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Dr K. Onoé, Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-0815 Japan. E-mail: firstname.lastname@example.org
Dendritic cells (DCs) are maturated by a variety of stimuli. However, the precise mechanisms underlying the maturation of DCs are not fully understood. In the present study, we analysed the effects of tumour necrosis factor-α (TNF-α) and 2,4-dinitrochlorobenzene (DNCB) on phenotypic maturation and p38 mitogen activated protein kinase (MAPK) activity, using a murine DC line. TNF-α markedly increased the surface expression of major histocompatibility complex (MHC) and costimulatory molecules, CD86 and CD80, on DCs. DNCB more markedly enhanced the surface expression of costimulatory molecules, but showed less stimulatory capability on MHC molecules, compared with TNF-α. Simultaneous treatment of DCs with TNF-α and DNCB showed additive enhancement of costimulatory molecule expression. TNF-α activated p38 MAPK in DCs only at an early time-point (15 min). In contrast, DNCB activated p38 MAPK at later time-points (3–6 hr). SB203580, a specific inhibitor of p38 MAPK, partially or markedly inhibited the phenotypic changes of DCs induced by TNF-α or DNCB, respectively. In addition, N-acetyl-l-cysteine, a reducing supplier, completely inhibited the DNCB-induced expression of MHC and costimulatory molecules, but not those induced by TNF-α. These findings demonstrate that TNF-α and DNCB activate the p38 MAPK pathway at an early and a late phase, respectively, and thereby induce DC maturation through different signal pathways.
stress-activated protein kinase/c-Jun N-terminal kinase
tumour necrosis factor-α.
Dendritic cells (DCs) are potent antigen-presenting cells (APC) and play major roles in the regulation of immune responses to various antigens.1–3 DCs exhibit a unique ability to activate naive T cells, which depends on their maturational stage. Immature DCs are present in almost all tissues and internalize antigens from the environment with high efficiency. Upon encountering foreign antigen, DCs are rapidly activated by complex processes and become mature.2,3 The mature DCs show a high expression of major histocompatibility complex (MHC) and costimulatory molecules (including CD80 and CD86) on their surface. During the maturational processes, DCs migrate from each tissue to the regional lymph nodes (LN); this migration is associated with the change of surface receptors for chemokines. Mature DCs located in the T-cell area of LN present MHC–peptide complexes with costimulatory molecules to T cells and thereby adaptive immune responses are initiated.1,3,4 Thus, it seems that DC maturation is an essential process for inducing adaptive immune responses in vivo.
So-called adjuvants are usually required to induce adaptive immune responses. It seems that adjuvants activate innate immune responses, including DC maturation. On the other hand, 2,4-dinitrochlorobenzene (DNCB), a contact sensitizer, can induce adaptive immune responses in the absence of any adjuvants. Thus, this chemical may directly activate DCs, including Langerhans' cells, in vivo.5 Recently, it has been reported that DNCB enhances the surface expression of MHC and costimulatory molecules on DCs in vitro.6,7 However, the precise mechanisms underlying the DNCB-induced enhancement of MHC and costimulatory molecule expression on DCs are not well understood.
