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Immunobiology 214 (2009) 350–358

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doi:10.1016/j.im

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The pivotal role of p38 and NF-jB signal pathways in the maturation of

human monocyte-derived dendritic cells stimulated by streptococcal

agent OK-432

Ke Pan1, Hui Wang1, Wan-li Liu, Hua-kun Zhang, Jun Zhou, Jian-jun Li,De-sheng Weng, Wei Huang, Jian-cong Sun, Xiao-ting Liang, Jian-chuan Xia�

The State Key Laboratory of Oncology in Southern China, Department of Experimental Research, Sun Yat-sen University Cancer

Center, 651 Dongfeng Road East, Yuexiu District, Guangzhou, Guangdong Province 510060, PR China

Received 18 August 2008; accepted 28 October 2008

Abstract

OK-432, a streptococcal preparation, has been shown as an effective activator to induce human monocyte-deriveddendritic cells maturation. During this process, the activation of Toll-like receptor 4 plays an important role. However,the signaling pathway involved in has not been fully understood. In the present study, we investigated the underlyingmechanisms, by which OK-432 induced maturation of human monocyte-derived DCs (MoDCs). We observed thatexposure of immature MoDCs to OK-432 activated the p38 MAPK and NF-kB pathway, accompanied up-regulatedthe surface expression of maturation markers CD80, CD83, CD86 and HLA-DR, increased secretion of TNF-a, IL-12and chemokine, IP-10. In addition, T cells stimulatory capacity was also enhanced. The maturation of MoDCsstimulated by OK-432 was inhibited by treatment with p38 pathway inhibitor, SB203580, or NF-kB pathway inhibitor,BAY-117082. Whereas, blocking of JNK pathway with SP600125 or ERK pathway with PD98059 did not influenceOK-432-induced DCs maturation. Taken together, our data indicated that OK-432-induced DCs maturation was due,at least partly to the activation of p38 and NF-kB pathway.r 2008 Elsevier GmbH. All rights reserved.

Keywords: p38 Pathway; NF-kB pathway; OK-432 stimulation; Dendritic cells

Introduction

OK-432, a penicillin-killed and lyophilized prepara-tion of a low-virulence strain of Streptococcus pyogenes

(group A) (Okamoto et al., 1967), has been successfullyused as an immunotherapeutic agent in many types of

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.10.008

ing author. Tel.: +86 20 87343173;

43392.

ess: xiajch@mail.sysu.edu.cn (J.-c. Xia).

authors contributed equally to this work.

malignancies without apparent side effects for over 20years (Torisu et al., 1983; Watanabe and Iwa, 1987;Kikkawa et al., 1993; Maehara et al., 1994; Kitaharaet al., 1996). The anti-tumour effects of OK-432 weredue to the activation of a variety of effector cells such asneutrophils, macrophages, natural killer (NK) cells,autotumour killer cells and cytotoxic T lymphocytes,and increased secretion of multiple cytokines includinginterleukin 1 (IL-1), IL-2, IL-6, tumor necrosis factor-a(TNF-a), and interferon-gamma (IFN-g) (Oshimi et al.,1980; Saito et al., 1982; Misaki et al., 1992). Recently, it

ARTICLE IN PRESSK. Pan et al. / Immunobiology 214 (2009) 350–358 351

has been demonstrated that OK-432 can induces thematuration of dendritic cells (DCs) that are professionalantigen-presenting cells (APCs), more strongly than theother known DCs-maturating agents such as lipopoly-saccharide (LPS) and inflammatory cytokines. In addi-tion, OK-432-stimulated DCs can induce tumor antigen-specific cytotoxic T lymphocytes (CTLs) in vitro as wellas in vivo (Itoh et al., 2003; Kuroki et al., 2003;Nakahara et al., 2003; Ahmed et al., 2004; Okamotoet al., 2004). Okamoto et al. demonstrated that theexpression of TLR4 on dendritic cells surface is veryimportant for the anticancer effect of OK-432-activateddendritic cells-based immunotherapy, and found thatOK-432 induced functional maturation of DCs via Toll-like receptor (TLR) 4 signal pathway (Okamoto et al.,2003, 2004, 2006).

