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, and Contact Sensitizers
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Department of Dermatology, DHURVD,
Division of Immunology and Allergy,
Division of Endocrinology and Diabetology, and
Division of Hematology, University Hospital, Geneva, Switzerland; and
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L’Oréal Advanced Recherche, Aulnay-sous-bois, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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-.
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-. Likewise, SB203580 partially
prevented the up-regulation of IL-1, IL-1
, IL-lRa, and TNF-
mRNA upon stimulation with LPS and TNF-, as well as the release of
bioactive TNF- 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1 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 and TNF-),
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- 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-- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- (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- 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
-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- (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- 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 [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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- 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- or 10 ng/ml LPS induced the phosphorylation of all of
the MAPK tested (Fig. 1
, A and
B). Upon TNF- 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- and LPS, respectively (mean ±
SD of eight experiments for TNF-, and of three experiments for LPS).
In addition, TNF- 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- preparation, we
used neutralizing polyclonal Ab raised against TNF-. This Ab
effectively blocked TNF--induced p38 MAPK phosphorylation (Fig. 1C) as well as TNF--stimulated ERK1/2 and p46/54 JNK
phosphorylation (data not shown). Thus, both TNF- and LPS induce
early phosphorylation of ERK1/2, p46/54 JNK, and p38 MAPK in
immature DC.
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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- or 10 ng/ml LPS. The up-regulation of CD1a, CD40, CD80, CD86,
HLA-DR, and CD83 induced by TNF- or LPS was prevented by
preincubating immature DC with SB203580 (Fig. 2, A–C and Table I). 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. 2 and
Table I). 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- or LPS (Fig. 3A). 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- or LPS were subjected to
Western blot analysis to detect phosphorylated forms of ERK1/2 and p38
MAPK. Both TNF- and LPS-induced ERK1/2 phosphorylation were
abrogated by 50 µM PD98059, whereas the level of activated p38 MAPK
was not affected (Fig. 3B). Conversely, 50 µM SB203580
neither affected the phosphorylation levels of ERK1/2 nor of p46/54 JNK
induced after TNF- or LPS stimulation (data not shown).
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- 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--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- or LPS (Fig. 4, A and B).
SB203580 added to immature DC before addition of LPS or TNF-
inhibited the allostimulatory capacity of DC in a
concentration-dependent manner. DC treated with a high concentration of
SB203580 (50 µM) before TNF- 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-. As shown in
Fig. 4C, 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--induced up-regulation of the allostimulatory capacity
of DC and partially interfered with the down-regulation of FITC-dextran
uptake.
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DC maturation is a process during which DC secrete many
inflammatory cytokines such as IL-1, IL-1, IL-6, IL8, and
TNF-. 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. 5A, 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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To extend our study to other proinflammatory cytokines that are
induced during maturation of DC stimulated by LPS and TNF-, we
performed multiplex RNase protection assay allowing the determination
of mRNA levels encoding various cytokines. Kinetic studies showed that
LPS-induced TNF- mRNA expression peaked at 1 h (Fig. 5
B,
upper left panel), whereas LPS-induced IL-1, IL-1, and
IL-1Ra mRNA expression peaked at 2 h (Fig. 5C, upper left
panel). SB203580 inhibited in a concentration-dependent manner the
LPS-induced increase of mRNA encoding for all the cytokines described
above (Fig. 5, B and C, upper right panels). The
induction of TNF-, Il-1, and IL-1Ra mRNA was only partially
inhibited using high doses of SB203580 (10–50 µM). SB203580 had the
same effect in TNF--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- 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. 6, 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. 6D, 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. 6D). 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
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- are mediated by two distinct cell surface receptors,
TNF-R1 and TNF-R2, and TNF-R1 has been implicated in TNF--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- 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- 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- 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- and IL-10 abolished
TNF--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-
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-B
nuclear translocation (42). Many proinflammatory cytokines
display NF-B-responsive elements in their promoters, conferring a
major role on NF-B in immune responses (43, 44).
Moreover, the p38 MAPK pathway was shown to contribute to
NF-B-mediated transactivation (33, 45, 46). Thus, it
was of interest to determine whether mRNA levels encoding for
NF-B-responsive proinflammatory cytokines were also affected by p38
MAPK inhibition. Indeed, we observed that the expression of TNF- 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-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- mRNA. In this case,
SB203580 may block TNF- expression at the translational level, as
already reported by Lee et al., who showed that pyridinyl imidazole
compounds primarily inhibited the translation of TNF- mRNA. p38 MAPK
action via NF-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-B binding
sites (50). One of them is essential for Th-induced CD86
gene transcription. Moreover, NF-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-, IL-1, and IL-1,
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,
IL-6, and TNF- 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.
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Acknowledgments |
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Footnotes |
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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.
Received for publication May 1, 2000. Accepted for publication January 5, 2001.
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A. Puig-Kroger, M. Relloso, O. Fernandez-Capetillo, A. Zubiaga, A. Silva, C. Bernabeu, and A. L. Corbi Extracellular signal-regulated protein kinase signaling pathway negatively regulates the phenotypic and functional maturation of monocyte-derived human dendritic cells Blood, October 1, 2001; 98(7): 2175 - 2182. [Abstract] [Full Text] [PDF]
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