It is well known that, although dendritic cells (DC) are the most potent antigen-presenting cells (APC) for helper T cells as well as cytotoxic T cells among various APC (Steinman, 1991), they reside in nonlymphoid organs and show extremely active endocytosis as well as antigen-processing but weak antigen-presenting functions (Romani et al, 1989a). In response to stimuli derived from microorganisms, such as lipopolysaccharide (LPS), these precursor DC migrate to T cell zones of lymph nodes (Macatonia et al, 1987;Kripke et al, 1990) and mature into effective APC by ceasing endocytosis as well as the antigen-processing function, but by stabilizing the expression of class II–peptide complexes (Stossel et al, 1990;Kampgen et al, 1991) and increasing the expression of CD80 or CD86 (Romani et al, 1989b;Symington et al, 1993;Girolomoni et al, 1994;Ozawa et al, 1996). The immune system also responds to simple chemicals. Whereas so-called adjuvants are usually required to activate the innate immune system, including DC in T cell-mediated immunity to protein antigens, the allergic contact hypersensitivity reaction to simple chemicals does not require the help of an adjuvant, but can be induced by the simple application of these chemicals to the skin. When we consider the similarities between immunity to simple chemicals and that to infectious agents, it is reasonable to speculate that hapten itself stimulates DC maturation.
Indeed, we reported that murine Langerhans cells upregulate their expression of class II major histocompatibility complex antigen and antigen-presenting function after hapten painting to the skin, whereas chemicals that simply irritate the skin rather than sensitize animals cannot induce this phenomenon (Aiba and Katz, 1990). Afterwards, we demonstrated that the application of haptens to murine skin was accompanied by the upregulation of several costimulatory molecules on Langerhans cells, i.e., CD40, CD54, CD80, and CD86 (Ozawa et al, 1996). In addition,Enk and Katz (1992) demonstrated a crucial role played by interleukin (IL)-1
secreted by Langerhans cells themselves in their increased expression of class II major histocompatibility complex antigen after exposure to hapten application.
In addition to these in vivo studies, using human monocyte-derived DC (MoDC),Aiba et al (1997) demonstrated in vitro that purified MoDC responded to haptens such as NiCl2 and 2,4-dinitrochlorobenzene (DNCB), but not to irritants such as benzalkonium chloride (BC) or sodium lauryl sulfate (SLS), by significantly augmenting their expression of CD54, CD86, and HLA-DR and by increasing their production of proinflammatory cytokines. Furthermore, using Langerhans cell-like DC induced from peripheral blood monocytes in the presence of transforming growth factor-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-4 (monocyte-derived Langerhans cells) according to a procedure reported byGeissmann et al (1998), it has been shown that in vitro treatment with haptens can induce the phenotypic and functional changes in monocyte-derived Langerhans cells seen in epidermal Langerhans cells during the initiation phase of contact hypersensitivity reaction in vivo (Aiba et al, 2000), e.g., the downregulation of E-cadherin (Schwarzenberger and Udey, 1996) and cutaneous lymphocyte antigen (Ebner et al, 1998), the induction of matrix metalloproteinase-9 expression (Kobayashi, 1997), and the augmentation of some of 1-integrins (Staquet et al, 1992;Aiba et al, 1993), CD44 and some of its variants (Weiss et al, 1997), as well as the expression of CCR7 mRNA that enable Langerhans cells to respond to MIP-3 (Dieu et al, 1998;Sallusto et al, 1998;Saeki et al, 1999).
In spite of these observations, however, it is still unknown how different haptens can stimulate MoDC to acquire a mature phenotype. So far, there have been few reports on the signal transduction elements stimulated by haptens.Kuhn et al (1998) demonstrated that strong sensitizers could induce the formation of phosphotyrosine in MoDC in vitro, suggesting that tyrosine phosphorylation plays an important part in the activation of MoDC by haptens. Recently,Ardeshna et al (2000) have reported the precise mechanisms for the survival and maturation of MoDC stimulated by LPS. According to their report, both p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-
B signal transduction pathways are responsible for the upregulation of CD80, CD83, and CD86 induced by LPS, whereas phosphoinositol 3-kinase is important in maintaining their survival. Therefore, it is conceivable that haptens also stimulate p38 MAPK, NF-B, or phosphoinositol 3-kinase. In fact, using MoDC,Arrighi et al (2001) have reported that 2,4-dinitrofluorobenzene and NiSO4 induce p38 MAPK phosphorylation, and that the augmentation of CD80 and CD83 induced by NiSO4 is partially suppressed by p38 MAPK inhibitor. The MAPK are an important group of serine/threonine signaling kinases that, by modulating the phosphorylation and hence activation status of transcription factors, link transmembrane signaling with gene induction events in the nucleus. There are at least three genetically distinct MAPK pathways in mammals, including the extracellular signal-regulated kinases (ERK), the c-jun N-terminal kinases (JNK), and the p38 MAPK. These kinases are activated by phosphorylation on both threonine and tyrosine residues in a regulatory TXY loop present in all MAPK. This phosphorylation is performed by distinct upstream dual-specificity MAPK kinases (MAPKK). Activated MAPK then phosphorylate their respective substrate on serine or threonine residues (Whitmarsh and Davis, 1996;Minden and Karin, 1997;Ip and Davis, 1998).