The TLRs are key initiators of innate and adaptiveimmune responses. It is well known that NF-kB andmitogen-activated protein kinase (MAPK) are twoimportant downstream signal pathways involved inTLRs-mediated action (Akira and Takeda, 2004). ManyTLR ligands, such as LPS, heat-shock protein 60(HSP60) and CpG-containing DNA, have been provedto activate effectively NF-kB and MAPK pathways inDCs. Blocking these two pathways activation interruptsthe ligand-induced maturation of DCs (Arrighi et al.,2001; Xie et al., 2003; Flohe et al., 2003; Osawa et al.,2006). OK-432 is also a ligand of TLR4. However, thesignaling mechanism of the OK-432-stimulated matura-tion of DCs has not been explored.

In the present study, we examine the role of NF-kBand MAPK signal pathways in the maturation ofhuman monocyte-derived dendritic cells (MoDC) sti-mulated by streptococcal agent OK-432. Our resultsshowed that OK-432-induced MoDC maturation de-pended, at least partly on the activation of p38 MAPKand NF-kB pathways, was not associated with theactivation of ERK and JNK pathways.

Materials and methods

Culture of human DCs

PBMC were isolated from peripheral blood of healthydonors by Ficoll–Hypaque gradient centrifugation. Andthen, monocytes were allowed to adhere in six-wellculture plates for 1 h in 37 1C. Nonadherent cells wereremoved and cultured in 20U/ml IL-2 medium forfurther using. Adherent cells were cultured in RPMI1640 complete medium supplemented with 2% heat-inactivated pool human AB serum, 50U/ml penicillin,50mg/ml streptomycin, 2mM L-glutamine, 10mMHepes, 1000U/ml GM-CSF and 300U/ml IL-4 (Bio-Source, USA). Half of the old medium was replaced

every 2 days with fresh medium. After culture for 6 days,immature dendritic cells (iDCs) were generated.

Chemical treatment of immature DC

On day 6, DC were harvested and treated with0.1KE/ml (10 mg/ml) OK432 (Shandong Lukang Phar-maceutical Co., China) for 48 h. In some experiments,DC were pre-treated for 30min by either SB203580(10 mM dissolved in DMSO, Sigma, USA), SP600125(10 mM dissolved in DMSO, Sigma), PD98059 (20 mMdissolved in DMSO, Sigma), BAY-117082 (10 mMdissolved in DMSO, Sigma), or DMSO 0.05% usedfor vehicle control of the inhibitors. Cell viability wasassessed by the Trypan blue exclusion method.

Flow cytometry analysis

Cultured DCs were re-suspended at 2.5� 105 cells in100 ml of culture medium and incubated for 30min at4 1C with monoclonal antibodies (mAbs) or appropriateisotypic controls. After three washes in cold phosphate-buffered saline (PBS) supplemented with 0.5% of bovineserum albumin (BSA), cells were fixed with 2%paraformaldehyde in PBS. The following mAbs wereused: FITC conjugated anti-HLA-DR (L243, BDBiosciences, USA), FITC conjugated anti-CD80(L307.4, BD Biosciences), FITC conjugated anti-CD83(HB15e, BD Biosciences) and FITC conjugated anti-CD86 (2331, BD Biosciences). Cells were analyzed on anFACSCaliburs cell analyzer (Becton Dickinson) usingCellQuests software (Becton Dickinson). Cellular deb-ris were eliminated from the analysis using a gate onforward and side scatter. For each sample 104 cells wereanalyzed. Results of phenotypic analysis are expressedas relative cell fluorescence intensity (relative FI), whichis the ratio between the total mean cell fluorescenceintensity obtained using the specific antibody directed tothe cell marker to the total mean cell fluorescenceintensity obtained using a control fluorescent antibodyof the same isotype as the specific antibody.

