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The Journal of Immunology, 2001, 166: 3837-3845.
Copyright © 2001 by The American Association of Immunologists

A Critical Role for p38 Mitogen-Activated Protein Kinase in the Maturation of Human Blood-Derived Dendritic Cells Induced by Lipopolysaccharide, TNF-{alpha}, and Contact Sensitizers

Jean-François Arrighi1,*,{dagger}, Michela Rebsamen{ddagger}, Françoise Rousset, Vincent Kindler§ and Conrad Hauser*,{dagger}

* Department of Dermatology, DHURVD, {dagger} Division of Immunology and Allergy, {ddagger} Division of Endocrinology and Diabetology, and § Division of Hematology, University Hospital, Geneva, Switzerland; and L’Oréal Advanced Recherche, Aulnay-sous-bois, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the involvement of mitogen-activated protein kinases (MAPKs) in the maturation of CD83- dendritic cells (DC) derived from human blood monocytes. Maturating agents such as LPS and TNF-{alpha} induced the phosphorylation of members of the three families of MAPK (extracellular signal-regulated kinase l/2, p46/54 c-Jun N-terminal kinase, and p38 MAPK). SB203580, an inhibitor of the p38 MAPK, but not the extracellular signal-regulated kinase l/2 pathway blocker PD98059, inhibited the up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and the DC maturation marker CD83 induced by LPS and TNF-{alpha}. In addition, SB203580 inhibited the enhancement of the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake induced by LPS and TNF-{alpha}. Likewise, SB203580 partially prevented the up-regulation of IL-1{alpha}, IL-1{beta}, IL-lRa, and TNF-{alpha} mRNA upon stimulation with LPS and TNF-{alpha}, as well as the release of bioactive TNF-{alpha} induced by LPS. DC maturation induced by the contact sensitizers 2,4-dinitrofluorobenzene and NiSO4, as seen by the up-regulation of CD80, CD86, and CD83, was also coupled to the phosphorylation of p38 MAPK, and was inhibited by SB203580. The irritants SDS and benzalkonium chloride that do not induce DC maturation did not trigger p38 MAPK phosphorylation. Together, these data indicate that phosphorylation of p38 MAPK is critical for the maturation of immature DC. These results also suggest that p38 MAPK phosphorylation in DC may become useful for the identification of potential skin contact sensitizers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)2 are unique professional APCs whose primary function is to capture, process, and present Ags to unprimed T cells (1, 2, 3). Immature DC reside in nonlymphoid tissues where they can capture and process Ags. Thereafter, DC migrate to the T cell areas of lymphoid organs where they lose Ag-processing activity and mature to become potent immunostimulatory cells (3). Fully mature DC show a high surface expression of MHC class II and costimulatory molecules (CD40, CD80, and CD86) but a decreased capacity to internalize Ags (4, 5, 6). Up-regulation of CD83, a specific marker for DC maturation, also occurs (7). Many inflammatory signals can induce DC maturation. LPS has been shown to fully activate DC both in vitro and in vivo (8, 9, 10). Cytokines such as TNF-{alpha} and IL-1{beta} are also potent DC maturation factors (8, 11). CD40 ligation of DC induces their maturation as well as the expression of IL-12 (5, 12). In addition, immunostimulatory oligodeoxynucleotides containing the CpG motif (13), viral double-stranded RNA (14), and contact sensitizers (15) also trigger maturation of DC.

The establishment of in vitro culture systems, allowing the induction of human DC from various precursors, offers the possibility to study molecular mechanisms involved in DC maturation (5, 16, 17). However, signal transduction pathways involved in DC maturation are still poorly characterized. A few reports describe mitogen-activated protein kinase (MAPK) activation in the process of human DC maturation (18, 19, 20), but the respective role of the three major MAPK pathways has not been elucidated yet. There are at least three distinct MAPK signaling pathways in mammals, including the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and the p38 MAPK (21). These kinases are activated by phosphorylation on both threonine and tyrosine residues in a regulatory TXY loop present in all MAPKs. This phosphorylation is performed by distinct upstream dual-specific MAPK kinases. Activated MAPKs are then translocated to the nucleus where they phosphorylate their respective substrates on serine or threonine residues. The ERK pathway responds mainly to mitogens and growth factors, and regulates cell proliferation and differentiation (22). The JNK and p38 MAPK cascades are referred to as stress-activated MAPK pathways, because they are strongly activated by stress-inducing agonists (23, 24). The availability of ERK- and p38-specific inhibitory drugs allowed rapid progress in the study of the role of these pathways in various biological processes (25, 26). In particular, the p38 MAPK-specific inhibitor, the pyridinyl imidazole compound SB203580, has facilitated extensive investigation of the role of the p38 MAPK pathway in inflammatory responses. This pathway is activated by proinflammatory cytokines (e.g., IL-1{beta} and TNF-{alpha}), LPS, and various environmental stresses (heat, osmotic stress, UV irradiation)(27, 28, 29, 30, 31). Moreover, p38 MAPK has been involved in the regulation of the expression of many proinflammatory genes (26, 32, 33, 34).

