of March 20, 2018.This information is current as

4Sensitizer NiSODendritic Cells Treated with the Chemical Mechanisms of IL-12 Synthesis by Human

Saadia Kerdine-Römer and Marc PallardyDiane Antonios, Philippe Rousseau, Alexandre Larangé,

http://www.jimmunol.org/content/185/1/89doi: 10.4049/jimmunol.09019922010;

2010; 185:89-98; Prepublished online 4 JuneJ Immunol 

MaterialSupplementary

2.DC1http://www.jimmunol.org/content/suppl/2010/06/02/jimmunol.090199

Referenceshttp://www.jimmunol.org/content/185/1/89.full#ref-list-1

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The Journal of Immunology

Mechanisms of IL-12 Synthesis by Human Dendritic CellsTreated with the Chemical Sensitizer NiSO4

Diane Antonios, Philippe Rousseau, Alexandre Larange, Saadia Kerdine-Romer, and

Marc Pallardy

Allergic contact dermatitis, caused by metallic ions, is a T cell-mediated inflammatory skin disease. IL-12 is a 70-kDa heterodimeric

protein composed of IL-12p40 and IL-12p35, playing a major role in the generation of allergen-specific T cell responses. Dendritic

cells (DCs) are APCs involved in the induction of primary immune responses, as they possess the ability to stimulate naive T cells. In

this study, we address the question whether the sensitizer nickel sulfate (NiSO4) itself or in synergy with other signals can induce the

secretion of IL-12p70 in human monocyte-derived DCs (Mo-DCs). We found that IL-12p40 was produced by Mo-DC in response

to NiSO4 stimulation. Addition of IFN-g concomitantly to NiSO4 leads to IL-12p70 synthesis. NiSO4 treatment leads to the

activation of MAPK, NF-kB pathways, and IFN regulatory factor 1 (IRF-1). We investigated the role of these signaling pathways

in IL-12 production using known pharmacological inhibitors of MAPK and NF-kB pathways and RNA interference-mediated

silencing of IRF-1. Our results showed that p38 MAPK, NF-kB, and IRF-1 were involved in IL-12p40 production induced by

NiSO4. Moreover, IRF-1 silencing nearly totally abrogated IL-12p40 and IL-12p70 production provoked by NiSO4 and IFN-g. In

response to NiSO4, we observed that STAT-1 was phosphorylated on both serine and tyrosine residues and participated to NiSO4-

induced IRF-1 activation. N-acetylcysteine abolished STAT-1 phosphorylation, suggesting that STAT-1 activation may be depen-

dent on NiSO4-induced alteration of the redox status of the cell. These results indicate that p38 MAPK, NF-kB, and IRF-1 are

activated by NiSO4 in Mo-DC and cooperate for IL-12 production. The Journal of Immunology, 2010, 185: 89–98.

Nickel is a metallic allergen causing contact dermatitisfollowingdermalexposure (1).Reports by theNorthAmer-ican Contact Dermatitis Group have consistently ranked

nickel as the number one allergen in frequency of positive patch testreactions. Nickel’s allergy is mainly due to jewelry, piercing, andcoins (2, 3).Allergic contact dermatitis (ACD) caused by metallic ions and

other reactive haptens is a T cell-mediated inflammatory skin dis-ease (4–6). ACD is characterized by two phases: a sensitizationphase and an elicitation phase. During the sensitization phase, skindendritic cells (DCs) capture the metal bound to self-protein, mi-grate through the lymphatic vessels, and present the hapten-protein complex to naive T cells in the draining lymph node(7, 8). Moreover, using human in vitro DC models, several groupshave showed that chemical sensitizer treatment can induce phe-notypic modifications with HLA-DR, CD86, CD40, CD83, andCCR7 upregulation and IL-12 and IL-8 production (9–12).IL-12 consists of two chains (p40 and p35) covalently linked to

give rise to a heterodimeric (p70)molecule. The induction and secre-tionofbioactiveIL-12are regulatedbythe independentproductionofp35 and p40 subunits. IL-12 is primarily produced by macrophages

and DCs mainly in response to danger signals such as TLR agonistsand plays amajor role in IFN-g production by immune cells, drivinga Th1/Tc1-type response (13–16).Many authors have demonstratedthat CD8+ cytotoxic T lymphocytes are the main effector cells ofACD and observed their early recruitment in the skin postchallengewith chemical sensitizers (6, 17). Injection of IL-12 during the sen-sitization phase favors CD8+ T cell differentiation and increases theACD reaction (18). In vivo studies also demonstrated that 2,4-dinitrofluorobenzene–mediated ACD was significantly blocked byanti–IL-12–neutralizing Abs, supporting the conclusion that IL-12is an important effector in the pathogenesis of ACD (19).The secretion of free IL-12p40 and IL-12p70 by activatedmacro-

phages and DCs is dependent on the regulation of the il-12p40 pro-moter by an array of transcription factors including C/EBP, NF-kB, AP-1, IFN regulatory factor (IRF)-1, and IRF-8 (20–25). IL-12p35 expression is also tightly regulated both at the transcrip-tional and translational level (26, 27). Specificity protein 1, AP-1, IRF-1, IRF-3, and IRF-8 have been described to play a role inthe expression of human IL12A (23, 28, 29). RNA interferenceexperiments showed a critical requirement for the NF-kB p50subunit in the production of IL-12 by human monocyte-derivedDCs (Mo-DCs) after CD40L and IL-1 stimulation (24). Moreover,IRF-1(2/2) splenic DCs were markedly impaired in their ability toproduce IL-12 after LPS stimulation (25). In vivo, a defectiveIL-12 production was also observed in LPS-treated macrophagesobtained from MAP kinase kinase 3-deficient mice (30). All ofthese reports suggest that MAPKs, NF-kB, and IRF pathways areinvolved in the production of IL-12 by DCs.NiSO4 has been shown to activate both MAPK and NF-kB

pathways in human Mo-DCs or in human DCs differentiated fromCD34+ cells (CD34-DC) (9–11, 31, 32). Inhibition of p38 MAPK,JNK, and ERK abrogated the upregulation of CD86, CD83, andCCR7expression inducedbyNiSO4,whereas expressionofHLA-DRand CD40 and the production of IL-6, IL-12p40, and IL-8 were

Universud, Institut National de la Sante et de la Recherche Medicale Unite Mixte deRecherche S 749 and 996, Faculte de Pharmacie, Chatenay-Malabry, France

Received for publication June 23, 2009. Accepted for publication April 19, 2010.

