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Interleukin (IL)-17A synergistically enhances CC chemokine ligand 20 production in IL-1β-stimulated human gingival fibroblasts
Human Immunology 73 (2012) 26-30
Contents lists available at SciVerse ScienceDirect
Interleukin (IL)-17A synergistically enhances CC chemokine ligand 20 production in IL-1-stimulated human gingival fibroblasts Yoshitaka Hosokawa a,*, Ikuko Hosokawa a, Kazumi Ozaki b, Hideaki Nakae a, Takashi Matsuo a a b
Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Department of Oral Health Care Promotion, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
A R T I C L E
I N F O
Article history: Received 11 April 2011 Accepted 3 October 2011 Available online 8 October 2011
Keywords: CCL20 Human gingival fibroblasts IL-1 IL-17A
A B S T R A C T
CC chemokine ligand 20 (CCL20) plays a pivotal role in the recruitment of T-helper (Th)-17 cells and thus in the development of periodontal disease, but the effect of simultaneous interleukin (IL)-17A and IL-1 stimulation on CCL20 production in human gingival fibroblasts (HGFs) are not known. In this study, we investigated the mechanisms of IL-1- and IL-17A-induced CCL20 production in HGFs. IL-17A synergistically enhanced CCL20 production from IL-1-stimulated HGFs in a concentration-dependent manner. Extracellular signal-regulated kinase (ERK) and inhibitor of nuclear factor (NF)-B-␣ phosphorylation were increased in IL-1- and IL-17A-stimulated HGFs. Inhibitors of or ERK and NF-B decreased IL-1- and IL-17A-induced CCL20 production. IL-1 stimulation elevated IL-17 receptor C expression on HGFs. These data suggest that IL-1 is actively related to Th17 cell migration into peripheral tissues to induce production of the Th17 chemokine, CCL20. Therefore, IL-1 might be a therapeutic target for Th17-related diseases, such as periodontal disease and arthritis. 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Periodontitis is a chronic bacterial infection of toothsupporting structures. It causes destruction of periodontal connective tissues and bone. The initiation and progression of the disease result from the host response to biofilm. Immunohistochemical studies have revealed dense inflammatory cell infiltration, involving T cells, B cells, and macrophages in periodontitisaffected regions [1–3]. Recently, a new type of T cell, T-helper (Th)-17, has been linked with several inflammatory diseases [4], and it was reported that Th17 cells are present in periodontally diseased tissues [5]. However, the effect of IL-17A, which is produced from Th17, on Th17 migration in periodontally diseased tissues is uncertain. Gingival fibroblasts, the major cell type in periodontal connective tissues, provide a tissue framework for tooth anchorage. Until recently, they were presumed to be immunologically inert. Currently, however, researchers recognize their active role in host defense. Upon stimulation with cytokines or bacterial pathogens, human gingival fibroblasts (HGFs) secrete various soluble mediators of inflammation, such as interleukin (IL)-8 and other chemokines [6 –11]. These fibroblast-derived mediators are thought to play an important role in the periodontal inflammatory response.
* Corresponding author. E-mail address: [email protected] (Y. Hosokawa).
It has been reported that the CC chemokine ligand 20 (CCL20) attracts activated Th17 cells through its interaction with CC chemokine receptor 6 (CCR6) [12]. We previously reported that IL-1 could induce CCL20 production in HGFs [8] and that CD4⫹CCR6⫹ cells, which are Th17 cells, infiltrate into periodontally diseased tissues [13]. Our reports [8,13] suggest that IL-1 induced IL-17A expression in periodontally diseased tissues, because CCL20 produced from IL-1-stimulated HGFs could attract Th17 cells in periodontal tissues. However, the effect of IL-17A on CCL20 production in IL-1-stimulated HGFs is unknown. The aim of this study was to examine the effect of IL-17A on CCL20 production in IL-1-stimulated HGFs. Moreover, we investigated the effects of IL-1 and IL-17A stimulation on signal transduction pathways in HGFs, including mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-B. 2. Subjects and methods 2.1. Gingival tissue biopsies and cell culture We used HGFs that were 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 100 mm2 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 U/mL; streptomycin, 100 g/
0198-8859/$36.00 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.10.004
Y. Hosokawa et al. / Human Immunology 73 (2012) 26-30
mL) at 37OC 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 experiments 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 The HGFs were stimulated with IL-17A (Peprotech, Rocky Hill, NJ) and IL-1 (Peprotech) for 24 hours. The supernatants from the HGFs were collected, and the CCL20 concentrations of the culture supernatants were measured in triplicate using enzymelinked immunosorbent assay (ELISA). Duoset (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 (20 M; Santa Cruz Biotechnology, Santa Cruz, CA), PD98059 (20 M; Santa Cruz Biotechnology), SP600125 (20 M; Sigma), Bay11⫺7085 (BIOMOL International, Plymouth Meeting, PA), or SC514 (Biomol international) before their incubation with IL17A and IL-1. Inhibitors were dissolved in dimethyl sulfoxide and diluted in culture medium. 