid transporter ABCA1.³ Moreover, PPAR- inhibits cholesterol esterification, resulting in an enhanced availability of free cholesterol for efflux through the ABCA1 pathway.³ PPAR- also interferes with the activation of a number of inflammatory response genes. PPAR-^–/– mice exhibit a prolonged inflammatory response when challenged with LTB4.⁴ In macrophages, PPAR- inhibits the production of inflammatory response markers, such as tissue factor, inducible nitric oxide synthase (iNOS), and metalloproteinases by negatively interfering with inflammatory transcription pathways, such as nuclear factor B and activator protein-1.³ In mouse peritoneal macrophages, PPAR- ligand treatment decreased tumor necrosis factor expression.⁵ However, PPAR-^–/– mice were more sensitive to the lethal effects of lipopolysaccharide (LPS), which suggests that PPAR- may also play a protective role during endotoxemia.⁶

Macrophages are phagocytic cells that scavenge a large variety of compounds and produce reactive oxygen species (ROS) during phagocytosis in a process called the "respiratory burst," which involves the activation of the NADPH oxidase complex.⁷ In addition to its bactericidal action, recent observations indicate that controlled, physiological production of ROS is required for proper functioning of growth factor and other receptor-mediated cell-signaling processes.⁸ By contrast, sustained overproduction of ROS may result in inflammation and tissue injury. Excessive and uncontrolled ROS production can damage vascular cells, induce the formation of oxidized LDL, and may, as such, participate in the initiation and development of atherosclerosis.

In the present study, we analyzed whether PPAR- controls the formation of ROS in human and murine macrophages. Our results demonstrate that PPAR- activation increases intracellular concentrations of ROS, through the activation of NADPH oxidase. This induction of NADPH oxidase by PPAR- agonists leads to a modification of LDL, likely resulting in the concomitant generation of oxidized LDL metabolites, which act as PPAR- ligands and inhibit the induction of the inflammatory marker iNOS in macrophages. These surprising observations indicate that induction of NADPH oxidase can possibly lead also to the generation of anti-inflammatory signals.

	Materials and Methods

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Reagents
DMEM, RPMI medium 1640, and fetal calf serum (FCS) were from Invitrogen (Cergy-Pontoise, France), Wy14643 from Chemsyn (Lenexa, Kan), and rosiglitazone from Janssen Research Foundation (Beerse, Belgium); all other chemical compounds were from Sigma (Saint-Quentin, France).

Cell Culture
Human mononuclear cells were isolated from blood⁹ and murine peritoneal macrophages from the peritoneal cavity.¹⁰ Murine bone marrow–derived macrophages were prepared from C57BL/6J PPAR-^+/+ or PPAR-^–/–,¹ gp91^phox+/+, or gp91^phox–/– and from Sv129 p47^phox+/+ or p47^phox–/– mice¹¹ as described.¹² (See the online data supplement available at http://circres.ahajournals.org.)

ROS Measurement
Adherent cells cultured in 6-well plates were incubated with dichlorofluorescin diacetate (DCFH-DA, 10 µmol/L) for 30 minutes. Medium was removed, cells washed with PBS, and solubilized in 1 mL 1 mol/L NaOH. DCF fluorescence was read at _exc=485 nm and _em=530 nm.¹³

RNA Analysis
Total RNA, isolated from cells using TRIzol (Invitrogen), was reverse transcribed using random hexamer primers and Superscript reverse transcriptase (Invitrogen). cDNAs were quantified by real-time PCR on a MX 4000 apparatus (Stratagene), using specific primers (see the online data supplement).

LDL Oxidation by Macrophages
THP-1 macrophages, incubated with Wy14643 (50 µmol/L for 12 hours), were washed 3 times with Earle saline solution and incubated in the same medium with dialyzed LDL (200 µg protein/mL) for 24 hours. In parallel, LDL was also incubated under similar conditions without cells. The extent of cell-mediated oxidation of LDL¹⁴ and the absence of Wy14643¹⁵ were analyzed in the supernatants (see the online data supplement).