In this study, we investigated the effects of LPS and TNF-{alpha} on the phosphorylation state of members of the three major MAPK pathways in human monocyte-derived immature DC. To obtain further insight into the roles of ERK and p38 MAPK pathways in TNF-{alpha}- or LPS-induced DC maturation, we used their specific inhibitors, PD98059 and SB203580, respectively. Finally, we tested the effects of irritants and contact sensitizers, the latter being inducers of DC maturation (15), on the phosphorylation of MAPK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DC generation from buffy coats

Buffy coats of healthy donors were obtained according to institutional guidelines. PBMC were prepared by density centrifugation using Ficoll-Paque (Pharmacia, Uppsala, Sweden). PBMC were seeded (50 x 106 cells) in 100-mm tissue culture plates (Nunc, Roskilde, Denmark) in 10 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all supplied by Life Technologies, Paisley, U.K.), and incubated for 1 h at 37°C in a humidified 5% CO2 atmosphere. Nonadherent cells were removed by extensive washing, and the remaining adherent cells were recovered by scraping. In some instances, residual contaminating cells were depleted using anti-CD3 and anti-CD19 mIgG-coated M450 Dynabeads obtained from Dynal (Oslo, Norway). The mean purity of purified CD14+ cells was greater than 90%. Cells were subsequently cultured in six-well plates (3 x 106 cells/well) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME (Life Technologies), 103 U/ml GM-CSF, and 103 U/ml IL-4 (Stratagen, Hanover, Germany). Cells were refed with 1 ml of fresh medium containing 3 x 103 U of GM-CSF and 3 x 103 U of IL-4 on days 2, 4, and 6. To get mature DC, the nonadherent cells (immature DC) were harvested on day 7 and stimulated with 100 U/ml recombinant human TNF-{alpha} (a gift of Glaxo Wellcome, Geneva, Switzerland), 10 ng/ml LPS (Escherichia coli, strain 055:B5; Difco, Detroit, MI), or with different chemical reagents as described below. For all of the following assays, 5 x 105 immature DC were cultured in 1 ml of medium containing 103 U/ml GM-CSF and 103 U/ml IL-4 in 24-well plates.

Chemical reagents

Nickel sulfate (NiSO4), 2,4-dinitrofluorobenzene (DNFB), benzalkonium chloride (BC), SDS, and DMSO were obtained from Sigma (Buchs, Switzerland). SB203580 and PD98059 were purchased from Alexis (Läufelfingen, Switzerland). Treatment of immature DC with SB203580 or PD98059 before stimulation was performed for 30 and 75 min, respectively. Because SB203580 and PD98059 were dissolved in DMSO, a 0.1% (v/v) concentration of DMSO was used as a negative control where indicated.

Abs and immunoreactants

Specific FITC-labeled mAbs. Anti-CD1a (mIgG1, clone OKT6) was obtained from Ortho (Raritan, NJ); anti-HLA-DR (mIgG2a, clone L243) from Becton Dickinson (Mountain View, CA); anti-CD40 (mIgG1, clone 5C3) and anti-CD80 (mIgM, clone BB1) from PharMingen (San Diego, CA); anti-CD19 (mIgG1, clone HD37) and anti-CD45RO (mIgG2a, clone UCHL1) from Dako (Glostrup, Denmark); and anti-CD45RA (mIgG1, clone 2H4) from Immunotech (Marseille, France).

Specific PE-labeled mAbs. Anti-CD14 (mIgG2b, clone MoP9) and anti-CD3 (mIgG1, clone SK7) were obtained from Becton Dickinson; anti-CD1a (mIgG1, clone BL6) and anti-CD83 (mIgG2b, clone HB15a) from Immunotech; and anti-CD86 (mIgG2b, clone IT2.2) from PharMingen.

Monoclonal isotype controls. FITC- and PE-labeled mIgG1 was obtained from Dako; FITC-labeled mIgG2a from Ancell (Bayport, MN), FITC-labeled mIgM from PharMingen, and PE-labeled mIgG2b from Immunotech.

Other reagents. Mouse polyclonal IgG reagent grade was obtained from Sigma, FITC-labeled dextran (molecular mass 40 kDa) from Molecular Probes (Eugene, OR), rabbit polyclonal IgG anti-TNF-{alpha} from Serotec (Oxford, U.K.), and control rabbit polyclonal IgG from Sigma.

Flow cytometric analysis

Cultured DC were washed, resuspended at 0.5–1 x 105 cells in 50 µl of cold PBS containing 0.1% sodium azide, 10 mg/ml BSA, and 200 µg/ml mouse IgG (Sigma), and incubated for 15 min on ice. Subsequent staining with labeled mAb or appropriate isotypic controls was performed for 30 min on ice. Cells were then washed and resuspended in 300 µl of cold PBS containing 0.1% sodium azide, 10 mg/ml BSA, and 10 µg/ml 7-amino-actinomycin D (Sigma). Stained cells were analyzed for three-color immunofluorescence with a FACSCalibur cell analyzer (Becton Dickinson). Cellular debris were eliminated from the analysis using a gate on forward and side scatter. A life gate was set using 7-amino-actinomycin D, which allows discrimination between viable, necrotic, and apoptotic cells (35). A minimum of 104 cells were analyzed for each sample. Results were processed using CellQuest software (Becton Dickinson).