2.3. Western blot analysis To confirm the IL-17A- and/or IL-1-induced phosphorylation of signal transduction molecules, Western blot analysis was performed. HGFs stimulated with IL-17A (100 ng/mL) and/or IL-1 (10 ng/mL) were washed once with cold phosphatebuffered saline (PBS) before being incubated on ice for 30 minutes with lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with protease inhibitors (Sigma). After removal of debris by centrifugation, the protein concentrations of the lysates were quantified with the Bradford protein assay using immunoglobulin G (IgG) as a standard. A 20-g protein sample was loaded onto a 4 –20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel before being electrotransferred to a polyvinylidene fluoride membrane. The activation of p38 MAPK, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and inhibitor of nuclear factor B (IB)-␣ was assessed using phospho-p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology), phospho-ERK rabbit monoclonal antibody (Cell Signaling Technology), phospho-JNK rabbit 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, JNK rabbit monoclonal antibody (Cell Signaling Technology), or IB-␣ mouse monoclonal antibody according to the manufacturer’s instructions. Protein bands were visualized by incubation with the horseradish peroxidase-conjugated secondary antibody (Sigma), followed by detection using the enhanced chemiluminescence system (GE Healthcare, Uppsala, Sweden). Quantitation of the chemiluminescent signal was analyzed using NIH image software (National Institute of Mental Health, Bethesda, MD). 2.4. Flow cytometric analyses Following the required culture time, the cells were washed twice with ice-cold PBS. The HGFs were harvested by incubation with PBS– 4 mmol/L EDTA. Most cells were rounded up following
27
this treatment and removed by gentle agitation. Any cells that failed to detach were removed with gentle scraping. The cells were washed twice with ice-cold PBS and incubated (for 20 minutes on ice) in PBS–1% bovine serum albumin (BSA; Sigma). The cells were incubated with mouse antihuman IL-17 receptor A (IL-17RA) antibody (R&D Systems; 5 g/mL), IL-17 receptor C (IL-17RC) antibody (R&D Systems; 5 g/mL), or an isotype control antibody on ice for 30 minutes. After being washed 3 times with PBS–1% BSA, the cells were incubated with an FITCconjugated rabbit anti-mouse F(ab=)2 fragment (Dako, Kyoto, Japan) for 30 minutes on ice. After being washed 3 times with PBS–1% BSA, the cells were immediately analyzed with flow cytometry (Epics XL-MCL; Coulter, Hialeah, FL). 2.5. Statistical analysis Statistical significance was analyzed using the Student t test. We used Statview software (Abacus Concepts, Berkeley, CA). p values ⬍ 0.05 were considered significant. 3. Results 3.1. The effect of IL-17A and IL-1 on CCL20 production by HGFs We previously reported that single stimulation of IL-1 or IL-17A could induce CCL20 production from HGFs [8,14]. However, the effect of simultaneous stimulation of IL-17A and IL-1 on CCL20 production from HGFs is uncertain. As illustrated in Fig. 1, IL-17A stimulation synergistically enhanced CCL20 production from IL-1-stimulated HGFs in a concentrationdependent manner. 3.2. P38 MAPK, ERK, and NF-B pathways are related to CCL20 production in IL-17A- and IL-1-stimulated HGFs We previously reported that MAPKs and NF-B are related to CCL20 production in HGFs [8]. Therefore, we examined the effects of a p38 MAPK inhibitor (SB203580), a MEK inhibitor (PD98059), a JNK inhibitor (SP600125), or NF-B inhibitors (Bay11⫺7085 and SC514) on CCL20 production in IL-17A- and IL-1-stimulated HGFs. Fig. 2 shows that SB203580, PD98059, Bay11-7085, and SC514 significantly suppressed CCL20 production in IL-17A- and IL-1-stimulated HGFs. Moreover, U0126 (MEK1 inhibitor) significantly inhibited CCL20 production (data not shown). The same concentration of dimethyl sulfoxide did
Fig 1. Effects of IL-1 and IL-17A on CCL20 production by HGFs. HGFs were treated with IL-1 (10 ng/mL) and/or IL-17A (1, 10, or 100 ng/mL) and the supernatants were collected after 24 hours. The expression levels of CCL20 in the supernatants were measured using ELISA. Data are representative of 3 different HGFs 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. #p ⬍ 0.05, significantly different from the nonstimulated HGFs.
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These results demonstrate that ERK is positively related to increased CCL20 production in IL-1- and IL-17A-stimulated HGFs. 3.4. Effects of IL-17A and/or IL-1 stimulation on IB-␣ phosphorylation in HGFs
Fig 2. Effects of signal transduction inhibitors on IL-17A-stimulated CCL20 release by HGFs. Cells were preincubated with SB203580 (20 M), PD98059 (20 M), SP600125 (20 M), Bay11-7085 (20 M), or SC514 (20 M) for 1 hour and then incubated with IL-1 (10 ng/mL) and IL-17A (100 ng/mL). After a 24-hour incubation, the supernatants were collected, and CCL20 production was measured by ELISA. 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, **p ⬍ 0.01, significantly different from the IL-1- and IL-17A-stimulated HGFs without inhibitors.
not change CCL20 production from IL-17A- and IL-1-stimulated HGFs (data not shown). 3.3. Effects of IL-17A and/or IL-1 stimulation on MAPK phosphorylation in HGFs Next, we examined the effects of IL-17A and IL-1 on MAPKs in HGFs. IL-17A treatment significantly enhanced JNK phosphorylation. However, IL-17A slightly induced p38 MAPK and ERK phosphorylation in HGFs. By contrast, IL-1 treatment apparently induced p38 MAPK, ERK, and JNK phosphorylation in HGFs. Simultaneous stimulation of IL-17A and IL-1 further enhanced ERK phosphorylation in HGFs compared with the single stimulation of IL-1. However, p38 MAPK phosphorylation in IL-17A- and IL1-stimulated HGFs did not change compared with IL-1stimulated HGFs (Fig. 3), although p38 MAPK inhibitor suppressed CCL20 production from IL-1- and IL-17A-stimulated HGFs (Fig. 2).
