of June 13, 2013.
This information is current as
Dendritic Cells Treated with the Chemical
Mechanisms of IL-12 Synthesis by Human
Saadia Kerdine-Römer and Marc Pallardy
Diane Antonios, Philippe Rousseau, Alexandre Larangé,
2010; 185:89-98; Prepublished online 4 June
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The Journal of Immunology
Mechanisms of IL-12 Synthesis by Human Dendritic Cells
Treated with the Chemical Sensitizer NiSO4
Diane Antonios, Philippe Rousseau, Alexandre Larange ´, Saadia Kerdine-Ro ¨mer, and
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) areAPCs 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) itselfor 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 NiSO4stimulation. Addition of IFN-g concomitantly to NiSO4leads to IL-12p70 synthesis. NiSO4treatment 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 NiSO4and 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 NiSO4in Mo-DC and cooperate for IL-12 production.
nickel as the number one allergen in frequency of positive patch test
reactions. Nickel’s allergy is mainly due to jewelry, piercing, and
coins (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 sensitization
phase and an elicitation phase. During the sensitization phase, skin
dendritic 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 groups
have showed that chemical sensitizer treatment can induce phe-
notypic modifications with HLA-DR, CD86, CD40, CD83, and
CCR7 upregulation and IL-12 and IL-8 production (9–12).
IL-12 consists of two chains (p40 and p35) covalently linked to
p35 and p40 subunits. IL-12 is primarily produced by macrophages
The Journal of Immunology, 2010, 185: 89–98.
ickel is a metallic allergen causing contact dermatitis
ican Contact Dermatitis Group have consistently ranked
and DCs mainly in response to danger signals such as TLR agonists
that CD8+cytotoxic T lymphocytes are the main effector cells of
ACD and observed their early recruitment in the skin postchallenge
with chemical sensitizers (6, 17). Injection of IL-12 during the sen-
ACD reaction (18). In vivo studies also demonstrated that 2,4-
dinitrofluorobenzene–mediated ACD was significantly blocked by
anti–IL-12–neutralizing Abs, supporting the conclusion that IL-12
is an important effector in the pathogenesis of ACD (19).
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 in
the expression of human IL12A (23, 28, 29). RNA interference
experiments showed a critical requirement for the NF-kB p50
subunit in the production of IL-12 by human monocyte-derived
DCs (Mo-DCs) after CD40L and IL-1 stimulation (24). Moreover,
IRF-1(2/2)splenic DCs were markedly impaired in their ability to
produce IL-12 after LPS stimulation (25). In vivo, a defective
IL-12 production was also observed in LPS-treated macrophages
obtained from MAP kinase kinase 3-deficient mice (30). All of
these reports suggest that MAPKs, NF-kB, and IRF pathways are
involved in the production of IL-12 by DCs.
NiSO4has been shown to activate both MAPK and NF-kB
pathways in human Mo-DCs or in human DCs differentiated from
CD34+cells (CD34-DC) (9–11, 31, 32). Inhibition of p38 MAPK,
JNK, and ERK abrogated the upregulation of CD86, CD83, and
and CD40 and the production of IL-6, IL-12p40, and IL-8 were
Universud, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ Mixte de
Recherche S 749 and 996, Faculte ´ de Pharmacie, Cha ˆtenay-Malabry, France
Received for publication June 23, 2009. Accepted for publication April 19, 2010.
Faculte de Pharmacie, Universite Paris-Sud 11, 5 Rue Jean-Baptiste Cle ´ment, Cha ˆte-
nay-Malabry 92290, France. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this paper: ACD, allergic contact dermatitis; DC, dendritic
cell; iDC, DC with immature phenotype; IRF, IFN regulatory factor; ISRE, IFN
sequence response element; Mo-DC, monocyte-derived DC; NAC, N-acetylcysteine;
N.S., nonspecific; siRNA, small interfering RNA.
by guest on June 13, 2013
mainly dependent on the NF-kB pathway (10, 11, 31–33). Current
chemical sensitizers could be perceived as a danger signal by
DCs, leading to MAPK and NF-kB activation and DC phenotypic
In this study, we address the question whether NiSO4itself or in
synergy with other signals can induce the secretion of IL-12p70 in
human Mo-DCs. We showed that: 1) nickel induced the produc-
tion of IL-12p40; 2) IL-12p70 synthesis needed the presence of
NiSO4and IFN-g, and both signals were also required for the
expression of the p35 subunit of IL-12; and 3) the production of
IL-12p70 in response to NiSO4and IFN-g was dependent on p38
activated in response to NiSO4in a STAT-1–dependent manner and
played a crucial role in NiSO4-induced IL-12 production.
