Tumor Necrosis Factor‐α Inhibits Transforming Growth Factor‐β–Stimulated Myofibroblastic Differentiation and Extracellular Matrix Production in Human Gingival Fibroblasts

Background: Fibroblasts play a critical role during wound healing and chronic inflammation through the synthesis and assembly of extracellular matrix (ECM) molecules. These responses may be modulated by soluble cytokines and growth factors present in tissues. In the present study, we evaluate whether transforming growth factor‐β1 (TGF‐β1) and tumor necrosis factor‐α (TNF‐α) modulate myofibroblastic differentiation and the production of ECM components. Methods: Primary cultures of human gingival fibroblasts (HGFs) were stimulated with recombinant TGF‐β1 and TNF‐α. Protein levels of α‐smooth muscle actin (α‐SMA), type I collagen, heat shock protein‐47 (HSP‐47), fibronectin (FN), ED‐A‐FN, and periostin and activation of the Smad pathway were evaluated through Western blot analysis. α‐SMA and actin fibers were identified by immunofluorescence. TGF‐β1, TNF‐α, and α‐SMA were identified by immunohistochemistry in biopsies of inflamed human gingival tissues. TGF‐β1 activity was evaluated using a plasminogen activator inhibitor‐1 (PAI‐1) reporter transfected in HGFs. Results: TGF‐β1 stimulated the differentiation of myofibroblasts as evidenced by strong expression of α‐SMA and ED‐A‐FN. Moreover, TGF‐β1 induced the production of type I collagen, HSP‐47, FN, and periostin. Costimulation with TNF‐α and TGF‐β1 significantly reduced the expression of all the above‐mentioned proteins. TNF‐α also inhibited the activation of the Smad2/3 pathway and the activity of the PAI‐1 reporter. Conclusions: TNF‐α inhibits several cell responses induced by TGF‐β1, including the differentiation of myofibroblasts, the activation of the Smad signaling pathway, and the production of key molecules involved in tissue repair, such as type I collagen, FN, and periostin. The interaction between cytokines may explain the delayed tissue repair observed in chronic inflammation of gingival tissues.     

Cytokines differentially regulate CXCL10 production by interferon-gamma-stimulated or tumor necrosis factor-alpha-stimulated human gingival fibroblasts

Background and Objective: CXC chemokine 10 (CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T‐helper 1 cells. In this study we examined the effects of cytokines on CXCL10 production by human gingival fibroblasts. Material and Methods: Human gingival fibroblasts were exposed to pro‐inflammatory cytokines (interleukin‐1β, tumor necrosis factor‐α), a T‐helper 1 cytokine (interferon‐γ), T‐helper 2 cytokines (interleukin‐4, interleukin‐13), T‐helper 17 cytokines (interleukin‐17A, interleukin‐22) and regulatory T‐cell cytokines (interleukin‐10, transforming growth factor‐β1) for 24 h. CXCL10 production by human gingival fibroblasts was examined by enzyme‐linked immunosorbent assay. Results: Human gingival fibroblasts produced CXCL10 protein upon stimulation with interleukin‐1β, tumor necrosis factor‐α and interferon‐γ. Treatment of human gingival fibroblasts with interferon‐γ in combination with tumor necrosis factor‐α or interleukin‐1β resulted in a synergistic production of CXCL10. However, interleukin‐4 and interleukin‐13 inhibited CXCL10 production by interferon‐γ‐stimulated or tumor necrosis factor‐α‐stimulated‐human gingival fibroblasts. On the other hand, interleukin‐17A and interleukin‐22 enhanced CXCL10 production by human gingival fibroblasts treated with interferon‐γ and inhibited CXCL10 production by tumor necrosis factor‐α‐stimulated human gingival fibroblasts. Furthermore, the anti‐inflammatory cytokine, interleukin‐10, inhibited CXCL10 production by both interferon‐γ‐ and tumor necrosis factor‐α‐stimulated human gingival fibroblasts, but transforming growth factor‐β1 enhanced interferon‐γ‐mediated CXCL10 production by human gingival fibroblasts. Conclusion: These results mean that the balance of cytokines in periodontally diseased tissue may be essential for the control of CXCL10 production by human gingival fibroblasts, and the production of CXCL10 might be important for the regulation of T‐helper 1 cell infiltration in periodontally diseased tissue.     