Next, we examined the effects of IL-17A and IL-1 on the NF-B pathway in HGFs. IL-17A treatment slightly enhanced IB-␣ phosphorylation. IL-1 treatment apparently induced IB-␣ phosphorylation and degradation in HGFs. Simultaneous stimulation of IL17A and IL-1 significantly enhanced IB-␣ phosphorylation in HGFs treated for 30 or 60 minutes compared with the single stimulation of IL-1 (Fig. 4A and 4B). Furthermore, simultaneous stimulation of IL-17A- and IL-1 induced IB-␣ degradation in HGFs treated for 15 minutes, although a single stimulation of IL-1 did not induce degradation (Fig. 4A). 3.5. Effects of IL-1 on IL-17 receptor expression on HGFs We hypothesized that IL-1 stimulation might modulate IL-17 receptor expression because simultaneous stimulation of IL-1 and IL-17A enhanced CCL20 production. As illustrated in Fig. 5, IL-1 stimulation slightly enhanced IL-17RC expression on HGFs, although the IL-17RA expression did not change. 4. Discussion In this study, we demonstrated for the first time that IL-17A and IL-1 are able to induce CCL20 production in HGFs. It has been reported that CCL20 is involved in the migration of Th17 cells because Th17 cells preferentially express CCR6 [12]. Moreover, it was suggested that Th17 cells are involved in the exacerbation of periodontal disease [15]. Therefore, IL-17A might induce periodontal tissue destruction by enhancing Th17 cell migration. It has been reported that IL-1 enhanced IL-17A production from Th17 cells [16] and ␥␦T cells [17]. Moreover, IL-17A induced IL-1 production from human macrophages [18]. Our report suggests that IL-1 and IL-17A synergistically enhanced production of the Th17 chemokine, CCL20. These data indicate that IL-1 is actively involved in IL-17A-related inflammation in peripheral tissues to induce IL-17A production and attracting Th17 cells. Furthermore, IL-17A from Th17 cells might enhance IL-1 production from
Fig. 3. Effects of IL-1 and IL-17A stimulation on MAPK phosphorylation in HGFs. The cultured cells were stimulated with IL-1 (10 ng/mL) with or without IL-17A (100 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to SDS–PAGE followed by Western blotting analysis with antibodies against phosphospecific p38 MAPK, p38 MAPK, phosphospecific ERK, ERK, phosphorspecific JNK, or JNK. A representative Western blot displaying phospho-p38 MAPK, total-p38 MAPK, phosphor-ERK, total-ERK, phosphoJNK, and total-JNK levels in HGFs from 3 independent experiments.
Y. Hosokawa et al. / Human Immunology 73 (2012) 26-30
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Fig. 4. Effects of IL-1 and IL-17A stimulation on IB-␣ phosphorylation and degradation in HGFs. (A) The cultured cells were stimulated with IL-1 (10 ng/mL) with or without IL-17A (100 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to SDS–PAGE followed by Western blotting analysis with antibodies against phosphospecific phosphorspecific IB-␣, IB-␣, or actin. A representative Western blot displaying phospho-IB-␣, total-IB-␣, and actin levels in HGFs from 3 independent experiments. (B) Bar graphs of phospho-IB-␣ expression were normalized to actin using NIH image software. The error bars indicate the SD of the values. *p ⬍ 0.05, significantly different from the IL-1-stimulated HGFs for 30 minutes. #p ⬍ 0.05, significantly different from from the IL-1-stimulated HGFs for 60 minutes.
