Toxicological Sciences

ToxSci Advance Access originally published online on July 16, 2007
Toxicological Sciences 2007 99(2):488-501; doi:10.1093/toxsci/kfm178

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NF-B Plays a Major Role in the Maturation of Human Dendritic Cells Induced by NiSO₄ but not by DNCB

Nadège Ade^*,, Diane Antonios^*,, Saadia Kerdine-Romer^*,, Fanny Boisleve^*,, Françoise Rousset and Marc Pallardy^*,,1

^* Univ Paris-Sud INSERM, 92296 Châtenay-Malabry, France L'Oréal Recherche, 92117 Clichy-La Garenne, France

¹ To whom correspondence should be addressed at INSERM UMR-S 749 and Toxicology, Faculté de Pharmacie, 5 rue JB Clément, 92296 Châtenay-Malabry Cedex, France. Fax: +33-1-46-83-54-96. E-mail: marc.pallardy{at}u-psud.fr.

Received May 14, 2007; accepted July 3, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
Dendritic cell (DC) activation is a critical event for the induction of an immune response to haptens. Although signaling 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-B) 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 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 IB- leading to the binding of the p65 subunit of NF-B on specific DNA probes. Inhibition of the NF-B pathway using BAY 11-7085 prevents both CD40 and HLA-DR expression and cytokine production induced by NiSO₄. However, BAY 11-7085 only partially inhibited CD86 and CD83 expression induced by NiSO₄. In addition, p38 MAPK and NF-B were independently activated by NiSO₄ since SB203580 did not inhibit NF-B activation by NiSO₄. Interestingly, we also showed that DNCB inhibited the degradation of IB- induced by tumor necrosis factor- 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-B activation by NiSO₄ but not by DNCB.

Key Words: contact hypersensitivity; NF-B; hapten; dendritic cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
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 naïve 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 CD34⁺ 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 upregulation 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)-, IL-8, and IL-1ß (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., 1998). 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 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-B). However, signaling involved in CS-induced DC maturation remains to be clarified. Kühn et al. (1998) have demonstrated an increase in tyrosine-phosphorylated proteins after stimulation 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-B pathway in response to CS is still poorly understood. Attar et al. (1998) have shown that mutation of the IB-ß 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-B binding activity in human endothelial cells (Goebeler et al., 1995). Aiba et al. (2003) have also shown that NiSO₄ can activate NF-B in MoDC. However, to date there is no report evaluating the role of NF-B in DC maturation upon hapten exposure.

In unstimulated cells, NF-B-family proteins exist as heterodimers and homodimers that are sequestered in the cytoplasm by members of the IB family. Upon cell stimulation, activation of IB kinase (IKK) is observed leading to phosphorylation of IB proteins on serine residues. The phosphorylation of IB- induced the dissociation of the NF-B/IB complex and subsequent ubiquitination and proteolysis of IB- by the proteasome. Free NF-B heterodimers (p65/p50) can then translocate to the nucleus where they bind to NF-B enhancer element of target genes. Signals such as TLR agonists or proinflammatory cytokines are known to activate NF-B in DC leading to production or expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface molecules (Verhasselt et al., 1999).

NF-B 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-B 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-B 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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
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 (2000 rpm 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 CD34⁺ 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% CD34⁺ cells.

