NF-kB Plays a Major Role in the Maturation of Human Dendritic

TOXICOLOGICAL SCIENCES 99(2), 488–501 (2007)

doi:10.1093/toxsci/kfm178

Advance Access publication July 16, 2007

NF-jB Plays a Major Role in the Maturation of Human DendriticCells Induced by NiSO4 but not by DNCB

Nadege Ade,*,† Diane Antonios,*,† Saadia Kerdine-Romer,*,† Fanny Boisleve,*,† Francxoise Rousset,‡and Marc Pallardy*,†,1

*Univ Paris-Sud, †INSERM, 92296 Chatenay-Malabry, France; and ‡L’Oreal Recherche, 92117 Clichy-La Garenne, France

Received May 14, 2007; accepted July 3, 2007

Dendritic cell (DC) activation is a critical event for the

induction of an immune response to haptens. Although signal-

ing pathways such as mitogen-activated protein kinase (MAPK)

family members have been reported to play a role in DC

activation by haptens, little is known about the implication of

the nuclear factor kappa B (NF-kB) pathway. In this work, we

showed that NiSO4 induced the expression of HLA-DR, CD83,

CD86, and CD40 and the production of interleukin (IL)-8, IL-6,

and IL-12p40 in human DCs, whereas 1-chloro-2,4-dinitrobenzene

(DNCB) induced mainly the expression of CD83 and CD86 and

the production of IL-8. NiSO4 but not DNCB was able to activate

the degradation of IkB-a leading to the binding of the p65 subunit

of NF-kB on specific DNA probes. Inhibition of the NF-kB path-

way using BAY 11-7085 prevents both CD40 and HLA-DR expres-

sion and cytokine production induced by NiSO4. However, BAY

11-7085 only partially inhibited CD86 and CD83 expression in-

duced by NiSO4. In addition, p38 MAPK and NF-kB were

independently activated by NiSO4 since SB203580 did not inhibit

NF-kB activation by NiSO4. Interestingly, we also showed that

DNCB inhibited the degradation of IkB-a induced by tumor

necrosis factor-a leading to alteration of CD40, HLA-DR, and

CD83 expression but not of CD86 and CCR7. Extensive

modifications of DC phenotype by NiSO4 in comparison to

DNCB are probably the consequence of NF-kB activation by

NiSO4 but not by DNCB.

Key Words: contact hypersensitivity; NF-jB; hapten;

dendritic cells.

Allergic contact hypersensitivity is a T-cell immune

response resulting from dendritic cell (DC) activation in the

skin by small molecular weight compounds termed haptens.

Once DCs capture the hapten in the skin, they migrate to the

draining lymph nodes and present the hapten associated with

major histocompatibility complex (MHC) Class II molecules

to naıve T lymphocytes leading to the clonal expansion of

hapten-specific T lymphocytes (Bour et al., 1995; Rougier

et al., 2000).

Langerhans cells (LC) isolated from skin explants treated

with haptens have shown upregulation of CD86, CD54, and

HLA-DR expression (Tuschl and Kovac, 2001). However, the

use of LC from skin samples is limited in term of low LC yields

and sometimes spontaneous maturation occurs during the

extraction procedure. The discovery of methods for

in vitro generation of immature DC constituted a useful advance

to study molecular mechanisms involved in DC maturation. DC

can be generated either from monocytes (Sallusto and

Lanzavecchia, 1994) or CD34þ hematopoietic progenitors

obtained from neonatal cord blood (Caux et al., 1996). These

models offered new insight for studying DC response to contact

sensitizers (CS). Upon treatment with CS, DCs show upregu-

lation of markers such as MHC Class II molecules, CD54,

CCR7, CD86, CD83, CD80, and CD40 (Aiba et al., 1997, 2000;

Arrighi et al., 2001; Boisleve et al., 2004; De Smedt et al., 2001,

2005; Staquet et al., 2004; Tuschl et al., 2000). These

phenotypical changes are accompanied by cytokine production

such as interleukin (IL)-6, tumor necrosis factor (TNF)-a, IL-8,

and IL-1b (Aiba et al., 1997; De Smedt et al., 2001, 2005;

Tuschl et al., 2000; Verheyen et al., 2005). In addition, not only

cultured human LC but also in vitro generated human DC could

prime T cells to induce a proliferative response to strong

allergens (Rougier et al., 2000). All these results show that CS

are able to induce the activation of human DC in spite of the lack

of skin environment, suggesting that DCs act as a crucial

‘‘sensor’’ of danger signals including low molecular weight

compounds (Gallucci and Matzinger, 2001).

One purpose of our work is to elucidate if simple chemicals

are able to induce DC maturation in a manner similar to the one

observed with well described danger signals such as lipopoly-

saccharides (LPS) and proinflammatory cytokines (Arrighi

et al., 2001; Boisleve et al., 2004; Verhasselt et al., 1997). To

activate DC, danger signals use several common signal

transduction pathways with major roles for mitogen-activated

protein kinases (MAPKs) and nuclear factor kappa B (NF-jB).

However, signaling involved in CS-induced DC maturation

remains to be clarified. Kuhn et al. (1998) have demonstrated

1 To whom correspondence should be addressed at INSERM UMR-S 749

and Toxicology, Faculte de Pharmacie, 5 rue JB Clement, 92296 Chatenay-

Malabry Cedex, France. Fax: þ33-1-46-83-54-96. E-mail: marc.pallardy@

u-psud.fr.

� The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected]

at Pennsylvania State University on February 23, 2013

http://toxsci.oxfordjournals.org/D

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an increase in tyrosine-phosphorylated proteins after stimula-

tion with strong CS. Several authors have also reported the role

of members of the MAPK family, namely, extracellular

regulated kinases, jun kinases (JNK), and p38 MAPK in DC

maturation induced by CS (Aiba et al., 2003; Arrighi et al.,2001; Boisleve et al., 2004, 2005). Indeed, in monocyte-

derived DC (MoDC), p38 MAPK has been demonstrated to be

critical for the upregulation of CD80, CD83, and to a lesser

extent CD86 but not for CD1a, CD40, and HLA-DR

expression (Arrighi et al., 2001).

