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Tumor necrosis factor–like weak inducer of apoptosis increases CC chemokine ligand 20 production in interleukin 1β–stimulated human gingival fibroblasts
Human Immunology 73 (2012) 470–473
Contents lists available at SciVerse ScienceDirect
Tumor necrosis factor–like weak inducer of apoptosis increases CC chemokine ligand 20 production in interleukin 1–stimulated human gingival fibroblasts Yoshitaka Hosokawa a,*, Ikuko Hosokawa a, Satoru Shindo a, Kazumi Ozaki b, Hideaki Nakae a, Takashi Matsuo a a b
Department of Conservative Dentistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8504, Japan Department of Oral Health Care Promotion, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan, Tokushima, Tokushima 770-8504, Japan
A R T I C L E
I N F O
Article history: Received 4 November 2011 Accepted 27 February 2012 Available online 6 March 2012
Keywords: CCL20 Human gingival fibroblasts IL-1 TWEAK
A B S T R A C T
CC chemokine ligand 20 (CCL20) is related to T-helper (Th)-17 cell migration, and Th17 cells play important roles in exacerbation in periodontal disease. However, the effect of tumor necrosis factor–like weak inducer of apoptosis (TWEAK) on CCL20 production is unknown. In this study, we examined the mechanisms of TWEAK in combination with interleukin (IL)-1-induced CCL20 production in human gingival fibroblasts (HGFs). TWEAK alone did not induce CCL20 production in HGFs. However, TWEAK enhanced CCL20 expression from IL-1-stimulated HGFs in a dose-dependent manner. Inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), protein kinase B (Akt), and nuclear factor B (NF-B) significantly inhibited CCL20 production in TWEAK and IL-1-stimulated HGFs. Western blot analysis revealed that phosphorylations of ERK, Akt, and inhibitor of NF-B were enhanced in TWEAK and IL-1-treated HGFs. These data suggest that TWEAK is positively related to Th17 cell migration in periodontally diseased tissues to enhance CCL20 production in IL-1-stimulated HGFs. 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) is a recently described member of the TNF superfamily [1]. TWEAK has some biological effects, such as the modulation of immune responses [2– 4] and angiogenesis [5,6]. We previously reported that TWEAK is expressed in periodontally diseased tissues and that recombinant TWEAK could induce interleukin (IL)-8 and vascular endothelial growth factor (VEGF) productions in human gingival fibroblasts (HGFs) [7]. Therefore, we propose that TWEAK is related to the pathogenesis of periodontal disease to induce proinflammatory cytokines in gingival cells. It has been reported that T-helper (Th)-17 cells are related to inflammatory bone destruction [8]. IL-17A produced from Th17 cells induces the expression of receptor activator of nuclear factor B (NF-B) in some cells, which in turn stimulates the differentiation and activation of osteoclasts in inflammatory tissues such as rheumatoid arthritis [8] and periodontal disease [9]. CC chemokine ligand 20 (CCL20) is a member of the CC chemokine family, and it has been demonstrated that the receptor of CCL20 is CC chemokine receptor 6 (CCR6) [10]. It has been reported that Th17 cells preferentially express CCR6 [10]. Therefore, CCL20 attracts Th17 cells through its interaction with CCR6. We previously reported that CCR6-positive and CD4-positive cells are infiltrated in periodon* Corresponding author. E-mail address: [email protected] (Y. Hosokawa).
tally diseased tissues [11], and proinflammatory cytokines, such as IL-1 and TNF-␣, could induce CCL20 production in HGFs [12]. However, the effect of TWEAK on CCL20 production in HGFs is unknown. The aim of this study was to examine the effect of TWEAK on CCL20 production in HGFs. Moreover, we examined the role of TWEAK on CCL20 production in IL-1-stimulated HGFs because we know IL-1 can induce CCL20 production in HGFs. Furthermore, we investigated the effects of TWEAK and IL-1 stimulation on signal transduction pathways in HGFs, including mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), and NF-B. 2. Subjects and methods 2.1. Gingival tissue biopsies and cell culture We used HGFs isolated from 3 clinically healthy gingiva during routine distal wedge surgical procedures. The gingival specimens were cut into small pieces and transferred to culture dishes. The HGFs that grew from the gingiva were primarily cultured on 100mm2 uncoated plastic dishes in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and antibiotics (penicillin G, 100 units/mL; streptomycin, 100 g/mL) at 37⬚C in humidified air with 5% CO2. Confluent cells were transferred and cultured for use in the present study. After 3 to 4 subcultures with trypsinization, the cultures contained homogeneous, slim, and spindle-shape cells growing in characteristic swirls. The cells were used for experi-
0198-8859/12/$36.00 - see front matter 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2012.02.021
Y. Hosokawa et al. / Human Immunology 73 (2012) 470–473
Fig. 1. Effects of tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and interleukin (IL)-1 cotreatment on human gingival fibroblasts (HGF) CC chemokine ligand 20 (CCL20) production. HGFs were activated for 24 hours with TWEAK, IL-1, or mixtures of both cytokines at designated concentrations. The expression levels of CCL20 in the supernatants were measured using an enzyme-linked immunosorbent assay. Data are representative of 3 different HGF samples from 3 different donors. The results are presented as the mean and SD of 1 representative experiment performed in triplicate. The error bars indicate the SD of the values. *p ⬍ 0.05, **p ⬍ 0.01, significantly different from the IL-1-stimulated HGFs.