FITC-dextran uptake

Cells were incubated with FITC-dextran (0.1 mg/ml) either at 4°C (internalization control) or at 37°C for 1 h. Then, cells were washed twice with a cold buffer consisting of PBS containing 0.1% sodium azide and 10 mg/ml BSA and analyzed with a FACSCalibur cell analyzer as described above.

Allogenic MLR

Allogenic T cells were obtained from peripheral blood of healthy adults after Ficoll-Paque gradient (Pharmacia), adherence to plastic for 1 h at 37°C, and passage over a nylon wool column (Biotest, Dreieich, Germany). Cells recovered after purification were on the mean greater than 90% CD3+, and were distributed at 5 x 104 cells per well into round-bottom 96-well microplates (Nunc). Cells were incubated for 5 days in the presence of graded numbers of irradiated DC stimulators (3000 rad, 137Cs source) in 200 µl of medium containing 10% FCS. T cell proliferation was assessed after 8–14 h incorporation of [3H]thymidine (1 µCi/well; New England Nuclear, Boston, MA) by using standard procedures. The results are expressed as the mean of quadruplicate cultures. The SEM of the results never exceeded 15%.

Western blot analysis of cellular MAPKs

Immature DC were exposed to various agonists for the indicated periods of time. Thereafter, cells were washed twice with cold PBS and incubated with 100 µl of lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 40 mM {beta}-glycerophosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 µM Na3VO4, 0.3 mM leupeptin, 1 µM pepstatin A, 1 mM PMSF, and 100 nM okadaic acid, pH 7.4). The homogenates were centrifuged at 14,000 rpm for 10 min at 4°C. Cell lysates (10–20 µg) were electrophoresed on 12% SDS-PAGE gels, and transferred to nitrocellulose membranes for Western blot analysis. Briefly, nitrocellulose membranes were incubated in a blocking buffer (50 mM Tris-HCl, 200 mM NaCl, 0.2% Tween 20, and 5% nonfat dried milk) for 1 h at room temperature, then incubated for 2 h with Abs raised against phosphorylated p42/44 ERK, p38 MAPK (New England Biolabs, Beverly, MA), or p46/54 JNK (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were washed and incubated for 1 h with HRP-labeled goat anti-rabbit (CovalAb, Oullins, France) or rabbit anti-goat (Sigma) Abs. Immunoreactive bands were visualized by ECL detection reagent (Amersham, Zürich, Switzerland) and quantified by scanning densitometry (Molecular Dynamics, Sunnyvale, CA). Abs to total p38 MAPK or ERK (Santa Cruz Biotechnology) were used to analyze p38 MAPK and ERK expression as loading controls.

Bioassay for TNF quantification

A subclone (WEHI 1.14) of the TNF-sensitive WEHI 164 clone 13 was used as previously described (36) with the following modifications. Fifty microliters of graded dilutions from culture supernatant were added to 50 µl (2 x 104) WEHI 1.14 cells in flat-bottom 96-well plates (Nunc) in duplicates and incubated for 24 h at 37°C. Twenty microliters of MTS (333 µg/ml; Promega, Madison, WI) and 1 µl of the electron-coupling reagent PMS (25 µM; Sigma) were subsequently added to each well. After 2 h of incubation, the resulting intensity of the coloration was measured at 490 nm in a Thermomax microplate reader (Molecular Devices, Menlo Park, CA) and analyzed using Softmax software from the same company. Recombinant human TNF-{alpha} (provided by J.-M. Dayer, University of Geneva, Geneva, Switzerland) was used as a standard. The sensitivity of this assay was 0.1 pg/ml. This assay does not distinguish TNF-{alpha} from lymphotoxin. Thus, the resulting activity is referred to as TNF. SB203580 had no effect on the TNF bioassay.

Multiprobe RNase protection assay

Total RNA was extracted from DC using TRIzol reagent (Life Technologies). Multiprobe template sets hCK2 and hCK3 were purchased from PharMingen. hCK2 and hCK3 DNA templates were used to synthesize the [{alpha}32P]UTP (800 Ci/mmol, 5 mCi/ml; Hartmann, Braunschweig, Germany)-labeled riboprobes in the presence of rNTPs using T7 RNA polymerase (Promega). Hybridization with 1 µg of each target RNA was performed overnight at 56°C followed by digestion with RNases A and T1 at 37°C for 30 min. The samples were then treated by a proteinase K/SDS mixture, extracted with phenol/chloroform/isoamylalcohol (25:24:1), and precipitated in the presence of carrier E. coli MRE 600 tRNA (Boehringer Mannheim, Mannheim, Germany). The samples were loaded onto an acrylamide-urea sequencing gel next to the labeled control probes treated or not with RNases A and T1, and run at 50 W for 1.5 h. The gel was absorbed to filter paper, dried under vacuum, and exposed on Kodak X-AR film with intensifying screens at -80°C.