Therefore, in this study, to elucidate further the mechanism for the induction of DC maturation by haptens, we examined the activation of three distinct MAPK pathways such as ERK, JNK, and p38 MAPK and NF-B on human MoDC by two representative haptens with different chemical structures, i.e., DNCB and NiCl2. By using PD98059 and SB203580, which are relatively specific inhibitors for ERK and p38 MAPK pathways, respectively, and pyrrolidine dithiocarbamate (PDTC), a NF-B inhibitor, we investigated which kinds of signal transduction pathways mediate the augmentation of CD80, CD83, or CD86 expression and the increased production of IL-1, IL-8, IL-12p40, or tumor necrosis factor (TNF)-
by MoDC stimulated with these haptens. In these studies, we found that DNCB and NiCl2 stimulated MoDC via different signal transduction pathways. Namely, DNCB preferentially stimulated p38 MAPK, whereas NiCl2 activated ERK and NF-B in addition to p38 MAPK.
Materials and Methods
Media and reagents
The medium used in the study was RPMI-1640, including 25 mM Hepes buffer (Sigma, St Louis, MO) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% penicillin, streptomycin, and fungizone antibiotic solution (Sigma) and 10% fetal bovine serum (Bioserum, Canterbury, Victoria, Australia) (complete medium). The buffer used for the purification of CD14+ monocytes from peripheral blood mononuclear cells was phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (less than 1 ng per mg of detectable endotoxin) (Sigma) and 5 mM ethylenediamine tetraacetic acid [Magnetic activated cell sorter (MACS) buffer]. NiCl2 (Sigma), and DNCB (Wako Pure Chemicals, Osaka, Japan) were used for the stimulation of MoDC. The endotoxin content of the final dilution used was <30 pg per ml, as determined by the Limulus amebocyte lysate assay (Seikagaku Co. Inc., Tokyo, Japan). We used the following monoclonal antibodies for immunostaining: fluorescein isothiocyanate (FITC)-conjugated-anti-CD40, FITC-conjugated-anti-CD86 antibodies, FITC or phycoerythrin (PE)-conjugated isotype-matched mouse control antibodies (IgG2a and IgG2b) (PharMingen, San Diego, CA), PE-conjugated anti-CD80, PE-conjugated anti-HLA-DR antibodies (Becton-Dickinson, San Jose, CA), PE-conjugated anti-CD83 antibody (Immunotech, Marseilles, France), FITC-conjugated anti-CD54 antibody (Ancell, Bayport, MN), and PE-conjugated anti-CD1a antibody (Coulter, Hialeah, FL). MACS colloidal supermagnetic microbeads conjugated with anti-human CD14 monoclonal antibody (CD14 microbeads) were purchased from Miltenyi Biotec Inc. (Sunnyvale, CA). Recombinant human (rh) GM-CSF was a gift from Kirin Brewery Co. (Tokyo, Japan), and rhIL-4 was purchased from Peprotech EC Ltd (London, U.K.). NiCl2, BC, or SLS (Sigma) was solubilized in distilled water, whereas DNCB was solubilized in dimethyl sulfoxide (DMSO) at a concentration of 1 M. MEK 1 inhibitor PD98059 and p38 MAPK inhibitor SB203580 were purchased from New England BioLabs Inc. (Beverly, MA) and Calbiochem (La Jolla, CA), respectively. Each inhibitor was reconstituted in DMSO at a stock concentration of 20 mM and 5 mM, respectively. The final concentration of DMSO was always less than 0.1% and cultures of MoDC with 0.1% DMSO were also examined as a control. A NF-B inhibitor, PDTC, was purchased from Calbiochem and solubilized in distilled water.
Culture of MoDC from peripheral blood monocytes and chemical treatment with haptens in the presence or absence of MAPK inhibitor
Peripheral blood mononuclear cells were isolated from heparinized fresh leukocyte-enriched buffy coats from different donors using Lymphoprep (Nycomed Pharma As, Oslo, Norway). After several washes with PBS, 1
108 peripheral blood mononuclear cells were treated with 150 
l of CD14 microbeads in 600 l of MACS buffer at 4°C for 30 min. After washing with MACS buffer, the cells coated with CD14 microbeads were separated by a magnetic cell separator, MACS (Miltenyi Biotech), according to the manufacturer's protocol. Before culturing, we examined the percentage of CD14+ cells in these preparations by flow cytometry and used cell specimens containing more than 98% of CD14+ cells in the experiments.
