Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells

archives of oral biology 59 (2014) 93–101

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Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells Mayuka Nakajima a,b, Tomoyuki Honda a,b, Sayuri Miyauchi a,b, Kazuhisa Yamazaki a,* a

Laboratory of Periodontology and Immunology, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan b Division of Periodontology, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

article info

abstract

Article history:

Objectives: This study aims to clarify whether gingival fibroblasts produce periostin in

Accepted 13 October 2013

response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism.

Keywords:

Design: Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial

Periostin

cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-a

Gingival fibroblast

(TNF-a) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was

Periodontitis

analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expres-

Th2 cytokine

sion of the IL-4 receptor a-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Results: While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-a or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found. Conclusion: The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin. # 2013 Elsevier Ltd. All rights reserved.

* Corresponding author at: Laboratory of Periodontology and Immunology, Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, 5274 Gakkocho 2-ban-cho, Chuo-ku, Niigata 951-8514, Japan. Tel.: +81 25 227 0744; fax: +81 25 227 0744. E-mail address: [email protected] (K. Yamazaki). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.10.004

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1.

archives of oral biology 59 (2014) 93–101

Introduction

Since the proposal of the T helper 1/T helper 2 (Th1/Th2) immune response model, a number of studies have attempted to explain the progression of gingivitis to periodontitis in the context of the Th1/Th2 paradigm in humans.1,2 In this context, the authors and others have shown that interleukin-4 (IL-4) and IL-6 are predominant mediators at the protein level,3–5 Furthermore, IL-13, which shows biological activities that are similar to those of IL-4 in many aspects, is reported to be elevated in periodontitis lesions.6,7 Although contradictory results have been reported regarding the predominance of Th1 or Th2 cytokines in periodontitis lesions, both Th1 and Th2 cytokines are at least elevated in the lesions compared with their levels in gingivitis or healthy tissues. Periostin, a matricellular protein belonging to the fasciclin family, is highly expressed by periodontal ligament cells.8 It was reported that periostin plays a crucial role during periodontal homeostasis, maintaining a healthy tooth-supporting apparatus against various biochemical challenges.9 In addition, periostin was reported to be highly inducible by IL-4 and IL-13 in lung fibroblasts, resulting in fibrosis in bronchial asthma by inducing inflammation.10,11 Subsequently, using a skin inflammation model, it was demonstrated that periostin plays a critical role in the amplification and chronicity of allergic skin inflammation.12 These studies led us to speculate that periostin is involved in Th2-mediated tissue destruction in the periodontium. In the present study, we analysed the expression of periostin in gingival fibroblasts in response to Th2 cytokines, a representative bacterial antigen Porphyromonas gingivalis lipopolysaccharide (LPS) and the pro-inflammatory cytokine tumour necrosis factor-a (TNF-a). The role of periostin in the induction of cytokines and chemokines in gingival fibroblasts and in gingival epithelial cells was also analysed.

2.

Materials and methods

2.1.

Reagents and antibodies

and approved by the Institutional Review Board of the Niigata University Graduate School of Medical and Dental Sciences. Human gingival fibroblasts (hGFs) were prepared from clinically normal gingival tissue obtained via extraction of a non-infected third molar from healthy volunteers, as previously described.13 Human periodontal ligament cells (hPDLs) were obtained from the middle third of the premolar root extracted for orthodontic reasons, as previously described.14 The cells were maintained in 25 mM HEPESbuffered Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal calf serum, 100 U ml 1 penicillin and 100 mg ml 1 streptomycin, hereafter referred to as medium, and used for the experiments at passages 6–12. A Simian virus 40 (SV40) T-antigen-immortalised gingival epithelial cell line, epi4, was kindly provided by Dr. Shinya Murakami (Osaka University Graduate School of Dentistry, Osaka, Japan) and maintained as described previously.15,16 For the stimulation experiments, cells were seeded into a 12-well culture plate (TPP, Trasadingen, Switzerland) at a concentration of 2  105 cells ml 1 media in each well. After 12 h of incubation, the attached cells were stimulated with IL-4 (10 ng ml 1), IL-13 (10 ng ml 1), TNF-a (1 ng ml 1) and P. gingivalis LPS (1 mg ml 1) for 8 and 24 h and with periostin (1 mg ml 1) for 12, 24, 48 and 72 h (in some experiments).