Materials and Methods
Generation of Mo-DCs
du Sang (Ivry-Sur-Seine, France) by density centrifugation with Ficoll
gradient (Eurobio, Les Ulis, France). Monocytes were isolated from the
mononuclear fraction through magnetic positive selection using MiniMacs
CD14 Abs coated on magnetic beads following the provider’s instructions
106cells/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
IFN-g(1000U/ml),ortheirassociation.A,IL-12p40productionafter NiSO4addition.IL-12p40wasmeasured byELISA.Resultsareexpressedin picograms
permilliliter(valuesare mean6 SDofthree independent experiments).B, IL-12p40productionpostaddition ofNiSO4(500mM) andIFN-g(1000U/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
Mo-DC treatment with NiSO4or NiSO4and IFN-g induced the production of IL-12. At day 5, iDCs were treated for 24 h with either NiSO4,
(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.
IL12A mRNA expression induced by NiSO4and IFN-g in Mo-DCs. At day 5, iDC were treated for 4 or 8 h with NiSO4(500 mM), IFN-g
90IL-12 PRODUCTION BY HUMAN DCs TREATED WITH NISO4
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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 106cells/ml. iDCs were stimulated or not with
different concentrations of NiSO4(Sigma-Aldrich, St. Louis, MO) with or
without IFN-g (1000 U/ml; Abcys) for the indicated time. To study the
involvement of signaling pathways in IL-12 production, Mo-DCs were
pretreated 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 from Merck Chemicals, Darmstadt, Germany). N-acetylcysteine (NAC)
was purchased from Sigma-Aldrich.
Western blot analysis
Afteran adequatetime ofstimulation,cells werewashedwith ice-coldPBS.
Tris (pH 7.4), 137 mM NaCl, 2 mM EDTA (pH 7.4), 1% Triton, 25 mM
b-glycerophophate, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 10% glyc-
50 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 against
the phosphorylated forms of p38 MAPK (Thr180/Tyr182), JNK 1/2 (Thr183/
chemiluminescence (ECL solution, Amersham Biosciences). p38 MAPK or
STAT-1 were used as a loading control and revealed with Abs raised against
p38 MAPK (p38 N20, Santa Cruz Biotechnology) or STAT-1 (Cell Signaling
Quant software. The intensity of the specific band was normalized to the in-
tensity of loading control protein band. Folds represent the ratio of the
normalized intensity of treated cells divided by the normalized intensity of
Preparation of whole cell extract and DNA-affinity protein-
Following treatment with NiSO4(500 mM) or IFN-g (1000 U/ml), cells were
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, 1
mM Na3VO4, 1 mM sodium pyrophosphate, 0.125 mM okadaic acid, 1 mM
1 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
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 NiSO4or NiSO4and 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 NiSO4or NiSO4and 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.
NF-kB, MAPKs, and IRF-1 pathways are activated upon addition of NiSO4or NiSO4and IFN-g. A, MAPK activation after NiSO4or NiSO4
The Journal of Immunology 91
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precipitation assay was carried out as described previously (34). In brief, the
following 59 biotin-labeled single-stranded oligonucleotides were hybridized:
AAG CGA AAT GTT CGC AGT-39 (MWG Biotech, Ebersberg, Germany)
according to the human IFN sequence response element (ISRE) consensus
sequence from the IL-12p35 promoter. Mutated oligonucleotides (59-ACT
GCG AAC ATT TCG CAA ACA TTT TGG-39 and 59-CCA AAA TGT
TTG CGA AAT GTT CGC AGT-39) were used for unspecific control bind-
ing. Also, the following 59-biotin-labeled single-stranded oligonucleotides
were hybridized: 59-TTG AGG GGA CTT TCC CAG G-39 and 59-CCT
GGG AAA GTC CCC TCA A-39 (MWG Biotech) according to the human
NF-kB consensus sequence. Mutated oligonucleotides (59-TTG AGG CGA
CTT TCC CAG G-39 and 59-CCT GGG AAA GTC GCC TCA A-39) were
usedforunspecific control binding.DNA-binding proteinswere isolatedfrom
200 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 eluted
8% SDS-PAGE followed by Western blot analysis using anti–IRF-1 (C20) or
anti-p65 (sc109) mouse mAb (Santa Cruz Biotechnology).