Transient Transfection Assays
Cos cells were transfected with Gal4-hPPAR or Gal4-hPPAR expression and reporter plasmids,¹⁶ refed with DMEM supplemented with 0.2% FCS, and the same volume of either nontreated supernatant or treated supernatant and luciferase and ß-galactosidase activities quantified (see the online data supplement).

iNOS mRNA induction
Peritoneal macrophages from PPAR-^+/+ or PPAR-^–/– mice, preincubated for 24 hours with RPMI medium 1640 containing Nutridoma (1% v/v), were treated for 2 hours with Earle saline solution or supernatants diluted in RPMI medium 1640 containing Nutridoma (1% v/v) and finally stimulated with LPS (1 µg/mL, 2 hours) to induce iNOS expression. Total RNA was isolated and iNOS gene expression was determined by quantitative PCR (see the online data supplement).

	Results

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PPAR- Activation Stimulates ROS Formation in Macrophages
Macrophages were incubated with the PPAR- agonist Wy14643 (EC₅₀ for murine PPAR-: 0.63; for human PPAR-: 5 µmol/L)¹⁷ and intracellular ROS measured using the nonfluorescent probe DCFH-DA. After deacetylation, DCFH quantitatively reacts with ROS (mainly H₂O₂) to produce the fluorescent dye DCF, which remains trapped within the cell. Incubation with Wy14643 (50 µmol/L, 24 hours) increased DCF fluorescence in human primary macrophages (87%). A similar increase was observed in human THP-1 monocytes (79%) and differentiated THP-1 macrophages (129%) (Figure 1A).

View larger version (20K): [in this window] [in a new window]   Figure 1. PPAR- activation stimulates ROS production in human and murine macrophages. Human (A) or murine (B) monocytes or macrophages were incubated with Wy14643 (50 µmol/L, 24 hours and 10 µmol/L, 8 hours, respectively). DCFH-DA (10 µmol/L) was subsequently added for 0.5 hour. After washing, cells were dissolved in 1 mol/L NaOH and fluorescence measured. Results are expressed as AFU/mg protein±SD of triplicate determinations.

In different murine macrophage cell models, basal levels of ROS production were lower (78 to 103 arbitrary fluorescence units [AFU]/mg protein) than in human macrophages (490 to 848 AFU/mg protein). Wy14643 (10 µmol/L) treatment induced ROS production also in murine bone marrow–derived macrophages (96%), peritoneal macrophages (78%), and in Raw 264.7 cells (190%) (Figure 1B).

Interestingly, Wy14643 treatment increased ROS generation in human THP-1 macrophages, gradually reaching a maximum after 24 hours, whereas treatment with phorbol ester, a known activator of NADPH oxidase, resulted in a peak at 2 hours (Figure 2A). In murine macrophages, a peak was reached after 8 hours incubation with Wy14643 (Figure 2B).

View larger version (24K): [in this window] [in a new window]   Figure 2. PPAR- activation induces ROS production and decreases intracellular GSH concentrations in human THP-1 and murine Raw 264.7 macrophages. Differentiated THP-1 and Raw 264.7 macrophages were incubated with Wy14643 (50 or 10 µmol/L, respectively) or phorbol myristate acetate (167 nmol/L). A and B, ROS production measured as in Figure 1. C and D, GSH content analyzed with high-performance liquid chromatography and electrochemical detection. Results are expressed in nmol/mg protein±SD of triplicate determinations.

Different PPAR-, but Not PPAR-, Agonists Induce Generation of ROS
To determine whether the increase in ROS was also observed with other PPAR agonists, THP-1 macrophages were treated with PPAR- or PPAR- agonists at concentrations specifically activating human PPAR- or PPAR-.^17,18 An increase in ROS production was observed with all PPAR- agonists tested, including Wy14643 (50 µmol/L, 87%), bezafibrate (50 µmol/L, 41%), and ciprofibrate (50 µmol/L, 30%). By contrast, the PPAR- agonists rosiglitazone (tested at concentrations up to 10 µmol/L; not shown) or GW1929 (600 nmol/L) were without effect. Therefore, PPAR-, but not PPAR-, activation increases intracellular ROS in macrophages (Figure 3A).

View larger version (21K): [in this window] [in a new window]   Figure 3. PPAR-, but not PPAR-, agonists induce ROS production in THP-1 macrophages. A, Differentiated THP-1 macrophages were incubated for 24 hours with GW1929 (600 nmol/L), rosiglitazone (100 nmol/L), Wy14643 (50 µmol/L), bezafibrate (50 µmol/L), or ciprofibrate (50 µmol/L) and analyzed as in Figure 1. B, Wild-type or PPAR-–/– bone marrow–derived macrophages were incubated for 8 hours with Wy14643 (10 µmol/L), bezafibrate (50 µmol/L), or ciprofibrate (50 µmol/L) and treated as in Figure 1.