2.3.

Total RNA was isolated from unstimulated and stimulated cells using TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was synthesised as described previously17 and amplified using TaqMan Gene Expression Assay primer/probe sets for messenger RNAs (mRNAs) (Applied Biosystems, Foster City, CA, USA) and EagleTaq Master Mix (Roche Molecular Systems, Branchburg, NJ, USA), according to the manufacturer’s instructions. Polymerase chain reactions (PCRs) reactions were conducted using the ABI Prism 7900 HT sequence detection system (Applied Biosystems) and ABI Prism SDS 2.0 software (Applied Biosystems). The relative expression level of each mRNA was normalised to that of GAPDH mRNA using the 2 DDCt cycle threshold method.18

2.4. Recombinant human periostin and TNF-a were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human IL-4 and IL-13 were purchased from eBioscience (San Diego, CA, USA). Blocking mouse anti-human IL-4Ra antibody and mouse immunoglobulin G2a (IgG2a) antibody (as control) were purchased from R&D Systems. Ultrapure LPS from P. gingivalis was obtained from InvivoGen (San Diego, CA, USA). Anti-human periostin (ab14041; Abcam, Cambridge, UK), antihuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab8245, Abcam) and the ECL Plus Western Blotting Reagent Pack (GE Healthcare, Buckinghamshire, UK) were used for Western blotting.

2.2.

Cell preparation and culture

Prior to inclusion in this study, all human subjects provided informed consent according to a protocol that was reviewed

Gene expression analysis

Western blotting

The cells were washed twice with ice-cold PBS, and protein was extracted using M-PER Mammalian Protein Extraction Reagent supplemented with Halt Protease Inhibitor Cocktail and Halt Phosphatase Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL, USA). The protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce Biotechnology). Ten micrograms of each sample were solubilised in sodium dodecyl sulphate (SDS) sample buffer, separated by sodium dodecyl sulphate–polyacryalamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Co., Bedford, MA, USA) and Western blotted with each antibody. The proteins were detected using ECL Plus Western blotting detection reagents (GE Healthcare) and a LumiVision PRO 400EX system (Aisin Seiki, Aichi, Japan).

archives of oral biology 59 (2014) 93–101

2.5.

Cytokine assay

The levels of IL-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) in the supernatants of each culture were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Pierce Biotechnology for IL-6 and IL-8; R&D Systems for MCP-1), according to the manufacturer’s instructions.

2.6.

Immunocytochemistry

The hGFs and epi4 cells were seeded in a Lab-Tek Chamber Slide (Nunc, Rochester, NY, USA) at 1  105 cells ml 1. After 12 h of incubation, the attached cells were stimulated with IL-4 (10 ng ml 1) for 2 h. The cells were then partially fixed with chloroform/acetone for 5 min, washed in phosphate-buffered saline (PBS) and stained with mouse monoclonal anti-human IL-4Ra IgG (H-4; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For secondary staining, we used Alexa Fluor 488 rabbit anti-mouse IgG (Invitrogen). The nuclei were stained using Vectashield mounting medium with 4,6-diamidino-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The cells were then imaged by fluorescence microscopy (Biozero BZ-8000; Keyence, Tokyo, Japan).

2.7.

Statistical analysis

All experiments were performed in triplicate wells for each set of conditions and were repeated at least twice. The results were expressed as the mean  standard deviation (SD). When two groups were compared, an unpaired t-test was used. Probability values of <0.05 were considered statistically significant.

3.