Electroporation of Mo-DCs with small interfering IRF-1
At day 4 of differentiation, Mo-DCs werewashed oncewith serum-free me-
dium and once with PBS. Cells were then resuspended in serum-free
medium at 4 3 107cells/ml; 10 mg nonsilencing control small interfering
IRF-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 of
100 ml with serum-free medium. The siIRF-1 ON-TARGETplus SMART-
pool, which contained four individual duplexes, was chosen because it
enhances siRNA specificity and reduces off-target effects. A total of 4 3
106cells 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 106cells/ml for
24 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 the
manufacturer. RNA was quantified by spectrophotometry. First-strand
cDNAwas synthesized from total RNA extracted in RNAse-freeconditions.
The reaction was performed on 2 mg total RNA with oligo(dT) primers
(MWG Biotech) and 2 U AMV RT (Promega, Charbonnie `res-les-Bains,
France). For semiquantitative PCR, the reaction was performed using 1 U
Taq polymerase (QBiogen, Montre ´al, Canada). The number of cycles and
the hybridization temperature used for the PCR were optimized for all the
genes studied: GAPDH (24 cycles, 60˚C), IRF1 (30 cycles, 54˚C), and
ted with either DMSO, SB203580 (20 mM), SP600125
(20 mM), or Bay 11-7085 (3 mM) and then stimulated
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.
Role of MAPKs and NF-kB in NiSO4-
with 10mg siRNA random (RDM) or siRNA IRF-1. Forthe next 24 h, cells were incubatedwith GM-CSF (550 U/ml) and IL-4(550 U/ml). Cell treatments were
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)
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.
IRF-1 is needed for IL-12p40 and IL-12p70 production in response to NiSO4and IFN-g. At day 4 of differentiation, cells were electroporated
92 IL-12 PRODUCTION BY HUMAN DCs TREATED WITH NISO4
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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 GTC
ATCG-39;and IL12A: 59- ACCACTCCCAAA ACC TGC-39 and59- CCA
GGC AAC TCC CATTAG-39.PCR productswerevisualizedby additionof
ethidium bromide on 1% agarose gel. GAPDH was used to control and
calibrate cDNA synthesis. Fold induction represents the normalized ratio
of treated samples divided by untreated samples.
Real-time PCR was performed using the SYBR Green technology on
ward and reverse primerswereeachdesigned on a different exon ofthe target
gene sequence, eliminating the possibility of amplifying genomic DNA. To
confirm the specificity of the amplification, the PCR product was subjected
was performed in duplicate in a total reaction volume of 10 ml. The reaction
mixture consisted of 5 ml diluted template, 2 ml FastStart DNA MasterPLUS
SYBR Green kit, and 0.5 mM forward and reverses primers. After an 8 s
activation 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˚C
for 5 s, and extension at 72˚C for 9 s. Specific primers were used in the PCR
reaction mixture (forward and reverse primers, respectively): IL12A: 59-ACC
ACT 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 CAC
GCA GCT CAT TGT AG-39. Results were expressed as fold induction
calculated using the standard curve method. b-actin was used to control
and calibrate cDNA synthesis. Fold induction represents the normalized
ratio of treated samples divided by untreated samples.
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 NiSO4or 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 mean 6 SD of three independent
experiments). p # 0.05.
IRF-1 is activated by NiSO4and regulates IL-12p40 production. A, IRF-1 activation in response to NiSO4at 4 and 6 h. Whole-cell extracts
The Journal of Immunology 93
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ylation in response to NiSO4or NiSO4and 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
IRF-1 expression in response to NiSO4is dependent on Jak-induced STAT-1 phosphorylation but not on p38 MAPK. A, STAT-1 phosphor-
94 IL-12 PRODUCTION BY HUMAN DCs TREATED WITH NISO4
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ELISA assay for measuring IL-12p40 and IL-12p70 production
iDCs were treated with NiSO4with or without IFN-g for 24 h. In some
experiments, cells were pretreated for 30 min with MAPK inhibitors or
with 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 were
performed according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN). The OD proportional to the intensity of the color
was measured at 450 nM and 540 nM with a microplate reader (Thermo
Labsystems-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.
