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Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Faslpr mice

2006; Elsevier BV; Volume: 47; Issue: 11 Linguagem: Inglês

10.1194/jlr.m600187-jlr200

1539-7262

Paolo Bergamo, Diomira Luongo, Francesco Maurano, Giuseppe Mazzarella, Rosita Stefanile, Mauro Rossi,

Inflammatory mediators and NSAID effects

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor γ (PPARγ) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Faslpr mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 μM CLA downregulated IFNγ synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 μM CLA did not significantly influence cell proliferation but enhanced the expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 μM was not inhibited by the PPARγ antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor κB activity independent of PPARγ activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs. Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor γ (PPARγ) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Faslpr mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 μM CLA downregulated IFNγ synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 μM CLA did not significantly influence cell proliferation but enhanced the expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 μM was not inhibited by the PPARγ antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor κB activity independent of PPARγ activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs. Abbreviations anti-dsDNAanti-double-stranded DNAanti-tTGanti-tissue transglutaminaseCLAconjugated linoleic acid15dPGJ215-deoxy-Δ12,14-prostaglandin J2GCLCγ-glutamylcysteine ligase catalytic subunitILinterleukinMRL/lprMRL/MpJ-FaslprNFκBnuclear factor κBO2•−superoxide anion radicalPPARγperoxisome proliferator-activated receptor γROSreactive oxygen speciesSLEsystemic lupus erythematosus anti-double-stranded DNA anti-tissue transglutaminase conjugated linoleic acid 15-deoxy-Δ12,14-prostaglandin J2 γ-glutamylcysteine ligase catalytic subunit interleukin MRL/MpJ-Faslpr nuclear factor κB superoxide anion radical peroxisome proliferator-activated receptor γ reactive oxygen species systemic lupus erythematosus Oxidative stress has been implicated in the pathogenesis of several degenerative diseases, such as Alzheimer disease, Parkinson disease, systemic lupus erythematosus (SLE), and cancer. In particular, its involvement in autoimmune disease has been supported by the increase of oxidative stress markers (1Morgan P.E. Sturgess A.D. Davies M.J. Increased levels of serum protein oxidation and correlation with disease activity in systemic lupus erythematosus..Arthritis Rheum. 2005; 52: 2069-2079Crossref PubMed Scopus (111) Google Scholar, 2Giustarini D. Lorenzini S. Rossi R. Chindamo D. Di Simplicio P. Marcolongo R. Altered thiol pattern in plasma of subjects affected by rheumatoid arthritis..Clin. Exp. Rheumatol. 2005; 23: 205-212PubMed Google Scholar) and by the preventive effect played by the dietary intake of antioxidants (3Sukkar S.G. Rossi E. Oxidative stress and nutritional prevention in autoimmune rheumatic diseases..Autoimmun. Rev. 2004; 3: 199-206Crossref PubMed Scopus (45) Google Scholar). The beneficial effect of diet enrichment with n-3 PUFAs in autoimmune disease-prone mice has been associated with improved antioxidant defenses (4Bhattacharya A. Lawrence R.A. Krishnan A. Zaman K. Sun D. Fernandes G. Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated autoimmune-prone NZB/W female mice..J. Am. Coll. Nutr. 2003; 22: 388-399Crossref PubMed Scopus (78) Google Scholar, 5Venkatraman J.T. Bysani C. Kim J.D. Fernandes G. Effects of n-3 and n-6 fatty acids on the activities and expression of hepatic antioxidant enzymes in autoimmune-prone NZBxNZW F1 mice..Lipids. 1994; 29: 561-568Crossref PubMed Scopus (130) Google Scholar) and apoptosis modulation (6Reilly C.M. Oates J.C. Sudian J. Crosby M.B. Halushka P.V. Gilkeson G.S. Prostaglandin J(2) inhibition of mesangial cell iNOS expression..Clin. Immunol. 