Address correspondence and reprint requests toDr.Marc Pallardy, INSERMUMR996,Faculte de Pharmacie, Universite Paris-Sud 11, 5 Rue Jean-Baptiste Clement, Chate-nay-Malabry 92290, France. E-mail address: marc.pallardy@u-psud.fr

The online version of this article contains supplemental material.

Abbreviations used in this paper: ACD, allergic contact dermatitis; DC, dendriticcell; iDC, DC with immature phenotype; IRF, IFN regulatory factor; ISRE, IFNsequence response element; Mo-DC, monocyte-derived DC; NAC, N-acetylcysteine;N.S., nonspecific; siRNA, small interfering RNA.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901992

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mainly dependent on the NF-kB pathway (10, 11, 31–33). Current

hypothesis in the field of contact allergy suggests that nickel and other

chemical sensitizers could be perceived as a danger signal byDCs, leading to MAPK and NF-kB activation and DC phenotypicmodifications.In this study, we address the question whether NiSO4 itself or in

synergy with other signals can induce the secretion of IL-12p70 inhuman Mo-DCs. We showed that: 1) nickel induced the produc-tion of IL-12p40; 2) IL-12p70 synthesis needed the presence ofNiSO4 and IFN-g, and both signals were also required for theexpression of the p35 subunit of IL-12; and 3) the production ofIL-12p70 in response to NiSO4 and IFN-g was dependent on p38MAPK, NF-kB, and IRF-1 activation.We also found that IRF-1was

activated in response to NiSO4 in a STAT-1–dependent manner andplayed a crucial role in NiSO4-induced IL-12 production.

Materials and MethodsGeneration of Mo-DCs

PBMCswerepurified frombuffy coats obtained fromEtablissement Francaisdu Sang (Ivry-Sur-Seine, France) by density centrifugation with Ficollgradient (Eurobio, Les Ulis, France). Monocytes were isolated from themononuclear fraction through magnetic positive selection using MiniMacsseparation columns (Miltenyi Biotec, BergishGladbach, Germany) and anti-CD14 Abs coated on magnetic beads following the provider’s instructions(DirectCD14 isolation kit,MiltenyiBiotec).Monocyteswere cultured at 13106 cells/ml in the presence of GM-CSF (550 U/ml) and IL-4 (550 U/ml)(both from Abcys, Paris, France) in RPMI 1640 containing Glutamax I

FIGURE 1. Mo-DC treatment with NiSO4 or NiSO4 and IFN-g induced the production of IL-12. At day 5, iDCs were treated for 24 h with either NiSO4,

IFN-g (1000 U/ml), or their association. A, IL-12p40 production after NiSO4 addition. IL-12p40 was measured by ELISA. Results are expressed in picograms

per milliliter (values are mean6 SD of three independent experiments). B, IL-12p40 production postaddition of NiSO4 (500mM) and IFN-g (1000 U/ml). IL-

12p40 was measured by ELISA. Results are expressed in picograms per milliliter (values are mean 6 SD of three independent experiments). C, IL-12p70

production postaddition of NiSO4 (500 mM) and IFN-g (1000 U/ml). IL-12p70 was measured by ELISA. Results are expressed in picograms per milliliter

(values aremean6SDof three independent experiments). pp# 0.05 comparedwith control; †p# 0.05 comparedwithNiSO4;‡p# 0.05 comparedwith IFN-g.

FIGURE 2. IL12A mRNA expression induced by NiSO4 and IFN-g in Mo-DCs. At day 5, iDC were treated for 4 or 8 h with NiSO4 (500 mM), IFN-g

(1000 U/ml), or their association. A, IL12A mRNA expression was measured using real-time PCR. Results were expressed as fold factor compared with

untreated samples (control) and corrected by the expression of the housekeeping gene b-actin as described in Materials and Methods. Results are the mean

of three independent experiments. B, IL12A mRNA expression was evaluated using RT-PCR. GAPDH was used as the housekeeping gene. Folds represent

the ratio of the normalized intensity (intensity of IL12A band/intensity of GAPDH band) of treated cells divided by the normalized intensity of nontreated

cells. Results are representative of three independent experiments. pp # 0.05.

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supplemented with 10% heat-inactivated FCS, 1 mM sodium pyruvate,

0.1 mg/ml streptomycine, and 100 U/ml penicillin (RPMIc) (all from Life

Technologies/ Invitrogen, Paisley, U.K.). Within 5 d, monocytes differenci-

ate into DCs with immature phenotype (iDCs).

Chemical treatment of iDCs

Mo-DCs (at day 5) were washed three times in RPMIc, and their concen-tration was adjusted to 1 3 106 cells/ml. iDCs were stimulated or not withdifferent concentrations of NiSO4 (Sigma-Aldrich, St. Louis, MO) with orwithout IFN-g (1000 U/ml; Abcys) for the indicated time. To study theinvolvement of signaling pathways in IL-12 production, Mo-DCs werepretreated for 30 min with SP600125 (20 mM) or SB203580 (20 mM)for 1 h with Bay 11-7085 (3 mM) or for 2 h with Jak inhibitor I (0.5 mM)(all fromMerck Chemicals, Darmstadt, Germany). N-acetylcysteine (NAC)was purchased from Sigma-Aldrich.