All data are represented as mean 6 SD of the mean (based on the entire
population). Differences between the production of IL-12p40 or IL-12p70
in chemical-treated cells compared with respective controls were evaluated
using an unpaired Student t test. p # 0.05 was considered to be statistically
significant. All tests were performed using the software GraphPad Instat 3
(GraphPad, San Diego, CA).
NiSO4triggers the production of IL-12p40 in human Mo-DC
not (control) with NiSO4(300, 400, and 500 mM) for 24 h. NiSO4
induced the production of IL-12p40 in a concentration-dependent
manner (Fig. 1A). IL-12p40 production was significantly aug-
mented at 300 mM when compared with control cells. A significant
described (35). In the same conditions, an average of 100 ng/ml IL-
12p40 was found after LPS addition (25 ng/ml) (data not shown).
IL-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 NiSO4is necessary
for the expression of IL12A mRNA
(1000 U/ml), or their association. IL12A mRNA expression was
measured using real-time PCR (Fig. 2A) and semiquantitative
PCR (Fig. 2B). These times of stimulation were chosen according
to preliminary kinetics experiments measuring IL12A mRNA ex-
pression (data not shown). IL12A mRNA expression was chosen as
a readoutasthere are norobustmethods tomeasure IL-12p35 atthe
protein level. Results showed that very low levels of IL12A mRNA
were detected when NiSO4or IFN-g were added. The presence of
both NiSO4and IFN-g was necessary to induce the expression of
detectable levels of IL12 mRNA. Moreover, results obtained with
inhibitors of the p38 MAPK, JNK, and NF-kB pathways showed
that in addition to IRF-1, other pathways are involved in IL-12 p35
mRNA expression (Supplemental Fig. 1).
NiSO4and IFN-g activate complementary signaling pathways:
MAPKs, NF-kB, and IRF-1
The promoters of p35 and p40 are composed of several DNA
binding sites for AP-1, NF-kB, and IRFs. These pathways have
also been previously described to participate in IL-12 synthesis in
DCs and macrophages (24, 25, 30). We first evaluated the kinetics
of JNK and p38 MAPK activation in iDCs stimulated by NiSO4,
IFN-g, or their combination. Our results showed that NiSO4and
NiSO4with IFN-g induced the phosphorylation of p38 MAPK and
JNK at 30 min. The association of both NiSO4and IFN-g slightly
upregulated p38 MAPK (at 30 min) and JNK phosphorylation (at
30 min and 60 min) compared with NiSO4 alone (Fig. 3A).
Binding of the p65 subunit of NF-kB to specific NF-kB probes
usinga DNA-bindingassay after 1 hof treatment was detected with
both NiSO4and NiSO4with IFN-g (Fig. 3B). We then assessed the
binding of the transcription factor IRF-1 to specific IL-12p35 ISRE
using a DNA-binding assay after 90 min of treatment with NiSO4
and NiSO4with 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 of
IRF-1 was detectable after 90 min of NiSO4treatment alone.
p38 MAPK, NF-kB, and IRF-1 contribute to the production of
To investigate the implication of MAPKs and NF-kB in IL-12p40
synthesis 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. At
day 5, cells were washed and pretreated with SB203580 (20 mM),
SP600125 (20 mM) for 30 min, or Bay 11-7085 (3 mM) for 1 h
and then stimulated for 24 h with NiSO4. These concentrations of
inhibitors were not cytotoxic, and the optimal concentration for
each inhibitor was determined based on preliminary experiments
(data not shown). As JNK and NF-kB inhibitors were diluted in
DMSO, cells were also pretreated for 1 h with DMSO (0.1%) as
an internal control. Results showed that IL-12p40 production in-
duced by NiSO4was mainly dependent on p38 MAPK and NF-kB
pathways; this production was independent of the JNK pathway
To investigate whether IRF-1 was critically required for IL-12
production induced by both signals, we used RNA interference to
inhibit IRF-1 expression in iDCs. The levels of IL-12p40 and IL-
12p70 production were measured by ELISA. RNA interference
experiments revealed that IRF-1 was critical for IL-12p40 and IL-
12p70 production induced by NiSO4associated 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 NiSO4is 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.