The relative amounts of proinflammatory cytokine encoding mRNA were measured by scanning densitometry (Molecular Dynamics) on subexposed autoradiograms and further normalized using both housekeeping gene values (L32 and GAPDH).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} and LPS induce phosphorylation of members of the three families of MAPK in immature DC

To investigate the intracellular MAPK signaling pathways involved in DC maturation by two potent DC maturation factors, TNF-{alpha} and LPS, we performed Western blot analysis to detect the phosphorylated forms of ERK1/2, p46/54 JNK, and p38 MAPK. Stimulation of immature DC with 100 U/ml TNF-{alpha} or 10 ng/ml LPS induced the phosphorylation of all of the MAPK tested (Fig. 1Go, A and B). Upon TNF-{alpha} stimulation, maximal MAPK phosphorylation was observed between 5 and 10 min, whereas the LPS-induced peak response occurred after 15–30 min. Densitometric quantification revealed a 3.5 ± 1.7-fold and a 4.8 ± 1.2-fold induction of p38 MAPK phosphorylation by TNF-{alpha} and LPS, respectively (mean ± SD of eight experiments for TNF-{alpha}, and of three experiments for LPS). In addition, TNF-{alpha} induced the phosphorylation of ERK1 (2.9 ± 0.4), ERK2 (2.5 ± 0.3), p46 JNK (1.6 ± 0.2), and p54 JNK (2.8 ± 0.2) (n = 3). LPS induced also the phosphorylation of ERK1 (3.8 ± 0.4), ERK2 (4.2 ± 0.6), p46 JNK (1.7 ± 0.3), and p54 JNK (4.8 ± 0.9) (n = 3). To exclude any LPS contamination in the TNF-{alpha} preparation, we used neutralizing polyclonal Ab raised against TNF-{alpha}. This Ab effectively blocked TNF-{alpha}-induced p38 MAPK phosphorylation (Fig. 1GoC) as well as TNF-{alpha}-stimulated ERK1/2 and p46/54 JNK phosphorylation (data not shown). Thus, both TNF-{alpha} and LPS induce early phosphorylation of ERK1/2, p46/54 JNK, and p38 MAPK in immature DC.



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  		FIGURE 1. Detection of phosphorylated MAPK induced by TNF-{alpha} and LPS in immature DC. A, Immature monocyte-derived DC were stimulated with TNF-{alpha} (100 U/ml) for the indicated periods of time or left untreated (c). Cell lysates were subjected to Western blotting. Membranes were incubated with Abs raised against the phosphorylated form of p38 MAPK (p38-P), of p42/44 MAPK (ERK1/2-P), or of p46/54 JNK (p46/54-P). Membranes were then reprobed with an Ab against total p38 MAPK (total p38) for loading control. B, Monocyte-derived DC were stimulated or not (c) with LPS (10 ng/ml) as described in A. C, Monocyte-derived DC were stimulated or not (c) with TNF-{alpha} (100 U/ml) for 5 min. TNF-{alpha} was preincubated for 30 min on ice with 20 µg of either neutralizing rabbit polyclonal Ab against TNF-{alpha} or control rabbit polyclonal Ab before DC stimulation. Cell lysates were then subjected to Western blot analysis. Membranes were incubated with Abs raised against the phosphorylated form of p38 MAPK (p38-P), and reprobed with an Ab against total p38 MAPK (total p38) for loading control. Blots are representative of results obtained in three separate experiments (A and B) or in two independent experiments (C).

 
Inhibition of p38 MAPK activity prevents the up-regulation of costimulatory molecules induced by TNF-{alpha} and LPS

To study the roles of the MAPK signaling pathways in DC maturation, we treated immature DC with PD98059, an inhibitor of the ERK1/2 pathway (25), and SB203580, a specific blocker of p38 MAPK (26), and analyzed the surface Ag expression profile of DC after 48 h of culture in the presence of 100 U/ml TNF-{alpha} or 10 ng/ml LPS. The up-regulation of CD1a, CD40, CD80, CD86, HLA-DR, and CD83 induced by TNF-{alpha} or LPS was prevented by preincubating immature DC with SB203580 (Fig. 2Go, A–C and Table IGo). The mean fluorescence intensity (MFI) rather than the percentage of HLA-DR expression was affected by SB203580 treatment. SB203580 inhibition on CD1a, CD80, CD86, and CD83 expression was concentration-dependent, and a significant inhibitory effect was still observed using doses between 1 and 10 µM. Indeed, in these experimental conditions both the percentage of positive cells and the MFI values were lower compared with nontreated cells (Fig. 2Go and Table IGo). The expression of CD40 was inhibited using high doses of SB203580 (10–50 µM). In contrast, a high concentration of PD98059 (50 µM) did not prevent up-regulation of CD86 and HLA-DR expression induced by TNF-{alpha} or LPS (Fig. 3GoA). The expression of other surface molecules including CD1a, CD40, CD80, and CD83 were not modified by PD98059 treatment (data not shown). To confirm that PD98059 was active, cell lysates of DC pretreated or not with 50 µM PD98059 and subsequently stimulated with TNF-{alpha} or LPS were subjected to Western blot analysis to detect phosphorylated forms of ERK1/2 and p38 MAPK. Both TNF-{alpha} and LPS-induced ERK1/2 phosphorylation were abrogated by 50 µM PD98059, whereas the level of activated p38 MAPK was not affected (Fig. 3GoB). Conversely, 50 µM SB203580 neither affected the phosphorylation levels of ERK1/2 nor of p46/54 JNK induced after TNF-{alpha} or LPS stimulation (data not shown).