Results

3.1. Effect of IL-4, IL-13, TNF-a, and P. gingivalis LPS on the expression of periostin The stimulatory effect of IL-4 and IL-13 on periostin production was the most pronounced, whereas the effect of TNF-a was suppressive at both the RNA (Fig. 1A) and protein levels (Fig. 1B), irrespective of the cell types. No stimulatory effect was observed for P. gingivalis LPS. With regard to the effect of IL-4 and IL-13 on different cell types, hGFs and hPDLs responded significantly to the stimulations; however, epi4 cells showed a very weak response. To further confirm that increased expression of periostin in hGF is mediated by IL-4R signalling, anti-IL-4Ra blocking antibody was added to the cultures prior to addition of IL-4. The anti-IL-4Ra antibody significantly suppressed the IL-4-induced periostin mRNA expression (Fig. 1C).

3.2.

Expression of IL-4R

Because IL-4 and IL-13 share a receptor subunit (IL-4Ra), the expression of IL-4Ra was analysed by immunocytochemistry. As shown in Fig. 2, hGF cells expressed IL-4Ra weakly but expressed it substantially on their surfaces (A). The

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expression level increased upon stimulation with IL-4 (B). By contrast, neither substantial expression (C) nor induction by IL-4 stimulation of IL-4Ra (D) was observed in epi4 cells.

3.3. Effect of periostin stimulation on the expression of inflammatory cytokines and matrix protein-related genes The cells were stimulated with either periostin or TNF-a, and gene expression and protein production of IL-6, IL-8, and MCP1 were analysed by real-time PCR and ELISA, respectively. The expression profile was significantly different among the different cell types. Even though the stimulatory effect of periostin was much weaker than that of TNF-a, the gene expression of IL-6, IL-8 and MCP-1 was significantly increased in hGF at 12 h. Under these experimental conditions, IL-8 gene expression increased by 3-fold. IL-6 and MCP-1 expression levels were also significantly elevated at 24 h. However, the increase in gene expression was less than that observed after 12 h. The stimulatory effect of periostin further declined at 48 h and no difference was observed compared with 12-h stimulation. By contrast, the stimulatory effect of TNF-a peaked at 48 h (Fig. 3A). Consistent with the gene expression data, protein production by the cells was also increased but it was very weak with periostin stimulation. The stimulatory effect of periostin was significant for IL-8, and the level was two times higher compared to that of the unstimulated cells. Periostin induced a marginal increase of IL-6 and MCP-1 production in hGF, although the difference was statistically significant (Fig. 3B). In hPDL, a significant but very weak stimulatory effect of periostin was observed for MCP-1 at 12 h only, although the stimulatory effect of TNF-a on the gene expression of IL-6, IL-8 and MCP-1 was similar to those in hGF (Fig. 4A). Protein analysis was carried out for MCP-1 alone because no stimulatory effect on gene expression was observed for IL-6 or IL-8. However, no increase of MCP-1 production was found at any time point analysed (Fig. 4B). Similar to hPDL, the stimulatory effect of periostin was significant for the gene expression of MCP-1 in epi4 cells only. However, a significant effect was observed at 48 h (Fig. 4C). Therefore, protein production was analysed at 72 and 48 h. Although no significant increase in the production of MCP-1 protein resulting from stimulation with periostin was observed at 12 or 24 h, significant increases in MCP-1 production were observed at 48 and 72 h. The stimulatory effect of TNF-a on epi4 cells was much weaker than that on hGF and hPDL, particularly for MCP-1 expression. At 48 h, no effect was observed (Fig. 4D). Because periostin has been demonstrated to play an important role in matrix metabolism and to be essential for tissue integrity, we also analysed the gene expression of transforming growth factor-b1 (TGF-b1), type 1 collagen (Col1a1) and matrix metalloproteinase-2 (MMP-2) in hGF (Fig. 5A) and epi4 cells (Fig. 5B) by real-time PCR. Whereas no stimulatory effect of periostin was observed for the tested molecules, TNF-a stimulation tended to down-regulate the gene expressions, although the effect was weak. The effect of the stimulants demonstrated little difference between hGF and epi4 cells.