2001; 98: 337-345Crossref PubMed Scopus (59) Google Scholar). Apoptosis is the process that leads to the ordered destruction of cells, avoiding the release of intracellular contents into the extracellular environments, where they have a powerful inflammatory effect. Defective functioning of the cell death program has been recognized to play a critical role in the pathogenesis of autoimmune diseases (7Kühtreiber W.M. Hayashi T. Dale E. Faustman D.L. Central role of defective apoptosis in autoimmunity..J. Mol. Endocrinol. 2003; 31: 373-399Crossref PubMed Scopus (46) Google Scholar), and a proapoptotic effect has been identified in some antineoplastic and anti-inflammatory agents used as medication (methotrexate and mycophenolate mofetil, respectively) (8Nakazawa F. Matsuno H. Yudoh K. Katayama R. Sawai T. Uzuki M. Kimura T. Methotrexate inhibits rheumatoid synovitis by inducing apoptosis..J. Rheumatol. 2001; 28: 1800-1808PubMed Google Scholar, 9Allison A.C. Eugui E.M. Mycophenolate mofetil and its mechanisms of action..Immunopharmacology. 2000; 47: 85-118Crossref PubMed Scopus (1119) Google Scholar). The MRL/MpJ-Faslpr (MRL/lpr) mouse is a prototypical model for human SLE in which the presence of a single gene mutation on the Fas (CD95) gene leads to reduced signaling for apoptosis (10Watanabe-Fukunaga R. Brannan C.I. Copeland N. Jenkins N.A. Nagata S. Lymphoproliferative disorder in mice explained by defects in Fas antigen that mediates apoptosis..Nature. 1992; 356: 314-317Crossref PubMed Scopus (2726) Google Scholar). Actually, the impaired clearance of apoptotic cells favors the self-antigen presentation and the production of multiple autoantibodies, which represents the central immunological disturbance in murine SLE (11Ravirajan C.T. Sarraf C.E. Ankilumar T.V. Golding M.C.H Alison M.R. Isenberg D.A. An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE)..Clin. Exp. Immunol. 1996; 105: 306-312Crossref PubMed Scopus (28) Google Scholar). Indeed anti-double-stranded DNA (anti-dsDNA) and anti-tissue transglutaminase (anti-tTG) IgGs (12Piredda L. Amendola A. Coalizzi V. Davies P. Farrace M.G. Fraziano M. Gentile V. Uray I. Piacentini M. Fesus L. Lack of “tissue” transglutaminase protein cross-linking leads to leakage of macromolecules from dying cells: relationship to development of autoimmunity in MRL/lpr mice..Cell Death Differ. 1997; 4: 463-472Crossref PubMed Scopus (75) Google Scholar), splenomegaly, and deregulated production of Th1 and Th2 cytokines (13Theofilopoulos A.N. Lawson B.R. Tumour necrosis factor and other cytokines in murine lupus..Ann. Rheum. Dis. 1999; 58: 49-55Crossref PubMed Scopus (77) Google Scholar) have been used as markers to monitor murine SLE disease progression. Conjugated linoleic acid (CLA) refers to a heterogeneous group of positional and geometric isomers of a conjugated diene of linoleic acid (C18:2n-6). The best sources for CLA in the human diet are ruminant meat and dairy products in which the two predominant isomers are cis9,trans11 (c9,t11) and trans10,cis12 (t10,c12) CLA. In rodents, dietary administration of a mixture of CLA isomers was shown to have anticarcinogenic, antidiabetic, and anti-inflammatory processes (14Wahle K.W.J Hayes S.D. Rotondo D. Conjugated linoleic acids: are they beneficial or detrimental to health?.Prog. Lipid Res. 2004; 43: 553-587Crossref PubMed Scopus (440) Google Scholar), but only limited effects have been observed on autoimmune disease-prone mice (15Yang M. Pariza M.W. Cook M.E. Dietary conjugated linoleic acid protects against end stage disease of systemic lupus erythematosus in the NZB/W F1 mouse..Immunopharmacol. Immunotoxicol. 2000; 22: 433-449Crossref PubMed Scopus (41) Google Scholar, 16Yang M. Cook M.E. Dietary CLA decreased weight loss and extended survival following the onset of kidney failure in NZB/W F1 mice..Lipids. 