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  		FIGURE 2. Effect of SB203580 on TNF-{alpha}- and LPS-induced up-regulation of cell surface molecules in monocyte-derived DC. Peripheral blood monocytes were differentiated into immature DC for 7 days with GM-CSF and IL-4. Cells were left untreated in the presence of GM-CSF and IL-4 for 48 h (A). At day 7, DC were pretreated or not for 30 min with increasing concentrations of SB203580 as indicated and then stimulated with 100 U/ml TNF-{alpha} (B) or 10 ng/ml LPS (C) for 48 h. Two-dimensional plots show the surface expression profile of DC for the indicated markers evaluated by flow cytometric analysis as described in Materials and Methods. These results are representative of four independent experiments.

 

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  		Table I. Effects of SB203580 on the phenotype of matured human DC1

 


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  		FIGURE 3. Effect of PD98059 on TNF-{alpha}- and LPS-induced DC maturation. A, Peripheral blood monocytes were differentiated into immature DC for 7 days with GM-CSF and IL-4. At day 7, DC were pretreated for 75 min with 50 µM PD98059 and then stimulated with 100 U/ml TNF-{alpha} or 10 ng/ml LPS for 48 h. Two-dimensional plots show the surface expression profile of DC for CD86 and HLA-DR evaluated by flow cytometric analysis as described in Materials and Methods. Numbers in each quadrant represent cell percentages. One representative experiment of three is shown. B, Immature monocyte-derived DC were stimulated or not (medium) with 100 U/ml TNF-{alpha} for 5 min or with 10 ng/ml LPS for 15 min. Before stimulation, cells were treated or not (control) for 75 min with 0.1% DMSO or 50 µM PD98059. Cell lysates were subjected to Western blotting. Membranes were incubated with Abs raised against the phosphorylated forms of p42/44 MAPK (ERK1/2-P) and of p38 MAPK (p38-P). Membranes were then reprobed with an Ab against total ERK1/2 for loading control. One experiment of two is shown.

 
Taken together, these results suggest that in TNF-{alpha}- and LPS-induced DC maturation early activation of p38 MAPK, but not of ERK1/2, correlated with the up-regulation of the T cell-costimulatory molecules CD80 and CD86, and with the expression of CD1a, CD83, and HLA-DR.

SB203580 prevents the up-regulation of the allostimulatory capacity and the down-regulation of endocytosis in LPS- or TNF-{alpha}-stimulated DC

Fully mature DC elicit higher levels of allogenic T cell proliferation than immature DC (5). Thus, we tested whether SB203580 had an effect on the allostimulatory capacity of immature DC stimulated for 48 h with TNF-{alpha} or LPS (Fig. 4Go, A and B). SB203580 added to immature DC before addition of LPS or TNF-{alpha} inhibited the allostimulatory capacity of DC in a concentration-dependent manner. DC treated with a high concentration of SB203580 (50 µM) before TNF-{alpha} or LPS stimulation showed an allostimulatory capacity equivalent to that of immature DC. An inhibitory effect was still observed using 10 µM of SB203580. During their maturation process, DC lose their high endocytic activity. This function can be measured by the uptake of FITC-dextran, which is mediated by the mannose receptor (6). Thus, we investigated the effect of SB203580 on the uptake of FITC-dextran by immature DC stimulated with LPS or TNF-{alpha}. As shown in Fig. 4GoC, SB203580 treatment partially preserved the FITC-dextran uptake lost in the absence of the drug. In the presence of 50 µM SB203580, approximately twice more of the cells could still endocytose FITC-dextran. These results show that SB203580 inhibited the LPS and TNF-{alpha}-induced up-regulation of the allostimulatory capacity of DC and partially interfered with the down-regulation of FITC-dextran uptake.



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  		FIGURE 4. SB203580 treatment decreases the T cell stimulatory capacity and prevents the down-regulation of FITC-dextran uptake in TNF-{alpha}- or LPS-activated DC. DC were treated or not with increasing concentrations of SB203580 as indicated, further stimulated with TNF-{alpha} (100 U/ml) (A) or LPS (10 ng/ml) (B), and allogenic T cell proliferation was evaluated. Means of quadruplicates obtained after 5 days of culture are shown. These results are representative of three independent experiments. C, At day 7, DC were treated or not for 30 min with 50 µM SB203580 before stimulation or not for 48 h with TNF-{alpha} (100 U/ml) or LPS (10 ng/ml). Cells were then incubated with FITC-dextran for 1 h at 4°C (thin lines) or 37°C (bold lines). The percentage of cells positive for FITC-dextran uptake is indicated. These results are representative of two independent experiments with similar results.