In contrast to the MAPK pathway, the involvement of the

NF-jB pathway in response to CS is still poorly understood.

Attar et al. (1998) have shown that mutation of the IjB-b gene

in mice impaired delayed type hypersensitivity in response to

fluorescence isothiocyanate (FITC). Nickel and cobalt, that are

well known sensitizers, have been shown to induce NF-jB

binding activity in human endothelial cells (Goebeler et al.,1995). Aiba et al. (2003) have also shown that NiSO4 can

activate NF-jB in MoDC. However, to date there is no report

evaluating the role of NF-jB in DC maturation upon hapten

exposure.

In unstimulated cells, NF-jB-family proteins exist as

heterodimers and homodimers that are sequestered in the

cytoplasm by members of the IjB family. Upon cell stim-

ulation, activation of IjB kinase (IKK) is observed leading to

phosphorylation of IjB proteins on serine residues. The

phosphorylation of IjB-a induced the dissociation of the

NF-jB/IjB complex and subsequent ubiquitination and pro-

teolysis of IjB-a by the proteasome. Free NF-jB heterodimers

(p65/p50) can then translocate to the nucleus where they bind

to NF-jB enhancer element of target genes. Signals such as

Toll-Like Receptors (TLR) agonists or proinflammatory cyto-

kines are known to activate NF-jB in DC leading to production

or expression of many inflammatory cytokines, chemokines,

immune receptors, and cell surface molecules (Verhasselt

et al., 1999).

NF-jB and MAPK pathways are the two major pathways

involved in DC maturation induced by danger signals such as

TLR agonists. Our hypothesis is that CS are perceived as danger

signals by DC inducing their maturation. In this study, we

investigated the role of NF-jB in phenotypic changes and

cytokine production induced in response to NiSO4 and DNCB.

We choose these two haptens as prototypes of metallic and

organic sensitizers. Our results show that NiSO4 but not DNCB

is able to induce NF-jB activation in CD34-derived DC (CD34-

DC). This observation may explain phenotypical differences

observed when DCs are treated with either NiSO4 or DNCB.

MATERIALS AND METHODS

Preparing DC from human cord blood. Normal human umbilical cord

blood was obtained from Biopredic (Rennes, France) and processed within 24 h.

Cord blood samples were diluted 1:6 in phosphate-buffered saline (PBS). After

Ficoll–Hypaque (Medium for Lymphocyte Isolation, Eurobio, Les Ulis,

France) centrifugation (300 g for 30 min at 20�C), mononuclear cells were

collected and washed three times in PBS supplemented with 2% of heat-

inactivated fetal bovine serum (FBS). Cord blood CD34þ hematopoietic cells

were isolated using MiniMACS separation columns (Miltenyi Biotec, Bergish,

Germany) through magnetic positive selection using the direct CD34

progenitor cell isolation kit (Miltenyi Biotec). After purification, the isolated

cells were 80–95% CD34þ cells.

CD34þ cells were adjusted to the concentration of 3 3 105 cells/ml and

cultured at 37�C in a humidified 5% CO2 atmosphere in RPMI 1640 Glutamax

I medium, 10% FBS, 100 U/ml penicillin, 100 lg/ml streptomycin, 1mM

sodium pyruvate (all from Gibco Invitrogen, Paisley, UK), and supplemented

with 200 U/ml granulocyte-macrophage-colony stimulating factor, (GM-CSF)

(Leucomax 400 a kind gift from Novartis, Rueil-Malmaison, France), 50 U/ml

rhTNF-a (kindly provided by Dr Schmidt, Mainz, Germany) and 50 ng/ml

Flt-3 Ligand (Flt-3L, PeproTech, Tebu, Le Perray-en-Yvelines, France). Cells

were then cultured for 7 days.

Chemical exposure of immature DCs. After a culture period of 7 days,

the cells were washed three times before treatment. Cells (1 3 106 cells/ml)

were exposed to either NiSO4 (Sigma, St Louis, MO) or DNCB (Sigma). After

performing dose–response experiments, optimal concentrations of the two

allergens were selected according to their impact on cell viability and on cell

surface marker determined after 24 h of treatment. For cell viability, the highest

concentration should not lead to a cell viability inferior to 75%. In the case of

DNCB (25lM dissolved in dimethyl sulfoxide [DMSO] at final concentration

of 0,05%), treatment was performed for only 30 min and cells were then

washed three times and reincubated for different periods of time. In the case

of NiSO4 cells were treated according to the different times mentioned for

each experiment. BAY 11-7085 (dissolved in DMSO, Calbiochem, AMD

Biosciences, Darmstadt, Germany) was added 1 h before any treatment.

SB203580 (dissolved in distilled water, Calbiochem) was added 30 min before

treatment with NiSO4.

Immunoblotting. Western blot analysis was performed according to stan-

dard procedure, as previously described (Boisleve et al., 2004). Briefly, cells

were washed with cold PBS 1X. Cell lysates were prepared by resuspending the

cell pellet containing 2 3 106 cells in lysis buffer (20mM Tris pH 7.4, 137mM

NaCl, 2mM ethylenediaminetetraacetic acid (EDTA), pH 7.4, 1% Triton X-

100, 25mM b-glycerophosphate, 1mM Na3VO4, 2mM sodium pyrophosphate,

10% glycerol, 1mM phenylmethanesulphonylfluoride (PMSF), 10 lg/ml

aprotinin, 10 lg/ml leupeptin, and 10 lg/ml pepstatin), incubated on ice for

20 min and followed by centrifugation at 17,600 g at 4�C for 20 min. Protein

concentration was determined using the bicinchoninic acid assay (Sigma).