ments after 5 passages. Informed consent was obtained from all subjects participating in this study. The study was performed with the approval and compliance of the University of Tokushima Ethical Committee (No. 329). 2.2. CCL20 production in HGFs HGFs were stimulated with recombinant human TWEAK (1, 10, or 100 ng/mL; Peprotech, Rocky Hill, NJ) and IL-1 (10 ng/mL; Peprotech) for 24 hours. The supernatants from the HGFs were collected, and the CCL20 concentrations of the culture supernatants were measured in triplicate using enzyme-linked immunosorbent assay (ELISA). Human CCL20 DuoSet (Cat. No. DY360, R&D Systems, Minneapolis, MN) was used for the determination. All assays were performed according to the manufacturer’s instructions, and cytokine levels were determined using the standard curve prepared for each assay. In selected experiments, the HGFs were cultured for 1 hour in the presence or absence of SB203580 (a p38 MAPK inhibitor, 20 M; Santa Cruz Biotechnology, Santa Cruz, CA), PD98059 (an ERK inhibitor, 20 M; Cayman Chemical, Ann Arbor, MI), SP600125 (a c-Jun N terminal kinase [JNK] inhibitor, 20 M; Enzo Life Sciences, Plymouth Meeting, PA), LY294002 (a phosphoinositide 3 kinase inhibitor, 20 M; Cayman Chemical), or Bay11-7085 (a nuclear factor B [NF-B] inhibitor, 20 M; BIOMOL International, Plymouth Meeting, PA) prior to their incubation with TWEAK and IL-1. Inhibitors were dissolved in dimethyl sulfoxide and diluted in the culture medium.
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monoclonal antibody (Cell Signaling Technology), phospho-ERK rabbit monoclonal antibody (Cell Signaling Technology), phosphoJNK rabbit monoclonal antibody (Cell Signaling Technology), phospho-Akt mouse monoclonal antibody (Cell Signaling Technology), phospho-IB-␣ mouse monoclonal antibody (Cell Signaling Technology), p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology), ERK rabbit monoclonal antibody (Cell Signaling Technology), JNK rabbit monoclonal antibody (Cell Signaling Technology), Akt mouse monoclonal antibody (Cell Signaling Technology), or IB-␣ mouse monoclonal antibody (Cell Signaling Technology) according to the manufacturer’s instructions. Protein bands were visualized by incubation with horseradish peroxidase– conjugated antimouse or rabbit immunoglobulin G goat antibody (Sigma), followed by detection using a chemiluminescence reagent (ECL western blotting detection reagents, GE Healthcare, Uppsala, Sweden). We analyzed HGFs from 3 different donors separately. 2.4. Statistical analysis Statistical significance was obtained using the Student t test. We compared IL-1-stimulated HGFs with IL-1 and TWEAK-stimulated HGFs (Fig. 1). We also compared IL-1 and TWEAK-stimulated HGFs without inhibitors to IL-1 and TWEAK-stimulated HGFs with an inhibitor (Fig. 2). We used Statview software (SAS institute inc., Cary, NC). P values ⬍0.05 were considered significant. 3. Results 3.1. Effects of TWEAK and/or IL-1 on CCL20 production in HGFs To determine the effects of TWEAK and/or IL-1 stimulation on CCL20 production in HGFs, HGFs were exposed to TWEAK (100 ng/mL) or IL-1 (10 ng/mL) with or without TWEAK (1, 10, or 100 ng/mL) for 24 hours, and the CCL20 production in the supernatant was measured. The single stimulation of TWEAK (100 ng/mL) did not induce CCL20 production. However, TWEAK enhanced IL-1 (10 ng/mL) induced CCL20 production in a dose-dependent fashion (Fig. 1). 3.2. Effects of chemical inhibitors of intracellular signaling pathways on CCL20 production from TWEAK and Il-1-stimulated HGFs Some studies have reported that MAPKs [13,14], Akt [14], and NF-B [14] are related to CCL20 production. Therefore, we exam-
2.3. Western blot analysis To confirm the TWEAK and/or IL-1-induced phosphorylation of signal transduction molecules, western blot analysis was performed. HGFs stimulated with TWEAK (100 ng/mL) and/or IL-1 (10 ng/mL) were washed once with cold phosphate-buffered saline before being incubated on ice for 30 minutes with lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with protease inhibitor cocktail (Sigma). After removal of debris by centrifugation, the protein concentrations of the lysates were quantified with the Bradford protein assay using immunoglobulin G as a standard. A 20-g protein sample was loaded onto a 4 –20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis before being electrotransfered to a polyvinylidene difluoride membrane. The activation of p38 MAPK, ERK, JNK, protein kinase B (Akt), and inhibitor of NF-B (IB)-␣ was assessed using phospho-p38 MAPK rabbit
Fig. 2. Effects of signal transduction inhibitors on CC chemokine ligand 20 (CCL20) production in tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and interleukin (IL)-1-stimulated human gingival fibroblasts (HGFs). The cells were preincubated with SB203580 (20 M), PD98059 (20 M), SP600125 (20 M), LY294002 (20 M), or Bay11-7085 (20 M) for 1 hour and then incubated with IL-1 (10 ng/mL) and TWEAK (100 ng/mL). After 24 hours of incubation, the supernatants were collected, and CCL20 production was measured by an nenzyme-linked immunosorbent assay. Data are representative of HGFs from 3 different donors. The results are presented as the mean and SD of 1 representative experiment performed in triplicate. The error bars indicate the SD of the values. *p ⬍ 0.05, significantly different from the IL-1 and TWEAK-stimulated HGFs without inhibitors.