Western blot analysis

iDCs (106 cells/ml) were exposed to NiSO4 (500mM) or IFN-g (1000U/ml).After an adequate time of stimulation, cells were washed with ice-cold PBS.Cell lysates were prepared by resuspending cell pellet in lysis buffer [20mMTris (pH 7.4), 137 mM NaCl, 2 mM EDTA (pH 7.4), 1% Triton, 25 mMb-glycerophophate, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 10% glyc-erol, 1mMPMSF,1mg/mlaprotinin, 1mg/ml leupeptin, and1mg/mlpepstatin].The homogenateswere centrifugated at 15,000 rpm for 20min at 4˚C.A total of50 mg denaturated protein were loaded onto 12.5% SDS-PAGE gel and

transferred on polyvinylidene difluoride membrane (Amersham Biosciences,Les Ulis, France). Membranes were then incubated with Abs directed againstthe phosphorylated forms of p38 MAPK (Thr180/Tyr182), JNK 1/2 (Thr183/Tyr185), STAT-1 (Tyr 701), or STAT-1 (Ser 727) (all fromCell Signaling Tech-nology,Ozyme,St-Quentin enYvelines, France) or against IRF-1 (C-20) (SantaCruzBiotechnology, Santa Cruz,CA). Immunoreactive bandswere detected bychemiluminescence (ECL solution, Amersham Biosciences). p38 MAPK orSTAT-1 were used as a loading control and revealed with Abs raised againstp38 MAPK (p38 N20, Santa Cruz Biotechnology) or STAT-1 (Cell SignalingTechnology, Ozyme). Bands were quantified by densitometry using the Image-Quant software. The intensity of the specific band was normalized to the in-tensity of loading control protein band. Folds represent the ratio of thenormalized intensity of treated cells divided by the normalized intensity ofnontreated cells.

Preparation of whole cell extract and DNA-affinity protein-binding assay

Following treatment with NiSO4 (500 mM) or IFN-g (1000 U/ml), cells werelysed inNonidet P-40 hypertonic lysis buffer. In brief, cell pellets from83106

cells were resuspended in a buffer adjusted at pH 7.9 containing 0.2% Non-idet P-40, 20% glycerol, 20 mMHEPES-KOH, 420 mMNaCl, 1 mMDTT, 1mM Na3VO4, 1 mM sodium pyrophosphate, 0.125 mM okadaic acid, 1 mMEDTA,1mMEGTA,0.5mMPMSF, 1mg/ml leupeptin, 1mg/mlaprotinin, and1 mg/ml pepstatin and incubated at 4˚C for 30 min. Cellular debris were re-moved by centrifugation at 4˚C, 15,000 rpm, for 20 min. The DNA affinity

FIGURE 3. NF-kB, MAPKs, and IRF-1 pathways are activated upon addition of NiSO4 or NiSO4 and IFN-g. A, MAPK activation after NiSO4 or NiSO4

and IFN-g treatment. At day 5, iDCs were treated or not for 30, 60, and 120 min with NiSO4 (500 mM), IFN-g (1000 U/ml), or their association.

Poststimulation, cells were lysed, and the level of phosphorylated p38 MAPK and JNK was evaluated by Western blotting using anti–phospho-p38 MAPK

or anti–phospho-JNK Abs. The membrane was then probed with anti-p38 MAPK Ab for loading control. Folds represent the ratio of the normalized

intensity (intensity of phosphorylated form of MAPK band/p38 MAPK band) of treated cells divided by the normalized intensity of nontreated cells.

Results are representative of three independent experiments. B, NF-kB activation by NiSO4 or NiSO4 and IFN-g at 60 min. At day 5, iDCs were treated for

60 min with NiSO4 (500 mM), IFN-g (1000 U/ml), or their association. Whole-cell extracts were prepared and incubated with either biotinylated NF-kB

probes (NF-kB) or mutated NF-kB probes (NF-kB–mut) and streptavidin-agarose beads. P65 was detected by Western blotting. Results are representative

of three independent experiments. C, IRF-1 activation by NiSO4 or NiSO4 and IFN-g at 90 min. At day 5, iDCs were treated for 90 min with NiSO4 (500

mM), IFN-g (1000 U/ml), or their association. Whole-cell extracts were prepared and incubated with either biotinylated ISRE probes (ISRE) or mutated

ISRE probes (ISRE-mut) and streptavidin-agarose beads. IRF-1 was detected by Western blotting. p38 MAPK was used as a loading control in whole-cell

extracts using Western blotting and anti-p38 MAPK Ab. Results are representative of three independent experiments. N.S., nonspecific.

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precipitation assay was carried out as described previously (34). In brief, thefollowing 59 biotin-labeled single-stranded oligonucleotides were hybridized:59-ACTGCGAACATTTCGCTTTCATTTTGG-39 and59-CCAAAATGAAAG CGA AAT GTT CGC AGT-39 (MWG Biotech, Ebersberg, Germany)according to the human IFN sequence response element (ISRE) consensussequence from the IL-12p35 promoter. Mutated oligonucleotides (59-ACTGCG AAC ATT TCG CAA ACA TTT TGG-39 and 59-CCA AAA TGTTTG CGA AAT GTT CGC AGT-39) were used for unspecific control bind-ing. Also, the following 59-biotin-labeled single-stranded oligonucleotideswere hybridized: 59-TTG AGG GGA CTT TCC CAG G-39 and 59-CCTGGG AAA GTC CCC TCA A-39 (MWG Biotech) according to the humanNF-kB consensus sequence. Mutated oligonucleotides (59-TTG AGG CGACTT TCC CAG G-39 and 59-CCT GGG AAA GTC GCC TCA A-39) wereused for unspecific control binding. DNA-binding proteins were isolated from200 mg whole-cell extracts at 4˚C for 90 min with 2 mg double-stranded 59-biotinylated oligonucleotides coupled to 50 ml streptavidin-agarose beads(Sigma-Aldrich). Complexes were washed with the binding buffer and elutedby boiling in a reducing sample buffer. The binding proteins were separated on8% SDS-PAGE followed by Western blot analysis using anti–IRF-1 (C20) oranti-p65 (sc109) mouse mAb (Santa Cruz Biotechnology).