inflammatory cells in inflamed tissues. Therefore, IL-1 might be a therapeutic target to Th17 cell–related diseases such as arthritis and periodontal disease [12,15]. We revealed that ERK and NF-B pathways are related to Th17 chemokine expression in HGFs. Brereton et al. recently reported that treatment with an ERK inhibitor attenuated experimental autoimmune encephalomyelitis, which is a Th17-related disease [19]. They also reported that the ERK pathway is involved in the production of IL-23, a cytokine related to Th17 cell expansion, by lipopolysaccharide-stimulated dendritic cells. In this report, we elucidated that ERK is important for CCL20 production from IL-1and IL-17A-stimulated HGFs. It is also reported that the production of IL-23, which is related to Th17 cell differentiation, from synovial fibroblasts is inhibited by NF-B inhibitors [20] Therefore, ERK and NF-B are positively related to Th17 inflammation not only through Th17 cell expansion but also by attracting Th17 cells into peripheral tissues. Zrioual et al. reported that synoviocytes expressed both IL-17RA and IL-17RC, and both receptors are necessary for IL-6 production from IL-17A-stimulated synoviocytes [21]. Our report indicates that IL-1 stimulation enhanced only IL-17RC expression on HGFs. This is the first report that demonstrates that IL-17RC expression is modified by cytokine stimulation. IL-17RC might be actively related to Th17-related inflammation to change its expression. Further
examination might be necessary to determine the function and character of IL-17RC. IL-17A attracts neutrophils to control bacterial challenge and prevents pathogen-initiated bone destruction [22]. However, IL17A could increase the RANKL expression and promote osteoclastic bone erosion in murine collagen arthritis [23]. We believe that CCL20 produced from HGFs could attract Th17 cells near alveolar bone. Therefore, IL-17A produced from Th17 cells enhanced RANKL around osteoclasts in periodontally diseased tissues. However, further study is necessary to clarify the role of IL-17A in periodontal disease. Recently, Duarte et al. reported that Th17 cells are enhanced in the periodontal sites, especially in diabetic patients [24]. It has also been reported that high glucose induces CCL20 production in renal proximal tubule cells [25]. Therefore, high glucose levels might be related to Th17 cell migration in peripheral tissues. Further examination is necessary. In summary, the current study demonstrates that IL-17A and IL-1 synergistically enhanced CCL20 release by cultured HGFs. We also revealed that ERK and NF-B pathways and enhanced IL-17RC expression were related to CCL20 production from IL-1- and IL17A-stimulated HGFs. We discovered that IL-1 is related to Th17 migration into peripheral tissues to induce CCL20, although IL-1 can induce Th17 cell differentiation [26] and IL-17A production
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Y. Hosokawa et al. / Human Immunology 73 (2012) 26-30
Fig. 5. Effects of IL-1 on IL-17 receptor expression on HGFs. HGFs were treated with IL-1 (10 ng/mL), and the cells were collected after 24 hours. The expression levels of IL-17RA and IL-17RC on HGFs were measured using flow cytometry as described under Subjects and Methods. The blue line represents the background level of fluorescence caused by the isotype-matched antibody. The red line represents IL-17RA or IL-17RC expression on nonstimulated HGFs. The green line represents IL-17RA or IL-17RC expression on IL-1-treated HGFs. The numbers in the graph are the mean fluorescence intensity of each graph. One of 3 experiments with similar results is presented.
from Th17 cells and ␥␦T cells [27]. HGFs in the presence of IL-17A and IL-1 may actively participate in the recruitment of Th17 cells into periodontally diseased tissues by enhanced production of CCL20. Acknowledgments This study was supported by a Grant-in-Aid for Young Scientists (19791616). References [1] Seymour GJ. Importance of the host response in the periodontium. J Clin Periodontol 1991;18:421– 6. [2] Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions. Periodontology 2000 1997;14:216 – 48. [3] Fujihashi K, Kono Y, Beagley KW, Yamamoto M, McGhee JR, Mestecky J, et al. Cytokines and periodontal disease: immunopathological role of interleukins for B cell responses in chronic inflamed gingival tissues. J Periodontol 1993;64: 400 – 6. [4] Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007;148:32– 46.
[5] Cardoso CR, Garlet GP, Crippa GE, Rosa AL, JÛnior WM, Rossi MA, et al. Evidence of the presence of T helper type 17 cells in chronic lesions of human periodontal disease. Oral Microbiol Immunol 2009;24:1– 6. [6] Takashiba S, Takigawa M, Takahashi K, Myokai F, Nishimura F, Chihara T, et al. Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha. Infect Immun 1992;60:5253– 8. [7] Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Murakami K, Miyake Y, et al. CXCL12 and CXCR4 expression by human gingival fibroblasts in periodontal disease. Clin Exp Immunol 2005;141:467–74. [8] 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. [9] Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T. CXC chemokine ligand 16 in periodontal diseases: expression in diseased tissues and production by cytokine-stimulated human gingival fibroblasts. Clin Exp Immunol 2007;149: 146 –54. [10] Hosokawa I, Hosokawa Y, Ozaki K, Nakae H, Matsuo T. Adrenomedullin suppresses tumour necrosis factor alpha-induced CXC chemokine ligand 10 production by human gingival fibroblasts. Clin Exp Immunol 2008;152:568 –75. [11] Hosokawa Y, Hosokawa I, Ozaki K, Nakae H, Matsuo T. CC chemokine ligand 17 in periodontal diseases: expression in diseased tissues and production by human gingival fibroblasts. J Periodontal Res 2008;43:471–7. [12] 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. [13] 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. [14] 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. [15] Gaffen SL, Hajishengallis G. A new inflammatory cytokine on the block: rethinking periodontal disease and the Th1/Th2 paradigm in the context of Th17 cells and IL-17. J Dent Res 2008;87:817–28. [16] Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007;8:942–9. [17] Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KH. Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity. Immunity 2009;31:331– 41. [18] Beklen A, Ainola M, Hukkanen M, Gu¨rgan C, Sorsa T, Konttinen YT. MMPs, IL-1, and TNF are regulated by IL-17 in periodontitis. J Dent Res 2007;86:347–51. [19] Brereton CF, Sutton CE, Lalor SJ, Lavelle EC, Mills KH. Inhibition of ERK MAPK suppresses IL-23- and IL-1-driven IL-17 production and attenuates autoimmune disease. J Immunol 2009;183:1715–23. [20] Kim HR, Cho ML, Kim KW, Juhn JY, Hwang SY, Yoon CH, et al. Up-regulation of IL-23 p19 expression in rheumatoid arthritis synovial fibroblasts by IL-17 through PI3-kinase-, NF-kB-, and p38 MAPK-dependent signalling pathways. Rheumatology (Oxford) 2007;46:57– 64. [21] Zrioual S, Toh ML, Tournadre A, Zhou Y, Cazalis MA, Pachot A, et al. IL-17RA and IL-17RC receptors are essential for IL-17A-induced ELR⫹ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood. J Immunol 2008;180:655– 63. [22] Yu JJ, Ruddy MJ, Wong GC, Sfintescu C, Baker PJ, Smith JB, et al. An essential role for IL-17 in preventing pathogen-initiated bone destruction: recruitment of neutrophils to inflamed bone requires IL-17 receptor-dependent signals. Blood 2007;109:3794 – 802. [23] Lubberts E, van den Bersselaar L, Oppers-Walgreen B, Schwarzenberger P, Coenen-de Roo CJ, Kolls JK, et al. IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kB ligand/osteoprotegerin balance. J Immunol 2003;170:2655– 62. [24] Duarte PM, Santos VR, Dos Santos FA, de Lima Pereira SA, Rodrigues DB, Napimoga MH. Role of smoking and type 2 diabetes in the immunobalance of advanced chronic periodontitis. J Periodontol 2011;82:429 –38. [25] Qi W, Chen X, Zhang Y, Holian J, Mreich E, Gilbert RE, et al. High glucose induces macrophage inflammatory protein-3 ␣ in renal proximal tubule cells via a transforming growth factor-beta 1 dependent mechanism. Nephrol Dial Transplant 2007;22:3147–53. [26] Stritesky GL, Yeh N, Kaplan MH. IL-23 promotes maintenance but not commitment to the Th17 lineage. J Immunol 2008;181:5948 –55. [27] Sutton CE, Lalor SJ, Sweeney CM, Brereton CF, Lavelle EC, Mills KH. Interleukin-1 and IL-23 induce innate IL-17 production from ␥␦ T cells, amplifying TH17 responses and autoimmunity. Immunity 2009;31:331– 41.
Contents lists available at SciVerse ScienceDirect
Interleukin (IL)-17A synergistically enhances CC chemokine ligand 20 production in IL-1-stimulated human gingival fibroblasts Yoshitaka Hosokawa a,*, Ikuko Hosokawa a, Kazumi Ozaki b, Hideaki Nakae a, Takashi Matsuo a a b
Department of Conservative Dentistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Department of Oral Health Care Promotion, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
A R T I C L E
I N F O
Article history: Received 11 April 2011 Accepted 3 October 2011 Available online 8 October 2011
Keywords: CCL20 Human gingival fibroblasts IL-1 IL-17A
A B S T R A C T
CC chemokine ligand 20 (CCL20) plays a pivotal role in the recruitment of T-helper (Th)-17 cells and thus in the development of periodontal disease, but the effect of simultaneous interleukin (IL)-17A and IL-1 stimulation on CCL20 production in human gingival fibroblasts (HGFs) are not known. In this study, we investigated the mechanisms of IL-1- and IL-17A-induced CCL20 production in HGFs. IL-17A synergistically enhanced CCL20 production from IL-1-stimulated HGFs in a concentration-dependent manner. Extracellular signal-regulated kinase (ERK) and inhibitor of nuclear factor (NF)-B-␣ phosphorylation were increased in IL-1- and IL-17A-stimulated HGFs. Inhibitors of or ERK and NF-B decreased IL-1- and IL-17A-induced CCL20 production. IL-1 stimulation elevated IL-17 receptor C expression on HGFs. These data suggest that IL-1 is actively related to Th17 cell migration into peripheral tissues to induce production of the Th17 chemokine, CCL20. Therefore, IL-1 might be a therapeutic target for Th17-related diseases, such as periodontal disease and arthritis. 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Periodontitis is a chronic bacterial infection of toothsupporting structures. It causes destruction of periodontal connective tissues and bone. The initiation and progression of the disease result from the host response to biofilm. Immunohistochemical studies have revealed dense inflammatory cell infiltration, involving T cells, B cells, and macrophages in periodontitisaffected regions [1–3]. Recently, a new type of T cell, T-helper (Th)-17, has been linked with several inflammatory diseases [4], and it was reported that Th17 cells are present in periodontally diseased tissues [5]. However, the effect of IL-17A, which is produced from Th17, on Th17 migration in periodontally diseased tissues is uncertain. Gingival fibroblasts, the major cell type in periodontal connective tissues, provide a tissue framework for tooth anchorage. Until recently, they were presumed to be immunologically inert. Currently, however, researchers recognize their active role in host defense. Upon stimulation with cytokines or bacterial pathogens, human gingival fibroblasts (HGFs) secrete various soluble mediators of inflammation, such as interleukin (IL)-8 and other chemokines [6 –11]. These fibroblast-derived mediators are thought to play an important role in the periodontal inflammatory response.
* Corresponding author. E-mail address: [email protected] (Y. Hosokawa).