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Fig. 1 – The effect of stimulation with IL-4, IL-13, TNF-a and P. gingivalis LPS on the expression of periostin in human gingival fibroblasts (hGF), human periodontal ligament cells (hPDL), and epi4 cells. The cells were either unstimulated or stimulated with the indicated stimulants for 8 h and 24 h, and the mRNA level of periostin (POSTN) was analysed by quantitative RTPCR (A). After stimulation with the indicated reagents for 24 h, the protein level of periostin was further analysed by Western blotting (B). To confirm the stimulating effect of IL-4, hGF were cultured in the presence of anti-human IL-4Ra antibody or control antibody at a concentration of 20 mg mlS1, and then stimulated with IL-4. The periostin mRNA levels were analysed by quantitative RT-PCR (C). The results shown are the mean W SD of triplicate cultures from a representative experiment. The gene expression levels in unstimulated cultures were set to one. *p < 0.05.

archives of oral biology 59 (2014) 93–101

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Fig. 2 – Expression of IL-4R in human gingival fibroblasts (hGF) and epi4 cells. hGF and epi4 cells were either unstimulated (A and C) or stimulated with 10 ng mlS1 of IL-4 (B and D) for 2 h, and the expression of IL-4Ra was analysed by immunocytochemistry. Surface IL-4Ra and nuclei of hGF and epi4 cells were labelled with Alexa Fluor 488 (green) and DAPI (blue), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

4.

Discussion

Periodontitis lesions are considered to be clinically progressive and are histologically characterised as B-cell/plasma cellpredominant lesions.19,20 While activated B cells are typically observed in the most apical portion of the inflammatory infiltrate beneath the epithelium of the developing pocket,21 the sulcular region is enriched with T cells.22,23 Thus, B cells and plasma cells in periodontitis lesions are localised in proximity to T cells to activate B cells. For B cells to be adequately activated, Th2-associated cytokines are required.24 The cells in the lesions have been reported to express these proteins, suggesting that they are produced locally. Although Th2-associated cytokines are known to be involved in the activation of B cells in periodontitis lesions, their association with pathogenesis, for example, the inflammation of periodontal tissue, remains unclear. The role of periostin, a matricellular protein, in the mediation of the inflammatory response has recently been clarified.11,12 Periostin is highly expressed in periodontal ligament cells and in the periosteum.8 It has been demonstrated that periostin plays an important role in tissue integrity, tooth development, the eruption process,9,25,26 recruitment and attachment of osteoblast precursors8 and osteoblast differentiation.27 These roles indicate that periostin

has a protective rather than a destructive function regarding the periodontium. In this study, we found that periostin can be produced by gingival fibroblasts in addition to periodontal ligament cells. Because periostin is primarily produced in the periodontium and is in fact produced by gingival fibroblasts as much as by periodontal ligament cells, it is reasonable to suppose that periostin is also involved in inflammation in gingival tissue. Periostin expression can be significantly upregulated by stimulation with IL-4 and IL-13 in hGF and hPDL, both of which are identified in periodontitis lesions. However, inflammatory stimuli such as TNF-a or P. gingivalis LPS demonstrated no such stimulatory effect. In contrast to fibroblastic cell types, IL-4 and IL-13 exhibited very weak stimulatory activity on the gingival epithelial cell line epi4. This finding is inconsistent with the effect of IL-13 on human bronchial epithelial cells, where IL-13 dramatically increased periostin gene expression.28 By contrast, the expression of the IL-4R gene was not different among the cell types examined in this study (data not shown). Therefore, the effect of IL-4 and IL-13 on the expression of periostin may be cell-type specific. IL-4R and IL-13R share a common receptor subunit IL-4Ra. The expression level of IL-4Ra on hGF was low without stimulation but increased with IL-4 stimulation. Therefore, it is conceivable that IL-4 stimulation further enhanced the production of periostin in response to IL-4

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archives of oral biology 59 (2014) 93–101