PPAR- Agonists Do Not Induce Cellular Toxicity nor Variation of Antioxidant Enzyme Activity but Decrease Intracellular Glutathione Levels in Macrophages
To determine whether the induction of ROS production by PPAR- agonists was associated with general toxic effects, several parameters were measured. Treatments with PPAR- agonists did not induce any morphological changes. Medium lactic dehydrogenase activity did not increase, indicating that cell permeability was not modified. Mitochondrial dehydrogenase activity, measured by methylthiazolyltetrazolium reduction, and cell energy balance, measured by ATP levels, were not altered. Thiobarbituric acid reactive substances (TBARS) in treated or control cells were identical, indicating no evidence of undue oxidative stress (data not shown). Finally, PPAR- agonists did not induce variations in gene expression of Cu-Zn–superoxide dismutase (SOD) or activities of Cu-Zn-SOD and Mn-SOD, enzymes that dismutate superoxide anions.

H₂O₂ levels are controlled by either activities of catalase or of enzymes linked to glutathione (GSH) metabolism (peroxidase and reductase). The activity of these enzymes did not change for incubations up to 48 hours (data not shown). By contrast, PPAR- agonist treatment led to a large time-dependent decrease in intracellular GSH levels in human, as well in murine, macrophages reaching a minimum at 24 hours (Figure 2C and 2D).

PPAR- Agonists Have No Intrinsic Prooxidant Activity
To test whether ROS induction was attributable to potential chemical prooxidant properties, all compounds were tested for their ability to increase conjugated diene formation during LDL oxidation by copper.¹⁹ All PPAR- agonists tested were without effect at concentrations up to 200 µmol/L, whereas the PPAR- agonist rosiglitazone displayed weak antioxidant activity (ED₅₀=100 µmol/L) (data not shown). This indicates that the induction of ROS release by PPAR- agonists is not attributable to intrinsic prooxidant properties of these compounds.

ROS Induction Requires PPAR- Expression
Next, it was tested whether PPAR- gene expression is required to mediate the effects of the agonists. Stimulation of PPAR-^+/+ bone marrow–derived macrophages with different PPAR- agonists induced, as expected, a significant increase in ROS. However, the response to the different PPAR- agonists was completely lost in bone marrow–derived macrophages from PPAR-^–/– mice (Figure 3B). The same results were obtained in peritoneal macrophages from PPAR-^+/+ and PPAR-^–/– mice (data not shown), indicating that the increase in ROS production is mediated by PPAR-.

PPAR- Induces ROS Production Through NADPH Oxidase Activation
Preincubation of THP-1 macrophages with carbonyl cyanide 3-chlorophenyl hydrazone (1 µmol/L, 1 hour), a mitochondrial uncoupler, did not prevent the induction of ROS by Wy14643 (Figure 4A), suggesting that this is not dependent on mitochondrial function. Thus, it was tested whether ROS induction occurs via activation of NADPH oxidase, a complex formed by five major subunits: a plasma membrane spanning cytochrome b558 composed of two subunits gp91^phox and p22^phox linked to a flavin, forming a flavoprotein, and cytosolic components such as Rac-2, p67^phox, and the subunit p47^phox, which requires phosphorylation to be active.⁷ Preincubation with the flavoprotein inhibitor diphenyleneiodinium (DPI) (10 µmol/L, 1 hour) prevented the induction of ROS production by Wy14643 (Figure 4B), pointing to a possible implication of a flavin-containing oxidoreductase.

View larger version (19K): [in this window] [in a new window]   Figure 4. The flavin inhibitor DPI prevents ROS and production induced by Wy14643 in human THP-1 macrophages. THP-1 macrophages were incubated for 0.5 hour with carbonyl cyanide 3-chlorophenyl hydrazone (A) (1 µmol/L) or DPI (B) (10 µmol/L), then treated with Wy14643 (50 µmol/L, 24 hours) and ROS measured as in Figure 1. C, After treatment with DPI and Wy14643, production was measured by reduction of cytochrome c (150 µmol/L) with NADPH (100 µmol/L) in the presence or absence of SOD (200 U/mL)

Therefore, NADPH oxidase activity was specifically assessed by the SOD-inhibitable cytochrome c reduction assay. NADPH significantly enhanced Wy14643 induction of production. Moreover, this induction was abolished by the flavoprotein inhibitor DPI (Figure 4C).