DCs are maturated by inflammatory cytokines, such as tumour necrosis factor (TNF)-α, and by bacterial components such as lipopolysaccharide (LPS).8–10 It has been reported that TNF-α and LPS activate p38 mitogen-activated protein kinase (MAPK) of DCs, and SB203580, a specific inhibitor of the p38 MAPK, partially inhibits the DC maturation induced by these stimuli.10–12 Thus, DC maturation appears to be, at least in part, regulated by the p38 MAPK pathway. p38 MAPK belongs to the MAPK family.13,14 Two groups of MAPK, other than the p38 MAPK subfamily, have been identified in mammals: the extracellular signal-related kinase (ERK) subfamily15,16 and the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) subfamily.17,18 Signal transduction via MAPK plays an important role in cellular responses, including growth factor-induced cell proliferation, differentiation and survival. MAPKs are activated, following engagement of a variety of cell-surface receptors, via dual tyrosine and threonine phosphorylation. Indeed, previous studies have shown that ligation of TNF-α with TNF-receptor 1 initiates the activation of ERK, SAPK/JNK and p38 MAPK, in several cells and cell lines.19–22
Winzler et al.23 established a growth factor-dependent immature DC line from splenocytes of adult C57BL/6 mice. Using the similar in vitro differentiation system of DCs, a number of important findings have been reported and verified.24–32 Although this DC line showed an immature phenotype, various activating signals, such as inflammatory cytokines and bacterial components, promoted maturation of the cells. Therefore, this in vitro differentiation system appears to be useful for precisely elucidating the signal-transduction system involved in DC maturation. According to the method of Winzler et al.,23 we also established a homogeneous immature DC line (BC1) from BALB/c splenocytes.29 In the present study, using the BC1 culture system, we analysed the effects of TNF-α and DNCB on the phenotypic maturation and p38 MAPK activity of DCs. We show, herein, that both TNF-α and DNCB activate the p38 MAPK pathway, but at an early and a late phase, respectively; activation is followed by phenotypic changes of the DCs. We also demonstrated that N-acetyl-l-cysteine (NAC), a reducing supplier, completely inhibits the DNCB-induced phenotypic change of DCs. It has been reported that DNCB reduces both the cellular glutathione (GSH) and thiol content in mammalian cells33,34 and thereby generates intracellular oxidative stress, including lipid peroxidation.35 This generation of oxidative stress may be attributed to increases in the number of intracellular reactive oxygen species. Therefore, DC maturation induced by DNCB may be related to the effect on the intracellular redox status.
Materials and methods
Reagents and antibodies
Recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) and TNF-α were purchased from PeproTech (London, UK). DNCB was purchased from Wako Pure Chemicals (Osaka, Japan). DNCB was dissolved in ethanol (EtOH), and a 0·04% (vol/vol) concentration of EtOH was used as a negative control. SB203580 was obtained from Calbiochem (La Jolla, CA) and used at 20 µm. Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11c monoclonal antibody (mAb) (HL3), phycoerythrin (PE)-conjugated anti-mouse CD80 mAb (16-10A1), PE-conjugated anti-mouse CD86 mAb (GL1), biotin-conjugated anti-mouse H-2Kd mAb (SF1-1.1), biotin-conjugated anti-mouse I-Ad mAb (AMS-32.1), and streptavidin Cy-Chrome™, were obtained from PharMingen (La Jolla, CA). As a control, FITC-conjugated hamster immunoglobulin G (IgG) was obtained from PharMingen. PE-conjugated rat IgG2a, biotin-conjugated mouse IgG2a and biotin-conjugated mouse IgG2b were purchased from Immunotech (Marseille, France). PE-conjugated hamster IgG was obtained from Caltag Laboratories (Burlingame, CA). NAC was purchased from Sigma Chemical Co. (St Louis, MO). NAC was dissolved in Iscove's modified Dulbecco's medium (IMDM).