The Journal of Immunology 95
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by both signals was nearly completely abolished in the presence
of IRF-1 siRNA (Fig. 5C). Western blotting confirmed that ON-
directed to IRF-1 reduced the IFN-g–induced IRF-1 protein level
by .70% (Fig. 5D).
Nickel induces the expression of IRF-1 which contributes to
We then evaluated the expression of IRF-1 by NiSO4alone in Mo-
DCs. Using a DNA-binding assay, we showed that NiSO4induced
the 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-binding
activity was lower in the presence of NiSO4compared with NiSO4
plus IFN-g. NiSO4was also able to augment the expression of IRF1
NiSO4-induced IL-12p40 production (Fig. 6C).
NiSO4-induced IRF-1 expression depends on STAT-1
contains 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 NiSO4alone 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). Using
a p38 MAPK inhibitor, we showed that p38 MAPK controlled the
phosphorylation of STAT-1 on specific serine residues (Fig. 7B). On
the basis of the observed correlation between IRF-1 expression and
phosphorylation of STAT1, we hypothesized that IRF-1 expression
could depend on STAT-1 activation by NiSO4. iDCs were treated for
2 h with Jak inhibitor I at 0.5 mM or DMSO and then stimulated with
NiSO4for 3 h. The optimal concentration of Jak inhibitor I was de-
termined based on preliminary experiments (data not shown), and we
also controlled that Jak inhibitor I and SB203580 did not affect
STAT-1 serine phosphorylation and STAT-1 tyrosine phosphoryla-
tion, respectively (Fig. 7C). Results showed that the abrogation of
STAT-1 tyrosine phosphorylation completely inhibited the expres-
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 tyrosine
phosphorylation resulted from a direct effect of NiSO4on Jak ac-
but IFN-b was undetectable in the supernatants of Mo-DC treated
for 24 h by NiSO4(data not shown). We then used puromycin,
a well-known protein synthesis inhibitor. Mo-DCs were pretreated
The optimal concentration of puromycin for protein synthesis in-
shown). We showed that STAT-1 tyrosine phosphorylation induced
by NiSO4was not modified by puromycin, suggesting that STAT-1
phosphorylation did not depend on an intermediate product synthe-
sized by Mo-DC in response to NiSO4(Fig. 8A). IRF-1 protein
expression was strongly inhibited as a consequence of puromycin
treatment, showing that puromycin was an effective inhibitor of
protein synthesis in the conditions used (Fig. 8A). Preincubation
of cells with NAC (a precursor of glutathione) is often used to re-
inforce the redox potential of cells. The optimal concentration of
NAC was determined based on preliminary experiments (data not
shown). We showed that pretreatment with NAC inhibited STAT-1
phosphorylation and completely abolished NiSO4-induced IRF-1
expression (Fig. 8B).
DCs are activated by danger signals such as proinflammatory
cytokines, bacterial products, viruses, and chemicals. Sensitizers
have also been demonstrated to induce the expression of markers
related to function and maturation of DCs, the production of pro-
inflammatory cytokines, and the phosphorylation of members of
the MAPK family and NF-kB pathway, thus mimicking the
effects of danger signals (9–11, 31–33).
(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), can
of IL-12 as previously described for danger signals.
We showed that NiSO4induced the production of IL-12p40 in
Mo-DCs in a concentration-dependent manner. We and others have
tyrosine phosphorylation induced by NiSO4was 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 NiSO4is 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.
STAT-1 phosphorylation (Tyr 701) in response to NiSO4is not dependent on de novo protein synthesis and is redox sensitive. A, STAT-1
96IL-12 PRODUCTION BY HUMAN DCs TREATED WITH NISO4
by guest on June 13, 2013
Mo-DCs and CD34-DC (10, 11, 33). However, NiSO4was not able
to induce IL-12p70 production. When IFN-g was added with
NiSO4, detectable amounts of IL-12p70 were then produced by
Mo-DCs. In addition, both signals induced a high production of
IL-12p40, showing an interesting synergistic effect compared with
NiSO4alone. Our results are in accordance with other works that
have previously shown that the production of IL-12p70 in Mo-DCs
required 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 NiSO4treatment in Mo-DCs. O’Leary et al. (42)
showed that NK cells can mediate contact hypersensitivity reaction
in mice with SCID or deficient in RAG2 (Rag22/2), which lack
T cells (but have NKs). NK cells are present in low frequency in
epidermal inflammatory infiltrates in human contact hypersensitiv-
ity (43). NiSO4has also been described to induce the production of
IFN-g in splenic NK cells (44). We suggest that IFN-g produced by
NK cells may participate to the production of IL-12 by NiSO4-
The production of IL-12p40 in activated professional APCs is
generally in great excess over that of the p35 chain, making the
latter molecule a limiting step in the formation of bioactive IL-12
(45). As NiSO4alone induced a high level of IL-12p40 but not of
in response to NiSO4and IFN-g. Our results showed that the
combination of NiSO4and IFN-g induced a high expression of
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). We
then addressed the question of the signaling pathways involved in
the regulation of IL-12p70 after NiSO4 and IFN-g treatment.