To unequivocally establish the involvement of NADPH oxidase in the induction of ROS production by PPAR- agonists, experiments were performed on bone marrow–derived macrophages from p47^phox–/– or gp91^phox–/– mice. Whereas wild-type bone marrow–derived macrophages responded as expected, no increase in ROS production in response to PPAR- agonists was observed in either p47^phox–/– or gp91^phox–/– cells (Figure 5A). These data indicate that PPAR- ligands increase intracellular ROS production in macrophages via the NADPH oxidase system.

View larger version (20K): [in this window] [in a new window]   Figure 5. NADPH oxidase subunits p47phox, p67phox, and gp91phox are involved in the ROS production by PPAR- activation. A, Wild-type or p47phox- or gp91phox-deficient bone marrow–derived macrophages were incubated for 8 hours with Wy14643 (10 µmol/L), bezafibrate (50 µmol/L), or ciprofibrate (50 µmol/L) and ROS analyzed as in Figure 1. B, THP-1 macrophages were treated for 24 hours with Wy14643 (50 µmol/L) or ciprofibrate (50 µmol/L). p47phox, p67phox, and gp91phox mRNA levels were analyzed by Q-PCR and normalized to cyclophilin mRNA (mean±SD of triplicates). C, Cells membranes were isolated from THP-1 cells treated with Wy14643 (50 µmol/L, 24 hours) and p47phox protein levels measured by Western blotting, as described in the online data supplement.

Because PPAR- agonists require several hours to induce ROS production, it was tested whether PPAR- agonists regulate NADPH oxidase subunit gene expression. Whereas p22^phox mRNA did not change, an increase in p47^phox, p67^phox, and gp91^phox mRNA levels was observed after 24 hours of treatment with Wy14643 and ciprofibrate (Figure 5B). By contrast, the PPAR- ligand rosiglitazone did not influence p47^phox mRNA levels (data not shown). Finally, incubation of THP-1 cells with Wy14643 significantly induced plasma membrane p47^phox protein levels (Figure 5C).

PPAR- Activation Accelerates LDL Oxidation by THP-1 Macrophages
To determine whether PPAR-–induced ROS production influences macrophage LDL oxidation, Wy14643-treated THP-1 macrophages were incubated with LDL. The extent of LDL oxidation was determined in the medium by measuring the increase of different markers of lipoprotein peroxidation: conjugated dienes (234 nm), ketocholesterols (250 nm), apolipoprotein modification by aldehydes (fluorescence intensity at 360 to 430 nm), and TBARS using a fluorimetric detection method.¹⁴ Because Wy14643 absorbs with a maximum at 244 nm, THP-1 cells were preincubated with Wy14643 for 12 hours to increase ROS production, and cells were subsequently extensively washed to remove any remaining Wy14643 before adding LDL. To facilitate LDL oxidation, complete RPMI medium 1640 containing FCS was replaced by Earle medium.²⁰ Although LDL oxidation is often induced in vitro by the addition of copper, THP-1 cells oxidize LDL even in the absence of added copper ions, therefore representing a more physiological system of LDL oxidization. Dienes, ketocholesterol, and Schiff bases (Figure 6 A through 6C) all increased in the supernatant of Wy14643-pretreated THP-1 cells, whereas TBARS (Figure 6D) increased mildly. A maximal induction was observed in cells incubated with Wy14643 for 12 hours (data not shown), which is in agreement with the kinetics of p47 mRNA induction, ROS formation, and GSH decrease. These results suggest that NADPH oxidase induction, and ROS thus generated by PPAR- activation, enhances THP-1–mediated LDL oxidation.

View larger version (22K): [in this window] [in a new window]   Figure 6. PPAR- activation increases THP-1–mediated oxidation of LDL. THP-1 macrophages were treated with or without Wy14643 (50 µmol/L) for 12 hours. The cells were washed with Earle saline solution and incubated in the same medium containing dialyzed LDL (200 µg protein/mL) for 0 to 24 hours. In parallel, LDL was also incubated under similar conditions in the absence of cells. At the indicated times, supernatants were removed to analyze cell-mediated LDL oxidation. Diene formation was measured at 234 nm (A), ketocholesterol formation at 250 nm (B), Schiff base formation by fluorescence emission at 360 to 430 nm (C), and aldehyde equivalents using the TBARS method (D).