Electroporation of Mo-DCs with small interfering IRF-1

At day 4 of differentiation, Mo-DCs were washed once with serum-free me-dium and once with PBS. Cells were then resuspended in serum-free

medium at 4 3 107 cells/ml; 10 mg nonsilencing control small interferingRNA (siRNA) (siRDM from Qiagen, Courtaboeuf, France) orIRF-1 siRNA (siIRF-1 ON-TARGETplus SMARTpool from Thermo Sci-entific Dharmacon, Lafayette, CO) were transferred into 4-mm cuvettes(Bio-Rad, Marnes-La-Coquette, France) and filled up to a final volume of100 ml with serum-free medium. The siIRF-1 ON-TARGETplus SMART-pool, which contained four individual duplexes, was chosen because itenhances siRNA specificity and reduces off-target effects. A total of 4 3106 cells was added and pulsed in a GenePulser II (Bio-Rad) (300 V,150 mF). Cells were then resuspended in complete medium with GM-CSF (550 U/ml) and IL-4 (550 U/ml) at a concentration of 106 cells/ml for24 h and then treated according to different protocols.

mRNA expression using semiquantitative and real-time PCR

Total RNA was extracted using TRIzol Reagent (Invitrogen, Cergy Pon-toise, France) by the guanidium thiocyanate method as mentioned by themanufacturer. RNA was quantified by spectrophotometry. First-strandcDNAwas synthesized from total RNA extracted in RNAse-free conditions.The reaction was performed on 2 mg total RNA with oligo(dT) primers(MWG Biotech) and 2 U AMV RT (Promega, Charbonnieres-les-Bains,France). For semiquantitative PCR, the reaction was performed using 1 UTaq polymerase (QBiogen, Montreal, Canada). The number of cycles andthe hybridization temperature used for the PCR were optimized for all thegenes studied: GAPDH (24 cycles, 60˚C), IRF1 (30 cycles, 54˚C), and

FIGURE 4. Role of MAPKs and NF-kB in NiSO4-

induced IL-12p40 production. At day 5, iDCs were trea-

ted with either DMSO, SB203580 (20 mM), SP600125

(20 mM), or Bay 11-7085 (3 mM) and then stimulated

with NiSO4 at 500mM.After 24 h, IL-12p40 production

was measured by ELISA. Results are expressed in pico-

grams per milliliter (values are mean 6 SD of three in-

dependent experiments). pp # 0.05 compared with

NiSO4-treated cells (control NiSO4);¥p # 0.05 com-

pared with NiSO4- and DMSO-treated cells.

FIGURE 5. IRF-1 is needed for IL-12p40 and IL-12p70 production in response to NiSO4 and IFN-g. At day 4 of differentiation, cells were electroporated

with 10mg siRNA random (RDM) or siRNA IRF-1. For the next 24 h, cells were incubated with GM-CSF (550 U/ml) and IL-4 (550 U/ml). Cell treatments were

proceeded 24 h postelectroporation.A, Effect of IRF-1 siRNA treatment on IL-12p40 production. Cellswere treatedwithNiSO4 (500mM)and IFN-g (1000U/ml).

After 24 h, IL-12p40 production was measured by ELISA. Results are expressed in pg/ml (values are mean 6 SD of three independent experiments). B, Effect

of IRF-1 siRNA treatment on IL-12p70 production. Cells were treated with NiSO4 (500 mM) and IFN-g (1000 U/ml). IL-12p70 production was measured after

24 h by ELISA. Results are expressed in picograms per milliliter (values are mean 6 SD of three independent experiments). C, IL12A mRNA (IL-12p35)

expression. Cells were treated with NiSO4 (500mM) and IFN-g (1000 U/ml) for 8 h. IL12AmRNA expressionwas evaluated using RT-PCR.GAPDHwas used as

the housekeeping gene. Results are representative of three independent experiments. D, siRNA IRF-1 effect on IRF-1 protein levels. Cells were stimulated with

IFN-g for two hours and lysed. IRF-1 expression was evaluated by Western blotting. Membrane was then probed with anti-p38 MAPK Ab for loading control.

Numbers represent the ratio of the intensity of IRF-1 band/intensity of p38 MAPK band. Results are representative of three independent experiments.

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IL12A (IL-12p35) (35 cycles, 59.2˚C). The specific primers used were(forward and reverse primers, respectively): GAPDH: 59-ACC ACA GTCCATGCCATCAC-39 and 59-TCCACCACCCTGTTGCTGTA-39; IRF1:59-ACCCTGGCTAGAGATGCAGA-39 and59-TTTTCCCCTGCTTGTATCG-39; and IL12A: 59- ACCACT CCCAAAACC TGC-39 and 59- CCAGGCAAC TCC CAT TAG-39. PCR products were visualized by addition ofethidium bromide on 1% agarose gel. GAPDH was used to control andcalibrate cDNA synthesis. Fold induction represents the normalized ratioof treated samples divided by untreated samples.

Real-time PCR was performed using the SYBR Green technology ona LightCycler rapid thermal cycler (Roche Diagnostics, Meylan, France). For-ward and reverse primers were each designed on a different exon of the targetgene sequence, eliminating the possibility of amplifying genomic DNA. Toconfirm the specificity of the amplification, the PCR product was subjected

to amelting curve analysis and agarose gel electrophoresis. PCR amplificationwas performed in duplicate in a total reaction volume of 10 ml. The reactionmixture consisted of 5 ml diluted template, 2 ml FastStart DNA MasterPLUSSYBR Green kit, and 0.5 mM forward and reverses primers. After an 8 sactivation of Taq polymerase at 95˚C, amplification was proceeded for 30–45 cycles, each consisting of denaturation at 95˚C for 5 s, annealing at 60˚Cfor 5 s, and extension at 72˚C for 9 s. Specific primers were used in the PCRreaction mixture (forward and reverse primers, respectively): IL12A: 59-ACCACT CCC AAA ACC TGC-39 and 59-CCA GGC AAC TCC CAT TAG-39;and b-actin: 59-GGC ATC CTC ACC CTG AAG TA-39 and 59-GCA CACGCA GCT CAT TGT AG-39. Results were expressed as fold inductioncalculated using the standard curve method. b-actin was used to controland calibrate cDNA synthesis. Fold induction represents the normalizedratio of treated samples divided by untreated samples.