Western blot analysis

Immature DCs were exposed to OK432 or LPS forthe indicated periods of time. Thereafter, cells werewashed twice with cold PBS and incubated with 100 mlof RIPA lysis buffer, and lysates were cleared bycentrifugation (14,000 rpm) at 4 1C for 10min. About40 mg protein samples were run on a 12% SDS-PAGEgel and transferred to a polyvinylidine difluoridemembrane (Pall, USA). Membranes were blocked for1 h in Tris-buffered saline with 0.05% Tween 20 (TBST)containing 5% skim milk. The membrane was probedwith antibodies raised against phosphorylated forms of

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Fig. 1. OK-432-activated MAPK and NF-kB signaling path-

way in human monocyte-derived dendritic cells. Immature

DCs were incubated for the indicated times with OK-432

(0.1KE/ml), LPS (100 ng/ml) or PBS. The levels of phosphor-

ylation of MAPK family or NF-kB protein were determined

by Western blotting of whole-cell lysates. The corresponding

Western blots for the total levels of these kinases are depicted

in the bottom panels.

K. Pan et al. / Immunobiology 214 (2009) 350–358352

IkB-a, p38 MAPK, JNK 1/2 or ERK1/2 (all from SantaCruz, USA, 1:500 diluted in blocking buffer) overnight.The membrane was then washed three times with TBSTfor 10min and incubated with HRP-conjugated goat-anti-mouse secondary antibody (Santa Cruz, 1:2000diluted in blocking buffer) for 1 h in room temperature.After being washed three times, the membrane wasdeveloped by an enhanced chemiluminescence system(ECL, Pierce, USA). Antibodies to total IkB-a, p38MAPK, JNK 1/2 or ERK1/2 (Santa Cruz) were used asloading controls.

Analysis of cytokines

Cytokines TNF-a, IL-12p70, IP-10 in the super-natants of differently treated DCs cultures weremeasured by ELISA kits (BioSource, USA) accordingto the manufacturer’s protocols.

Mixed lymphocyte reaction (MLR)

For proliferation assays, nonadherent cells (2� 105)collected from freshly isolated PBMC were co-culturedwith 1� 104 of DCs in triplicate in round-bottom 96-wells for 5 days. The proliferation assay was carried outin RPMI 1640 medium supplemented with 2% humanAB serum, 100U/ml penicillin, and 100 mg/ml strepto-mycin. Dye solution was added to each well andincubated for 2 h according to the Protocol of Cell Titer96 Nonradioactive Cell Proliferation Assay kit (Prome-ga, USA). For measurement, we used the MicroplateImaging System (Bio-Rad) at an OD of 490 nm.

Statistical analysis

Data were compared using the Student’s t test, anddifferences of po0.05 were considered to be significant.Each experiment was repeated at least three times.

Results

OK-432-induced phosphorylation of members of

MAPK and NF-jB

Activation of MAPK or NF-kB signal pathwayrequires the phosphorylation of p38, JNK, ERK orIkB-a molecule. Thus, we first measured phosphoryla-tion of p38, JNK, ERK involved in the MAPK pathwayand IkB-a in the NF-kB pathway in response to the OK-432 stimulation by western blot analysis. Our dateshowed that OK-432-induced activation of NF-kB, p38MAPK and ERK MAPK pathway (Fig. 1). Treatmentof immature DCs with OK-432 induced the phosphor-ylation of IkB-a, p38 MAPK and ERK1/2 within

30min. However, unlike LPS, phosphorylated formsof JNK 1/2 could not be detected after OK-432treatment, indicating that OK-432 activates similar butnot identical pathways to LPS in MoDCs.

Inhibition of p38 MAPK and NF-jB activity

prevented the OK-432-induced maturation of DCs

In following experiments, we evaluated the effects ofspecific inhibitors of MAPK or NF-kB activation on theexpression of DC maturation markers. As showed inFig. 2, OK-432 stimulation significantly up-regulatedCD80, CD83 and CD86 expression, but slightly up-regulated HLA-DR expression. Blocking the p38MAPK pathway with SB203580 significantly inhibitedthe OK-432 induced the up-regulation of CD80, CD83,CD86, and down-regulated HLA-DR. In contrast,blocking the JNK and ERK pathway with SP600125and PD98059, respectively, slightly up-regulated CD80and CD83, and had no effect on CD86 and HLA-DRexpression. BAY-117082, a specific inhibitor of NF-kB,also inhibited these costimulatory molecules and HLA-DR expression, albeit to a lesser extent than thatobserved with SB203580-treated DCs (Fig. 2).