2003; 38: 21-24Crossref PubMed Scopus (24) Google Scholar). The mechanisms whereby CLA could influence immune systems have not been established, although two main mechanisms have been proposed: the first hypothesizes the ability of CLA to alter eicosanoid signaling by modifying cell membrane composition (17Pariza M.W. Park Y. Cook M.E. Mechanisms of action of conjugated linoleic acid: evidence and speculation..Proc. Soc. Exp. Biol. Med. 2000; 223: 8-13Crossref PubMed Scopus (269) Google Scholar); the other assumes its ability to modulate genes through peroxisome proliferator-activated receptors (PPARs) (18Bassaganya-Riera J. Hontecillas R. Beitz D. Colonic anti-inflammatory mechanisms of conjugated linoleic acid..Clin. Nutr. 2002; 21: 451-459Abstract Full Text PDF PubMed Scopus (126) Google Scholar). PPARγ agonists includes some long-chain n-3 and n-6 PUFAs (docosaheaxaenoic acid, eicosapentaenoic acid, and CLA) and the prostanoid prostaglandin D2 dehydration product, 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2); their potential use of PPARγ agonists in the treatment of inflammatory disease was reviewed recently (19Oates J.C. Reilly C.M. Crosby M.B. Gilkeson G.S. Peroxisome proliferator-activated receptor gamma agonists: potential use for treating chronic inflammatory diseases..Arthritis Rheum. 2002; 46: 598-605Crossref PubMed Scopus (29) Google Scholar). In addition, the ability of PPARγ ligands (15dPGJ2 and docosaheaxaenoic acid) to upregulate the expression of stress-responding enzymes and/or to enhance cellular redox status by increasing the concentration of intracellular GSH has been demonstrated (20Levonen A.L. Dickinson D.A. Moellering D.R. Mulcahy R.T. Forman H.J. Darley-Usmar V.M. Biphasic effects of 15-deoxy-delta(12,14)-prostaglandin J(2) on glutathione induction and apoptosis in human endothelial cells..Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1846-1851Crossref PubMed Scopus (145) Google Scholar, 21Lim S.Y. Jang J.H. Na H.K. Lu S.C. Rahman I. Surh Y.J. 14-Deoxy-Δ12,14-prostaglandin J2 protects against nitrosative PC12 cell death through up-regulation of intracellular glutathione synthesis..J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The association of CLA-induced pro-oxidant activity with its immunomodulatory and proapoptotic ability was demonstrated previously in Jurkat T-cells (22Luongo D. Bergamo P. Rossi M. Effect of conjugated linoleic acid on growth and cytokine expression in Jurkat T cells..Immunol. Lett. 2003; 90: 195-201Crossref PubMed Scopus (41) Google Scholar, 23Bergamo P. Luongo D. Rossi M. Conjugated linoleic acid-mediated apoptosis in Jurkat T cells involves the production of reactive oxygen species..Cell. Physiol. Biochem. 2004; 14: 57-64Crossref PubMed Scopus (39) Google Scholar). In this study, on the basis of the ability of CLA to modify the functioning of different biochemical mechanisms (PPARγ, apoptosis, and redox status) involved in the pathogenesis of autoimmune disease, its efficacy in attenuated mouse SLE signs was evaluated. In particular, a first set of experiments addressed the question of whether CLA exposure may modulate redox status and cell differentiation of mouse splenocytes from both MRL/lpr and congeneic control mice (MRL/+). Then, its beneficial effects on typical pathological signs of MRL/lpr mice were investigated, and the involvement of biochemical mechanisms was examined. Splenocyte treatment with 100 μM CLA resulted in enhanced apoptosis and significantly decreased IFNγ and proliferation synthesis. More interestingly, cell exposure to a lower CLA concentration (25 μM) was accompanied by enhanced cytoprotective defense [increased expression of γ-glutamylcysteine ligase catalytic subunit (GCLC) and increased intracellular GSH concentration]. Significant amelioration of typical pathological signs (splenomegaly, cytokines, and autoantibody synthesis) in MRL/lpr by short- and long-term CLA administration was accompanied by nuclear