Equal amount of proteins were subjected to 12.5% sodium dodecyl sulfate–

polyacrylamide electrophoresis (SDS-PAGE). The proteins were transferred

onto polyvinylidene difluoride membranes (Amersham Biosciences, Les Ulis,

France) and the membranes were probed with the rabbit anti-IjB-a poly-

clonal Ab (C-21, Santa Cruz Biotechnology, Santa Cruz, CA) or the rabbit

anti-phospho-p38 MAPK (Thr 180/Tyr 182) monoclonal Ab (3D7, Cell

Signaling Technology, Ozyme, St-Quentin-en-Yveline, France) followed by

goat anti-rabbit polyclonal Ab conjugated to horse radish peroxidase (Cell

Signaling Technology). The membranes were stripped for the primary Abs

and reprobed with Ab raised against total p38 MAPK as a loading control

(p38 N20, Santa Cruz Biotechnology). The immunoblots were visualized by

enhanced chemiluminescence (Amersham Biosciences). Densitometric analysis

of the blots was performed using the Quantity One Software (Bio-Rad

Laboratories, CA).

Flow cytometric analysis. Selected monoclonal antibodies (MoAb) were

used to phenotype the DC population. DCs were analyzed with dual-color flow

cytometry using PE-Cy5–labeled mouse anti-human CD86 MoAb (2331 (FUN-1)),

PE-labeled mouse anti-human CD83 MoAb (HB15e), Allophycocyanin (APC)-

labeled mouse anti-human CD40 MoAb (5C3), FITC-labeled mouse anti-

human HLA-DR (G46-6 (L243)), PE-Cy5-labeled mouse IgG1,j isotype

control, PE-labeled mouse IgG1,j isotype control, APC-labeled mouse IgG1,

ROLE OF NF-jB IN HAPTEN-ACTIVATED DC 489



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j isotype control and FITC-labeled mouse IgG2a,j isotype control provided by

BD Biosciences Pharmingen (San Diego, CA). The PE-labeled mouse anti-

human CCR7 MoAb (150503) and the PE-labeled mouse IgG2a isotype control

was purchased from R&D Systems (Lille, France). The labeling procedure was

as follows: cells were washed in cold PBS (containing 0.5% bovine serum

albumin [BSA]) and stained with antibodies in the dark on ice for 30 min,

washed twice with cold PBS (containing 0.5% BSA), once with PBS without

BSA, and resuspended in PBS. Cells were then analyzed on a FACScalibur cell

analyzer (Becton Dickinson, San Jose, CA) using the CellQuest Software

(Becton Dickinson). Cellular debris were eliminated from the analysis using

a gate on the forward and side scatter. For each sample, 10,000 cells were

collected.

Cytokine measurements. The production of IL-8, IL-6, and IL-12p40 was

measured in supernatants collected 24 h after treatment. Levels of IL-8 and IL-6

in supernatants were measured using the BD CBA system and flow cytometry

using a FACSCalibur flow cytometer (BD Biosciences). Data analysis was

performed using the FCAP Array Software (BD Biosciences).

The production of IL-12p40 was measured using an enzyme-linked

immunosorbent assay (ELISA) kit from R&D Systems. The levels of IL-8,

IL-6, and IL-12p40 were calculated by using a standard curve obtained with

recombinant IL-8 (5–5000 pg/ml), recombinant IL-6 (20–5000 pg/ml), or

recombinant IL-12p40 (31.2–2000 pg/ml). The sensitivity of the methods was

5, 52, and 31 pg/ml for IL-8, IL-6, and IL-12p40, respectively.

Preparation of whole cell extract and DNA-binding assay. Following

treatment with NiSO4 or TNF-a, cells were lysed in Nonidet P-40 (NP-40)

hypertonic lysis buffer. In brief, cell pellets from 8 3 106 cells were

resuspended in buffer containing 0.2% (vol/vol) NP-40, 20% (vol/vol)

Glycerol, 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH,

pH 7.9, 420mM NaCl, 1mM dithiothreitol, 1mM sodium orthovanadate,

1mM sodium pyrophosphate, 0.125lM okadaic acid, 62.5mM EDTA, 40lM

ethyleneglycol-bis(2-aminoethylether)-N,N,N#,N#-tetraacetic acid, 0.5mM

PMSF, 1 lg/ml leupeptin, 1 lg/ml aprotinin, 1 lg/ml pepstatin, and incubated

at 4�C for 30 min. Cellular debris were removed by centrifugation at 4�C,

17,600 g for 20 min. The DNA-binding assay was carried out as described

previously (Wery-Zennaro et al., 1999). In brief, the following 5# biotin-

labeled single-stranded oligonucleotides were hybridized: 5#-TTG AGG GGA

CTT TCC CAG G-3#, and 5#-CCT GGG AAA GTC CCC TCA A-3# (MWG)

according to the human NF-jB promoter sequence. Mutated oligonucleotides

(5#-TTG AGG CGA CTT TCC CAG G-3# and 5#-CCT GGG AAA GTC GCC

TCA A-3#) were used for unspecific control binding. DNA-binding proteins

were isolated from 200 lg of whole cell extracts at 4�C for 90 min with 2 lg

double-stranded 5#-biotinylated oligonucleotides coupled to 50 ll of

streptavidin–agarose beads (Sigma). Complexes were washed in binding buffer

and eluted by boiling in reducing sample buffer. The binding proteins were

separated on 8% SDS-PAGE followed by Western blot analysis using an anti-

p65 mouse monoclonal Antibody (Santa Cruz Biotechnology).

Statistical analysis. Dunett’s multicomparison modification of the Stu-

dent’s t-test was used to assess the statistical significance of experimental data

for continuous variables. Experimental data were considered statistically

different from control at p < 0.05.