The DC line, BC1, from BALB/c splenocytes, was generated as described previously.23,29 BC1 cells were cultured and expanded in IMDM containing 10% fetal calf serum, 30% NIH/3T3 supernatant, and mouse recombinant GM-CSF (henceforth referred to as R1 medium). BC1 cells are CD11c+ MHC class I+ MHC class II+ CD80+ CD86+. Although BC1 cells show an immature phenotype, various activating signals, such as TNF-α and LPS, promote maturation of this precursor line. The maturated BC1 cells highly express MHC and costimulatory molecules and show potent allostimulatory and antigen-presenting capability as compared with immature BC1 cells.29,32
Analysis of cell-surface markers by flow cytometry
BC1 cells (2 × 105 cells/ml) were treated with DNCB and/or TNF-α for 24 hr in R1 medium. To examine the effect of SB203580 or NAC, cells were pretreated with SB203580 or NAC for 1 hr and then treated with DNCB or TNF-α in the presence of SB203580 or NAC. The cells were detached with 3 mm EDTA (5 min at 37°). The cells were incubated with 2.4G2 (rat anti-mouse FcR II/III receptor, CD32) supernatant to prevent binding to FcR II/III and then stained using FITC-, PE-, or biotin-conjugated mAb and streptavidin-Cy-Chrome™. Flow cytometry was performed on an EPICS® XL (Coulter Co., Miami, FL), as described in a previous study.36 Results shown represent the mean fluorescence intensity (MFI), after subtraction of the MFI of the isotype-matched control Ab.
In vitro p38 MAPK assay
BC1 cells (4 × 106) in R1 medium were preincubated for 1 hr, then stimulated with TNF-α (40 ng/ml) or DNCB (2·0 µm) for the indicated times at 37°. Reactions were stopped by rapidly cooling on ice. The cells were washed in ice-cold phosphate-buffered saline (PBS) containing 2 mm EDTA. The kinase activity of p38 MAPK was determined using a commercially available kinase assay kit (New England BioLabs, Beverly, MA), according to the manufacturer's protocol. The cell lysate was subjected to immunoprecipitation with immobilized anti-phospho-p38 MAPK mAb. The complex was then subjected to the kinase reaction with a substrate, activating transcription factor (ATF)-2. After the kinase reaction, phospho-ATF-2 was detected by Western blotting.
The Student's t-test was used to analyse data for significant differences. P-values of < 0·05 were regarded as significant.
Effect of TNF-α and DNCB on the expression of MHC and costimulatory molecules on BC1 cells
A murine DC line, BC1, from BALB/c splenocytes has been previously established.29 Unstimulated BC1 cells maintain the form and qualities that are characteristic of immature DCs. Inflammatory cytokines and bacterial components promote phenotypical and functional maturation of these cells.29–32 We first analysed the effects of DNCB on phenotypic changes of BC1 cells by flow cytometry and compared the results with those obtained using TNF-α (Fig. 1a). In agreement with previous studies,29,32 an optimal dose of 40 ng/ml TNF-α increased the surface expression of costimulatory molecules (CD86 and CD80) and MHC molecules [MHC class II (I-Ad) and MHC class I (H-2Kd)] on BC1 cells. DNCB also markedly increased the surface expression of CD86, CD80 and MHC class II on BC1 cells, in a dose-dependent manner, while showing little effect on that of MHC class I (Fig. 1b). DNCB markedly enhanced the expression of CD80 and CD86, but showed only a moderate effect on the expression of MHC compared with TNF-α.
We then examined the effect of simultaneous treatment with TNF-α (40 ng/ml) and DNCB (2·0 µm) on the expression of MHC and costimulatory molecules on BC1 cells (Fig. 2). In the presence of both TNF-α and DNCB, costimulatory molecule expression was markedly augmented. The expression levels of CD86 and CD80 were significantly higher than those induced by an optimal dose of TNF-α alone. Although the level of MHC molecules on BC1 cells treated with both TNF-α and DNCB were slightly higher than those on BC1 cells treated with TNF-α alone, no statistically significant differences were detected between the two groups.