CD34⁺ cells were adjusted to the concentration of 3 x 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 µg/ml streptomycin, 1mM sodium pyruvate (all from Gibco Invitrogen, Paisley, UK), and supplemented with 200 U/ml GM-CSF (Leucomax 400 a kind gift from Novartis, Rueil-Malmaison, France), 50 U/ml rhTNF- (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 x 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 (25µM 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 standard 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 x 10⁶ cells in lysis buffer (20mM Tris pH 7.4, 137mM NaCl, 2mM ethylenediaminetetraacetic acid (EDTA), pH 7.4, 1% Triton X-100, 25mM ß-glycerophosphate, 1mM Na₃VO₄, 2mM sodium pyrophosphate, 10% glycerol, 1mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin), incubated on ice for 20 min and followed by centrifugation at 15,000 rpm 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-IB- polyclonal 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), APC-labeled mouse anti-human CD40 MoAb (5C3), FITC-labeled mouse anti-human HLA-DR (G46-6 (L243)), PE-Cy5-labeled mouse IgG1, isotype control, PE-labeled mouse IgG1, isotype control, APC-labeled mouse IgG1, isotype control and FITC-labeled mouse IgG2a, 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-, cells were lysed in Nonidet P-40 (NP-40) hypertonic lysis buffer. In brief, cell pellets from 8 x 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, 0.125µM okadaic acid, 62.5mM EDTA, 40µM ethyleneglycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, 0.5mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and incubated at 4°C for 30 min. Cellular debris were removed by centrifugation at 4°C, 15,000 rpm 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-B 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 µg of whole cell extracts at 4°C for 90 min with 2 µg double-stranded 5'-biotinylated oligonucleotides coupled to 50 µl 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 Student'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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
NiSO₄ and DNCB Induce Phenotypical Changes in 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 CD34⁺ hematopoietic progenitors were cultured for 7 days with GM-CSF, TNF-, 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 500µM. In this experiment, cell viability remained unchanged at all the concentrations tested (data not shown).

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   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, 500µM) or DNCB (12.5, 25, 50µM). 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.

 
DNCB was tested at 12.5, 25, and 50µM. The expression of CD83, CD86, HLA-DR, and CD40 was increased since 12.5µM and reached a maximum at 25µM of DNCB (Fig. 1B). Although cell viability was not affected by the treatment with DNCB tested at 12.5µM, it was slightly diminished with 25µM of DNCB (75%) and dramatically affected at 50µM (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 25µM seemed to be the concentration that induced the strongest expression of cell surface markers, we choose 25µM for testing DNCB for all the following experiments.

NiSO₄ (500µM) and DNCB (25µM) 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.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   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 (500µM). For DNCB treatment, cells were incubated during 30 min with DNCB (25µM), 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.

 

View this table: [in this window] [in a new window]   TABLE 1 Expression of Surface Markers on NiSO4- or DNCB-Treated DC

 

View this table: [in this window] [in a new window]   TABLE 2 Expression of Surface Markers on NiSO4- and DNCB-Treated DC: Mean of Three Independent Experiments

 
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.

View this table: [in this window] [in a new window]   TABLE 3 Production of Cytokines by DC after Chemical Allergen Exposure

 
NiSO₄ but not DNCB Activates the NF-B 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-B pathway (Laderach et al., 2003). We therefore evaluated whether NiSO₄ or DNCB was able to activate the NF-B pathway. Levels of IB- were determined by Western blotting on NiSO₄- or DNCB-stimulated CD34-DC. Results showed that IB- was degraded 15 min of NiSO₄-treatment with a maximal degradation at 1 h (Fig. 3A). IB- level was then gradually restored reaching control level at 4 h (Fig. 3C). In contrast, no IB- degradation was observed in CD34-DC stimulated with DNCB at any time points measured (Figs. 3B and 3D). Although IB- degradation was different between NiSO₄- and DNCB-stimulated cells, phosphorylation of p38 MAPK was detectable in response to both treatments demonstrating that DC responded to DNCB treatment. Results from Figures 3A–D are from different donors that may explain the variations in fold observed between experiments.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 3. The NF-B pathway is activated by NiSO4 but not by DNCB. At day 7, CD34-DC were treated for different time period with NiSO4 (500µM). For DNCB treatment, cells were incubated during 30 min with DNCB (25µM), washed thrice for 30 min, and reincubated for various time period. Cells were treated with TNF- (500 U/ml) for 15 min as a positive control for NF-B activation in Figures 3B and 3D. The level of IB- 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) IB- Western blot after short-time treatments of DC with haptens. (C, D) IB- 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, CD34+ derived DCs were treated with NiSO4 (500µM) or DNCB (25µM) for 1 h. Cells were treated with TNF- (500 U/ml) for 15 min as a positive control for NF-B activation. Whole cell extracts were prepared and incubated with biotinylated NF-B promoter probes (NF-B) or with mutated NF-B probes (NF-B-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.