NiSO4 has been described to activate MAPKs and NF-kB in
CD34-DCsandMo-DCs(9, 11,32).Our results showed thatNiSO4
provoked p38 MAPK and JNK phosphorylation and the binding of
p65 on specific DNA probes in Mo-DCs. The association of NiSO4
and IFN-g slightly augmented p38 MAPK and JNK activation
compared with NiSO4-treated DCs. Moreover, IFN-g or NiSO4
associated to IFN-g induced a detectable DNA-binding activity of
IRF-1 at 90 min. The early activation of p38 MAPK, JNK, NF-kB,
and IRF-1 by NiSO4and IFN-g is probably responsible for the
synergistic production of IL-12p40 and IL-12p70 induced by both
signals compared with NiSO4alone.
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 NiSO4was dependent on p38 MAPK
and NF-kB. Ade et al. (10) have recently showed that IL-12p40
production induced by NiSO4was inhibited by pharmacological
inhibitors of p38 MAPK, JNK, and NF-kB pathways in CD34-
DCs. Aiba et al. (11) have also shown an inhibition of IL-12p40
production by SB203580, a p38 MAPK inhibitor, in Mo-DCs.
Moreover, in murine bone marrow-derived DCs, the production of
IL-12p40 induced by CpG-DNA or PAM3CSK4depends on p38
MAPK 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 LPS
and IFN-g treatment (20, 23, 28, 48). Gabriele et al. (25) have also
observed that IRF-1(2/2)splenic DCs were markedly impaired for
their ability to produce IL-12 after LPS treatment. Our results
showed that both the levels of IL-12p40 and IL-12p70 were signif-
suggesting a crucial role for IRF-1 in IL-12p40 and IL-12p70
production induced by NiSO4and IFN-g. The downregulation of
IL12A mRNA expression with IRF-1 siRNA correlated with the
strong inhibition of IL-12p70 production. Interestingly, we showed
that NiSO4alone was able to induce IRF1 mRNA expression at 2 h
and a DNA-binding activity of IRF-1 to ISRE after 4 h of treatment
compared with 90 min for NiSO4and IFN-g. Downregulation of
IRF-1 using siRNA significantly decreased IL-12p40 production
induced by NiSO4. These results suggested that IRF-1 was also
a key factor for IL-12p40 production in DCs in response to NiSO4
alone. This report is the first one describing IRF-1 activation by
NiSO4in 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-DCs
were 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 on
STAT-1 tyrosine phosphorylation. In contrast, NiSO4-induced
STAT-1 phosphorylation on serine 727 was regulated by p38
MAPK, but p38 MAPK did not play any role in NiSO4-induced
IRF-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 question
of whether NiSO4-induced STAT-1 phosphorylation was depen-
dent on type I IFN production. STAT-1 tyrosine phosphorylation
induced by NiSO4was not affected by a pretreatment with puro-
mycin, suggesting a direct activation of STAT-1 by NiSO4. Simon
et 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-
gestingapossiblelink betweenactivationoftheJak-STAT pathway
by NiSO4and oxidative stress (51–54). We showed that NiSO4-
induced oxidative stress could regulate STAT-1 phosphorylation
and IRF-1 expression, suggesting that NiSO4-induced IL-12p40
production following NiSO4 treatment could be redox sensitive.
Finally, our results describe the specific signaling pathways in-
duced by NiSO4for IL-12 production and found similarity with the
results on DC maturation induced by NiSO4(9–11, 31–33), we can
speculate that NiSO4can be perceived by DCs as a danger signal,
thus playing a double role of a hapten and a danger signal.
The authors have no financial conflicts of interest.
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