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Fig. 3. Effects of interleukin (IL)-1 and tumor necrosis factor–like weak inducer of apoptosis (TWEAK) stimulation on mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and inhibitor of nuclear factor B-␣ (IB-␣) phosphorylation in human gingival fibroblasts (HGFs). The cultured cells were stimulated with TWEAK (100 ng/mL), IL-1 (10 ng/mL), or combination treatment with TWEAK(100 ng/mL) and IL-1 (10 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by. western blotting analysis with antibodies against phosphospecific p38 MAPK, p38 MAPK, phosphospecific extracellular signal-related kinase (ERK), ERK, phosphorspecific JNK, JNK, phosphospecific Akt, Akt, phosphospecific IB-␣, or actin. A representative western blot displays phospho-p38 MAPK, total-p38 MAPK, phospho-ERK, totalERK, phospho-JNK, total-JNK, phospho-Akt, total-Akt, phospho-IB-␣, and actin levels in HGFs from 3 different experiments from 3 different donors.
ined the effects of a p38 MAPK inhibitor SB203580), a MEK inhibitor (PD98059), a JNK inhibitor (SP600125), an Akt inhibitor (LY294002), or a NF-B inhibitor (Bay11-7085) on CCL20 production in TWEAK and IL-1-stimulated HGFs. Fig. 2 indicates that SB203580, PD98059, LY294002, and Bay11-7085 significantly suppressed CCL20 production in TWEAK and IL-1-stimulated HGFs. The same concentration of dimethyl sulfoxide did not change CCL20 production from TWEAK and IL-1-stimulated HGFs (data not shown). 3.3. Effects of TWEAK and/or IL-1 stimulation on MAPKs, Akt, and IB-␣ phosphorylation in HGFs Finally, to determine the effects of TWEAK and/or IL-1 stimulation on the activation of intracellular signal transduction pathways in HGFs, HGFs were exposed to TWEAK (100 ng/mL), IL-1 (10 ng/mL), or TWEAK (100 ng/mL) in combination with IL-1 (10 ng/mL) for 15, 30, or 60 minutes; HGFs were lysed, and western blot analysis was performed using antibodies against MAPKs (p38 MAPK, ERK, and JNK), Akt, or IB-␣. TWEAK and IL-1 treatment significantly enhanced ERK, JNK, Akt, and IB-␣ phosphorylation compared with the single stimulation of TWEAK or IL-1 (Fig. 3). 4. Discussion It has been reported that Th17 cells are related to exacerbation of periodontal disease [9]. However, it is unknown whether TWEAK is involved in Th17 cell accumulation in periodontal disease, although it is certain that TWEAK could induce proinflammatory cytokines, such as IL-8 and VEGF, in HGFs [9]. Therefore, we examined whether TWEAK could induce production of CCL20, which is a Th17 chemokine, in HGFs. In this study, we first revealed that TWEAK could induce CCL20 production in IL-1-stimulated fibroblasts. This result indicates that TWEAK is involved in Th17-related diseases to induce Th17 chemokine and CCL20 production in in-
flammatory sites in cooperation with IL-1. We then examine the roles of TWEAK on Th17 cell functions. It has been reported that TWEAK stimulation alone activates MAPK, Akt, and NF-B pathways. Ando et al. reported that TWEAK stimulation induced phosphorylation of ERK and Akt in mouse osteoblastic MC3T3-E1 cells [15]. Vincent et al. reported that TWEAK alone induced the activation of ERK, JNK, and NF-B pathways in MC3T3-E1 cells [16]. We revealed that TWEAK activated p38 MAPK, ERK, JNK, and Akt pathways in HGFs, although we could not detect NF-B pathway activation. The difference in signal transduction pathway activation in previous reports and our result might depend on the type of cells. We believe that we could not detect CCL20 production in TWEAK-stimulated HGFs because the activation of the NF-B pathway was weak in TWEAK-treated HGFs. No report has examined the effect of TWEAK on the phosphorylation of MAPKs, Akt, and IB-␣ in IL-1-treated cells. Recently, Vincent et al. reported that TWEAK in combination with TNF-␣ enhanced the phosphorylation of ERK, JNK, and