FIGURE 6. IRF-1 is activated by NiSO4 and regulates IL-12p40 production. A, IRF-1 activation in response to NiSO4 at 4 and 6 h. Whole-cell extracts

were prepared and incubated with biotinylated ISRE probes (ISRE) or mutated ISRE probes (ISRE-mut) and streptavidin-agarose beads. IRF-1 was

detected by Western blotting. Results are representative of three independent experiments. p38 MAPK was used as a loading control in whole-cell extracts

using Western blotting and anti-p38 MAPK. B, IRF1 mRNA expression in NiSO4 or NiSO4- and IFN-g–treated cells. At day 5, iDCs were treated or not

(control) with either NiSO4 (500 mM), IFN-g (1000 U/ml), or their association. IRF1 mRNA expression was evaluated using RT-PCR at 2, 4, and 6 h.

GAPDH was used as the housekeeping gene. Folds represent the ratio of the normalized intensity of treated cells divided by the normalized intensity of

nontreated cells. Results are representative of two independent experiments. C, IRF-1 regulates NiSO4-induced IL-12p40 production. At day 4 of

differentiation, cells were electroporated with 10 mg siRNA random (RDM) or siRNA IRF-1. Twenty-four hours later, cells were treated with NiSO4

(500 mM), and IL-12p40 production was measured by ELISA. Results are expressed in picograms per milliliter (values are mean6 SD of three independent

experiments). p # 0.05.

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FIGURE 7. IRF-1 expression in response to NiSO4 is dependent on Jak-induced STAT-1 phosphorylation but not on p38 MAPK. A, STAT-1 phosphor-

ylation in response to NiSO4 or NiSO4 and IFN-g. At day 5, iDCs were treated or not (control) with either NiSO4 (500 mM), IFN-g (1000 U/ml), or their

association for 2, 3, or 4 h. Cells were lysed, and the level of STAT-1 phosphorylation (p-tyr 701 and p-Ser 727) was evaluated by Western blotting.

Membrane was then probed with an anti–STAT-1 Ab for loading control. Folds represent the ratio of the normalized intensity of treated cells divided by the

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ELISA assay for measuring IL-12p40 and IL-12p70 production

iDCs were treated with NiSO4 with or without IFN-g for 24 h. In someexperiments, cells were pretreated for 30 min with MAPK inhibitors orwith Bay 11-7085 or DMSO for 1 h and then stimulated. Culture super-natants were collected after 24 h and stored at 280˚C. ELISA assays wereperformed according to the manufacturer’s instructions (R&D Systems,Minneapolis, MN). The OD proportional to the intensity of the colorwas measured at 450 nM and 540 nM with a microplate reader (ThermoLabsystems-Multiskan, Philadelphia, PA). Results were expressed in pico-grams per milliliter. The sensitivity of the method was 31 pg/ml for IL-12p40 and 7.8 pg/ml for IL-12p70 according to the manufacturer.

Data analysis

All data are represented as mean 6 SD of the mean (based on the entirepopulation). Differences between the production of IL-12p40 or IL-12p70in chemical-treated cells compared with respective controls were evaluatedusing an unpaired Student t test. p# 0.05 was considered to be statisticallysignificant. All tests were performed using the software GraphPad Instat 3(GraphPad, San Diego, CA).

ResultsNiSO4 triggers the production of IL-12p40 in human Mo-DCcells

In the first part of the study,wemeasured the production of IL-12p40and IL-12p70 afterNiSO4 addition inMo-DCs. iDCswere treated ornot (control) with NiSO4 (300, 400, and 500 mM) for 24 h. NiSO4

induced the production of IL-12p40 in a concentration-dependentmanner (Fig. 1A). IL-12p40 production was significantly aug-mented at 300 mMwhen compared with control cells. A significantamount of IL-12p40was present in untreatedMo-DCs as previouslydescribed (35). In the same conditions, an average of 100 ng/ml IL-12p40 was found after LPS addition (25 ng/ml) (data not shown).When IFN-gwas added to NiSO4, IL-12p40 production was greatlyaugmented (Fig. 1B). NiSO4 was unable to induce the production ofIL-12p70, but the concomitant presence of IFN-g and NiSO4

induced the production of IL-12p70 in Mo-DCs (Fig. 1C).

The presence of IFN-g in association with NiSO4 is necessaryfor the expression of IL12A mRNA

IL-12p70 depends on the synthesis of both p40 and p35 chains. iDCswere treatedor not (control) for 4or 8hwithNiSO4 (500mM), IFN-g(1000 U/ml), or their association. IL12A mRNA expression wasmeasured using real-time PCR (Fig. 2A) and semiquantitativePCR (Fig. 2B). These times of stimulation were chosen accordingto preliminary kinetics experiments measuring IL12A mRNA ex-pression (data not shown). IL12AmRNA expression was chosen asa readout as there are no robust methods to measure IL-12p35 at theprotein level. Results showed that very low levels of IL12A mRNAwere detected when NiSO4 or IFN-g were added. The presence ofboth NiSO4 and IFN-g was necessary to induce the expression ofdetectable levels of IL12 mRNA. Moreover, results obtained with

inhibitors of the p38 MAPK, JNK, and NF-kB pathways showedthat in addition to IRF-1, other pathways are involved in IL-12 p35mRNA expression (Supplemental Fig. 1).