CD14+ monocytes (2106 per ml) were cultured in complete medium containing 50 ng per ml of rhGM-CSF and 100 ng per ml rhIL-4 for 5 d. One half of the culture medium was changed on day 3 and day 5. On the fifth day, the cells were treated with different concentrations of NiCl2, DNCB, SLS, or BC. To analyze ERK, JNK, p38 MAPK, and IB phosphorylation, these cells were cultured for 30 min or 1 h. To examine the effects of MAPK inhibitors or NF-B inhibitor, MoDC were exposed to various concentrations of PD98059, SB203580, or PDTC, 1 h prior to the stimulation by chemicals, and were then cultured for 48 h before collection of the supernatants or flow cytometrical analysis.
Flow cytometry
Forty-eight hours after treatment with the chemicals, the surface expression of CD40, CD54, CD80, CD83, CD86, or HLA-DR antigen was analyzed by flow cytometry. Cell staining was performed using a combination of FITC-conjugated anti-CD86 (10 g per ml), FITC-conjugated anti-CD40 (10 g per ml), FITC-conjugated anti-CD54 (10 g per ml), or FITC-conjugated isotype control (10 g per ml) antibody, and PE-conjugated anti-HLA-DR antigen (10 g per ml), PE-conjugated anti-CD83 (10 g per ml), PE-conjugated anti-CD80 (10 g per ml), or PE-conjugated isotype control (10 g per ml) antibody. After washing with PBS supplemented with 1% bovine serum albumin and 0.02% NaN3 (fluorescence-activated cell sorter buffer), the cells were analyzed by FACScan using CellQuest software (Becton Dickinson). Dead cells were gated out after staining with 0.5 g propidium iodide solution per ml.
Enzyme-linked immunosorbent assay for cytokine production
The culture supernatants of MoDC treated with haptens in the presence or absence of MAKP inhibitors or PDTC were recovered 48 h after culture. Their production of IL-1, TNF-, IL-8, and IL-12p40 was measured by enzyme-linked immunosorbent assay kits obtained from Endogen, Inc. (Woburn, MA) for TNF- and IL-8 and from R&D Systems (Minneapolis, MN) for IL-1, IL-12p40, using 96-well microtiter plates, according to each manufacturer's instructions. The levels of TNF-, IL-8, and IL-12p40 were calculated by using a standard curve obtained with rhIL-1 (from 3.9 to 250 pg per ml), rhTNF- (from 0 to 400 pg per ml), rhIL-8 (from 0 to 1000 pg per ml), and rhIL-12p40 (from 0 to 2000 pg per ml). When the culture supernatants contained cytokines over the upper limits of the standard curves, they were measured again after optimal dilution.
Immunoblotting
MoDC were either unstimulated or stimulated with 10 g per ml, 30 g per ml of SLS, 10 g per ml, 30 g per ml of BC, 10 M, 30 M, and 100 M of DNCB, 100–300 M, and 1000 M of NiCl2 for 1 h. Immunoblotting of phosphorylated p42/44 ERK, SAPK/JNK, p38 MAPK, or IB was performed using p42/44 ERK, SAPK/JNK, p38 MAPK, or IB immunoblotting kits purchased from New England Biolabs. The cells were washed twice in cold PBS and resuspended in 100 l of lysis buffer (1% Nonidet P-40, 20 mM Tris–HCl (pH 8.0), 137 M NaCl, 10% glycerol, 2 mM ethylenediamine tetraacetic acid, 10 g per ml leupeptin, 10 g per ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovandate). The nuclei and the insoluble cell debris were removed by centrifugation at 4°C for 10 min at 14,000g. The postnuclear extracts were collected and used as total cell lysates. Total cell lysates were suspended in 2 sodium dodecyl sulfate sample buffer (313 mM Tris–HCl, pH 6.8, 10% sodium dodecyl sulfate, 2-mercaptoethanol, 50% glycerol, and 0.01% bromophenol blue) and heated at 95°C for 3 min. The protein samples were fractionated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The nonspecific antibody binding sites on the membranes were blocked with 1% bovine serum albumin, 0.01% Tween 20 in saline (10 mM Tris–HCl (pH 7.4) 100 mM NaCl) for 20 min at 37°C. Immunoblotting of ERK, SAPK/JNK, p38 MAPK, and IB was performed according to the instruction manual. Briefly, the membranes were incubated for 16 h at 4°C with rabbit polyclonal antibodies to anti-tyrosine phosphorylated p42/44 ERK, SAPK/JNK, p38 MAPK, or anti-serine (Ser32/36) phosphorylated IB, washed for 15 min, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Blots were visualized by enhanced chemiluminescence. To ensure that there were similar amounts of MAPK in each sample, the same membranes were stripped off, reprobed with monoclonal antibodies to p42/44 ERK, SAPK/JNK, or p38 MAPK, and developed with HRP-conjugated secondary antibodies by enhanced chemiluminescence. For IB, we prepared two immunoblotted membranes, which were treated using the same amount of total cell lysate of each sample, and immunostained with anti-non-phosphorylated IB antibody and anti-phosphorylated IB antibody, respectively.