IB-␣ compared with stimulation with TNF-␣ alone [16], similar to our report. Therefore, TWEAK might have the ability to increase activation of MAPK, Akt, and NF-B pathways in proinflammatory cytokinetreated cells. In this report, inhibitors of p38 MAPK, ERK, Akt, and NF-B decreased CCL20 production in TWEAK and IL-1-stimulated HGFs. However, the inhibitor of JNK did not modulate CCL20 production. Ghosh et al. reported that a p38 MAPK inhibitor and an ERK inhibitor decreased CCL20 production from IL-1- or TNF-␣-stimulated human oral epithelial cells, although a JNK inhibitor did not change CCL20 production [17] Kao et al. reported that inhibitors of p38 MAPK, ERK, and NF-B decreased CCL20 production in IL-17Atreated human trancheobronchial epithelial cells, although inhibitors of JNK and Akt did not inhibit CCL20 production [18], similar to our results. However, we observed that the activation of JNK in HGFs was enhanced by TWEAK and IL-1 stimulation. This finding means that JNK activation might be related to the production of some unknown factor in TWEAK and IL-1-stimulated HGFs. Further investigation might be necessary. Taken together, the data here strongly suggest that TWEAK in combination with IL-1 is related to Th17 cell migration in diseased periodontal tissues to induce CCL20 production in HGFs. Th17 cells are involved in bone erosion in inflammatory sites such as periodontal disease and arthritis. Therefore, TWEAK might be a target for the treatment of inflammatory bone resorption in periodontal disease. Acknowledgments This study was supported by a Grant-in-Aid for Young Scientists (19791616) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Burkly LC, Michaelson JS, Hahm K, Jakubowski A, Zheng TS. TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease. Cytokines 2007;40:1–16. [2] Chicheportiche Y, Chicheportiche R, Sizing I, Thompson J, Benjamin CB, Ambrose C, et al. Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies. Arthritis Res 2002;4:126 –33. [3] Kim SH, Kang YJ, Kim WJ, Woo DK, Lee Y, Kim DI, et al. TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages. Circ J 2004;68:396 –9. [4] Kamijo S, Nakajima A, Kamata K, Kurosawa H, Yagita H, Okumura K. Involvement of TWEAK/Fn14 interaction in the synovial inflammation of RA. Rheumatol Oxf Engl 2008;47:442–50. [5] Lynch CN, Wang YC, Lund JK, Chen YW, Leal JA, Wiley SR. TWEAK induces angiogenesis and proliferation of endothelial cells. J Biol Chem 1999;274: 8455–9. [6] Donohue PJ, Richards CM, Brown SA, Hanscom HN, Buschman J, Thangada S, et al. TWEAK is an endothelial cell growth and chemotactic factor that also
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potentiates FGF-2 and VEGF-A mitogenic activity. Arterioscler Thromb Vasc Biol 2003;23:594 – 600. Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T. Proinflammatory effects of tumour necrosis factor-like weak inducer of apoptosis (TWEAK) on human gingival fibroblasts. Clin Exp Immunol 2006;146:540 –9. Doodes PD, Cao Y, Hamel KM, Wang Y, Farkas B, Iwakura Y, et al. Development of proteoglycan-induced arthritis is independent of IL-17. J Immunol 2008; 181:329 –37. Kramer JM, Gaffen SL. Interleukin-17: a new paradigm in inflammation, autoimmunity, and therapy. J Periodontol 2007;78:1083–93. Hirota K, Yoshitomi H, Hashimoto M, Maeda S, Teradaira S, Sugimoto N, et al. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model. J Exp Med 2007;204: 2803–12. Hosokawa Y, Nakanishi T, Yamaguchi D, Takahashi K, Yumoto H, Ozaki K, et al. Macrophage inflammatory protein 3alpha-CC chemokine receptor 6 interactions play an important role in CD4⫹ T-cell accumulation in periodontal diseased tissue. Clin Exp Immunol 2002;128:548 –54. Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T. Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin. Clin Exp Immunol 2005;142:285–91.