NiSO4 and IFN-g activate complementary signaling pathways:MAPKs, NF-kB, and IRF-1

The promoters of p35 and p40 are composed of several DNAbinding sites for AP-1, NF-kB, and IRFs. These pathways havealso been previously described to participate in IL-12 synthesis inDCs and macrophages (24, 25, 30). We first evaluated the kineticsof JNK and p38 MAPK activation in iDCs stimulated by NiSO4,IFN-g, or their combination. Our results showed that NiSO4 andNiSO4 with IFN-g induced the phosphorylation of p38 MAPK andJNK at 30 min. The association of both NiSO4 and IFN-g slightlyupregulated p38 MAPK (at 30 min) and JNK phosphorylation (at30 min and 60 min) compared with NiSO4 alone (Fig. 3A).Binding of the p65 subunit of NF-kB to specific NF-kB probesusing a DNA-binding assay after 1 h of treatment was detected withboth NiSO4 and NiSO4 with IFN-g (Fig. 3B). We then assessed thebinding of the transcription factor IRF-1 to specific IL-12p35 ISREusing a DNA-binding assay after 90 min of treatment with NiSO4

and NiSO4 with IFN-g. Results showed that IFN-g and NiSO4

associated to IFN-g showed comparable binding of IRF-1 on spe-cific ISRE DNA probes (Fig. 3C). However, no binding ofIRF-1 was detectable after 90 min of NiSO4 treatment alone.

p38 MAPK, NF-kB, and IRF-1 contribute to the production ofIL-12

To investigate the implication of MAPKs and NF-kB in IL-12p40synthesis induced by NiSO4, we used three well-described phar-macological inhibitors: SB203580, a p38 MAPK inhibitor, SP60-0125, a JNK inhibitor, and Bay 11-7085, an NF-kB inhibitor. Atday 5, cells were washed and pretreated with SB203580 (20 mM),SP600125 (20 mM) for 30 min, or Bay 11-7085 (3 mM) for 1 hand then stimulated for 24 h with NiSO4. These concentrations ofinhibitors were not cytotoxic, and the optimal concentration foreach inhibitor was determined based on preliminary experiments(data not shown). As JNK and NF-kB inhibitors were diluted inDMSO, cells were also pretreated for 1 h with DMSO (0.1%) asan internal control. Results showed that IL-12p40 production in-duced by NiSO4 was mainly dependent on p38 MAPK and NF-kBpathways; this production was independent of the JNK pathway(Fig. 4).To investigate whether IRF-1 was critically required for IL-12

production induced by both signals, we used RNA interference toinhibit IRF-1 expression in iDCs. The levels of IL-12p40 and IL-12p70 production were measured by ELISA. RNA interferenceexperiments revealed that IRF-1 was critical for IL-12p40 and IL-12p70 production induced by NiSO4 associated to IFN-g (Fig. 5A,

normalized intensity of nontreated cells. Results are representative of three independent experiments. B, STAT-1 phophorylation (p-Ser 727) in response

to NiSO4 is controlled by p38 MAPK. At day 5, iDCs were pretreated or not (control) with SB203580 at 20 mM for 30 min and then treated with NiSO4

(500 mM) for 3 h. Cells were lysed, and the level of phosphorylation of STAT-1 (p-Ser 727) and the level of IRF-1 was evaluated by Western blotting.

Membrane was then probed with STAT-1 Ab for loading control. Results are representative of three independent experiments. C, Specificity of Jak inhibitor

1 and SB203580 for Ser and Tyr STAT-1 phosphorylation, respectively. At day 5, iDCs were pretreated or not (control) with SB203580 at 20 mM for 30 min

or Jak inhibitor at 0.5 mM for 2 h and then treated with NiSO4 (500 mM) for 3 h. Cells were lysed, and the level of phosphorylated STAT-1 was evaluated by

Western blotting. Membrane was then probed with STAT-1 Ab for loading control. Results are representative of two independent experiments. D, Jaks

control IRF-1 expression and STAT-1 tyrosine phosphorylation induced by NiSO4. At day 5, iDCs were pretreated or not (control) with either DMSO or the

Jak inhibitor at 0.5 mM for 2 h and then treated with NiSO4 (500 mM) for 3 h. Cells were lysed, and the level of phosphorylation of STAT-1 (p-Tyr 701) and

IRF-1 were evaluated by Western blotting. Membrane was then probed with STAT-1 Ab for loading control. Results are representative of three independent

experiments. E, p38 MAPK inhibition does not control IRF-1 expression. At day 5, iDCs were pretreated or not (control) with SB203580 at 20 mM for

30 min and then treated with NiSO4 (500 mM) for 3 h. Cells were lysed, and the level of IRF-1 was evaluated by Western blotting. Membrane was then

probed with STAT-1 Ab for loading control. Results are representative of the mean of two independent experiments.

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5B). Furthermore,we showed that IL12AmRNAexpression inducedby both signals was nearly completely abolished in the presenceof IRF-1 siRNA (Fig. 5C). Western blotting confirmed that ON-TARGETplus SMARTpool siRNA (Thermo Scientific Dharmacon)directed to IRF-1 reduced the IFN-g–induced IRF-1 protein levelby .70% (Fig. 5D).

Nickel induces the expression of IRF-1 which contributes toIL-12p40 production

We then evaluated the expression of IRF-1 by NiSO4 alone in Mo-DCs. Using a DNA-binding assay, we showed that NiSO4 inducedthe binding of IRF-1 to specific ISRE at 4 h and 6 h (Fig. 6A),whereas this was not the case at 120 min (Fig. 3C). DNA-bindingactivity was lower in the presence of NiSO4 compared with NiSO4

plus IFN-g. NiSO4 was also able to augment the expression of IRF1at the mRNA level, suggesting an augmentation of its synthesis (Fig.6B). IRF-1 downregulation using IRF-1 siRNA reduced significantlyIL-12p40productionbyNiSO4, suggesting that IRF-1 participated toNiSO4-induced IL-12p40 production (Fig. 6C).