Oxidized LDL Increases PPAR-, but Not PPAR-, Activity
Because PPAR- and PPAR- are activated by in vitro oxidized LDL,^16,21 it was tested whether cellular LDL modification resulted in the generation of PPAR agonists. Using a conventional transactivation assay, supernatants from Wy14643-treated LDL-incubated THP-1 cells increased PPAR- activity (6-fold) to a comparable extent as Wy14643 tested as positive control. By contrast, supernatants from nontreated LDL-incubated cells failed to activate PPAR- (Figure 7A). Interestingly, LDL-containing supernatants from Wy14643-treated THP-1 cells failed to induce PPAR- activity, whereas rosiglitazone increased it (Figure 7B). High-performance liquid chromatographic analysis indicated that the induction of PPAR- by the LDL-containing supernatant was not attributable to residual Wy14643 in the medium (retention time=19 minutes; signal/noise=20 for the injection of 100 µL of a 10^–9 mol/L Wy14643 standard solution). These results indicate that pretreatment of THP-1 cells with Wy14643 induces a modification of LDL resulting in the formation of derivatives that selectively activate PPAR-.

View larger version (27K): [in this window] [in a new window]   Figure 7. LDL-containing supernatant from Wy14643 pretreated THP-1 macrophages activates PPAR-, but not PPAR-, and inhibits LPS-induced iNOS gene expression in a PPAR-–dependent manner. Cos cells were transiently transfected with reporter and expression plasmids for PPAR- (A) or PPAR- (B). After 2 hours, cells were refed with DMEM supplemented with 0.2% FCS and the same volume of THP-1 supernatants obtained as described in Material and Methods. After 18 hours, cells were collected and luciferase and ß-galactosidase assays were performed. Wy14643 (10 µmol/L) or rosiglitazone (1 µmol/L) was used as a positive control and luciferase activity expressed as fold-induction over vehicle control±SD of triplicate determinations. C, PPAR-+/+ or PPAR-–/– peritoneal macrophages pretreated for 2 hours with RPMI medium 1640 containing 1% Nutridoma and the same volume of Earle saline, treated supernatant or Wy14643, were activated with LPS (1 µg/mL, 2 hours). iNOS mRNA was analyzed by Q-PCR and normalized to 28S.

Oxidized LDL Inhibits LPS-Induced iNOS Gene Expression in a PPAR-–Dependent Manner
Because PPAR- agonists decrease LPS-induced iNOS expression, it was tested whether LDL-containing supernatant from Wy14643 shares this property, in PPAR-^+/+ and PPAR-^–/– mice peritoneal macrophages. As expected, incubation of peritoneal macrophages with LPS increased iNOS mRNA, whereas treatment with Wy14643 (10 µmol/L) inhibited this increase in iNOS expression, an effect completely lost in PPAR-^–/– macrophages. Incubation with LDL-containing supernatant from Wy14643-treated THP-1 macrophages also inhibited iNOS mRNA induction by LPS in PPAR-^+/+ macrophages. By contrast, iNOS mRNA rather increased more pronouncedly when PPAR-^–/– macrophages were incubated with LDL-containing supernatant (Figure 7C). Therefore, modified LDL metabolites generated by Wy14643-treated THP-1 macrophages decrease LPS-induced iNOS expression, an inflammatory marker gene, in a PPAR-–dependent manner. These surprising data indicate that NADPH oxidase activation can result in the generation of LDL metabolites possessing certain properties of synthetic PPAR- agonists, namely inhibition of LPS induced iNOS expression.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
ROS are essential in the physiological response of vascular cells, where they are transiently produced at relatively low levels in response to cellular activation signals and play a role as second messengers to regulate signal transduction pathways that ultimately control gene expression and posttranslational protein modifications.^8,22,23 In addition, ROS generation by inflammatory cells such as neutrophils and macrophages is essential in host defense. By contrast, sustained and pronounced ROS production has been implicated in a variety of diseases, including Alzheimer disease, cancer, and vascular diseases such as atherosclerosis.²³

In this study, we demonstrate that PPAR- activation increases ROS production in human and murine macrophages. This induction in ROS requires the presence of a functional PPAR- gene and occurs via the NADPH oxidase pathway.