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[13] Hosokawa Y, Hosokawa I, Ozaki K, Nakanishi T, Nakae H, Matsuo T. Catechins inhibit CCL20 production in IL-17A-stimulated human gingival fibroblasts. Cell Physiol Biochem 2009;24:391– 6. [14] Kim YS, Min KS, Jeong DH, Jang JH, Kim HW, Kim EC. Effects of fibroblast growth factor-2 on the expression and regulation of chemokines in human dental pulp cells. J Endod 2010;36:1824 –30. [15] Ando T, Ichikawa J, Wako M, Hatsushika K, Watanabe Y, Sakuma M, et al. TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells. Arthritis Res Ther 2006;8:R146. [16] Vincent C, Findlay DM, Welldon KJ, Wijenayaka AR, Zheng TS, Haynes DR, et al. Pro-inflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts. J Bone Miner Res 2009;24:1434 – 49. [17] Ghosh SK, Gupta S, Jiang B, Weinberg A. Fusobacterium nucleatum and human beta defensins modulate the release of antimicrobial chemokine CCL20/ Mip-3{alpha}. Infect Immun 2011;79:4578 – 87. [18] Kao CY, Huang F, Chen Y, Thai P, Wachi S, Kim C, et al. Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-kappaB-dependent signaling pathway. J Immunol 2005;175:6676 – 85.
Contents lists available at SciVerse ScienceDirect
Tumor necrosis factor–like weak inducer of apoptosis increases CC chemokine ligand 20 production in interleukin 1–stimulated human gingival fibroblasts Yoshitaka Hosokawa a,*, Ikuko Hosokawa a, Satoru Shindo a, Kazumi Ozaki b, Hideaki Nakae a, Takashi Matsuo a a b
Department of Conservative Dentistry, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8504, Japan Department of Oral Health Care Promotion, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan, Tokushima, Tokushima 770-8504, Japan
A R T I C L E
I N F O
Article history: Received 4 November 2011 Accepted 27 February 2012 Available online 6 March 2012
Keywords: CCL20 Human gingival fibroblasts IL-1 TWEAK
A B S T R A C T
CC chemokine ligand 20 (CCL20) is related to T-helper (Th)-17 cell migration, and Th17 cells play important roles in exacerbation in periodontal disease. However, the effect of tumor necrosis factor–like weak inducer of apoptosis (TWEAK) on CCL20 production is unknown. In this study, we examined the mechanisms of TWEAK in combination with interleukin (IL)-1-induced CCL20 production in human gingival fibroblasts (HGFs). TWEAK alone did not induce CCL20 production in HGFs. However, TWEAK enhanced CCL20 expression from IL-1-stimulated HGFs in a dose-dependent manner. Inhibitors of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), protein kinase B (Akt), and nuclear factor B (NF-B) significantly inhibited CCL20 production in TWEAK and IL-1-stimulated HGFs. Western blot analysis revealed that phosphorylations of ERK, Akt, and inhibitor of NF-B were enhanced in TWEAK and IL-1-treated HGFs. These data suggest that TWEAK is positively related to Th17 cell migration in periodontally diseased tissues to enhance CCL20 production in IL-1-stimulated HGFs. 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Tumor necrosis factor (TNF)–like weak inducer of apoptosis (TWEAK) is a recently described member of the TNF superfamily [1]. TWEAK has some biological effects, such as the modulation of immune responses [2– 4] and angiogenesis [5,6]. We previously reported that TWEAK is expressed in periodontally diseased tissues and that recombinant TWEAK could induce interleukin (IL)-8 and vascular endothelial growth factor (VEGF) productions in human gingival fibroblasts (HGFs) [7]. Therefore, we propose that TWEAK is related to the pathogenesis of periodontal disease to induce proinflammatory cytokines in gingival cells. It has been reported that T-helper (Th)-17 cells are related to inflammatory bone destruction [8]. IL-17A produced from Th17 cells induces the expression of receptor activator of nuclear factor B (NF-B) in some cells, which in turn stimulates the differentiation and activation of osteoclasts in inflammatory tissues such as rheumatoid arthritis [8] and periodontal disease [9]. CC chemokine ligand 20 (CCL20) is a member of the CC chemokine family, and it has been demonstrated that the receptor of CCL20 is CC chemokine receptor 6 (CCR6) [10]. It has been reported that Th17 cells preferentially express CCR6 [10]. Therefore, CCL20 attracts Th17 cells through its interaction with CCR6. We previously reported that CCR6-positive and CD4-positive cells are infiltrated in periodon* Corresponding author. E-mail address: [email protected] (Y. Hosokawa).