Microwell colorimetric NF-
B assay
The NF-B p65 activity of MoDC either unstimulated or stimulated with 30 M of DNCB, 300 M of NiCl2, or 10 ng per ml of LPS was examined using a NF-B p65 transcriptional factor assay kit (Active Motif North America, Carlsbad, CA). Thirty minutes and 1 h after stimulation, cells were rinsed twice with cold PBS and centrifuged for 10 min at 1400 r.p.m. (360g). The pellet was then resuspended in 100 l of Lysis Buffer containing a protease inhibitor cocktail and 5 mM dithiothreitol. After 10 min on ice, the lysate was centrifuged for 20 mM at 14,000 r.p.m. (10,976g). The supernatant constitutes the total protein extract and can be kept frozen at -70°C. The activity of NF-B in each sample was measured by a microwell colorimetric NF-B assay using a NF-B p65 transcriptional factor assay kit. To confirm the specificity of binding, the wild-type or mutated consensus oligonucleotide was added to the corresponding wells.
Quantitative real-time reverse transcription–PCR
Quantitative, fluorescent PCR was performed using the TaqMan system (ABI 7700; PE Applied Biosystems, Foster City, CA). Sequences for human GAPDH, CD86, and cytokines were obtained from GenBank. We chose forward primers and reverse primers to span exon–intron boundaries. TaqMan probes were chosen to be used with these primers with Primer Express version. 1.0 (PE Applied Biosystems). Forward and reverse primers were made by Operon (Nihon Gene Research Laboratories Inc., Sendai, Japan), whereas TaqMan probes were made by PE Applied Biosystems. The primers and probes used in these studies are shown in Table I. RNA was extracted by using the guanidinium thiocyanate method described by the manufacturer (ISOGEN; Nippon gene Inc., Toyama, Japan) from MoDC either unstimulated or stimulated with 30 M of DNCB or 300 M of NiCl2 for 2 h and 6 h in the presence or absence of MAPK inhibitors or PDTC. First strand cDNA was synthesized from total RNA extracted in RNAse-free conditions. cDNA was obtained from total RNA using TaKaRa RNA PCR kit (AMV) (Takara Biochemicals, Osaka, Japan), as described by the manufacturer's protocol. PCR for GAPDH, CD86, and cytokines were performed in duplicate in 30 l total reaction volumes with 66 nM TaqMan probe, 400 nM forward primers, 400 M reverse primers, and 2TaqMan universal PCR Master (PE Applied Biosystems). Thermal cycling was performed with 2 min at 50°C for depleting contaminated RNA, 10 min denaturation at 95°C, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min in the ABI Prism 7700 detection system (PE Applied Biosystems). Levels of cDNA for GAPDH, CD86, or each cytokine generated from cellular RNA were calculated by using standard curves generated with bona fide human cDNA for GAPDH, CD86, or each cytokine where there was a linear relationship between the number of cycles required to exceed the threshold and the number of copies of cDNA added (Anonymous, 1997).
Results
DNCB and NiCl2 activated p38 MAPK and ERK differently
To study the signal transduction elements for haptens, we examined the phosphorylation of three MAPK in MoDC 1 h after hapten, LPS, or irritant stimulation (Figure 1). MoDC stimulated with DNCB induced strong phosphorylation of p38 MAPK and SAPK/JNK in a concentration-dependent manner, 1 h after treatment. DNCB also phosphorylated p44/42 ERK at some restricted concentrations; however, p44/42 ERK were also phosphorylated in MoDC treated with 0.1% DMSO that served as a solvent control for DNCB. Therefore, it is impossible to evaluate the effect of DNCB on the activation of p44/42 ERK. On the other hand, NiCl2 induced strong phosphorylation of p44/42 ERK in a concentration-dependent manner, and that of p38 MAPK and SAPK/JNK. As we solubilized NiCl2 in distilled water, we could evaluate phosphorylation of p44/42 ERK in MoDC treated with NiCl2 by comparing that in nontreated DC. We stimulated MoDC with LPS as a positive control. LPS phosphorylated all three MAPK. In contrast, BC used as a representative for primary irritants did not augment any MAPK at its nontoxic concentrations, 30 g per ml or less. SLS, another irritant used at less than 100 g per ml, also did not induce the phosphorylation of any MAPK (data not shown).
Figure 1.
Haptens induce phosphorylation of MAPK in MoDC. MoDC were either unstimulated or stimulated with different concentrations of BC, NiCl2, DNCB, or 10 ng per ml of LPS for 1 h. MoDC were also cultured with 0.1% of DMSO as a solvent control. Immunoblotting of phosphorylated ERK1/2, SAPK/JNK, or p38 MAPK was performed using ERK1/2, SAPK/JNK, and p38 MAPK immunoblotting kits. Blots were developed with HRP-conjugated secondary antibodies and visualized by enhanced chemiluminescence. To ensure similar amounts of MAPK for each sample, the same membrane was stripped off and reprobed with monoclonal antibodies to ERK1/2, SAPK/JNK, or p38 MAPK. Similar results were obtained in three different experiments (a). The optical densities of the bands were determined by scanning radiograms (b).