 
SB203580 treatment abrogates TNF secretion by LPS-stimulated DC

DC maturation is a process during which DC secrete many inflammatory cytokines such as IL-1{alpha}, IL-1{beta}, IL-6, IL8, and TNF-{alpha}. Thus, we studied TNF secretion by DC stimulated with LPS, and tested the effect of SB203580 on this biological response. Immature DC were treated or not with increasing concentrations of SB203580, and then stimulated for 24 h with LPS. Bioactive TNF was measured after 24 h in the culture supernatants using a bioassay. As shown in Fig. 5GoA, SB203580 abrogated LPS-induced TNF secretion in a concentration-dependent manner with an ID50 ranging between 0.1 and 1 µM.



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  		FIGURE 5. Effect of SB203580 on LPS-induced proinflammatory cytokine expression in immature DC. A, Seven-day cultured immature DC were pretreated or not for 30 min with increasing concentrations of SB203580 as indicated, and further stimulated with LPS (10 ng/ml) for 24 h. Bioactive TNF was determined in 24-h culture supernatants using a TNF-sensitive cell line as described in Materials and Methods. These data represent means ± SD of triplicates, and are representative of three independent experiments. B and C, Effect of SB203580 on mRNA levels encoding for proinflammatory cytokines induced in LPS-stimulated DC was evaluated by RNase protection assay as described in Materials and Methods. Upper left autoradiograms show kinetic studies of induction of proinflammatory cytokines in LPS-treated cells using multiprobe template set hCK3 (B) and hCK2 (C). Upper right autoradiograms show the effect of increasing concentrations of SB203580 on mRNA levels encoding for proinflammatory cytokines. B and C (lower panels), mRNA levels for two distinct housekeeping genes (L32 and GAPDH). These results are representative of three independent experiments.

 
SB203580 treatment affects mRNA levels encoding proinflammatory cytokines induced by LPS

To extend our study to other proinflammatory cytokines that are induced during maturation of DC stimulated by LPS and TNF-{alpha}, we performed multiplex RNase protection assay allowing the determination of mRNA levels encoding various cytokines. Kinetic studies showed that LPS-induced TNF-{alpha} mRNA expression peaked at 1 h (Fig. 5GoB, upper left panel), whereas LPS-induced IL-1{alpha}, IL-1{beta}, and IL-1Ra mRNA expression peaked at 2 h (Fig. 5GoC, upper left panel). SB203580 inhibited in a concentration-dependent manner the LPS-induced increase of mRNA encoding for all the cytokines described above (Fig. 5Go, B and C, upper right panels). The induction of TNF-{alpha}, Il-1{beta}, and IL-1Ra mRNA was only partially inhibited using high doses of SB203580 (10–50 µM). SB203580 had the same effect in TNF-{alpha}-stimulated immature DC (data not shown). These results indicate that inhibition of p38 MAPK activity partially prevents the increase of proinflammatory cytokines mRNA induced by LPS and TNF-{alpha} in immature DC.

Contact sensitizers but not irritants induce p38 MAPK phosphorylation

Recent reports demonstrated that tyrosine phosphorylation is an early molecular event in the activation of APCs by skin contact sensitizers (37, 38). This was not observed with irritants. Furthermore, Aiba et al. showed that human monocyte-derived DC responded to contact sensitizers but not to irritants by increased expression of the costimulatory molecule CD86 and of HLA-DR (15). In this context, we first asked whether contact sensitizers and irritants could differentially activate MAPK signaling pathways. To achieve this, we chose two potent and chemically unrelated sensitizers, NiSO4 and DNFB, as well as two irritants, SDS and BC. We stimulated immature DC with the above chemicals and performed Western blot analysis to detect phosphorylated forms of MAPK. As shown in Fig. 6Go, A–C, NiSO4 and DNFB, but not the two irritants, led to p38 MAPK phosphorylation. The phosphorylation was already detectable after 5 min and remained during 1 h of stimulation. Maximal responses were obtained using 500 µM and 2.5 µg/ml of NiSO4 and DNFB, respectively. Densitometric quantification revealed a 4.3 ± 0.4-fold and a 6.5 ± 0.7-fold induction of p38 MAPK phosphorylation by NiSO4 and DNFB, respectively (mean ± SD of three experiments). A slight increase in ERK1/2 phosphorylation level was also observed after 15–30 min following NiSO4 but not DNFB stimulation, whereas p46/54 JNK phosphorylation was not affected by either of the two contact sensitizers (data not shown). Moreover, the two irritants tested did not induce ERK1/2 and p46/54 JNK phosphorylation (data not shown). These results show that p38 MAPK is the MAPK pathway activated by both contact sensitizers tested. In contrast, none of the MAPKs studied were phosphorylated by irritants in our cellular model. Next, we analyzed the surface Ag profile of DC after a 48-h NiSO4 stimulation. As shown in Fig. 6GoD, we observed a strong up-regulation of CD40, CD80, CD86, CD83, and HLA-DR. To determine whether the p38 MAPK pathway was involved in the up-regulation of these surface molecules induced by NiSO4 in DC, cells were pretreated with the p38 MAPK inhibitor. SB203580 blocked almost completely the increase in CD80 and CD83 and, to a lesser extent, the increase of CD86 (Fig. 6GoD). The surface densities of CD1a, CD40, and HLA-DR were not affected by SB203580. Altogether, these results indicate that p38 MAPK phosphorylation also plays a role in the up-regulation of the surface molecules CD80, CD86, and CD83 induced by contact sensitizers in maturating DC.