tally diseased tissues [11], and proinflammatory cytokines, such as IL-1 and TNF-␣, could induce CCL20 production in HGFs [12]. However, the effect of TWEAK on CCL20 production in HGFs is unknown. The aim of this study was to examine the effect of TWEAK on CCL20 production in HGFs. Moreover, we examined the role of TWEAK on CCL20 production in IL-1-stimulated HGFs because we know IL-1 can induce CCL20 production in HGFs. Furthermore, we investigated the effects of TWEAK and IL-1 stimulation on signal transduction pathways in HGFs, including mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), and NF-B. 2. Subjects and methods 2.1. Gingival tissue biopsies and cell culture We used HGFs isolated from 3 clinically healthy gingiva during routine distal wedge surgical procedures. The gingival specimens were cut into small pieces and transferred to culture dishes. The HGFs that grew from the gingiva were primarily cultured on 100mm2 uncoated plastic dishes in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and antibiotics (penicillin G, 100 units/mL; streptomycin, 100 g/mL) at 37⬚C in humidified air with 5% CO2. Confluent cells were transferred and cultured for use in the present study. After 3 to 4 subcultures with trypsinization, the cultures contained homogeneous, slim, and spindle-shape cells growing in characteristic swirls. The cells were used for experi-
0198-8859/12/$36.00 - see front matter 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2012.02.021
Y. Hosokawa et al. / Human Immunology 73 (2012) 470–473
Fig. 1. Effects of tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and interleukin (IL)-1 cotreatment on human gingival fibroblasts (HGF) CC chemokine ligand 20 (CCL20) production. HGFs were activated for 24 hours with TWEAK, IL-1, or mixtures of both cytokines at designated concentrations. The expression levels of CCL20 in the supernatants were measured using an enzyme-linked immunosorbent assay. Data are representative of 3 different HGF samples from 3 different donors. The results are presented as the mean and SD of 1 representative experiment performed in triplicate. The error bars indicate the SD of the values. *p ⬍ 0.05, **p ⬍ 0.01, significantly different from the IL-1-stimulated HGFs.
ments after 5 passages. Informed consent was obtained from all subjects participating in this study. The study was performed with the approval and compliance of the University of Tokushima Ethical Committee (No. 329). 2.2. CCL20 production in HGFs HGFs were stimulated with recombinant human TWEAK (1, 10, or 100 ng/mL; Peprotech, Rocky Hill, NJ) and IL-1 (10 ng/mL; Peprotech) for 24 hours. The supernatants from the HGFs were collected, and the CCL20 concentrations of the culture supernatants were measured in triplicate using enzyme-linked immunosorbent assay (ELISA). Human CCL20 DuoSet (Cat. No. DY360, R&D Systems, Minneapolis, MN) was used for the determination. All assays were performed according to the manufacturer’s instructions, and cytokine levels were determined using the standard curve prepared for each assay. In selected experiments, the HGFs were cultured for 1 hour in the presence or absence of SB203580 (a p38 MAPK inhibitor, 20 M; Santa Cruz Biotechnology, Santa Cruz, CA), PD98059 (an ERK inhibitor, 20 M; Cayman Chemical, Ann Arbor, MI), SP600125 (a c-Jun N terminal kinase [JNK] inhibitor, 20 M; Enzo Life Sciences, Plymouth Meeting, PA), LY294002 (a phosphoinositide 3 kinase inhibitor, 20 M; Cayman Chemical), or Bay11-7085 (a nuclear factor B [NF-B] inhibitor, 20 M; BIOMOL International, Plymouth Meeting, PA) prior to their incubation with TWEAK and IL-1. Inhibitors were dissolved in dimethyl sulfoxide and diluted in the culture medium.