Full figure and legend (59K)NiCl2, but not DNCB phosphorylated IB and activated NF-B
Next, we investigated whether these two haptens, LPS, or irritants activated the NF-B pathway. When we examined the phosphorylation of IB, only NiCl2 and LPS used as a positive control, but not DNCB, phosphorylated IB 1 h after stimulation (Figure 2). Neither BC or SLS phosphorylated IB (data not shown). Consistent with the phosphorylation of IB, both NiCl2 and LPS induced the binding activity to NF-B p65 consensus sequence in the total protein extracts of MoDC 30 min and 1 h after treatment. The specificity of the binding was confirmed by the competitive inhibition with the wild-type but not by mutated consensus oligonucleotides (Figure 3).
Figure 2.
NiCl2, but not DNCB phosphorylates IB in MoDC. MoDC were either unstimulated or stimulated with different concentrations of NiCl2, DNCB, or 10 ng per ml of LPS for 1 h. Immunoblotting of phosphorylated IB was performed using IB immunoblotting kits. In this experiment, we prepared two immunoblotted membranes, which were treated using the same amount of total cell lysate of each sample, and immunostained with anti-nonphosphorylated IB antibody and anti-phosphorylated IB antibody, respectively. Blots were developed with HRP-conjugated secondary antibodies and visualized by enhanced chemiluminescence. Similar results were obtained in three different experiments.
Figure 3.
NiCl2, but not DNCB activates NF-B. MoDC were either unstimulated or stimulated with 30 M of DNCB, 300 M of NiCl2, or 10 ng per ml of LPS. The total cell extracts were prepared from MoDC 30 min and 1 h after stimulation. The activity of NF-B in each sample was measured by microwell colorimetric NF-B assay using a NF-B p65 transcriptional factor assay kit. To confirm the specificity of binding, the wild-type or mutated consensus oligonucleotide was added to the corresponding wells. Similar results were obtained in two different experiments.
SB203580, but not PD98059, suppressed some of the phenotypic changes of activated MoDC induced by haptens
Because DNCB and NiCl2 distinctly activated MAPK in MoDC, we studied the effects of MAPK inhibitors on the phenotypic changes of MoDC induced by these haptens. We examined the following surface markers that are related to the function and maturation of DC, i.e., CD40, CD54, CD80, CD83, CD86, and HLA-DR. Consistent with our previous observation (Aiba et al, 1997), DNCB augmented the expression of CD86 and HLA-DR antigen, and, although weakly, that of CD83, whereas NiCl2 augmented that of CD86, HLA-DR antigen, and CD83 (Figure 4). When we added MAPK inhibitors in this system, we found that SB203580 suppressed the augmented expression of CD86, HLA-DR antigen, and CD83 induced by DNCB, and that of HLA-DR antigen and CD83 induced by NiCl2 in a concentration-dependent manner. Figure 4 is a representative flow cytometry of MoDC stimulated with the haptens in the presence or absence of different concentrations of SB203580. Figure 5 shows summarized data from four different subjects that demonstrate the changes of mean fluorescence intensity of CD86, CD83, and HLA-DR on hapten-treated MoDC plotted against different concentrations of SB203580. The augmentation of CD86 on MoDC induced by NiCl2 was insensitive to the treatment of SB203580. The treatment with SB203580 did not induce any significant changes on the DC phenotypes in the absence of the haptens.
Figure 4.
SB203580 suppresses the augmentation of CD86 and HLA-DR antigen expression on MoDC treated with DNCB. MoDC were pretreated with various concentrations of SB203580 for 1 h and then cultured with 300 M of NiCl2, 30 M of DNCB, or 10 ng per ml of LPS. After 2 d of culture, their surface expression of costimulatory molecules and HLA-DR antigen was analyzed by flow cytometry. The histogram of hapten-treated MoDC expressing each costimulatory molecule or HLA-DR antigen in the absence or the presence of various concentrations of SB203580 is expressed as a dotted line (0.1 M), a solid-dotted line (1 M), and a thick solid line (10 M) or a solid line (0 M). A shaded line indicates the histogram of nontreated DC. This is a representative flow cytometry of four independent experiments.
Figure 5.
The summarized data of the effects of SB203580 on the augmented expression of CD86 and HLA-DR antigen by MoDC treated with haptens. MoDC were pretreated with various concentrations of SB203580 for 1 h and then cultured with 300 M of NiCl2 or 30 M of DNCB. After 2 d of culture, their surface expression of CD83, CD86, and HLA-DR antigen was analyzed by flow cytometry. Changes in the mean fluorescence intensity were plotted against the concentrations of SB203580. Different symbols correspond to different experiments.