RESULTS

NiSO4 and DNCB Induce Phenotypical Changesin CD34-DC

Capacity of chemicals to induce the maturation of CD34-DC

was assessed by determining the expression of key membrane

proteins. CD34-DC generated from CD34þ hematopoietic

progenitors were cultured for 7 days with GM-CSF, TNF-a,

and Flt-3L (Boisleve et al., 2004) and incubated in the presence

of either the water-soluble metal NiSO4 or the DMSO-soluble

organic chemical DNCB. CD83, CD86, HLA-DR, and CD40

expression was measured after 24 h. We first performed dose–

response studies to determine the optimal concentration of

chemical for DC activation. The optimal concentrations could

be defined as a concentration that modify DC phenotype with

cell viability not under 75% viable cells. As seen in Figure 1A,

CD83, CD86, HLA-DR, and CD40 expression was induced

dose dependently after treatment with NiSO4. The maximal

activation level was reached when NiSO4 was tested at 500lM.

In this experiment, cell viability remained unchanged at all the

concentrations tested (data not shown).

DNCB was tested at 12.5, 25, and 50lM. The expression of

CD83, CD86, HLA-DR, and CD40 was increased since

12.5lM and reached a maximum at 25lM of DNCB (Fig.

1B). Although cell viability was not affected by the treatment

with DNCB tested at 12.5lM, it was slightly diminished with

25lM of DNCB (75%) and dramatically affected at 50lM

(47%), and corresponded to a loss of marker expression (Fig.

1B). Considering that a minimum of 75% of cell must be viable

to analyze the expression of cell surface markers and that

25lM seemed to be the concentration that induced the

strongest expression of cell surface markers, we choose

25lM for testing DNCB for all the following experiments.

NiSO4 (500lM) and DNCB (25lM) were then tested on the

same donor. In Figure 2 and Table 1, NiSO4 displayed a major

effect on CD83 and CD86 but also upregulated significantly

HLA-DR and CD40. DNCB also induced the expression of

CD83 and CD86. However, HLA-DR and CD40 were only

slightly and not significantly affected in response to DNCB

(Table 2). Our results showed that NiSO4 had a strong effect on

CD83, CD86, HLA-DR, and CD40 expression, whereas

DNCB affected mainly CD83 and CD86 expression.

Cytokine Production after Treatment with NiSO4 or DNCB

Effect of various chemicals on cytokine production by

CD34-DC was assessed using the same protocol as for

phenotype analysis. Results showed that untreated DC did

not produce IL-6 nor IL-12p40 whereas a basal level of IL-8

was detected (Table 3). Upon NiSO4 addition, DC produced

large amounts of IL-6 and IL-12p40 and IL-8 secretion was

significantly increased (p < 0.05). However, in the presence of

DNCB, only IL-8 production was augmented (p < 0.01).

These results clearly showed that NiSO4 was able to induce the

production of cytokines involved in DC function, whereas

DNCB impact was limited to IL-8.

NiSO4 but not DNCB Activates the NF-jB Pathway

Our observations indicated that NiSO4 but not DNCB was

able to upregulate protein expression such as surface CD40 or

cytokine production such as IL-12p40, two events known to

depend strongly on the NF-jB pathway (Laderach et al.,

490 ADE ET AL.



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FIG. 1. Dose-dependent effect of NiSO4 and DNCB on the phenotype of DCs. At day 7, CD34-DC were treated with various concentrations of NiSO4 for 24 h

(A) (300, 400, 500lM) or DNCB (12.5, 25, 50lM). For DNCB treatment, cells were incubated during 30 min with DNCB, washed thrice, and reincubated in

supplemented medium for 24 h. CD83, CD86, HLA-DR, and CD40 surface expression was then measured using flow cytometry. Numbers represent the

percentage of positive cells after hapten treatment. Results were obtained from two different donors (Fig. 1A is from one donor and Fig. 1B is from a second donor)

and are representative experiments out of two for NiSO4 and out of four for DNCB.




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2003). We therefore evaluated whether NiSO4 or DNCB was

able to activate the NF-jB pathway. Levels of IjB-a were

determined by Western blotting on NiSO4- or DNCB-

stimulated CD34-DC. Results showed that IjB-a was degraded

15 min of NiSO4-treatment with a maximal degradation at 1 h

(Fig. 3A). IjB-a level was then gradually restored reaching

control level at 4 h (Fig. 3C). In contrast, no IjB-a degradation

was observed in CD34-DC stimulated with DNCB at any time

points measured (Figs. 3B and 3D). Although IjB-adegradation was different between NiSO4- and DNCB-

stimulated cells, phosphorylation of p38 MAPK was detectable

in response to both treatments demonstrating that DC

FIG. 2. Phenotypic changes of DCs from the same donor following chemical allergen exposure. At day 7, CD34-DC from the same donor were treated with

NiSO4 for 24 h (500lM). For DNCB treatment, cells were incubated during 30 min with DNCB (25lM), washed thrice, and reincubated in supplemented medium

for 24 h. CD83, CD86, HLA-DR, and CD40 surface expression was measured using flow cytometry. Numbers represent the percentage of positive cells after

hapten treatment; numbers in brackets represent the percentage of positive cells of nonstimulated DC. Results of a representative experiment out of five.

492 ADE ET AL.



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responded to DNCB treatment. Results from Figures 3A–D are

from different donors that may explain the variations in fold

observed between experiments.

All these results demonstrated that in contrast to NiSO4,

DNCB failed to induce the degradation of IjB-a although the

kinetic of p38 MAPK phosphorylation was similar in response

to both chemicals. All these results were confirmed using

a DNA-binding assay using specific NF-jB probes. This

experiment showed the binding of the p65 subunit after NiSO4-

treatment but not following DNCB treatment (Fig. 3E).