It has been reported that the CC chemokine ligand 20 (CCL20) attracts activated Th17 cells through its interaction with CC chemokine receptor 6 (CCR6) [12]. We previously reported that IL-1 could induce CCL20 production in HGFs [8] and that CD4⫹CCR6⫹ cells, which are Th17 cells, infiltrate into periodontally diseased tissues [13]. Our reports [8,13] suggest that IL-1 induced IL-17A expression in periodontally diseased tissues, because CCL20 produced from IL-1-stimulated HGFs could attract Th17 cells in periodontal tissues. However, the effect of IL-17A on CCL20 production in IL-1-stimulated HGFs is unknown. The aim of this study was to examine the effect of IL-17A on CCL20 production in IL-1-stimulated HGFs. Moreover, we investigated the effects of IL-1 and IL-17A stimulation on signal transduction pathways in HGFs, including mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-B. 2. Subjects and methods 2.1. Gingival tissue biopsies and cell culture We used HGFs that were 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 100 mm2 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 U/mL; streptomycin, 100 g/
0198-8859/$36.00 䉷 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.10.004
Y. Hosokawa et al. / Human Immunology 73 (2012) 26-30
mL) at 37OC 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 experiments 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 The HGFs were stimulated with IL-17A (Peprotech, Rocky Hill, NJ) and IL-1 (Peprotech) for 24 hours. The supernatants from the HGFs were collected, and the CCL20 concentrations of the culture supernatants were measured in triplicate using enzymelinked immunosorbent assay (ELISA). Duoset (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 (20 M; Santa Cruz Biotechnology, Santa Cruz, CA), PD98059 (20 M; Santa Cruz Biotechnology), SP600125 (20 M; Sigma), Bay11⫺7085 (BIOMOL International, Plymouth Meeting, PA), or SC514 (Biomol international) before their incubation with IL17A and IL-1. Inhibitors were dissolved in dimethyl sulfoxide and diluted in culture medium. 2.3. Western blot analysis To confirm the IL-17A- and/or IL-1-induced phosphorylation of signal transduction molecules, Western blot analysis was performed. HGFs stimulated with IL-17A (100 ng/mL) and/or IL-1 (10 ng/mL) were washed once with cold phosphatebuffered saline (PBS) before being incubated on ice for 30 minutes with lysis buffer (Cell Signaling Technology, Danvers, MA) supplemented with protease inhibitors (Sigma). After removal of debris by centrifugation, the protein concentrations of the lysates were quantified with the Bradford protein assay using immunoglobulin G (IgG) as a standard. A 20-g protein sample was loaded onto a 4 –20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel before being electrotransferred to a polyvinylidene fluoride membrane. The activation of p38 MAPK, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and inhibitor of nuclear factor B (IB)-␣ was assessed using phospho-p38 MAPK rabbit monoclonal antibody (Cell Signaling Technology), phospho-ERK rabbit monoclonal antibody (Cell Signaling Technology), phospho-JNK rabbit 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, JNK rabbit monoclonal antibody (Cell Signaling Technology), or IB-␣ mouse monoclonal antibody according to the manufacturer’s instructions. Protein bands were visualized by incubation with the horseradish peroxidase-conjugated secondary antibody (Sigma), followed by detection using the enhanced chemiluminescence system (GE Healthcare, Uppsala, Sweden). Quantitation of the chemiluminescent signal was analyzed using NIH image software (National Institute of Mental Health, Bethesda, MD). 2.4. Flow cytometric analyses Following the required culture time, the cells were washed twice with ice-cold PBS. The HGFs were harvested by incubation with PBS– 4 mmol/L EDTA. Most cells were rounded up following
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this treatment and removed by gentle agitation. Any cells that failed to detach were removed with gentle scraping. The cells were washed twice with ice-cold PBS and incubated (for 20 minutes on ice) in PBS–1% bovine serum albumin (BSA; Sigma). The cells were incubated with mouse antihuman IL-17 receptor A (IL-17RA) antibody (R&D Systems; 5 g/mL), IL-17 receptor C (IL-17RC) antibody (R&D Systems; 5 g/mL), or an isotype control antibody on ice for 30 minutes. After being washed 3 times with PBS–1% BSA, the cells were incubated with an FITCconjugated rabbit anti-mouse F(ab=)2 fragment (Dako, Kyoto, Japan) for 30 minutes on ice. After being washed 3 times with PBS–1% BSA, the cells were immediately analyzed with flow cytometry (Epics XL-MCL; Coulter, Hialeah, FL). 2.5. Statistical analysis Statistical significance was analyzed using the Student t test. We used Statview software (Abacus Concepts, Berkeley, CA). p values ⬍ 0.05 were considered significant. 3. Results 3.1. The effect of IL-17A and IL-1 on CCL20 production by HGFs We previously reported that single stimulation of IL-1 or IL-17A could induce CCL20 production from HGFs [8,14]. However, the effect of simultaneous stimulation of IL-17A and IL-1 on CCL20 production from HGFs is uncertain. As illustrated in Fig. 1, IL-17A stimulation synergistically enhanced CCL20 production from IL-1-stimulated HGFs in a concentrationdependent manner. 3.2. P38 MAPK, ERK, and NF-B pathways are related to CCL20 production in IL-17A- and IL-1-stimulated HGFs We previously reported that MAPKs and NF-B are related to CCL20 production in HGFs [8]. Therefore, we examined the effects of a p38 MAPK inhibitor (SB203580), a MEK inhibitor (PD98059), a JNK inhibitor (SP600125), or NF-B inhibitors (Bay11⫺7085 and SC514) on CCL20 production in IL-17A- and IL-1-stimulated HGFs. Fig. 2 shows that SB203580, PD98059, Bay11-7085, and SC514 significantly suppressed CCL20 production in IL-17A- and IL-1-stimulated HGFs. Moreover, U0126 (MEK1 inhibitor) significantly inhibited CCL20 production (data not shown). The same concentration of dimethyl sulfoxide did
Fig 1. Effects of IL-1 and IL-17A on CCL20 production by HGFs. HGFs were treated with IL-1 (10 ng/mL) and/or IL-17A (1, 10, or 100 ng/mL) and the supernatants were collected after 24 hours. The expression levels of CCL20 in the supernatants were measured using ELISA. Data are representative of 3 different HGFs 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. #p ⬍ 0.05, significantly different from the nonstimulated HGFs.