In contrast to the treatment with SB203580, that with PD98059 did not show any suppressive effects on the phenotypic changes induced by the haptens on MoDC, but rather augmented the expression of several phenotypic markers, such as CD86, CD83, and HLA-DR antigen. Figure 6 is a representative flow cytometry of MoDC stimulated with the haptens in the presence or absence of different concentrations of PD98059. The stimulatory effects of PD98059 on the expression of CD86, CD83, and HLA-DR antigen by MoDC was remarkable with a suboptimum stimulation of the haptens, such as 300 M of NiCl2, or 10 M of DNCB. Figure 7 shows summarized data of the mean fluorescence intensity of CD86 on MoDC obtained from two different subjects, which were unstimulated or stimulated with the suboptimum concentrations of the haptens in the presence of different concentrations of PD98059. Unexpectedly, PD98059 alone induced augmented expression of CD86 in a concentration-dependent manner. Furthermore, it increased the expression of CD86 synergistically with the haptens. Similarly, PD98059 also augmented CD83 and HLA-DR antigen on MoDC by itself (data not shown).
Figure 6.
PD98059 does not suppress the augmented expression of costimulatory molecules or HLA-DR antigen on MoDC treated with haptens. MoDC were pretreated with various concentrations of PD98059 for 1 h and then cultured with 300 M or 1 mm of NiCl2 or 10 M or 30 M of DNCB. After 2 d of culture, their surface expression of CD86, CD83, and HLA-DR antigen was analyzed by flow cytometry. The histogram of hapten-treated MoDC expressing CD83, CD86, or HLA-DR antigen in the absence or the presence of various concentrations of PD98059 is expressed as a dotted line (10 M), a solid-dotted line (30 M), and a thick solid line (100 M) or a solid line (0 M). A shaded line indicates the histogram of nontreated DC. This is a representative flow cytometry of two independent experiments.
Figure 7.
The summarized data of the effects of PD98059 on the augmented expression of CD86 and HLA-DR antigen by MoDC treated with haptens. MoDC were pretreated with various concentrations of PD98059 for 1 h and then cultured with 300 M of NiCl2 or 10 M of DNCB. After 2 d of culture, their surface expression of CD86 was analyzed by flow cytometry. Changes in the mean fluorescence intensity were plotted against the concentrations of PD98059.
In contrast to the significant changes observed with flow cytometry in CD86 expression between the nontreated MoDC and MoDC treated with the haptens or between the MoDC treated with DNCB in the presence and absence of the MAPK inhibitors, we could not find any significant changes in the expression of CD86 mRNA among them (Figure 9).
Figure 9.
SB203580 and PD98059 differently suppress cytokine mRNA expression by MoDC stimulated with haptens. MoDC were pretreated with 10 M of SB203580 or with 50 M of PD98059 or SB203580 for 1 h and then cultured with 30 M of DNCB or 300 M of NiCl2 for 2 h or 6 h. Total RNA was extracted from these MoDC and then cDNA was reverse-transcribed from total RNA. Quantitative, fluorescent PCR was performed using the TaqMan system. Levels of cDNA (copy number) for GAPDH, CD86 or each cytokine generated from cellular RNA (50ng) were calculated by using standard curves generated with bona fide human cDNA for GAPDH, CD86, or each cytokine where there is a linear relationship between the number of cycles required to exceed threshold and the number of copies of cDNA added. This figure shows representative data from three different experiments.
SB203580 and PD98059 differently regulate the cytokine production and the expression of cytokine mRNA by MoDC stimulated with the haptens
Next we examined the effects of these MAPK inhibitors on the cytokine production and the expression of cytokine mRNA by MoDC stimulated with the haptens. Figure 8, Figure 9 demonstrate the production of cytokines and their mRNA expression, respectively. When we added the haptens to the culture of MoDC, DNCB induced significant production of only IL-8, whereas NiCl2 induced that of IL-1, IL-8, IL-12p40, and TNF-. None of these haptens induced the production of IL-12p70 by MoDC (data not shown). In these cultures, SB203580 suppressed IL-8 production by MoDC stimulated with DNCB, and the IL-12p40 production by MoDC stimulated with NiCl2 in a concentration-dependent manner. On the other hand, PD98059 suppressed the IL-1, IL-8, and TNF- production by MoDC stimulated with NiCl2. Again, the treatment of MoDC with PD98059 enhanced the production of IL-12p40 synergistically with NiCl2, although the treatment with PD98059 alone did not increase it (Figure 8).
Figure 8.
SB203580 and PD98059 differently suppress the cytokine production by MoDC stimulated with haptens. To examine the effects of MAPK inhibitors on the production of cytokines by MoDC stimulated with the haptens, MoDC were pretreated with various concentrations of SB203580 or PD98059 for 1 h and then cultured with 300 M NiCl2 or 30 M of DNCB for 48 h. After culture, the supernatants were recovered and examined for the production of IL-1, IL-8, TNF- and IL-12p40 by an enzyme-linked immunosorbent assay. Different symbols correspond to different experiments. The data shown are the mean of triplicate determinations with SEM <10% (data not shown).