factor κB (NFκB) activity suppression and improved cytoprotective defenses independent of apoptosis induction and PPARγ activation. Stock solutions of an isomeric CLA mixture containing 40% c9,t11 and 45% t10,c12 (Sigma, St. Louis, MO), hereafter called CLA, was used. Pure (⩾98%) c9,t11, t10,c12, and trans9,trans11 (t9,t11) CLA isomers were purchased from Cayman Chemical (Ann Arbor, MI). CLA and isomer stock solutions were prepared in ethanol to a final concentration of 1 M and stored at −20°C. Fatty acids were diluted just before use in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μM 2-mercaptoethanol, and 1% non-essential amino acids (NEAA) (complete RPMI). Cultures exposed to the same amount of ethanol were used as a control. For in vivo trials, CLA isomeric mixture (containing 38.5% t10,c12, 37.4% c9,t11, 3.4% palmitic acid, 3.8% stearic acid, 15.4% oleic acid, and 1.5% linoleic acid) was purchased from Natural, Inc. (CLA-T). Individual doses consisting of 30 mg of CLA-T in 200 μl of olive oil were prepared just before administration. Olive oil was given to control animals. MRL/lpr and congeneic control MRL/+ mice purchased from the Jackson Laboratory (Bar Harbor, ME) were bred and maintained under standard conditions of temperature and light in specific pathogen-free conditions at the Istituto di Scienze dell'Alimentazione animal facility. Animals were fed ad libitum with standard mouse chow. Three to four predisease MRL/lpr and congeneic control mice (7–8 weeks old) were used as cell sources for each in vitro experiment. Splenocytes from 7–8 (predisease) and 20–22 (diseased) week old MRL/lpr and age-matched MRL/+ mice (n = 16) were used to evaluate the age-dependent decrease of intracellular GSH and the antiproliferative effect of CLA. For in vivo trials, 48 MRL/lpr mice were gavaged with CLA-T (13Theofilopoulos A.N. Lawson B.R. Tumour necrosis factor and other cytokines in murine lupus..Ann. Rheum. Dis. 1999; 58: 49-55Crossref PubMed Scopus (77) Google Scholar) four times per week up to 21 weeks of age. Four homogeneous groups (sex and body weight; n = 12 animals each) were used to evaluate the effect CLA on MRL/lpr mice administered for short (2 weeks) and long (15 weeks) periods with CLA-T or olive oil as a control. Blood was taken from each mouse, and serum was prepared and stored at −20°C for analysis of autoantibodies. After euthanasia, spleen was aseptically removed and kept on ice in sterile complete RPMI. Spleen weights were expressed as a percentage of total body weight [spleen weight (g) × 100/body weight (g)]. A portion of spleen was snap-frozen in liquid nitrogen and stored at −80°C for Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. Single cell suspensions were prepared according to a published protocol (24Senger S. Maurano F. Mazzeo M.F. Gaita M. Fierro O. David C.S. Troncone R. Auricchio S. Siciliano R.A. Rossi M. Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization..J. Immunol. 2005; 175: 8087-8095Crossref PubMed Scopus (46) Google Scholar). Cells were divided into aliquots at 107 cells/ml and used for in vitro incubations (at 37°C in humidified atmosphere of 5% CO2) or analyzed for intracellular GSH and cytokine levels. Cytoplasmic protein extracts were also prepared (25Zvonic S. Cornelius P. Stewart W.C. Mynatt R.L. Stephens J.M. Effects of cardiotrophin on adipocytes..J. Biol. Chem. 2004; 279: 47572-47579Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and stored at −80°C. Splenocytes (1 × 105 cells/well) were cultured on 96-well plates in complete RPMI supplemented with 10 μg/l PMA and 5 μM ionomycin (PMA/ion). Cells were incubated (37°C, 5% CO2) for 72 h in the presence of pure isomers or CLA. In some experiments, splenocytes were pretreated for 2 h with 5 μM GW9662. Eighteen hours before harvesting, cells were pulsed with 1 μCi/well [3H]thymidine. At the end of the incubation, cultures were harvested on filters using a semiautomatic cell harvester (Filtermate; Packard, Danvers, MA) and incorporated [3H]thymidine was assessed with a microplate liquid scintillator (Top Count NXTTM; Packard). Results were expressed as cpm, and culture incubated without CLA (mixture or pure isomers) was used as a control. The experimental conditions used (mitogen type, concentrations, and incubation times) were based on preliminary in vitro experiments. Splenocytes (6 × 106/well), cultured with varying CLA concentrations (0–200 μM) for 72 h in the presence of PMA/ion, were monitored for cell viability by measuring released lactate dehydrogenase activity with the CytoTox 96® detection kit (Promega, Biosciences, Inc.) according to the manufacturer's protocol. Values were expressed as percentages of those obtained by exposure to 10% NP40 (100%). Cells incubated without CLA were used as a control. Splenocytes (8 × 106 per well) were preincubated (3 h) in complete RPMI containing 100 μM CLA before stimulation with PMA/ion. After 18 h of incubation, IFNγ concentration was quantified in the supernatant by indirect ELISA according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN). IFNγ concentration was expressed as pg/ml, and cells incubated without CLA were used as a control. At the end of the in vivo trial, IFNγ, interleukin-4 (IL-4), and IL-10 mRNA levels were assessed in spleen cells by semiquantitative RT-PCR, as described (26Senger S. Luongo D. Maurano F. Mazzeo M.F. Siciliano R.A. Gianfrani C. David C. Troncone R. Auricchio S. Rossi M. Intranasal administration of a recombinant alpha-gliadin down-regulates the immune response to wheat gliadin in DQ8 transgenic mice..Immunol. Lett. 2003; 88: 127-134Crossref PubMed Scopus (63) Google Scholar). PCR products were analyzed on a 2% agarose gel stained with VISTRA Green (Amersham International). Quantitative analysis of detected bands was carried out on the STORM 860 system with ImageQuant software (Molecular Dynamics, Inc.). Results were expressed as cytokine/β-actin mRNA ratio. Caspase-8 and -3 are key enzymes in the initiation and execution stages of the apoptotic pathway, and their activities were measured in cytoplasmic protein extract using the Fluorometric Assay Kit (Sigma-Aldrich) according to the manufacturer's instructions. Splenocytes (2 × 107 cells/well) were seeded on 12-well plates and incubated for 16 h with 50 ng/ml anti-CD95/Fas/Apo1 monoclonal antibody (clone CH 11; Immunotech) in complete RPMI-supplemented PMA/ion. At the end of the incubation, caspase-3 was measured, and cells incubated without anti-CD95 antibody were used as a control. The time-dependent activities of caspase-3 and -8 were determined on splenic cell aliquots (2 × 107 cells/well) from MRL/+ and MRL/lpr exposed to 100 μM CLA for different time periods. Protease activity was calculated by subtracting the value measured in cells incubated without CLA (spontaneous) for the same time period; after normalization of the protein content (27Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), its activity was expressed as nmol Amino-4-Methyl Coumarin (AMC)/mg protein. To confirm the proapoptotic ability of CLA in vitro, FITC-Annexin (BD Discovery Labware) and DNA fragmentation were evaluated in spleen cells exposed for 4 h to 100 μM CLA. At the end of the incubation, cells were analyzed by flow cytometry using a FACSCalibur and CellQuest software (BD Biosciences) and by TUNEL assay, respectively. Cell cultures incubated without CLA were used as a control, and cultures exposed to 10 μM actinomycin D for 6 h were used as a positive control. Cytoplasmic protein extracts, prepared from mouse splenocytes at the end of in vivo or in vitro treatment, were used to evaluate PPARγ and GCLC expression. After normalization of protein content (27Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), aliquots (20 μg of protein) were fractionated by 10% SDS-PAGE and electroblotted onto Immobilon™ polyvinylidene difluoride membranes (Millipore). Membranes