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Fig. 2. Effects of inhibitor of MAPK or NF-kB signaling pathway on OK-432-induced DCs maturation. Immature DCs were

incubated for 30min with 10 mM SB203580, 10mM SP600125, 20mM PD98059, 10 mM BAY-117082, or DMSO (vehicle) and then

cultured for 2 days with OK-432 (0.1KE/ml). The expression of cell surface molecules was measured using the flow cytometry. (A)

The product of the percentage of cells expressing various cell surface antigens. (B) The mean cell fluorescence of the whole

population of cells under scrutiny. Bars represent SE on the mean. MFI, means fluorescent intensity.

K. Pan et al. / Immunobiology 214 (2009) 350–358 353

stimulated DCs-induced T cell responses

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Fig. 2. (Continued)

K. Pan et al. / Immunobiology 214 (2009) 350–358354

Effect of MAPK and NF-jB inhibitor on OK-432-

induced cytokines production from mature DCs

Cytokines secreted by DCs are instrumental to theprocess of priming of native CD4+ and CD8+ T cellsto initiate an immune response. Thus, we evaluated therelative role of MAPK and NF-kB in cytokinesproduction of OK-432-stimulated DCs. Immature DCswere stimulated for 48 h with OK-432, supernatantswere collected from cultured cells and ELISA wasemployed to measure the concentration of DC-maturingcytokines, TNF-a, Th1-inducing cytokines, IL-12p70and chemokine for Th1 cells, IFN-g-inducible protein-10 (IP-10). The results in Fig. 3 showed that OK-432stimulation-induced detectable secretion of TNF-a, IL-12p70 and IP-10 by DCs. Blocking either JNK signalingpathway with SP600125 or ERK signaling pathway withPD98059 showed little influence on the productionof TNF-a, IL-12p70 and IP-10. However, blockingthe p38 MAPK with SB203580 or NF-kB withBAY-117082 significantly inhibited secretion of IL-12p70 and IP-10. Both SB203580 and BAY-117082treatment only partially inhibited TNF-a production,indicating that TNF-a secretion from OK-432-stimulatedDCs might be partly involved in these two signalingpathway.

Roles of MAPK and NF-jB pathways in OK-432-

Mature DCs have the capacity to induce proliferationin T cells at a much higher level than immature DCs. Wetherefore tested the roles of MAPK and NF-kB pathwaysin the ability of OK-432-activated DCs to stimulate Tlymphocytes in MLR. As showed in Fig. 4, OK-432-treated DCs strongly enhanced T cell proliferation.Blocking the NF-kB or p38 MAPK pathways withBAY-117082 or SB203580 significantly reduced thecapacity of OK-432-induced T cell proliferation, and wefound SB203580 treatment completely inhibited matureDCs to induce T cell proliferation. However, blockingJNK or ERK pathway with SP600125 or PD98059 didnot interrupt T cell proliferation. Our data indicated thatthe NF-kB and p38 MAPK pathways, especially p38pathway, play an important role in the stimulatorycapacity of OK-432-activated DCs.

Discussion

The in vitro maturation process of human MoDC canbe initiated by various stimuli. Many stimuli such as

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Fig. 3. Effects of inhibitor of MAPK or NF-kB signaling pathway on the cytokines release of OK-432-induced DCs. Immature DCs were pretreated or not with indicated

inhibitors for 30min and then stimulated with OK-432 (0.1KE/ml) for 2 days. The supernatant were collected for TNF-a, IL-12p70 and IP-10 production determine by using

ELISA method. Data are means7SD of triplicates from one experiment.

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Fig. 4. Effects of signaling transduction via MAPK or NF-kB pathways on autostimulatory ability of OK-432-activated DCs.