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These results demonstrate that ERK is positively related to increased CCL20 production in IL-1- and IL-17A-stimulated HGFs. 3.4. Effects of IL-17A and/or IL-1 stimulation on IB-␣ phosphorylation in HGFs
Fig 2. Effects of signal transduction inhibitors on IL-17A-stimulated CCL20 release by HGFs. Cells were preincubated with SB203580 (20 M), PD98059 (20 M), SP600125 (20 M), Bay11-7085 (20 M), or SC514 (20 M) for 1 hour and then incubated with IL-1 (10 ng/mL) and IL-17A (100 ng/mL). After a 24-hour incubation, the supernatants were collected, and CCL20 production was measured by ELISA. 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, **p ⬍ 0.01, significantly different from the IL-1- and IL-17A-stimulated HGFs without inhibitors.
not change CCL20 production from IL-17A- and IL-1-stimulated HGFs (data not shown). 3.3. Effects of IL-17A and/or IL-1 stimulation on MAPK phosphorylation in HGFs Next, we examined the effects of IL-17A and IL-1 on MAPKs in HGFs. IL-17A treatment significantly enhanced JNK phosphorylation. However, IL-17A slightly induced p38 MAPK and ERK phosphorylation in HGFs. By contrast, IL-1 treatment apparently induced p38 MAPK, ERK, and JNK phosphorylation in HGFs. Simultaneous stimulation of IL-17A and IL-1 further enhanced ERK phosphorylation in HGFs compared with the single stimulation of IL-1. However, p38 MAPK phosphorylation in IL-17A- and IL1-stimulated HGFs did not change compared with IL-1stimulated HGFs (Fig. 3), although p38 MAPK inhibitor suppressed CCL20 production from IL-1- and IL-17A-stimulated HGFs (Fig. 2).
Next, we examined the effects of IL-17A and IL-1 on the NF-B pathway in HGFs. IL-17A treatment slightly enhanced IB-␣ phosphorylation. IL-1 treatment apparently induced IB-␣ phosphorylation and degradation in HGFs. Simultaneous stimulation of IL17A and IL-1 significantly enhanced IB-␣ phosphorylation in HGFs treated for 30 or 60 minutes compared with the single stimulation of IL-1 (Fig. 4A and 4B). Furthermore, simultaneous stimulation of IL-17A- and IL-1 induced IB-␣ degradation in HGFs treated for 15 minutes, although a single stimulation of IL-1 did not induce degradation (Fig. 4A). 3.5. Effects of IL-1 on IL-17 receptor expression on HGFs We hypothesized that IL-1 stimulation might modulate IL-17 receptor expression because simultaneous stimulation of IL-1 and IL-17A enhanced CCL20 production. As illustrated in Fig. 5, IL-1 stimulation slightly enhanced IL-17RC expression on HGFs, although the IL-17RA expression did not change. 4. Discussion In this study, we demonstrated for the first time that IL-17A and IL-1 are able to induce CCL20 production in HGFs. It has been reported that CCL20 is involved in the migration of Th17 cells because Th17 cells preferentially express CCR6 [12]. Moreover, it was suggested that Th17 cells are involved in the exacerbation of periodontal disease [15]. Therefore, IL-17A might induce periodontal tissue destruction by enhancing Th17 cell migration. It has been reported that IL-1 enhanced IL-17A production from Th17 cells [16] and ␥␦T cells [17]. Moreover, IL-17A induced IL-1 production from human macrophages [18]. Our report suggests that IL-1 and IL-17A synergistically enhanced production of the Th17 chemokine, CCL20. These data indicate that IL-1 is actively involved in IL-17A-related inflammation in peripheral tissues to induce IL-17A production and attracting Th17 cells. Furthermore, IL-17A from Th17 cells might enhance IL-1 production from
Fig. 3. Effects of IL-1 and IL-17A stimulation on MAPK phosphorylation in HGFs. The cultured cells were stimulated with IL-1 (10 ng/mL) with or without IL-17A (100 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to SDS–PAGE followed by Western blotting analysis with antibodies against phosphospecific p38 MAPK, p38 MAPK, phosphospecific ERK, ERK, phosphorspecific JNK, or JNK. A representative Western blot displaying phospho-p38 MAPK, total-p38 MAPK, phosphor-ERK, total-ERK, phosphoJNK, and total-JNK levels in HGFs from 3 independent experiments.