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Fig. 3 – The effect of periostin stimulation on the expression of inflammatory cytokines in human gingival fibroblasts (hGF). hGF were stimulated with either 1 mg mlS1 of periostin or 1 ng mlS1 of TNF-a for the indicated times, and the expression levels of IL-6, IL-8 and MCP-1 mRNAs were analysed by quantitative RT-PCR (A). The levels of IL-6, IL-8 and MCP-1 in the culture supernatants were analysed by ELISA (B). The results shown are the mean W SD of triplicate cultures from a representative experiment. The gene expression levels in unstimulated cultures were set to one. *p < 0.05.

or IL-13. The lack of the stimulating effect of IL-4 on the epi4 cell expression of periostin could be due to very low expression of IL4R on the cell surface. A recent study demonstrated that periostin produced by fibroblasts stimulates keratinocyte inflammation and mediates chronic allergic inflammation resulting from atopic dermatitis.12 However, epi4 cells showed weak elevation of only MCP-1 gene expression at 48 h after stimulation by periostin. At the protein level, very weak stimulatory effect was observed at 48 and 72-h stimulations. Although these weak elevations were statistically significant, periostin was considered to have little biological effect in epi4 cells. Therefore, it is reasonable to assume that skin keratinocytes and gingival epithelial cells have differential responsiveness to periostin.

The differential responsiveness of airway epithelial cells and gingival epithelial cells to periostin was further confirmed by the expression of TGF-b1 and Col1a1 mRNA. Recombinant periostin induced type 1 collagen and TGF-b in airway epithelial cells that are important molecules in airway fibrosis in asthma,28 whereas these effects were not observed in gingival epithelial cells. Moreover, although periostin is reported to downregulate MMP-2 in hPDL,29 the effect was not found in hGF. These results further indicate that cellular interaction between epithelial and mesenchymal cells via periostin is different between gingiva and skin or airway. Although we clearly demonstrated that gingival fibroblasts are a source of periostin, which does not mediate

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archives of oral biology 59 (2014) 93–101

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Fig. 4 – The effect of periostin stimulation on the expression of inflammatory cytokines in human periodontal ligament cells (hPDL) (A and B) and epi4 cells (C and D). The cells were stimulated with either 1 mg mlS1 of periostin or 1 ng mlS1 of TNF-a for indicated times, and the expression levels of IL-6, IL-8 and MCP-1 mRNAs were analysed by quantitative RT-PCR (A and C). The levels of MCP-1 in the culture supernatants were analysed by ELISA (B and D). The results shown are the mean W SD of triplicate cultures from a representative experiment. The gene expression levels in unstimulated cultures were set to one. *p < 0.05.

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archives of oral biology 59 (2014) 93–101

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Fig. 5 – The effect of periostin stimulation on the expression of matrix protein-related genes in hGF (A) and epi4 cells (B). The cells were stimulated with either 1 mg mlS1 of periostin or 1 ng mlS1 of TNF-a for indicated times, and the expression levels of MMP-2, TGF-b1 and type 1 collagen (Col1a1) mRNAs were analysed by quantitative RT-PCR. The results shown are the mean W SD of triplicate cultures from a representative experiment. The gene expression levels in unstimulated cultures were set to one. *p < 0.05.

inflammatory responses in either gingival epithelial cells or gingival fibroblasts, this study has a limitation in that the expression and the effect of periostin were analysed by using only one cell line for each cell culture. Further study is clearly needed to determine whether periostin is indeed produced by connective tissue fibroblasts in human periodontitis lesions, and if so, the exact role of periostin in chronic inflammation of the periodontium.

Funding The work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (23390476 and the Young Researcher Overseas Visits Program for Vitalizing Brain Circulation, S2203) and the Promotion of Niigata University Research Project.

Conflict of interest There is no conflict of interest.

Ethical approval The study protocol was reviewed and approved by the Institutional Review Board of the Niigata University Graduate School of Medical and Dental Sciences.

Acknowledgements We thank S. Murakami from the Department of Periodontology, Division of Oral Biology and Disease Control, Osaka

archives of oral biology 59 (2014) 93–101

University Graduate School of Dentistry, Osaka, Japan, for providing the human gingival epithelial cell line, epi4.

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