NiSO4-induced IRF-1 expression depends on STAT-1phosphorylation

Accordingtothepublishedpromotersequence(36), theIRF-1promotercontains a conserved IFN-g activation site. Furthermore, IFN-g–induced expression of IRF-1 is completely abolished in STAT-12/2

cells (37). Our results showed that NiSO4 alone induced the phos-phorylation of STAT-1 on both serine and tyrosine residues at 2 h.STAT-1 phosphorylation was still observed at 4 h (Fig. 7A). Usinga p38 MAPK inhibitor, we showed that p38 MAPK controlled thephosphorylation of STAT-1 on specific serine residues (Fig. 7B). Onthe basis of the observed correlation between IRF-1 expression andphosphorylation of STAT1, we hypothesized that IRF-1 expressioncould depend on STAT-1 activation by NiSO4. iDCs were treated for2 h with Jak inhibitor I at 0.5 mM or DMSO and then stimulated withNiSO4 for 3 h. The optimal concentration of Jak inhibitor I was de-termined based on preliminary experiments (data not shown), and wealso controlled that Jak inhibitor I and SB203580 did not affectSTAT-1 serine phosphorylation and STAT-1 tyrosine phosphoryla-tion, respectively (Fig. 7C). Results showed that the abrogation ofSTAT-1 tyrosine phosphorylation completely inhibited the expres-sion of IRF-1 induced byNiSO4. These results suggested that NiSO4

activated STAT-1 tyrosine phosphorylation, leading to IRF-1 ex-pression in Mo-DCs (Fig. 7D). However, STAT-1 phosphorylation

and IRF-1 expression were not dependent on p38 MAPK activity(Fig. 7E). The next question was to elucidate if STAT-1 tyrosinephosphorylation resulted from a direct effect of NiSO4 on Jak ac-tivity or through the synthesis of an intermediate factor. Indeed, typeI IFNs arewell-known inducers of STAT-1 tyrosine phosphorylationinMo-DCs (38).We found thatNiSO4 upregulated the ifn-bmRNA,but IFN-b was undetectable in the supernatants of Mo-DC treatedfor 24 h by NiSO4 (data not shown). We then used puromycin,a well-known protein synthesis inhibitor. Mo-DCs were pretreatedfor 1 hwith puromycin at 10mMand then treated for 3 hwithNiSO4.The optimal concentration of puromycin for protein synthesis in-hibitionwas determined based on preliminary experiments (data notshown). We showed that STAT-1 tyrosine phosphorylation inducedby NiSO4 was not modified by puromycin, suggesting that STAT-1phosphorylation did not depend on an intermediate product synthe-sized by Mo-DC in response to NiSO4 (Fig. 8A). IRF-1 proteinexpression was strongly inhibited as a consequence of puromycintreatment, showing that puromycin was an effective inhibitor ofprotein synthesis in the conditions used (Fig. 8A). Preincubationof cells with NAC (a precursor of glutathione) is often used to re-inforce the redox potential of cells. The optimal concentration ofNAC was determined based on preliminary experiments (data notshown). We showed that pretreatment with NAC inhibited STAT-1phosphorylation and completely abolished NiSO4-induced IRF-1expression (Fig. 8B).

DiscussionDCs are activated by danger signals such as proinflammatorycytokines, bacterial products, viruses, and chemicals. Sensitizershave also been demonstrated to induce the expression of markersrelated to function and maturation of DCs, the production of pro-inflammatory cytokines, and the phosphorylation of members ofthe MAPK family and NF-kB pathway, thus mimicking theeffects of danger signals (9–11, 31–33).IL-12 is a key factor in the generation of Th1 and cytotoxic T cells

(14–16), thus playing a major role in the generation of allergen-specific T cell response in ACD. In this study, we address the ques-tion if nickel, the most prevalent of all metal sensitizers (39), cantrigger specific signaling pathways inDCs, leading to the productionof IL-12 as previously described for danger signals.We showed that NiSO4 induced the production of IL-12p40 in

Mo-DCs in a concentration-dependent manner. We and others havepreviously shown that NiSO4 induced the production of IL-12p40 in

FIGURE 8. STAT-1 phosphorylation (Tyr 701) in response to NiSO4 is not dependent on de novo protein synthesis and is redox sensitive. A, STAT-1

tyrosine phosphorylation induced by NiSO4 was not modified by puromycin. At day 5, Mo-DCs were pretreated for 1 h with 10 mM puromycin and then

treated for 3 h with NiSO4. Cells were lysed, and the level of STAT-1 phosphorylation (p-Tyr 701) was evaluated by Western blot. Membrane was probed

with an anti–IRF-1 to evaluate IRF-1 protein synthesis inhibition or with an anti–STAT-1 Ab for loading control. Results are representative of three

independent experiments. B, STAT-1 phosphorylation in response to NiSO4 is redox sensitive. At day 5, iDCs were pretreated or not (control) with NAC at

25 mM for 30 min. Postremoval of NAC containing medium, Mo-DCs were treated with NiSO4 (500 mM) for 4 h. Cells were lysed, and the level of

phosphorylation of STAT-1 (p-Tyr 701, p-Ser 727) and IRF-1 expression were evaluated by Western blotting. Membrane was then probed with STAT-1 Ab

for loading control. Results are representative of three independent experiments.