Y. Hosokawa et al. / Human Immunology 73 (2012) 26-30
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Fig. 4. Effects of IL-1 and IL-17A stimulation on IB-␣ phosphorylation and degradation in HGFs. (A) The cultured cells were stimulated with IL-1 (10 ng/mL) with or without IL-17A (100 ng/mL) for 15, 30, or 60 minutes. The cell extracts were subjected to SDS–PAGE followed by Western blotting analysis with antibodies against phosphospecific phosphorspecific IB-␣, IB-␣, or actin. A representative Western blot displaying phospho-IB-␣, total-IB-␣, and actin levels in HGFs from 3 independent experiments. (B) Bar graphs of phospho-IB-␣ expression were normalized to actin using NIH image software. The error bars indicate the SD of the values. *p ⬍ 0.05, significantly different from the IL-1-stimulated HGFs for 30 minutes. #p ⬍ 0.05, significantly different from from the IL-1-stimulated HGFs for 60 minutes.
inflammatory cells in inflamed tissues. Therefore, IL-1 might be a therapeutic target to Th17 cell–related diseases such as arthritis and periodontal disease [12,15]. We revealed that ERK and NF-B pathways are related to Th17 chemokine expression in HGFs. Brereton et al. recently reported that treatment with an ERK inhibitor attenuated experimental autoimmune encephalomyelitis, which is a Th17-related disease [19]. They also reported that the ERK pathway is involved in the production of IL-23, a cytokine related to Th17 cell expansion, by lipopolysaccharide-stimulated dendritic cells. In this report, we elucidated that ERK is important for CCL20 production from IL-1and IL-17A-stimulated HGFs. It is also reported that the production of IL-23, which is related to Th17 cell differentiation, from synovial fibroblasts is inhibited by NF-B inhibitors [20] Therefore, ERK and NF-B are positively related to Th17 inflammation not only through Th17 cell expansion but also by attracting Th17 cells into peripheral tissues. Zrioual et al. reported that synoviocytes expressed both IL-17RA and IL-17RC, and both receptors are necessary for IL-6 production from IL-17A-stimulated synoviocytes [21]. Our report indicates that IL-1 stimulation enhanced only IL-17RC expression on HGFs. This is the first report that demonstrates that IL-17RC expression is modified by cytokine stimulation. IL-17RC might be actively related to Th17-related inflammation to change its expression. Further
examination might be necessary to determine the function and character of IL-17RC. IL-17A attracts neutrophils to control bacterial challenge and prevents pathogen-initiated bone destruction [22]. However, IL17A could increase the RANKL expression and promote osteoclastic bone erosion in murine collagen arthritis [23]. We believe that CCL20 produced from HGFs could attract Th17 cells near alveolar bone. Therefore, IL-17A produced from Th17 cells enhanced RANKL around osteoclasts in periodontally diseased tissues. However, further study is necessary to clarify the role of IL-17A in periodontal disease. Recently, Duarte et al. reported that Th17 cells are enhanced in the periodontal sites, especially in diabetic patients [24]. It has also been reported that high glucose induces CCL20 production in renal proximal tubule cells [25]. Therefore, high glucose levels might be related to Th17 cell migration in peripheral tissues. Further examination is necessary. In summary, the current study demonstrates that IL-17A and IL-1 synergistically enhanced CCL20 release by cultured HGFs. We also revealed that ERK and NF-B pathways and enhanced IL-17RC expression were related to CCL20 production from IL-1- and IL17A-stimulated HGFs. We discovered that IL-1 is related to Th17 migration into peripheral tissues to induce CCL20, although IL-1 can induce Th17 cell differentiation [26] and IL-17A production
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Fig. 5. Effects of IL-1 on IL-17 receptor expression on HGFs. HGFs were treated with IL-1 (10 ng/mL), and the cells were collected after 24 hours. The expression levels of IL-17RA and IL-17RC on HGFs were measured using flow cytometry as described under Subjects and Methods. The blue line represents the background level of fluorescence caused by the isotype-matched antibody. The red line represents IL-17RA or IL-17RC expression on nonstimulated HGFs. The green line represents IL-17RA or IL-17RC expression on IL-1-treated HGFs. The numbers in the graph are the mean fluorescence intensity of each graph. One of 3 experiments with similar results is presented.
from Th17 cells and ␥␦T cells [27]. HGFs in the presence of IL-17A and IL-1 may actively participate in the recruitment of Th17 cells into periodontally diseased tissues by enhanced production of CCL20. Acknowledgments This study was supported by a Grant-in-Aid for Young Scientists (19791616). References [1] Seymour GJ. Importance of the host response in the periodontium. J Clin Periodontol 1991;18:421– 6. [2] Page RC, Offenbacher S, Schroeder HE, Seymour GJ, Kornman KS. Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions. Periodontology 2000 1997;14:216 – 48. [3] Fujihashi K, Kono Y, Beagley KW, Yamamoto M, McGhee JR, Mestecky J, et al. Cytokines and periodontal disease: immunopathological role of interleukins for B cell responses in chronic inflamed gingival tissues. J Periodontol 1993;64: 400 – 6. [4] Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007;148:32– 46.
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