Treatment of macrophages with PPAR- ligands resulted in an increase in ROS, in the absence of any detectable cellular toxicity, measured using biochemical markers of cellular injury (lactic dehydrogenase leakage, methylthiazolyltetrazolium reduction, and cellular ATP content). These results are in agreement with observations in human HepG2 cells showing that the PPAR- agonist fenofibrate only induces cytotoxic effects at concentrations >100 µmol/L, which is well above its EC₅₀ for PPAR-.²⁴ The ambient ROS level is determined by the balance between enzymatic ROS formation and degradation and may, therefore, be modulated by changes in activity of enzymes able to regulate ROS. Produced superoxide anion is reduced by SOD to produce H₂O₂. We did not observe any variation in Cu-Zn-SOD expression or in the activity of Mn-SOD that is located primarily in the mitochondrial matrix. Enzymes that decompose H₂O₂, such as catalase and enzymes linked to GSH metabolism, did not vary either. The most important change was a decrease in GSH, the substrate of GSH peroxidase and an important cellular antioxidant, which reached a minimum between 12 to 24 hours. Previous reports demonstrated increase of SOD expression or activity after treatment with PPAR- agonists in human liver cells with clofibric acid²⁵ or in human endothelial cells with bezafibrate.²⁶ However, these effects were observed at much higher doses or longer treatment periods. A decrease in GSH level was also previously reported in human HepG2 cells treated with high concentrations of fenofibrate.²⁴ Our results show that the PPAR- agonist–induced increase in ROS is not toxic for macrophages, and the decreased GSH levels are likely a reflection of the increased ROS production, but does not compromise cellular-oxidant defenses nor lead to a redox imbalance.

The main sources of ROS are the peroxisomes, mitochondria, and NADPH oxidases. The mechanism of ROS production varies between among and cell types. In rodents, PPAR- agonists act as peroxisome proliferators and increase the expression of the H₂O₂-generating peroxisomal fatty acyl-coenzyme A oxidase (ACO) in liver²⁷ or J774.3 macrophages.²⁸ In contrast to rodents, there is no evidence that fibrates induce peroxisome proliferation in humans and primates. These data are corroborated by the lack of induction of ACO gene expression by fibrates in human cells.²⁹ Thus, ROS increase by PPAR- ligands in human macrophages is unlikely to be attributable to an increase in ACO activity. A mitochondrial origin of H₂O₂ production induced by PPAR- ligands could also be eliminated, because the production of ROS was not decreased by a cyanide inhibitor. However, the increase in ROS appeared to be attributable to the induction of the membrane NADPH oxidase, because it was inhibited by a specific inhibitor of flavin, an important component of NADPH oxidase.³⁰ The increase in ROS production was associated with an increase in the expression of the NADPH oxidase protein subtypes p47^phox, p67^phox, and gp91^phox but not p22^phox, as well as membrane p47^phox protein levels. Moreover, PPAR- agonists failed to activate ROS generation in p47^phox–/– or gp91^phox–/– bone marrow–derived macrophages, which indicates that the generation of ROS by PPAR- depends on p47^phox and gp91^phox expression.

Our results obtained in rodent and human macrophages are in line with data reported on rodent Kupffer cells, the resident hepatic macrophages. In these cells, short-term treatment with peroxisome proliferators increased superoxide production nearly 7-fold,³¹ and this activity was lost in p47^phox–/– mice.³² However, rodent Kupffer cells do not express PPAR-, indicating that other, PPAR-–independent, mechanisms than those reported in the present report must be operative in these cells.³³ By contrast, in human primary endothelial cells (HUVEC and HAEC), treatment with bezafibrate decreased p22^phox mRNA levels and p47^phox protein levels.²⁶ Our results in macrophages showed moderate increases in ROS, measured with DCF-DA not only with Wy14643 but also with all other tested PPAR- agonists. Moreover, the implication of NADPH oxidase was demonstrated not only by its inhibition by DPI and the lack of effect in macrophages of p47^phox–/– and gp91^phox–/– mice but also by an increase in the expression of the NADPH oxidase protein p47^phox. PPAR- agonists have no intrinsic oxidant effect; therefore, the increase in ROS is not attributable to a chemical prooxidant effect. The absence of increase of ROS in macrophages derived from PPAR-^–/– mice clearly shows that this effect is mediated by this nuclear receptor, which contrasts with the situation in Kupffer cells. The effects of PPAR- agonists on ROS production are clearly different: in our experiments, rosiglitazone did not change ROS or p47^phox mRNA levels, whereas in the human macrophage cell line U937, NO-dependent PPAR- activation lowered p47^phox phagocyte oxidase activity, whereas p22^phox was unchanged.³⁴