Consistent with the production of cytokines, the expression of IL-1, IL-8, and TNF- mRNA by MoDC was augmented by the treatment with both haptens, although NiCl2 induced these mRNA more strongly than did DNCB. Furthermore, SB203580 significantly suppressed the augmented expression of IL-1 and IL-8 mRNA by MoDC treated with DNCB, whereas PD98059 suppressed that of IL-1, IL-8, and TNF- by MoDC treated with NiCl2. Neither hapten augmented the expression of IL-12p40 mRNA. Again, the treatment of MoDC with PD98059 enhanced the expression of IL-12p40 of MoDC treated with either DNCB or NiCl2. In these samples, there was no significant change in GAPDH mRNA expression among MoDC with various treatments (Figure 9).
NiCl2 does not induce the augmentation of CD86 expression and the cytokine production by MoDC through the NF-B pathway
As NiCl2 induced the activation of NF-B, we next examined the effects of NF-B inhibitor, PDTC, on the augmentation of CD86 and the cytokine production. Figure 10 shows representative data from three different experiments. One hundred and fifty micromoles per liter of PDTC marginally suppressed the augmentation of CD86 expression by MoDC stimulated with NiCl2, whereas it did not influence that of CD83. For cytokine production, PDTC did not suppress the production of IL-1, IL-8, TNF-, or IL-12p40 (data not shown).
Figure 10.
PDTC does not significantly suppress the augmentation of CD86 or CD83 expression on MoDC treated with NiCl2. MoDC were pretreated with 150 M of PDTC for 1 h and then cultured without or with 300 M of NiCl2. After 2 d of culture, their surface expression of CD86 and CD83 was analyzed by flow cytometry. The histogram of nontreated or NiCl2-treated MoDC expressing CD86 or CD83 in the absence or the presence of 150 M of PDTC is expressed as a solid line, or a thick solid line, respectively, whereas that of DC without NiCl2 or PDTC treatment is expressed as a shaded line. A dotted line indicates DC stained with isotype control antibody. This shows representative data from four different experiments.
Discussion
Although the signal transduction pathways that mediate each phenotypic and functional change that accompanies DC maturation are not fully clarified, several recent papers have addressed this issue.Ardeshna et al (2000) demonstrated the role of p38 MAPK and NF-B pathways in the phenotypic changes of DC that occur under the stimulation of LPS. They found that p38 MAPK was responsible for the augmentation of CD80, CD83, and CD86, whereas the NF-B pathway mediated that of CD40, CD80, CD83, CD86, and HLA-DR. The role of p38 MAPK in the augmentation of CD80, CD83, and CD86 was confirmed byArrighi et al (2001), although they also detected its role in the augmentation of CD1a, CD40, and HLA-DR. In contrast to the phenotypic changes induced by LPS, our understanding of the role of MAPK or NF-B in cytokine production by DC is only fragmentary.Rescigno et al (1998) demonstrated by using a murine GM-CSF-dependent DC line that TNF- production by DC that were stimulated by LPS was suppressed by PD98059.Lu et al (1999) showed that the mice lacking MKK-3, which activates p38 MAPK, were defective in IL-12p40 production. These findings can be summarized as follows: (i) both p38 MAPK and NF-B mediate at least the augmentation of CD80, CD83, and CD86, and (ii) TNF- and IL-12p40 production is induced by ERK and p38 MAPK, respectively.
Most of these studies, however, analyzed DC stimulated by LPS or TNF-. It is well known that LPS or TNF- introduces the signals into the cells via their specific receptors, i.e., CD14 and Toll-like receptor 4 for LPS (Guha and Mackman, 2001) and TNF-R1 and TNF-R2 for TNF- (Wajant and Scheurich, 2001). In contrast, we do not have any information about how haptens stimulate DC or whether haptens have their own specific receptors. Therefore, we cannot speculate on the signal transduction pathways that mediate DC maturation after hapten stimulation simply by analogy with those after LPS stimulation. So far, studies on the signal transduction pathways that mediate DC maturation after hapten stimulation are very limited. Recently,Arrighi et al (2001) have reported that 2,4-dinitrofluorobenzene and NiSO4 induced p38 MAPK phosphorylation, whereas NiSO4 also slightly increased the phosphorylation of ERK, and that the augmentation of CD80 and CD83 induced by NiSO4 and, to a lesser extent, that of CD86, was blocked by SB203580.