were incubated (1 h at 37°C) with rabbit polyclonal primary antibody directed against PPARγ (dilution, 1:5,000; Cayman Chemical) or GCLC (dilution, 1:5,000; LabVision). After immunodetection with goat anti-rabbit biotinylated secondary IgGs and streptavidin-conjugated peroxidase (dilution, 1:2,000; Dako Cytomation), chemiluminescence was performed according to the manufacturer's protocol (ECL; Amersham Biosciences). The pro-oxidant effect of CLA was evaluated on splenocytes for short time periods and without the addition of mitogens to avoid additional stress on cells. Superoxide (O2•−) reduction of ferricytochrome c was measured as described by Arroyo et al. (28Arroyo A. Modriansky M. Serinkan F.B. Bello R.I. Matsura T. Jiang J. Tyurin V.A. Tyurina Y.Y. Fadeel B. Kagan V.E. NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO differentiated HL-60 cells. Role in phagocytic clearance..J. Biol. Chem. 2002; 277: 49965-49975Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Total GSH concentration (GSH + GSSG) was determined in splenocytes by measuring the reaction of GSH and DTNB coupled to the recycling of GSSG to GSH by glutathione reductase, as described previously (29Bergamo P. Luongo D. Maurano F. Rossi M. Butterfat fatty acids differentially regulate growth and differentiation in Jurkat T-cells..J. Cell. Biochem. 2005; 96: 349-360Crossref PubMed Scopus (10) Google Scholar). GSSG was measured according to Davies, Birt, and Schnell (30Davies M.H. Birt D.F. Schnell R.C. Direct enzymatic assay for reduced and oxidized glutathione..J. Pharmacol. Methods. 1984; 12: 191-194Crossref PubMed Scopus (54) Google Scholar). Intracellular GSH concentration in mouse splenocytes exposed to different CLA concentrations was expressed as the percentage of that measured in cells incubated without CLA for the same time period. For in vivo evaluation of intracellular GSH content, a standard curve was used to calculate the GSH amount and the concentration was normalized to the protein content and expressed as nmol GSH/mg protein/min. The titers of anti-dsDNA and anti-tTG IgGs in mouse sera were quantified by ELISA according to published methods (12Piredda L. Amendola A. Coalizzi V. Davies P. Farrace M.G. Fraziano M. Gentile V. Uray I. Piacentini M. Fesus L. Lack of “tissue” transglutaminase protein cross-linking leads to leakage of macromolecules from dying cells: relationship to development of autoimmunity in MRL/lpr mice..Cell Death Differ. 1997; 4: 463-472Crossref PubMed Scopus (75) Google Scholar). The amount of NFκB p65 was evaluated in nuclear protein extracts of MRL/lpr mouse splenocytes at the end of the in vivo study using the Trans-AM™ NFκB p65 enzyme-linked immunosorbent assay (Active Motif) accordingly to the manufacturer's instructions. NFκB activation was expressed as absorbance at 450 nm. Differences were assessed using Student's t-test, and the levels of significance were designated as follows: * P < 0.001, ** P < 0.01, *** P < 0.05. The in vitro antiproliferative ability of CLA or of pure isomers in mouse splenocytes from MRL/lpr mice was preliminarily investigated. A significant inhibitory effect on cell proliferation was exhibited by CLA and c9,t11 and c10,t12 CLA (P < 0.001); by contrast, no change in cell proliferation of cells exposed to the t9,t11 isomer was detected (Fig. 1A). Incubation with different amounts of CLA resulted in a dose-response effect (Fig. 1B); in particular, our data indicate 60% inhibition of cell proliferation produced by 50, 100, and 200 μM CLA in both MRL/lpr and control mice. On the other hand, we found no evidence for CLA cytotoxicity even at the higher concentrations tested (200 μM) (data not shown). The influence of age/disease activity on the antiproliferative effect of CLA was also evaluated upon the exposure of spleen cells from animals in the range of 7–22 weeks of age. These results demonstrate that the antiproliferative effect of CLA was not affected by