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monoclonal antibody (Cell Signaling Technology), phospho-ERK rabbit monoclonal antibody (Cell Signaling Technology), phosphoJNK rabbit monoclonal antibody (Cell Signaling Technology), phospho-Akt mouse monoclonal antibody (Cell Signaling Technology), phospho-IB-␣ mouse monoclonal antibody (Cell Signaling Technology), p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology), ERK rabbit monoclonal antibody (Cell Signaling Technology), JNK rabbit monoclonal antibody (Cell Signaling Technology), Akt mouse monoclonal antibody (Cell Signaling Technology), or IB-␣ mouse monoclonal antibody (Cell Signaling Technology) according to the manufacturer’s instructions. Protein bands were visualized by incubation with horseradish peroxidase– conjugated antimouse or rabbit immunoglobulin G goat antibody (Sigma), followed by detection using a chemiluminescence reagent (ECL western blotting detection reagents, GE Healthcare, Uppsala, Sweden). We analyzed HGFs from 3 different donors separately. 2.4. Statistical analysis Statistical significance was obtained using the Student t test. We compared IL-1-stimulated HGFs with IL-1 and TWEAK-stimulated HGFs (Fig. 1). We also compared IL-1 and TWEAK-stimulated HGFs without inhibitors to IL-1 and TWEAK-stimulated HGFs with an inhibitor (Fig. 2). We used Statview software (SAS institute inc., Cary, NC). P values ⬍0.05 were considered significant. 3. Results 3.1. Effects of TWEAK and/or IL-1 on CCL20 production in HGFs To determine the effects of TWEAK and/or IL-1 stimulation on CCL20 production in HGFs, HGFs were exposed to TWEAK (100 ng/mL) or IL-1 (10 ng/mL) with or without TWEAK (1, 10, or 100 ng/mL) for 24 hours, and the CCL20 production in the supernatant was measured. The single stimulation of TWEAK (100 ng/mL) did not induce CCL20 production. However, TWEAK enhanced IL-1 (10 ng/mL) induced CCL20 production in a dose-dependent fashion (Fig. 1). 3.2. Effects of chemical inhibitors of intracellular signaling pathways on CCL20 production from TWEAK and Il-1-stimulated HGFs Some studies have reported that MAPKs [13,14], Akt [14], and NF-B [14] are related to CCL20 production. Therefore, we exam-
2.3. Western blot analysis To confirm the TWEAK and/or IL-1-induced phosphorylation of signal transduction molecules, western blot analysis was performed. HGFs stimulated with TWEAK (100 ng/mL) and/or IL-1 (10 ng/mL) were washed once with cold phosphate-buffered saline before being incubated on ice for 30 minutes with lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with protease inhibitor cocktail (Sigma). After removal of debris by centrifugation, the protein concentrations of the lysates were quantified with the Bradford protein assay using immunoglobulin G as a standard. A 20-g protein sample was loaded onto a 4 –20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis before being electrotransfered to a polyvinylidene difluoride membrane. The activation of p38 MAPK, ERK, JNK, protein kinase B (Akt), and inhibitor of NF-B (IB)-␣ was assessed using phospho-p38 MAPK rabbit
Fig. 2. Effects of signal transduction inhibitors on CC chemokine ligand 20 (CCL20) production in tumor necrosis factor–like weak inducer of apoptosis (TWEAK) and interleukin (IL)-1-stimulated human gingival fibroblasts (HGFs). The cells were preincubated with SB203580 (20 M), PD98059 (20 M), SP600125 (20 M), LY294002 (20 M), or Bay11-7085 (20 M) for 1 hour and then incubated with IL-1 (10 ng/mL) and TWEAK (100 ng/mL). After 24 hours of incubation, the supernatants were collected, and CCL20 production was measured by an nenzyme-linked immunosorbent assay. Data are representative of HGFs from 3 different donors. The results are presented as the mean and SD of 1 representative experiment performed in triplicate. The error bars indicate the SD of the values. *p ⬍ 0.05, significantly different from the IL-1 and TWEAK-stimulated HGFs without inhibitors.
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Fig. 3. Effects of interleukin (IL)-1 and tumor necrosis factor–like weak inducer of apoptosis (TWEAK) stimulation on mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and inhibitor of nuclear factor B-␣ (IB-␣) phosphorylation in human gingival fibroblasts (HGFs). The cultured cells were stimulated with TWEAK (100 ng/mL), IL-1 (10 ng/mL), or combination treatment with TWEAK(100 ng/mL) and IL-1 (10 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by. western blotting analysis with antibodies against phosphospecific p38 MAPK, p38 MAPK, phosphospecific extracellular signal-related kinase (ERK), ERK, phosphorspecific JNK, JNK, phosphospecific Akt, Akt, phosphospecific IB-␣, or actin. A representative western blot displays phospho-p38 MAPK, total-p38 MAPK, phospho-ERK, totalERK, phospho-JNK, total-JNK, phospho-Akt, total-Akt, phospho-IB-␣, and actin levels in HGFs from 3 different experiments from 3 different donors.