 
All these results demonstrated that in contrast to NiSO₄, DNCB failed to induce the degradation of IB- 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-B probes. This experiment showed the binding of the p65 subunit after NiSO₄-treatment but not following DNCB treatment (Fig. 3E).

Inhibition of NF-B Activation Alters NiSO₄-Induced DC Maturation
Using BAY 11-7085, a newly described inhibitor of the NF-B pathway (Richter et al., 2001), we investigated the consequences of NF-B 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 5µM of BAY 11-7085 significantly abolished the degradation of IB- induced by NiSO₄. We chose to use 3µM BAY 11-7085 since this concentration had no effect on cell viability and on the phenotype of control untreated cells (Fig. 5), whereas 5µM altered significantly cell viability (data not shown).

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 4. BAY 11-7085 inhibits IB- degradation. CD34-DC were pretreated for 1 h with several concentrations of BAY 11-7085 and then incubated in the presence or in the absence of NiSO4 (500µM) for 1 h. Cells were then lysed and probed for IB- 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.

 

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 5. Inhibition of NF-B activation altered surface marker expression on NiSO4-treated DC. CD34-DC were pretreated with BAY 11-7085 (3µM) for 1 h and further stimulated or not with NiSO4 (500µM). 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.

 
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 3µM (Table 5). These results suggest that the NF-B 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.

View this table: [in this window] [in a new window]   TABLE 4 Inhibition of the NF-B Pathway Altered the Phenotype of NiSO4-Stimultated CD34-DC

 

View this table: [in this window] [in a new window]   TABLE 5 Inhibition of the NF-B Pathway Altered Cytokine Production Induced by NiSO4

 
Effect of Inhibition of p38 MAPK on the IB- Degradation 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-B pathways upon addition of NiSO₄, CD34-DC were pretreated for 30 min with SB203580, a well-described pharmacological inhibitor of p38 MAPK, and further stimulated with NiSO₄ (500µM) for 1 h. As shown in Figure 6, SB203580 had no effect on IB- degradation induced by NiSO₄. This result showed that p38 MAPK did not regulate NiSO₄-induced NF-B activation suggesting that the two pathways are activated independently.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 6. p38 MAPK does not regulate NiSO4-induced NF-B activation. CD34-DC were pretreated for 30 min with or without 20µM SB203580 and further treated in the presence or in the absence of 500µM NiSO4 for 1 h. Cells were then lysed and probed for IB- 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.

 
DNCB Inhibits NF-B Activation by TNF-
To further elucidate the absence of NF-B activation in response to DNCB, we investigated the responsiveness of DC after TNF- addition in the presence of DNCB. CD34-DC were treated for 30 min with DNCB (25µM) and then further incubated for 15 min with 500 U/ml TNF-, a concentration known to activate NF-B (see Fig. 3). As shown in Figure 7, treatment with DNCB of CD34-DC before TNF- addition drastically decreased the IB degradation observed in response to TNF- alone. On the other hand, TNF-–induced phosphorylation of p38 MAPK was not affected by DNCB treatment.

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 7. DNCB inhibits TNF-–induced IB- degradation. CD34-DC were pretreated in the presence or in the absence of DNCB (25µM) for 30 min and then further stimulated or not with TNF- (500 U/ml) for 15 min. Cells were then lysed and probed for IB- 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 nontreated cells. Blots are representative of results obtained in three independent experiments.

 
Since DNCB inhibited NF-B activation, we asked the question about the impact of DNCB treatment on TNF-–induced phenotypical changes of DC (Fig. 8). Pretreatment of CD34-DC with DNCB, totally abolished the effect of TNF- on HLA-DR expression (Table 6). A partial inhibition of TNF- effect was observed with CD83 and CD40 in the presence of DNCB whereas TNF- and DNCB displayed additive effects on CD86 and CCR7 expression.