Inhibition of NF-jB Activation Alters NiSO4-InducedDC Maturation

Using BAY 11-7085, a newly described inhibitor of the

NF-jB pathway (Richter et al., 2001), we investigated the

consequences of NF-jB inhibition on NiSO4-stimulated

CD34-DC phenotype. We first determined the optimal

subtoxic dose of BAY 11-7085. As shown in Figure 4,

concentrations of 3 and 5lM of BAY 11-7085 significantly

abolished the degradation of IjB-a induced by NiSO4. We

chose to use 3lM BAY 11-7085 since this concentration had

no effect on cell viability and on the phenotype of control

untreated cells (Fig. 5), whereas 5lM altered significantly cell

viability (data not shown).

As reported in Figure 5 and Table 4, BAY 11-7085 nearly

abolished CD40 and HLA-DR increase generated by NiSO4

and partially inhibited CD83 and CD86 upregulation (45 and

51%, respectively, when reported to the percentage of positive

cells). In parallel, the effect of BAY 11-7085 was evaluated

on cytokine production induced by NiSO4. BAY 11-7085

alone augmented the basal level of IL-8, whereas basal levels

of IL-6 and IL-12p40 were not modified (Table 5).

Interestingly, the production of all these three cytokines

induced by NiSO4 was completely abolished by the treatment

with BAY 11-7085 at 3lM (Table 5). These results suggest

that the NF-jB pathway is strongly involved in cytokine

production as well as in HLA-DR and CD40 expression and

to a lesser extent in CD83 and CD86 expression in CD34-DC

stimulated by NiSO4.

Effect of Inhibition of p38 MAPK on the IjB-aDegradation Induced by NiSO4

As previously described, both NiSO4 and DNCB induced

the phosphorylation of p38 MAPK (Boisleve et al., 2004). To

evaluate a potential relationship between the p38 MAPK and

the NF-jB pathways upon addition of NiSO4, CD34-DC were

pretreated for 30 min with SB203580, a well-described

pharmacological inhibitor of p38 MAPK, and further stimu-

lated with NiSO4 (500lM) for 1 h. As shown in Figure 6,

SB203580 had no effect on IjB-a degradation induced by

NiSO4. This result showed that p38 MAPK did not regulate

NiSO4-induced NF-jB activation suggesting that the two

pathways are activated independently.

DNCB Inhibits NF-jB Activation by TNF-a

To further elucidate the absence of NF-jB activation in

response to DNCB, we investigated the responsiveness of DC

after TNF-a addition in the presence of DNCB. CD34-DC

TABLE 1

Expression of Surface Markers on NiSO4- or DNCB-Treated DC

Cell surface

marker Control NiSO4

NiSO4-fold

induction

DMSO

0.05% DNCB

DNCB-fold

induction

CD83 1.0 4.7 4.7 1.5 3.2 2.1

CD86 3.5 29.3 8.4 3.8 15.9 4.2

HLA-DR 14.6 52.9 3.6 32.4 42.2 1.3

CD40 1.6 3.6 2.3 1.9 2.6 1.4

Note. Results are the mean fluorescence intensity of the cytometry study

shown in Figure 2 corrected for the background intensity of isotype control

antibodies (cMFI). Fold induction has been calculated as follows: cMFI of

hapten-treated cells/cMFI of vehicle-treated cells.

TABLE 2

Expression of Surface Markers on NiSO4- and DNCB-Treated

DC: Mean of Three Independent Experiments

Control NiSO4 DMSO DNCB

cMFI CD83 0.8 ± 0.1 6.0 ± 4.1* 1.5 ± 0.2 3.2 ± 0.7**

CD86 3.1 ± 0.4 20.1 ± 6.2** 3.7 ± 0.1 15.0 ± 1.2***

HLA-DR 17.7 ± 5.4 39.6 ± 17.4* 20.9 ± 15.2 29.4 ± 20.1

CD40 1.6 ± 0.3 3.2 ± 1.3* 1.8 ± 0.3 3.7 ± 2.4

% Positive

cells

CD83 4 ± 1 59 ± 7*** 14 ± 3 29 ± 7*

CD86 23 ± 2 77 ± 7*** 34 ± 6 64 ± 6**

HLA-DR 75 ± 19 85 ± 13 78 ± 22 84 ± 19

CD40 15 ± 3 38 ± 8** 23 ± 9 26 ± 10

Note. Results are the mean ± SD of three independent experiments. The

mean fluorescence intensity was corrected for the background intensity of

isotype control antibodies (cMFI). ***p < 0.001, **p < 0.01, *p < 0.05.

TABLE 3

Production of Cytokines by DC after Chemical Allergen

Exposure

ng/ml Untreated DMSO 0.05% NiSO4 DNCB

IL-8 0.41 ± 0.07 1.00 ± 0.07 175.77 ± 121.50 14.3 ± 1.78

IL-6 < 0.05 < 0.05 9.80 ± 2.94 < 0.05

IL-12p40 < 0.03 < 0.03 4.23 ± 2.56 < 0.03

Note. At day 7, DC were treated with NiSO4 (500lM) or with DNCB

(25lM). IL-8 and IL-6 productions were determined in supernatants using flow

cytometry as described in ‘‘Materials and Methods’’ section. IL-12p40

production was measured by ELISA. Cytokine production was measured

after 24 h of treatment following the same protocol as for phenotype evaluation.

Results represent the mean ± SD of three independent experiments. Results

were expressed in ng/ml.




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were treated for 30 min with DNCB (25lM) and then further

incubated for 15 min with 500 U/ml TNF-a, a concentration

known to activate NF-jB (see Fig. 3). As shown in Figure 7,

treatment with DNCB of CD34-DC before TNF-a addition

drastically decreased the IjB degradation observed in response

to TNF-a alone. On the other hand, TNF-a–induced phosphory-

lation of p38 MAPK was not affected by DNCB treatment.