Regulation of NADPH oxidase activity occurs at two levels. Rapid oxidation of the oxidase is mediated by PKC, leading to the phosphorylation of p47^phox and the association of cytosolic subunits with the membrane flavocytochrome.^11,35,36 The most extensively studied activator is angiotensin II, which induces phosphorylation of p47^phox in vascular smooth muscle cells,²² in endothelial cells,³⁶ and in macrophages.³⁷ Secondly, and more gradually, oxidase activity can also be modulated by upregulation of the expression levels of one or more NADPH oxidase component subunits. To date, it is unclear whether stimulation of PKC activity or increase in fatty acid catabolism³⁸ could be implicated in the effects of PPAR- agonists. However, phorbol myristate acetate or angiotensin II activation of macrophages increases ROS production within 15 to 20 minutes, whereas PPAR- activators require a longer time with a maximum around 16 to 24 hours, suggesting that p47^phox, p67^phox and gp91^phox mRNA levels may be functionally significant.

The physiological function of PPAR- in macrophages is complex. Several lines of evidence indicate a protective activity of PPAR- agonists against atherosclerosis by regulating systemic and macrophage cholesterol homeostasis and via antiinflammatory actions. However, PPAR- agonists specifically activate NADPH oxidase that promotes ROS generation, an effect which may have both physiological and pathological consequences. Reports have spurred an interest in examining the role of PPARs in the host immune response.⁶ Superoxide anion and H₂O₂ are required participants of normal physiological cellular signaling, a process referred to as redox signaling.⁸ H₂O₂ appears essential for the proper development and proliferation of cells and acts as a second messenger for signal transduction and amplification.²² H₂O₂ is a rather mild oxidant that primarily targets proteins, such as tyrosine phosphatases containing in their catalytic center a reactive and redox-regulated cysteine.²³ On the other hand, superoxide production in macrophages may result in LDL oxidation^39–41 which can induce a proatherogenic cascade. In the absence of PPAR-, the proinflammatory response of oxidized LDL metabolites generated via NADPH oxidase activation is evident (Figure 7C). However, oxidized LDL not only elicits an oxidative burst, but also contains lipid peroxidation products such as oxidized phospholipids, 9- and 13-hydroxyoctadecadienoic acids, which are PPAR- agonists¹⁶ and have been shown to promote antiinflammatory responses.⁴¹ Interestingly, when the PPAR- gene is intact, NADPH oxidase–generated, oxidized LDL–derived metabolites act as natural PPAR- agonists and decrease LPS-induced iNOS expression.

Altogether, these results confirm a regulatory role for PPAR- in the inflammatory response, as measured here by LPS-induced iNOS gene expression in macrophages. When PPAR- is absent or defective, LDL oxidation induced by NADPH oxidase leads to an enhanced induction of iNOS expression by LPS, but when PPAR- is functional, these effects are counterbalanced via its activation by oxidized LDL. Such a mechanism may result in a return to equilibrium (Figure 8). These observations lead to the surprising concept that NADPH oxidase activation may also generate certain antiinflammatory activities.

View larger version (19K): [in this window] [in a new window]   Figure 8. Schematic representation of potential consequences of NADPH oxidase induction by PPAR- agonists in macrophages. PPAR- agonists induce an increase in intracellular ROS in a PPAR-–dependent and p47phox-dependent manner. This oxidative stress facilitates the oxidation of LDL, a proatherogenic lipoprotein, but also leads to the generation of PPAR- activators that inhibit LPS induction of iNOS mRNA.

	Acknowledgments

 
We kindly acknowledge grant support from the Fondation Leducq, the British Heart Foundation (to A.S.), and the European Vascular Genomics Network (LSHM-CT-2003-503254).

	Footnotes

 
Original received April 27, 2004; resubmission received September 27, 2004; revised resubmission received October 29, 2004; accepted November 2, 2004.

	References

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