Involvement of p38 MAPK in TNF-α- or DNCB-induced phenotypic change of BC1 cells
Several reports have suggested that p38 MAPK is involved in phenotypic and functional maturation of DCs.9–12 Therefore, we next examined the effect of TNF-α or DNCB on the p38 MAPK activity of BC1 cells (Fig. 3). BC1 cells were incubated with TNF-α or DNCB for 15 min or 3 hr, and the in vitro kinase assay for p38 MAPK activation was performed using ATF-2 as a substrate for p38 MAPK. Phosphorylation of ATF-2 was determined by Western blotting using anti-phospho-ATF-2 mAb. A modest, but distinct, band of phospho-ATF-2 was detected in unstimulated BC1 cells. TNF-α significantly enhanced the p38 MAPK activity in BC1 cells 15 min after treatment, whereas DNCB showed no effect on the p38 MAPK activity at this early time-point. DNCB induced no significant enhancement of p38 MAPK activity until 1 hr after stimulation (data not shown). By contrast, when the p38 MAPK activity in BC1 cells was analysed 3 hr after stimulation, the activity in TNF-α-treated BC1 cells showed a decrease similar to that observed in the control. The p38 MAPK activity in BC1 cells treated with DNCB showed a significant increase at this time-point as compared with the p38 MAPK activity in control cells. The DNCB-induced enhancement of p38 MAPK activity was maintained for at least 6 hr after stimulation (data not shown). Thus, TNF-α and DNCB induced early- and late-phase activation, respectively, of p38 MAPK in DCs.
To elucidate the role of p38 MAPK activation by TNF-α or DNCB in DC maturation, we then analysed the effect of SB203580, a specific inhibitor of the p38 MAPK pathway, on the TNF-α- or DNCB-induced phenotypic change of BC1 cells. Figure 4 shows that pretreatment of BC1 cells with SB203580 (20 µm) alone slightly decreased the constitutive expression of I-Ad, but exerted no significant influence on the expression of CD86 and CD80 on unstimulated BC1 cells. SB203580 slightly, but significantly, decreased CD86 expression on TNF-α-treated BC1 cells, whereas SB203580 showed no significant influence on the expression of CD80 and I-Ad. In contrast, SB203580 markedly reduced the expression of CD86, CD80 and I-Ad on DNCB-treated BC1 cells. The viability of BC1 cells was unaltered by the pretreatment with SB203580 (20 µm) (data not shown). Therefore, p38 MAPK activation appears to be partially or considerably involved in the phenotypic change of BC1 cells stimulated with TNF-α or DNCB, respectively.
Effect of NAC on the DNCB-induced phenotypic change of BC1 cells
It has been reported that DNCB decreases the intracellular GSH content and thereby results in the development of intracellular oxidative stress.34,35 We considered that DNCB-induced oxidative stress might be involved in the phenotypic maturation of BC1 cells, and examined the effect of NAC, which can potently decrease intracellular oxidative stress,37,38 on the DNCB-induced phenotypic change of BC1 cells (Fig. 5). NAC decreased the constitutive expression of CD80, but had no effect on the expression of CD86 and I-Ad on unstimulated BC1 cells. NAC completely inhibited the DNCB-induced expression of CD80, CD86 and I-Ad, but had no significant effect on the TNF-α-induced enhancement of CD86, CD80 and I-Ad expression.
Activation and maturation states of DCs are regulated by various extracellular stimuli, including cytokines, costimulatory molecules and bacterial products. These events are accompanied with alterations of the morphological, phenotypical and functional properties of DCs.8–10,29,30 TNF-α, an inflammatory cytokine, potently promotes the activation and maturation of DCs.12,29 In the present study, we examined the effect of DNCB on phenotypic changes of DCs and compared the effects with those stimulated by TNF-α. DNCB markedly enhanced the expression of CD86 and CD80 on BC1 cells, while showing only a moderate effect on the expression of MHC molecules, as compared with TNF-α. Thus, DNCB may predominantly promote the signal pathway involved in the up-regulation of CD80 and CD86 expression, rather than MHC expression. This finding indicates that the expression of costimulatory molecules and MHC molecules is regulated by different pathways in DCs. In addition, the markedly up-regulated expression of costimulatory molecules, stimulated by TNF-α alone, was further augmented in the presence of DNCB. These findings suggest that the mechanism underlying the DNCB-induced up-regulation of costimulatory molecules is different from that of TNF-α.