Since DNCB inhibited NF-jB activation, we asked the

question about the impact of DNCB treatment on TNF-a–

induced phenotypical changes of DC (Fig. 8). Pretreatment of

CD34-DC with DNCB, totally abolished the effect of TNF-aon HLA-DR expression (Table 6). A partial inhibition of TNF-

a effect was observed with CD83 and CD40 in the presence of

DNCB whereas TNF-a and DNCB displayed additive effects

on CD86 and CCR7 expression.

FIG. 3. The NF-jB pathway is activated by NiSO4 but not by DNCB. At day 7, CD34-DC were treated for different time period with NiSO4 (500lM). For

DNCB treatment, cells were incubated during 30 min with DNCB (25lM), washed thrice for 30 min, and reincubated for various time period. Cells were treated

with TNF-a (500 U/ml) for 15 min as a positive control for NF-jB activation in Figures 3B and 3D. The level of IjB-a and the phosphorylation level of p38

MAPK were evaluated by Western blotting as described in ‘‘Materials and Methods’’ section. Total p38 MAPK level was determined as a loading control. *Fold

induction was determined as follows: normalized intensity of specific band in chemical-treated cells at different time points divided by the normalized intensity of

untreated cells. (A, B) IjB-a Western blot after short-time treatments of DC with haptens. (C, D) IjB-a Western blot after long-time treatment with haptens.

Figures (A), (B), (C), and (D) are from different experiments. (E) p65 DNA-binding assay after treatment with NiSO4 or DNCB. At day 7, CD34þ derived DCs

were treated with NiSO4 (500lM) or DNCB (25lM) for 1 h. Cells were treated with TNF-a (500 U/ml) for 15 min as a positive control for NF-jB activation.

Whole cell extracts were prepared and incubated with biotinylated NF-jB promoter probes (NF-jB) or with mutated NF-jB probes (NF-jB-mut) and streptavidin–

agarose beads. p65 captured in whole cell extracts was detected by Western blotting. Results shown on (A), (B), (C), and (D) are from a representative experiment

out of three from different donors.

FIG. 4. BAY 11-7085 inhibits IjB-a degradation. CD34-DC were pre-

treated for 1 h with several concentrations of BAY 11-7085 and then incubated

in the presence or in the absence of NiSO4 (500lM) for 1 h. Cells were then

lysed and probed for IjB-a level by Western blotting. Total p38 MAPK level

was determined as a loading control. *Fold induction was determined as

follows: normalized intensity of specific band in NiSO4-treated cells divided by

the normalized intensity of nontreated cells. Results of a representative

experiment out of two.

494 ADE ET AL.



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All these results suggested that the NF-jB pathway is

important for CD83, CD40, and HLA-DR expression on DC

stimulated with TNF-a. However, CD86 and CCR7 expression

induced by TNF-a seemed to be regulated in a NF-jB–

independent manner.

DISCUSSION

The key properties required for successful skin sensitization

are the following: ability of the chemical to gain access to the

viable epidermis, potential to form conjugates with protein to

FIG. 5. Inhibition of NF-jB activation altered surface marker expression on NiSO4-treated DC. CD34-DC were pretreated with BAY 11-7085 (3lM) for 1 h

and further stimulated or not with NiSO4 (500lM). Twenty-four hours after stimulation, cells were analyzed using flow cytometry for the expression of CD83,

CD86, HLA-DR, and CD40. Results of a representative experiment out of four.




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create an immunogen, dermal trauma, and expression of skin

cytokines involved in the induction of cutaneous immune

response and recognition by T lymphocytes of the allergen

complex displayed by immunostimulatory DCs.

DCs are activated by danger signals such as proinflammatory

cytokines, bacterial products, and viruses. Haptens have also

been demonstrated to induce the expression of markers related

to function and maturation of DC, the production of proin-

flammatory cytokines, and the phosphorylation of members of

the MAPK family. However, it is not clear if haptens are also

able to activate the NF-jB pathway like danger signals.

In this study, NiSO4 upregulated the expression of CD83,

CD86, HLA-DR, and CD40 in CD34-DC. However, the effect

of DNCB was limited to the expression of CD83 and CD86.

Using MoDC, Staquet et al. (2004) have also observed that, in

contrast to NiSO4, DNCB did not induce the expression of

CD40. Several authors have also reported that DNCB

compared to NiSO4 is a weak inducer of HLA-DR expression

(Aiba et al., 1997; De Smedt et al., 2005; Staquet et al., 2004).

Moreover, Aiba et al. (2000) showed that MoDC differentiated

in the presence of transforming growth factor-b responded to

DNCB by increasing the expression of CD86 and CD83 but

not of HLA-DR.

In parallel to phenotypical changes, NiSO4 showed a good

capacity to induce the production of cytokines such as IL-6,

IL-8, and IL-12p40 although DNCB only increased the produc-

tion of IL-8. Several authors have also reported discrepancies

in cytokine production between Nickel and DNCB. Aiba et al.(2003) have reported that NiCl2 induced the production of IL-8

FIG. 6. p38 MAPK does not regulate NiSO4-induced NF-jB activation.

CD34-DC were pretreated for 30 min with or without 20lM SB203580 and

further treated in the presence or in the absence of 500lM NiSO4 for 1 h. Cells

were then lysed and probed for IjB-a level by Western blotting. Total p38

MAPK level was determined as a loading control. *Fold induction was

determined as follows: normalized intensity of specific band in treated cells

divided by the normalized intensity of in nontreated cells. Blots are

representative of results obtained in three independent experiments.