ined the effects of a p38 MAPK inhibitor SB203580), a MEK inhibitor (PD98059), a JNK inhibitor (SP600125), an Akt inhibitor (LY294002), or a NF-B inhibitor (Bay11-7085) on CCL20 production in TWEAK and IL-1-stimulated HGFs. Fig. 2 indicates that SB203580, PD98059, LY294002, and Bay11-7085 significantly suppressed CCL20 production in TWEAK and IL-1-stimulated HGFs. The same concentration of dimethyl sulfoxide did not change CCL20 production from TWEAK and IL-1-stimulated HGFs (data not shown). 3.3. Effects of TWEAK and/or IL-1 stimulation on MAPKs, Akt, and IB-␣ phosphorylation in HGFs Finally, to determine the effects of TWEAK and/or IL-1 stimulation on the activation of intracellular signal transduction pathways in HGFs, HGFs were exposed to TWEAK (100 ng/mL), IL-1 (10 ng/mL), or TWEAK (100 ng/mL) in combination with IL-1 (10 ng/mL) for 15, 30, or 60 minutes; HGFs were lysed, and western blot analysis was performed using antibodies against MAPKs (p38 MAPK, ERK, and JNK), Akt, or IB-␣. TWEAK and IL-1 treatment significantly enhanced ERK, JNK, Akt, and IB-␣ phosphorylation compared with the single stimulation of TWEAK or IL-1 (Fig. 3). 4. Discussion It has been reported that Th17 cells are related to exacerbation of periodontal disease [9]. However, it is unknown whether TWEAK is involved in Th17 cell accumulation in periodontal disease, although it is certain that TWEAK could induce proinflammatory cytokines, such as IL-8 and VEGF, in HGFs [9]. Therefore, we examined whether TWEAK could induce production of CCL20, which is a Th17 chemokine, in HGFs. In this study, we first revealed that TWEAK could induce CCL20 production in IL-1-stimulated fibroblasts. This result indicates that TWEAK is involved in Th17-related diseases to induce Th17 chemokine and CCL20 production in in-
flammatory sites in cooperation with IL-1. We then examine the roles of TWEAK on Th17 cell functions. It has been reported that TWEAK stimulation alone activates MAPK, Akt, and NF-B pathways. Ando et al. reported that TWEAK stimulation induced phosphorylation of ERK and Akt in mouse osteoblastic MC3T3-E1 cells [15]. Vincent et al. reported that TWEAK alone induced the activation of ERK, JNK, and NF-B pathways in MC3T3-E1 cells [16]. We revealed that TWEAK activated p38 MAPK, ERK, JNK, and Akt pathways in HGFs, although we could not detect NF-B pathway activation. The difference in signal transduction pathway activation in previous reports and our result might depend on the type of cells. We believe that we could not detect CCL20 production in TWEAK-stimulated HGFs because the activation of the NF-B pathway was weak in TWEAK-treated HGFs. No report has examined the effect of TWEAK on the phosphorylation of MAPKs, Akt, and IB-␣ in IL-1-treated cells. Recently, Vincent et al. reported that TWEAK in combination with TNF-␣ enhanced the phosphorylation of ERK, JNK, and IB-␣ compared with stimulation with TNF-␣ alone [16], similar to our report. Therefore, TWEAK might have the ability to increase activation of MAPK, Akt, and NF-B pathways in proinflammatory cytokinetreated cells. In this report, inhibitors of p38 MAPK, ERK, Akt, and NF-B decreased CCL20 production in TWEAK and IL-1-stimulated HGFs. However, the inhibitor of JNK did not modulate CCL20 production. Ghosh et al. reported that a p38 MAPK inhibitor and an ERK inhibitor decreased CCL20 production from IL-1- or TNF-␣-stimulated human oral epithelial cells, although a JNK inhibitor did not change CCL20 production [17] Kao et al. reported that inhibitors of p38 MAPK, ERK, and NF-B decreased CCL20 production in IL-17Atreated human trancheobronchial epithelial cells, although inhibitors of JNK and Akt did not inhibit CCL20 production [18], similar to our results. However, we observed that the activation of JNK in HGFs was enhanced by TWEAK and IL-1 stimulation. This finding means that JNK activation might be related to the production of some unknown factor in TWEAK and IL-1-stimulated HGFs. Further investigation might be necessary. Taken together, the data here strongly suggest that TWEAK in combination with IL-1 is related to Th17 cell migration in diseased periodontal tissues to induce CCL20 production in HGFs. Th17 cells are involved in bone erosion in inflammatory sites such as periodontal disease and arthritis. Therefore, TWEAK might be a target for the treatment of inflammatory bone resorption in periodontal disease. Acknowledgments This study was supported by a Grant-in-Aid for Young Scientists (19791616) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] Burkly LC, Michaelson JS, Hahm K, Jakubowski A, Zheng TS. TWEAKing tissue remodeling by a multifunctional cytokine: role of TWEAK/Fn14 pathway in health and disease. Cytokines 2007;40:1–16. [2] Chicheportiche Y, Chicheportiche R, Sizing I, Thompson J, Benjamin CB, Ambrose C, et al. Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies. Arthritis Res 2002;4:126 –33. [3] Kim SH, Kang YJ, Kim WJ, Woo DK, Lee Y, Kim DI, et al. TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages. Circ J 2004;68:396 –9. [4] Kamijo S, Nakajima A, Kamata K, Kurosawa H, Yagita H, Okumura K. Involvement of TWEAK/Fn14 interaction in the synovial inflammation of RA. Rheumatol Oxf Engl 2008;47:442–50. [5] Lynch CN, Wang YC, Lund JK, Chen YW, Leal JA, Wiley SR. TWEAK induces angiogenesis and proliferation of endothelial cells. J Biol Chem 1999;274: 8455–9. [6] Donohue PJ, Richards CM, Brown SA, Hanscom HN, Buschman J, Thangada S, et al. TWEAK is an endothelial cell growth and chemotactic factor that also
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