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Mo-DCs and CD34-DC (10, 11, 33). However, NiSO4 was not ableto induce IL-12p70 production. When IFN-g was added withNiSO4, detectable amounts of IL-12p70 were then produced byMo-DCs. In addition, both signals induced a high production ofIL-12p40, showing an interesting synergistic effect compared withNiSO4 alone. Our results are in accordance with other works thathave previously shown that the production of IL-12p70 in Mo-DCsrequired the presence of two complementary signals such as LPS,IFN-g, or CD40L to induce a high amount of IL-12p70 synthesis(35, 40, 41).These results showed the necessity of IFN-g for high IL-12 pro-

duction after NiSO4 treatment in Mo-DCs. O’Leary et al. (42)showed that NK cells can mediate contact hypersensitivity reactionin mice with SCID or deficient in RAG2 (Rag22/2), which lackT cells (but have NKs). NK cells are present in low frequency innormal human skin, and an influx of NK cells is found in dermal andepidermal inflammatory infiltrates in human contact hypersensitiv-ity (43). NiSO4 has also been described to induce the production ofIFN-g in splenic NK cells (44). We suggest that IFN-g produced byNK cells may participate to the production of IL-12 by NiSO4-stimulated DCs.The production of IL-12p40 in activated professional APCs is

generally in great excess over that of the p35 chain, making thelatter molecule a limiting step in the formation of bioactive IL-12(45). As NiSO4 alone induced a high level of IL-12p40 but not ofIL-12p70, we evaluated the expression of IL12AmRNA (IL-12p35)in response to NiSO4 and IFN-g. Our results showed that thecombination of NiSO4 and IFN-g induced a high expression ofIL12A mRNA.The regulation of both the IL-12p40 and IL12A genes is mainly

controlled by AP-1, NF-kB, and IRF-1 factors (20, 23, 28, 46). Wethen addressed the question of the signaling pathways involved inthe regulation of IL-12p70 after NiSO4 and IFN-g treatment.NiSO4 has been described to activate MAPKs and NF-kB inCD34-DCs andMo-DCs (9, 11, 32). Our results showed that NiSO4

provoked p38 MAPK and JNK phosphorylation and the binding ofp65 on specific DNA probes in Mo-DCs. The association of NiSO4

and IFN-g slightly augmented p38 MAPK and JNK activationcompared with NiSO4-treated DCs. Moreover, IFN-g or NiSO4

associated to IFN-g induced a detectable DNA-binding activity ofIRF-1 at 90 min. The early activation of p38 MAPK, JNK, NF-kB,and IRF-1 by NiSO4 and IFN-g is probably responsible for thesynergistic production of IL-12p40 and IL-12p70 induced by bothsignals compared with NiSO4 alone.To investigate the implication of MAPK and NF-kB in the reg-

ulation of IL-12p40 by NiSO4, we used well-described pharmaco-logical inhibitors of these pathways. Our results showed that IL-12p40 production induced by NiSO4 was dependent on p38 MAPKand NF-kB. Ade et al. (10) have recently showed that IL-12p40production induced by NiSO4 was inhibited by pharmacologicalinhibitors of p38 MAPK, JNK, and NF-kB pathways in CD34-DCs. Aiba et al. (11) have also shown an inhibition of IL-12p40production by SB203580, a p38 MAPK inhibitor, in Mo-DCs.Moreover, in murine bone marrow-derived DCs, the production ofIL-12p40 induced by CpG-DNA or PAM3CSK4 depends on p38MAPK and JNK pathways (47).IRF-1, which has been shown to play a crucial role in IL-12p70

production, is known to regulate IL12A gene expression after LPSand IFN-g treatment (20, 23, 28, 48). Gabriele et al. (25) have alsoobserved that IRF-1(2/2) splenic DCs were markedly impaired fortheir ability to produce IL-12 after LPS treatment. Our resultsshowed that both the levels of IL-12p40 and IL-12p70 were signif-icantly inhibitedwhen IRF-1was downregulated byRNA silencing,suggesting a crucial role for IRF-1 in IL-12p40 and IL-12p70

production induced by NiSO4 and IFN-g. The downregulation ofIL12A mRNA expression with IRF-1 siRNA correlated with thestrong inhibition of IL-12p70 production. Interestingly, we showedthat NiSO4 alone was able to induce IRF1mRNA expression at 2 hand a DNA-binding activity of IRF-1 to ISRE after 4 h of treatmentcompared with 90 min for NiSO4 and IFN-g. Downregulation ofIRF-1 using siRNA significantly decreased IL-12p40 productioninduced by NiSO4. These results suggested that IRF-1 was alsoa key factor for IL-12p40 production in DCs in response to NiSO4

alone. This report is the first one describing IRF-1 activation byNiSO4 in human DCs.IRF-1 synthesis in Mo-DCs has been described to be mainly reg-

ulated by STAT-1 in response to type I IFN (49). When Mo-DCswere treated with NiSO4, we found that STAT-1 was phosphory-lated on both serine and tyrosine residues. Using Jak inhibitor I,we found that NiSO4-induced IRF-1 expression was dependent onSTAT-1 tyrosine phosphorylation. In contrast, NiSO4-inducedSTAT-1 phosphorylation on serine 727 was regulated by p38MAPK, but p38 MAPK did not play any role in NiSO4-inducedIRF-1 expression. As it is well described that TLR agonists in-duced type I IFN synthesis leading to STAT-1 phosphorylation,IRF-1 activation, and IL-12 production, we addressed the questionof whether NiSO4-induced STAT-1 phosphorylation was depen-dent on type I IFN production. STAT-1 tyrosine phosphorylationinduced by NiSO4 was not affected by a pretreatment with puro-mycin, suggesting a direct activation of STAT-1 by NiSO4. Simonet al. (50) have shown in fibroblasts that the activation of the Jak-STAT pathway was dependent on the redox status of cells. NiSO4

treatment can modify the redox status of human DCs in vitro, sug-gesting a possible link between activation of the Jak-STAT pathwayby NiSO4 and oxidative stress (51–54). We showed that NiSO4-induced oxidative stress could regulate STAT-1 phosphorylationand IRF-1 expression, suggesting that NiSO4-induced IL-12p40production following NiSO4 treatment could be redox sensitive.Finally, our results describe the specific signaling pathways in-

duced by NiSO4 for IL-12 production and found similarity with theone already described for danger signals. According to all publishedresults on DC maturation induced by NiSO4 (9–11, 31–33), we canspeculate that NiSO4 can be perceived by DCs as a danger signal,thus playing a double role of a hapten and a danger signal.

DisclosuresThe authors have no financial conflicts of interest.

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