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  		FIGURE 6. Contact sensitizers and irritants differentially activate immature DC. A-C, Immature monocyte-derived DC were stimulated or not with the indicated concentrations of contact sensitizers (NiSO4 and DNFB) or irritants (SDS and BC). Upper panels from (A) and (B) and panels from (C) show kinetic studies of induction of p38 MAPK phosphorylation analyzed by Western blotting. A and B (lower panels), Dose response of contact sensitizers on the activation of p38 MAPK. c, Unstimulated cells; TNF, 5-min stimulation of cells with 100 U/ml TNF-{alpha}. Blots showing kinetic studies are representative of results obtained in three separate experiments. Blots dealing with contact sensitizer dose-responses are representative of results obtained in two independent experiments. D, The effect of SB203580 (50 µM) on NiSO4 (500 µM)-induced cell surface molecule expression in immature DC was evaluated by flow cytometric analysis as described in Materials and Methods. Numbers in each quadrant represent cell percentages. These results are representative of results obtained in two separate experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The biological process of DC maturation represents a crucial step in the initiation of an adaptive immune response (39). This process is regulated by various extracellular stimuli including cytokines, bacterial products, and membrane-bound ligands (6, 8, 11). DC maturation is accompanied by changes of their morphological, phenotypic, and functional properties (3). However, little is known about the molecular mechanisms responsible for the regulation of DC in their maturation state. In this study, we show that TNF-{alpha} and LPS, two potent DC maturation factors, induced early phosphorylation of ERKl/2, p46/54 JNK, and p38 in monocyte-derived DC. LPS-induced MAPK activation was delayed in time compared with TNF-{alpha} activation. This delayed response may be simply explained by the use of distinct receptors for these molecules to trigger DC maturation. The effects of TNF-{alpha} are mediated by two distinct cell surface receptors, TNF-R1 and TNF-R2, and TNF-R1 has been implicated in TNF-{alpha}-induced phenotypic and functional changes in DC (6). In the case of LPS, several molecules could be implicated. First, the CD14 surface Ag is one candidate because residual CD14 expression can occur on immature DC. Second, a soluble CD14-dependent pathway has been described (10). Third, the recently described family of human Toll-like receptors may play a role in the response to LPS (40). Our results corroborate recent reports using murine as well as human DC in vitro models showing activation of all three MAPK pathways during maturation (18, 20, 41). Interestingly, Aicher et al. showed that in human monocyte-derived DC CD40 ligation also activated all three MAPK pathways (18).

The availability of specific inhibitory drugs for the p38 and ERK1/2 pathways prompted us to investigate the respective roles of these MAPKs in DC maturation. Although PD98059 was shown to inhibit the ERK1/2 pathway in our experiments, we did not observe inhibitory effects of this compound on the up-regulation of costimulatory molecule expression in the process of DC maturation triggered by TNF-{alpha} or LPS. These results are in agreement with a recent report by Rescigno et al., who described the signaling pathways activated by LPS in a murine DC culture system (41). The authors found that LPS activated all three MAPK signaling pathways, and that specific inhibition of the ERK1/2 pathway by PD98059 did not inhibit DC maturation but rather regulated DC survival. In contrast to PD98059, inhibition of the p38 MAPK by SB203580 before TNF-{alpha} or LPS stimulation profoundly affected the phenotypic changes normally induced during DC maturation. Indeed, the up-regulation of CD1a, CD40, CD80, CD86, CD83, and HLA-DR cell surface expression was prevented in a concentration-dependent manner by SB203580 treatment. The expression of CD40 was inhibited using the high concentrations of SB203580. In addition, the cell surface Ag profile of immature DC cultured with GM-CSF and IL-4 in the presence of 50 µM SB203580 for 48 h was not affected and was thus comparable with vehicle (DMSO)-treated DC (data not shown). Moreover, the inhibitory effects of SB203580 were not due to nonspecific toxicity because the viability of DC was not modified by SB203580 (data not shown). The fact that the inhibitory effects of SB203580 were observed at concentrations as low as 1–10 µM indicates that p38 MAPK is involved, and virtually excludes nonspecific effects. The inhibition of costimulatory molecule expression represents an important and novel target of action for the anti-inflammatory effects of p38 MAPK inhibitors.