FIG. 7. DNCB inhibits TNF-a–induced IjB-a degradation. CD34-DC

were pretreated in the presence or in the absence of DNCB (25lM) for 30 min

and then further stimulated or not with TNF-a (500 U/ml) for 15 min. Cells

were then lysed and probed for IjB-a level and phosphorylation of p38 MAPK

by Western blotting. Total p38 MAPK level was determined as a loading

control. *Fold induction was determined as follows: normalized intensity of

specific band in treated cells divided by the normalized intensity of non-

treated cells. Blots are representative of results obtained in three independent

experiments.

TABLE 4

Inhibition of the NF-kB Pathway Altered the Phenotype of

NiSO4-Stimultated CD34-DC

Control NiSO4

BAY

11-7085

NiSO4 þBAY 11-7085

%

Inhibition

cMFI CD83 1.0 13.1 1.2 4.3 74

CD86 2.3 16.7 2.7 8.1 62

HLA-DR 60.3 105.2 53.1 63.9 76

CD40 1.5 5.4 1.9 2.2 92

% Positive

cells

CD83 3.8 70.1 5.6 42.2 45

CD86 24.6 80.7 35.2 62.9 51

HLA-DR 95.6 98.1 96.1 95.2 0

CD40 11.0 43 10.3 12.7 92

Note. The results showed in this table are the cMFI and the percentage of

positive cells (% (þ) cells) from the cytometry study shown in Figure 5. Cells

were not treated (A) or treated with either NiSO4 (B), BAY 11-7085 (C), or

NiSO4 with BAY 11-7085 (D). % Inhibition was calculated using the following

formula: 100 � [(D � C)/(B � A)] 3 100.

TABLE 5

Inhibition of the NF-kB Pathway Altered Cytokine Production

Induced by NiSO4

ng/ml Untreated

BAY

11-7085 NiSO4

NiSO4 þBAY 11-7085

IL-8 0.3 42.2 112.8 46.3

IL-6 < 0.05 < 0.05 8.5 < 0.05

IL-12p40 < 0.03 < 0.03 1.8 < 0.03

Note. CD34-derived DCs were pretreated in the presence or in the absence of

BAY 11-7085 (3lM) for 1 h and further stimulated or not with NiSO4

(500lM). IL-8 and IL-6 productions were determined in supernatants using

flow cytometry as described in ‘‘Materials and Methods’’ section. IL-12p40

production was measured by ELISA. IL-8, IL-6, and IL-12p40 productions

were measured after 24 h of treatment. Results of a representative experiment

out of four. Results were expressed in ng/ml.

496 ADE ET AL.



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FIG. 8. Effect of DNCB treatment on TNF-a-induced DC maturation. CD34-DC were pretreated in the presence or in the absence of DNCB (25lM) for 30

min, washed thrice, and further incubated for 24 h with TNF-a (500 U/ml). Cells were then analyzed using flow cytometry for the expression of CD83, CD86,

HLA-DR, CD40, and CCR7. Results of a representative experiment out of three.




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and IL-12p40 in MoDC, whereas DNCB only affected the

production of IL-8. Furthermore, IL-6 has been also shown to

be produced by MoDC in response to NiSO4 but not to DNCB

(Tuschl et al., 2000). Recently, Verheyen et al. (2005) showed

similar results in CD34-DC based on the messenger RNA

expression of IL-8 and IL-6 in response to DNCB and NiCl2;

NiCl2 induced both IL-8 and IL-6 gene expressions, whereas

DNCB induced only IL-8 gene expression.

Based on these results, we hypothesized that differences

observed between NiSO4 and DNCB in DC maturation could

involve the NF-jB pathway. IL-8, IL-12p40, CD83, and CD86

promoters contain NF-jB consensus sequences that are

required for their expression (Berchtold et al. 2002; Hoffmann

et al., 2002; Li et al., 2000; Yoshimoto et al., 1996).

Furthermore, using adenoviral transfert or inhibitors of IjB-aor RNAi for p50, NF-jB has been shown to be essential for

HLA-DR, CD86, CD83, and CD40 expression as well as

cytokine production such as IL-6, IL-12, and IL-8 in MoDC in

response to LPS, TNF-a, and CD40L (Laderach et al., 2003;

O’Sullivan and Thomas, 2002; Yoshimura et al., 2001). Our

results showed that NiSO4 was able to induce the degradation

of IjB-a as well as the binding of the p65 subunit on specific

DNA probes. This effect was maximal after 1 h of stimulation

with NiSO4. However, DNCB neither induced the degradation

of IjB-a nor the binding of p65 on specific DNA probes. Aiba

et al. (2003) have also observed in MoDC that nickel but not

DNCB activated the phosphorylation of IjB-a with compara-

ble kinetics. These results suggested that the difference in

NF-jB activation observed between NiSO4 and DNCB

correlated to the differences in phenotype and in cytokine

production observed with these two chemicals.

To test whether activation of NF-jB was involved in hapten-

induced DC maturation, we used the inhibitor BAY 11-7085,

known to block the phosphorylation of IjB-a and to prevent its

degradation (Richter et al., 2001). Experiments were conducted

only on NiSO4-treated cells since DNCB does not activate NF-

jB in our model. We found that BAY 11-7085 effectively

blocked NiSO4-induced IjB-a degradation in our model. In

DC activated by NiSO4 and treated with BAY 11-7085,

increased expressions of HLA-DR and CD40 as well as IL-8,

IL-6, and IL-12p40 production were nearly completely

abrogated, but CD83 and CD86 potentialization was only

partially inhibited. Using pyrrolidinedithiocarbamate, another

inhibitor of NF-jB, Aiba et al. (2003) showed in MoDC

a slight inhibition of CD86 and no effect on CD83 expression

induced by nickel. To further elucidate the signaling pathway

involved in NF-jB activation, we studied the relationship

between p38 MAPK activation and NF-jB. It has previously

been reported that stimulators of p38 MAPK, such as TNF-acan augment the activity of the NF-jB pathway via cross-talk

between MKK6 and IKKb (Craig et al., 2000). Our results

showed that inhibition of p38 MAPK did not alter the

degradation of IjB-a induced by NiSO4 suggesting that p38

MAPK did not participate to this NF-jB activation by NiSO4

in CD34-DC. This result is in agreement with recent papers

reporting that p38 MAPK and NF-jB pathways are in-

dependent (Cloutier et al., in press).