We found that TNF-α enhanced p38 MAPK activity in BC1 cells at an early stage, and that SB203580 (a specific inhibitor of the p38 MAPK pathway), partially inhibited the TNF-α-induced phenotypic change of the BC1 cells. Similar results have been demonstrated in previous studies using human monocyte-derived DCs.10,12 Therefore, TNF-α-induced DC maturation appears to be, in part, related to the activation of p38 MAPK. Recently, it has been reported that DNCB also enhances p38 MAPK activity in human monocyte-derived DCs, and thereby induces phenotypic change of the cells.39 However, the time course of p38 MAPK activity was not analysed in this report and the difference in the p38 MAPK activity between stimulation with TNF-α or DNCB was not demonstrated.
In the present study, we compared p38 MAPK activities at early and late time-points after stimulation with TNF-α or DNCB. Unlike TNF-α, DNCB had no effect on the p38 MAPK activity at an early time-point. However, DNCB significantly enhanced p38 MAPK activity 3–6 hr after stimulation. In addition, the DNCB-induced phenotypic change was markedly inhibited by SB203580. Therefore, the DNCB-induced phenotypic change appears to be promoted by the late-phase activation of p38 MAPK in DCs. This is a striking difference from that induced by TNF-α. As described above, the TNF-α-induced activation of p38 MAPK occurred at an early time-point and SB203580 exerted only a marginal effect on the phenotypic change of BC1 cells induced by TNF-α. The involvement of p38 MAPK signalling in the phenotypic change of DCs may differ among the various stimuli provided and timing after the stimulation.
Recently, several studies have demonstrated that DNCB irreversibly inhibits thioredoxin reductase and enhances NADPH oxidase activity in vitro, which results in a marked increase in the number of reactive oxygen species.40,41 The intracellular production of reactive oxygen species appears to be related to intracellular signal transductions via p38 MAPK and/or nuclear factor (NF)-κB pathways in mammalian cells.42,43 Although several studies have demonstrated a DNCB-induced phenotypic change in DCs, no studies have examined the relationship between DNCB-induced redox events and the phenotypic changes in DCs. In the present study, we found that NAC almost completely inhibited the DNCB-induced phenotypic changes in BC1 cells. Hence, these phenotypic changes appear to be mediated via a redox-sensitive pathway. Although the possibility that NAC reacts directly with DNCB could not completely be eliminated, we postulate that DNCB-induced redox events enhance p38 MAPK activity, which results in phenotypic changes of DCs.44 On the other hand, NAC showed no significant effects on the TNF-α-induced phenotypic changes of BC1 cells. Therefore, it seems that the expression of costimulatory molecules and MHC on DCs are regulated by, at least, redox-sensitive and -insensitive pathways, depending on the nature of the stimuli.
In conclusion, we have demonstrated two different maturational signals induced by TNF-α and DNCB in DCs. The TNF-α-induced maturational signal appears to be redox independent and partially dependent on p38 MAPK activation; this signal markedly enhanced the cell-surface expression of MHC and costimulatory molecules in DCs. On the other hand, the DNCB-induced maturational signal appears to be mediated via both redox events and the p38 MAPK pathway; this signal markedly enhanced the cell-surface expression of costimulatory molecules, but not MHC molecules. The expression pattern of MHC and costimulatory molecules on DCs during the presentation of antigen influences both the quality and the quantity of adaptive immunity of antigen-specific T cells. Thus, elucidation of the rather complex pathways controlling the expression of MHC and costimulatory molecules on DCs may lead to the development of clinical applications exploiting this new regulation system for the treatment of various infectious diseases and immune disorders.
This study was supported by a Grant-in-Aid for Scientific Research (S), a Grant-in-Aid for Scientific Research on a Priority Area (C) and a Grant-in-Aid for Enocuragement of Young Scientists (B) by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This study was also supported by the Tomakomai East Hospital Foundation.