the age/disease activity in MRL/lpr and control mice (data not shown). Next, to investigate the involvement of PPARγ, cells were preincubated with a specific PPARγ antagonist (GW9662) before exposure to 100 μM CLA. The data show that the CLA-induced antiproliferative effect was not influenced by GW9662, indicating PPARγ-independent mechanisms (Fig. 1C). Finally, 100 μM CLA caused an inhibition of IFNγ expression in both MRL/lpr and MRL/+ splenocytes (Fig. 1D). The defective Fas functioning in MRL/lpr mice was preliminarily confirmed by finding different caspase-3 activities in MRL/lpr and MRL/+ spleen cells upon anti-Fas IgG treatment (Fig. 2A). Next, the time-dependent apoptosis induction of 100 μM CLA was evaluated by analyzing the classical effectors of programmed cell death. As expected, caspase-3 activity was increased significantly in MRL/lpr splenocytes after 1–2 h of incubation (Fig. 2B) compared with MRL/+ cells, and the number of Annexin V- and dUTP-positive cells was also increased in MRL/lpr cells exposed to 100 μM CLA (3 h) compared with untreated culture (Fig. 2C, D). By contrast, caspase-8 activation was not induced by CLA (data not shown). Collectively, these results support the notion that the proapoptotic ability of CLA is independent of Fas involvement and is mainly responsible for its antiproliferative effect. As reactive oxygen species (ROS) involvement in CLA-induced apoptosis was shown previously, O2•− yield was measured in spleen cells incubated with 100 μM CLA. MRL/lpr and MRL/+ CLA-treated cells yielded significantly higher O2•− concentrations than untreated cells (Fig. 3A). Moreover, because another PPARγ ligand, 15dPGJ2, was shown to induce GSH production via GCLC activation (23Bergamo P. Luongo D. Rossi M. Conjugated linoleic acid-mediated apoptosis in Jurkat T cells involves the production of reactive oxygen species..Cell. Physiol. Biochem. 2004; 14: 57-64Crossref PubMed Scopus (39) Google Scholar), we next determined the concentration- and time-dependent induction of GSH synthesis by CLA in MRL/lpr splenocytes. Interestingly, exposure to 25 μM CLA for 3 h produced a significant intracellular GSH increase, compared with untreated cells (P < 0.01). On the contrary, a marked time-dependent decrease of cellular GSH concentration resulted from incubation with 100 μM CLA (Fig. 3B), whereas the GSH/GSSG ratio was not altered (data not shown). Noteworthy, increased PPARγ expression also occurred in MRL/lpr cells incubated with 100 μM CLA (Fig. 3C). A 4-fold increase of GCLC expression resulted upon 3 h of incubation with 25 μM CLA, whereas no difference in PPARγ expression was seen (data not shown). The proapoptotic ability of CLA at 25 μM was not completely abolished, as indicated by the cleavage of GCLC to a 60 kDa fragment, visualized after 3 h of incubation (Fig. 3C). Together, these findings suggest that CLA, depending on its concentration, may exhibit different abilities. Indeed, 100 μM CLA enhanced PPARγ protein expression and exhibited pro-oxidant properties; on the other hand, antioxidant activities may be triggered by lower CLA concentrations (25 μM). Next, the effects in vivo of short- and long-term CLA treatment were assessed. As splenomegaly is a typical sign of murine SLE, we determined the effect of the treatment on spleen weight in MRL/lpr mice. Animals receiving CLA-T for 2 weeks exhibited body weights that were indistinguishable from those of controls (38.3 ± 2.84 and 38.8 ± 6.2, respectively; P = 0.99), whereas a significant decrease resulted upon long-term administration of CLA-T compared with controls (37.6 ± 2.8 and 41.4 ± 3.4, respectively; P = 0.032). Consequently, we decided to express spleen weight as a percentage of total body weight. A significant reduction of splenomegaly resulted from both short- and long-term treatments with CLA-T (1.26 ± 0.44 and 1.23 ± 0.30, respectively) compared with vehicle-treated animals (1.52 ± 0.36 and 1.55 ± 0.44; P < 0.05)