This study confirmed the p38 MAPK phosphorylation in MoDC stimulated with haptens, such as DNCB and NiCl2, and the suppressive effect of SB203580 on the augmentation of CD83 by NiCl2. In addition, our data on the effects of irritants on MAPK or NF-B were consistent with those reported byArrighi et al (2001). Furthermore, we found that SB203580 suppressed the augmentation of CD86, CD83, and HLA-DR, and the production of IL-8 as well as their increased mRNA expression by MoDC stimulated with DNCB, and the IL-12p40 production by MoDC stimulated with NiCl2, whereas PD98059 suppressed the production of IL-1, IL-8, and TNF-, and their increased mRNA expression by MoDC treated with NiCl2. Consistent with the significant suppressive effects of PD98059, we detected strong phosphorylation of ERK by NiCl2, which is discrepant with the data reported byArrighi et al (2001). In our study, we also detected the phosphorylation of ERK by DNCB at a restricted concentration. As the DMSO that we used to solubilize DNCB also phosphorylated ERK, however, we considered that the phosphorylation of ERK by DNCB was not due to its biologic effect. Indeed, we could not detect any inhibitory effects of PD98059 on the cytokine production or augmentation of costimulatory molecules stimulated by DNCB. In addition, we found that only NiCl2, but not DNCB, activated NF-B, although we could not demonstrate any significant inhibitory effects of PDTC on the augmentation of CD86 or CD83, or the production of IL-1, IL-8, TNF-, or IL-12p40 induced by NiCl2.
When we compared these data with those obtained from the analysis of the signal transduction pathways in DC maturation after LPS stimulation (Rescigno et al, 1998;Lu et al, 1999;Ardeshna et al, 2000;Arrighi et al, 2001), it becomes clear that haptens share the similar pathways to induce phenotypic and functional changes as those so far reported in DC stimulated by LPS, e.g., p38 MAPK for the augmentation of CD83, CD86, and HLA-DR, the production of IL-8 and IL-12p40, and ERK for the production of IL-1 and TNF-. In our study, we could not succeed in demonstrating the role of the activation of NF-B in the augmentation of CD83 or CD86, or the production of IL-1, IL-8, IL-12p40, or TNF- by MoDC treated with NiCl2. So far we have not found any reasonable explanation for this observation.
Interestingly, in contrast to LPS and TNF- that activate both MAPK and NF-B, DNCB stimulated only p38 MAPK, but not ERK or NF-B. As cross-talk between MAPK and NF-B is highly possible, it is difficult to understand the role of each pathway in the phenotypic and functional changes in DC maturation induced by LPS, even by using specific inhibitors of each pathway. Indeed, there is a controversy about the role of p38 MAPK in NF-B activation by LPS. Using SB203580,Chen and Wang (1999) has reported that activation of p38 MAPK but not ERK by LPS resulted in the stimulation of NF-B-specific DNA-protein binding, whereasBaldassare et al (1999) andGarrett et al (1999) demonstrated that LPS-stimulated NF-B activation was not affected by the activation of p38 MAPK. By using DNCB, our studies could clearly demonstrate that p38 MAPK mediated the augmentation of CD86 and HLA-DR, and the increased production of IL-8 without NF-B activation. On the other hand, NiCl2 activated p38 MAPK, ERK, and NF-B. The augmentation of CD83 in MoDC stimulated by NiCl2, however, was suppressed by SB203580, but not by either PD98059 or PDTC, which suggested that p38 MAPK mediates this phenomenon. It is not clear why DNCB that also stimulates p38 MAPK cannot augment CD83, whereas NiCl2 increases it via activation of p38 MAPK. The augmentation of CD83 may require other signals distinct from p38 MAPK. In addition, we found that NiCl2 induced the augmentation of CD86, which was not suppressed by SB203580, PD98059, or even by PDTC. So far we have not known how NiCl2 augments the expression of CD86.
Unexpectedly, in our experiments, PD98059 augmented the expression of costimulatory molecules and HLA-DR antigen without any other exogenous stimulations, and it enhanced the expression of these molecules and the production of IL-12p40 synergistically with haptens. These data suggest that ERK may function as a suppressive signal for DC maturation. Our culture system that contained fetal bovine serum, IL-4, and GM-CSF might have induced a suppressive signal that is mediated by ERK.
Finally, in the sensitization phase of allergic contact hypersensitivity, the application of haptens induces several phenotypic and functional changes in DC that have been reported by several authors (Staquet et al, 1992;Aiba et al, 1993;Schwarzenberger and Udey, 1996;Kobayashi, 1997;Weiss et al, 1997;Dieu et al, 1998;Ebner et al, 1998;Sallusto et al, 1998;Saeki et al, 1999).Enk and Katz (1992) indicated the crucial role played by IL-1 secreted by Langerhans cells themselves in these phenomena. Our present data suggest that, before IL-1 secretion by DC, haptens stimulate MAPK or NF-B, which subsequently stimulates the augmentation of various surface molecules and the production of cytokines, such as IL-1 or TNF-.
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Acknowledgments
This study was supported in part by grant12670803 from the Ministry of Education, Science and Culture of Japan, by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of the Japanese Government, and by the Cell Science Research Foundation.