Immature DCs were incubated for 30min with indicated inhibitors or DMSO and then stimulated with 0.1KE/ml OK-432 for 2

days. Autologous T lymphocytes were mixed with different treated DCs at a ratio of 20:1 (DCs to T cells) and cocultured for 5 days.

T cell proliferation was performed in indicated numbers of T cells by the Cell Titer 96 Nonradioactive Cell Proliferation Assay kit

according to the protocol.

K. Pan et al. / Immunobiology 214 (2009) 350–358356

LPS or inflammation cytokines induce activation ofNF-kB and MAPK signaling pathway in DCs duringmaturation. The aforementioned findings promoted usto investigate whether NF-kB and MAPK signalingpathways are also involved in the process of OK-432-induced maturation of DCs.

In consistent with LPS (Ardeshna et al., 2000), OK-432 treatment quickly induced activation of p38 MAPKand NF-kB pathway, indicating that DCs maturationinduced by OK-432 might be depend, at least partly onthe activation of p38 MAPK and NF-kB pathway. Thishypothesis was further confirmed by using the specificinhibitor treatment. Treatment with p38 inhibitor,SB203580 or NF-kB inhibitor, BAY-117082 signifi-cantly blocked OK-432-induced MoDCs maturation,accompanied with down-regulation of maturation mar-ker and decrease-secretion of cytokines of DCs. More-over, T cells proliferation response was also inhibited.Our results support the notion that p38 MAPK and NF-kB pathways were highly conserved in regulating theimmune function of DCs.

The involvement of ERK activation in DC matura-tion remained unclear. Recent studies showed thatthe ERK signaling pathway does not affect or negativelyregulate the maturation of MoDCs in terms ofphenotypic maturation, IL-12 production or functionalmaturation (Yu et al., 2004; Hacker et al., 1999;Rescigno et al., 1998; Puig-Kroger et al., 2001).However, Miyazawa et al. (2008) and Nukada et al.(2008) found that inhibition of ERK pathway withPD98059 suppressed CD86, CD54 and CD40 expressionand IL-8 production of DCs induced by 2, 4-dinitro-chlorobenzene (DNCB) and nickel sulfate (NiSO4),

indicating that ERK pathway positively regulated thesetwo hapten-induced DCs activation. In our study, weobserved that OK-432 treatment also activated ERKpathway. But, blocking ERK pathway with PD98059did not affected the phenotypic maturation, cytokineproduction and stimulatory capacity of MoDCs, in-dicating that the activation of ERK pathway might notbe associated with OK-432-induced DCs maturation.The precise role of ERK pathway need to be furtherstudied in near future.

We also found that both inhibition p38 and NF-kBpathway did not reduce dramatically secretion of TNF-a, which is inconsistent with the results of LPS or otherstimulators treatment (Palova-Jelınkova et al., 2005;Termeer et al., 2002), suggesting that p38 and NF-kBpathway might be only partly involved in TNF-aproduction of OK-432-stimulated DCs. Miyazawa etal. (2008) also found that inhibition of p38 pathwayactivation selectively blocked DNCB-induced TNF-arelease, but not NiSO4-induced release. Philpott et al.(2004) found that unlike TNF-a production associatedwith exposure to LPS, adenovirus induction of TNF-awas not dependent on the MyD88 signaling pathway,but dependent on signaling by phosphoinositide-3-OHkinase (PI3K), as determined by wortmannin andLY294002 blocking experiments. Further work isrequired to clarify whether OK-432 induced TNF-aproduction via PI3K pathway.

OK-432 has been shown could be applied to developDCs-based cancer vaccine. In this report we have shownthat the NF-kB and p38 MAPK play an important rolein MoDCs mature triggered by OK-432. Our findsmight be useful to manipulate the OK-432-activated

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DCs vaccines for the improving immunotherapy ofcancer. Further understanding of the signaling pathwaysresponsible for may provide new information for thedevelopment of new therapeutic strategies.

Acknowledgments

This work was supported by the National NaturalScience Foundation of China (30571717, 30700985). Wethank Dr. Hong-wei Liu for critical reading of thismanuscript.

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