We further tested the effect of p38 MAPK inhibition on functional features induced during DC maturation. As expected, the decreased level of CD80 and CD86 correlated with reduced allostimulatory capacity of SB203580-treated DC. The down-regulation of FITC-dextran uptake was only partially prevented by SB203580, suggesting that other pathways may regulate mannose receptor-mediated uptake of FITC-dextran. Along the process of maturation, DC secrete many proinflammatory cytokines. p38 MAPK has been implicated in the regulation of expression of many cytokine genes (26, 32, 33, 34). We showed here that LPS-induced TNF secretion was abrogated in a concentration-dependent manner upon SB203580 treatment of immature DC. The low ID50 (0.1–1 µM) of the drug for this biological response is comparable to that observed in LPS-induced TNF production by macrophages (26, 32). In the same way, Häcker et al. showed that stress kinase activation is essential for CpG-DNA-induced release of TNF-{alpha} and IL-12 by murine DC, because inhibition of p38 MAPK resulted in a severe impairment of this biological response (13). Interestingly, Sato et al. reported that dual stimulation of TNF-{alpha} and IL-10 abolished TNF-{alpha}-induced activation of all three MAPK pathways in human DC (20). The treatment of DC with IL-10 resulted in the reduction of the cell surface expression levels of CD86, HLA-DR, and of the T cell stimulatory activity. However, these authors did not show which MAPKs were involved in the IL-10-induced inhibition. It is tempting to speculate that p38 MAPK is one of the targets of the IL-10 effect.

Recent reports show that NF-{kappa}B is responsible for LPS-induced DC maturation in an in vitro murine model (41), and that cytokine-induced maturation of human DC results in increased NF-{kappa}B nuclear translocation (42). Many proinflammatory cytokines display NF-{kappa}B-responsive elements in their promoters, conferring a major role on NF-{kappa}B in immune responses (43, 44). Moreover, the p38 MAPK pathway was shown to contribute to NF-{kappa}B-mediated transactivation (33, 45, 46). Thus, it was of interest to determine whether mRNA levels encoding for NF-{kappa}B-responsive proinflammatory cytokines were also affected by p38 MAPK inhibition. Indeed, we observed that the expression of TNF-{alpha} and members of the IL-1 family was partially reduced by SB203580 treatment. It remains to be determined whether these inhibitory effects are transcriptional or posttranscriptional, because recent experiments demonstrate that inhibition of p38 MAPK can lead to specific mRNA destabilization (47, 48). One possible explanation is that p38 MAPK inhibition interfered with the normal process of DC maturation by affecting proinflammatory cytokine expression through NF-{kappa}B negative modulation. However, other transcription factors such as C/EBP/NFIL-6, which is involved in IL-1 transcription (49), may also be regulated by p38 MAPK. In addition, the level of inhibition of LPS-induced TNF secretion cannot solely be explained by the degree of reduction of TNF-{alpha} mRNA. In this case, SB203580 may block TNF-{alpha} expression at the translational level, as already reported by Lee et al., who showed that pyridinyl imidazole compounds primarily inhibited the translation of TNF-{alpha} mRNA. p38 MAPK action via NF-{kappa}B may not only pertain to cytokine genes, but also to other genes important in inflammation. Indeed, the human CD86 promoter has been recently cloned, and revealed two canonical NF-{kappa}B binding sites (50). One of them is essential for Th-induced CD86 gene transcription. Moreover, NF-{kappa}B activation has been previously shown to drive CD80 transcription (51, 52). A recent report describes the generation of MKK3-deficient mice to study the role of the p38 MAPK pathway in vivo (53). Using this animal model, the authors showed that p38 MAPK is required for the production of IL-12 by macrophages and DC. However, MKK3 deficiency did not affect the expression of IL-6, TNF-{alpha}, IL-1{alpha}, and IL-1{beta}, suggesting a role for MKK6, the other specific MAPK kinase in the p38 cascade (54, 55).

Contact sensitizers have been shown to trigger DC maturation in vitro. Indeed, Aiba et al. showed that stimulation of human monocyte-derived DC with contact sensitizers such as NiCl2 and 1-chloro 2,4-dinitrobenzene increased DC immunostimulatory capacity (15). Up-regulation of CD54, CD86, and HLA-DR was observed following stimulation with contact sensitizers. Induction of IL-1{beta}, IL-6, and TNF-{alpha} secretion upon NiCl2 stimulation was also reported. Moreover, recent reports showed that an early molecular event during the activation of APC by contact sensitizers is the induction of tyrosine phosphorylation (37, 38). We show herein that contact sensitizers induce activation of p38 MAPK, whereas irritants do not. Furthermore, p38 MAPK inhibition before NiSO4 stimulation of DC inhibited up-regulation of costimulatory molecule expression. We found that NiSO4 also induced CD83 expression, and that this response was affected by SB203580 treatment. Thus, contact sensitizers and irritants differentially activate p38 MAPK. These results also suggest that p38 MAPK phosphorylation in DC may become a useful tool for the screening of potential skin contact sensitizers.


   Acknowledgments
 
We thank D. Wohlwend for his excellent assistance concerning flow cytometric analysis, and M. Rey for her excellent technical assistance concerning Western blotting.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Jean-François Arrighi, Allergy Unit, Hôpital Universitaire de Genève, rue Micheli-du-Crest 24, 1211 Genève 14, Switzerland. Back

2 Abbreviations used in this paper: DC, dendritic cell(s); MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; DNFB, 2,4-dinitrofluorobenzene; BC, benzalkonium chloride; MFI, mean fluorescence intensity. Back

Received for publication May 1, 2000. Accepted for publication January 5, 2001.


   References
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 Materials and Methods
 Results
 Discussion
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