Our next question was to understand why DNCB did not

activate the NF-jB pathway, whereas p38 MAPK was fully

activated by this molecule. In the present study, we showed

that pretreatment with DNCB blocked the degradation of IjB-ainduced by TNF-a suggesting that DNCB acts as a potent

inhibitor of the NF-jB pathway. Brennan and O’Neill (1998)

have reported that DCNB, a structural analog of DNCB known

to bind on thiol functions, blocked the activation of NF-jB by

TNF-a in Jurkat cells. Other authors have also reported that

curcumin and nordihydroguaiaritic acid, two CS, inhibited the

degradation of IjB-a as we observed with DNCB (Brennan

and O’Neill, 1998). DNCB has also been previously described

to be an irreversible inhibitor of thioredoxin reductase, leading

to a strong activation of nicotinamide adenine dinucleotide

phosphate, reduced oxidase, production of reactive oxygen

intermediates (ROI), and suppression of NF-jB–dependent

transcription in HELA cells (Arner et al., 1995; Nordberg

et al., 1998). As previously described for high concentrations

of H2O2, strong production of ROI by DNCB could alter

NF-jB activation by inhibiting nuclear translocation of NF-jB

through preservation of cytoplasmic IjB-a levels (Korn et al.,2001).

Alteration of the NF-jB pathway induced by DNCB resulted

in a decrease in CD83, CD40, and HLA-DR expressions in DC

treated with TNF-a. These results confirmed that expressions

of CD83, CD40, and HLA-DR were dependent on NF-jB.

Interestingly, CD86 expression induced by TNF-a was further

augmented in the presence of DNCB. CD86 was the marker the

less affected by treatment with BAY 11-7085 in NiSO4-treated

DC suggesting that CD86 expression is less dependent on

NF-jB. We have previously described that CCR7 expression

was upregulated following exposure of CD34-DC to DNCB,

TABLE 6

Effect of DNCB on TNF-a–Induced Surface Marker Expression

Control

(DMSO) DNCB TNF-aDNCB þ

TNF-a

cMFI CD83 1.3 7.3 16.5 11.0

CD86 4.0 6.3 6.8 12.8

HLA-DR 87.5 103.8 221.8 91.6

CD40 1.5 1.7 3.1 2.6

CCR7 2.0 3.5 2.8 3.9

% Positive cells CD83 8.9 25.8 63.3 41.3

CD86 29.9 55.2 56.8 65.9

HLA-DR 98.4 98.5 98.3 98.1

CD40 19.4 16.6 49.3 33.4

CCR7 13.0 30.7 20.7 36.5

Note. The results showed in this table are the isotype control antibodies

(cMFI) and the percentage of positive cells from the cytometry study shown in

Figure 8.

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and that an autocrine loop involving TNF-a was necessary

(Boisleve et al., 2004). Here we showed that DNCB did not

affect CCR7 expression induced by TNF-a confirming that

TNF-a plays a role in CCR7 expression in a NF-jB–

independent manner.

One important question remains if the effects we observed

could reflect that DNCB is a potent allergen and irritant,

whereas nickel is a moderate allergen and irritant. In human

DCs, results obtained with irritants such as SDS or benzalko-

nium chloride did not show any phenotype modification or

activation of signaling pathways such as MAPK or NF-jB

(Aiba et al., 1997; De Smedt et al., 2001; Mizuashi et al.,2005). In a recent paper, Toebak et al. (2005) have shown in

MoDC that allergens were able to increase IL-8 secretion,

whereas irritants rather decrease it. These results may suggest

that DCs can distinguish a chemical irritant from a chemical

sensitizer leading to the activation of specific signaling

pathways. However, the strong irritant potential of DNCB is

due to its capacity of inducing oxidative stress leading to the

activation of JNK and p38 MAPK and this event is directly

linked to DC phenotype alteration (Aiba et al., 2003; Boisleve

et al., 2004). Indeed, Aiba et al. have shown in MoDC that

DNCB is more potent than NiCl2 for reducing the GSH/GSSG

ratio (Mizuashi et al., 2005) and the authors suggested

a correlation with p38 MAPK activation. On the other hand,

Handley et al. (2005) have shown that H2O2 generates a strong

oxidative stress without altering DC phenotype. All these

results suggest that sensitizers probably need to produce some

oxidative stress to activate DC but this property is not sufficient

to induce full DC maturation.

In summary, our results suggested a causal relationship

between activation of NF-jB by chemical sensitizers and

alteration of DC phenotype. NiSO4 activates NF-jB and

inducing extensive modifications of DC phenotype. However,

DNCB induced the expression of CD86, CD83, CCR7, and the

production of IL-8 without activating NF-jB suggesting that

other signaling pathways such as MAPK are mainly involved.

The signaling pathways involved upon NiSO4 or DNCB

exposure provide an explanation for the phenotypical differ-

ences observed in DC maturation induced by these two CS.

Finally, a figure showing the signaling pathways already

identified as being involved in the activation of DC by CS, as

well as their possible interactions, is proposed in Figure 9.

FUNDING

Agence Francxaise de Securite Sanitaire des Produits de Sante

(AFSSAPS).

ACKNOWLEDGMENTS

The authors would like to acknowledge Silvia Martinozzi-Teissier,

Alexandre Larange, and Jacques Bertoglio for their technical advices and

helpful suggestions.

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NF-kB Plays a Major Role in the Maturation of Human Dendritic