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 8. Effect of DNCB treatment on TNF--induced DC maturation. CD34-DC were pretreated in the presence or in the absence of DNCB (25µM) for 30 min, washed thrice, and further incubated for 24 h with TNF- (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.

 

View this table: [in this window] [in a new window]   TABLE 6 Effect of DNCB on TNF-–Induced Surface Marker Expression

 
All these results suggested that the NF-B pathway is important for CD83, CD40, and HLA-DR expression on DC stimulated with TNF-. However, CD86 and CCR7 expression induced by TNF- seemed to be regulated in a NF-B–independent manner.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
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 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 proinflammatory cytokines, and the phosphorylation of members of the MAPK family. However, it is not clear if haptens are also able to activate the NF-B 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-ß 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 production 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 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-B pathway. IL-8, IL-12p40, CD83, and CD86 promoters contain NF-B 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 IB- or RNAi for p50, NF-B 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-, 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 IB- 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 IB- 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 IB- with comparable kinetics. These results suggested that the difference in NF-B 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-B was involved in hapten-induced DC maturation, we used the inhibitor BAY 11-7085, known to block the phosphorylation of IB- and to prevent its degradation (Richter et al., 2001). Experiments were conducted only on NiSO₄-treated cells since DNCB does not activate NF-B in our model. We found that BAY 11-7085 effectively blocked NiSO₄-induced IB- 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-B, 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-B activation, we studied the relationship between p38 MAPK activation and NF-B. It has previously been reported that stimulators of p38 MAPK, such as TNF- can augment the activity of the NF-B pathway via cross-talk between MKK6 and IKKß (Craig et al., 2000). Our results showed that inhibition of p38 MAPK did not alter the degradation of IB- induced by NiSO₄ suggesting that p38 MAPK did not participate to this NF-B activation by NiSO₄ in CD34-DC. This result is in agreement with recent papers reporting that p38 MAPK and NF-B pathways are independent (Cloutier et al., in press).

Our next question was to understand why DNCB did not activate the NF-B pathway, whereas p38 MAPK was fully activated by this molecule. In the present study, we showed that pretreatment with DNCB blocked the degradation of IB- induced by TNF- suggesting that DNCB acts as a potent inhibitor of the NF-B 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-B by TNF- in Jurkat cells. Other authors have also reported that curcumin and nordihydroguaiaritic acid, two CS, inhibited the degradation of IB- 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-B–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-B activation by inhibiting nuclear translocation of NF-B through preservation of cytoplasmic IB- levels (Korn et al., 2001).

Alteration of the NF-B pathway induced by DNCB resulted in a decrease in CD83, CD40, and HLA-DR expressions in DC treated with TNF-. These results confirmed that expressions of CD83, CD40, and HLA-DR were dependent on NF-B. Interestingly, CD86 expression induced by TNF- 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-B. We have previously described that CCR7 expression was upregulated following exposure of CD34-DC to DNCB, and that an autocrine loop involving TNF- was necessary (Boisleve et al., 2004). Here we showed that DNCB did not affect CCR7 expression induced by TNF- confirming that TNF- plays a role in CCR7 expression in a NF-B–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 benzalkonium chloride did not show any phenotype modification or activation of signaling pathways such as MAPK or NF-B (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-B by chemical sensitizers and alteration of DC phenotype. NiSO₄ activates NF-B 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-B 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 differences 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.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 9. Diagram representation of the pathways involved in CD34-DC activation following NiSO4 or DNCB treatment (from this work and from Boisleve et al., 2004, 2005). NiSO4 induces the expression of CD83, CD86, HLA-DR, and CD40 and the production of IL-8, IL-6, and IL-12p40 through the activation of both NF-B and MAPK (p38 MAPK, JNK). CCR7 is induced by NiSO4 with MAPK playing an important role. The role of NF-B is not known in this case. DNCB treatment upregulates CD86, CD83, and CCR7 and increases the secretion of IL-8. An autocrine loop of TNF- dependent on MAPK activation is necessary for the expression of CCR7 and CD83.

 

	FUNDING

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FUNDING REFERENCES

 
Agence Française de Sécurité Sanitaire des Produits de Santé (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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