Tumor necrosis factor-alpha stimulates gingival epithelial cells to release high mobility-group box 1

Background and Objective: High‐mobility‐group box 1 functions as a late‐phase inflammatory mediator. It can be released extracellularly by macrophages and necrotic cells through lipopolysaccharide and tumor necrosis factor‐α. The objective of this study was to clarify the source of high‐mobility‐group box 1 in chronic periodontitis tissues and tumor necrosis factor‐α‐stimulated gingival epithelial cells, and subsequently elucidate its inducible inflammatory pathway. Material and Methods: Chronic periodontitis and healthy gingival sections were stained for high‐mobility‐group box 1 by immunohistochemistry and immunofluorescence. The amounts of high‐mobility‐group box 1 released into the gingival crevicular fluid and supernatants from gingival epithelial cells stimulated by tumor necrosis factor‐α were examined by western blot. The phosphorylation of mitogen‐activated protein kinases (MAPKs) in gingival epithelial cells was also examined. Results: High‐mobility‐group box 1 was detected in the cytoplasm and nucleus of gingival epithelial cells with periodontitis. Western blotting revealed a significant increase in high‐mobility‐group box 1 expression in the gingival crevicular fluid from periodontitis patients. High‐mobility‐group box 1 production in gingival epithelial cells was increased following stimulation with tumor necrosis factor‐α. The molecular dialogue between tumor necrosis factor‐α and gingival epithelial cells involved modulation of the activities of p38MAPK, Jun N‐terminal kinase and p44/42. Interestingly, only phosphorylation of p38MAPK contributed to more than half of the signaling initiated by tumor necrosis factor‐α‐elicited high‐mobility‐group box 1 release. Conclusion: High‐mobility‐group box 1 is continuously released from the gingival epithelial cells modulated by tumor necrosis factor‐α. These findings imply that high‐mobility‐group box 1 expression and possibly p38MAPK constitute important features in periodontitis.     

Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Mechanical force augments the anti-osteoclastogenic potential of human gingival fibroblasts in vitro

Background and Objective: The cellular response of human gingival fibroblasts to a mechanical force is considered to be primarily anti‐osteoclastic because they produce relatively high levels of osteoprotegerin. However, there is little information available on the effects of compression force on the production of osteoprotegerin and osteoclastic differentiation by these cells. In this study, we examined how mechanical force affects the nature of human gingival fibroblasts to produce osteoprotegerin and inhibit osteoclastogenesis. Material and Methods: Human gingival fibroblasts were exposed to mechanical force by centrifugation for 90 min at a magnitude of approximately 50 g/cm². The levels of osteoprotegerin, receptor activator of nuclear factor‐κB ligand (RANKL), interleukin‐1β and tumor necrosis factor‐α were measured at various time‐points after applying the force. The effect of the centrifugal force on the formation of osteoclast‐like cells was also determined using a co‐culture system of human gingival fibroblasts and bone marrow cells. Results: Centrifugal force stimulated the expression of osteoprotegerin, RANKL, interleukin‐1β and tumor necrosis factor‐α by the cells, and produced a relatively high osteoprotegerin to RANKL ratio at the protein level. Both interleukin‐1β and tumor necrosis factor‐α accelerated the force‐induced production of osteoprotegerin, which was inhibited significantly by the addition of anti‐(interleukin‐1β) immunoglobulin Ig isotype; IgG (rabbit polyclonal). However, the addition of anti‐(tumor necrosis factor‐α) immunoglobulin Ig isotype; IgG1 (mouse monoclonal) had no effect. Centrifugal force also had an inhibitory effect on osteoclast formation. Conclusion: Application of centrifugal force to human gingival fibroblasts accelerates osteoprotegerin production by these cells, which stimulates the potential of human gingival fibroblasts to suppress osteoclastogenesis. Overall, human gingival fibroblasts might have natural defensive mechanisms to inhibit bone resorption induced by a mechanical stress.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Cytokine profile in the gingival crevicular fluid of rheumatoid arthritis patients with chronic periodontitis

The aim was to assess the cytokine profile in the gingival crevicular fluid (GCF) of rheumatoid arthritis (RA) patients with chronic periodontitis (CP). Databases were searched from 1991 to August 2013 using a combination of various keywords. Eight studies were included. The GCF concentrations of interleukin (IL)‐1β, IL‐4, IL‐10, matrix metalloproteinase (MMP)‐8, MMP‐13 and tumor necrosis factor‐alpha (TNF‐α) were reported to be higher in patients with RA than in healthy controls (HC) without CP. In one study, TNF‐α levels in GCF were significantly higher in HC than in RA patients receiving anti‐TNF‐α therapy. One study reported no significant difference in GCF TNF‐α levels among RA patients and HC regardless of anti‐TNF‐α therapy. One study reported no difference in IL‐1β and prostaglandin E₂ levels among RA patients and HC with CP. Raised levels of proinflammatory cytokines are exhibited in the GCF of RA patients with CP.     

Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor-α-stimulated human gingival epithelial cells

Fujita T, Yumoto H, Shiba H, Ouhara K, Miyagawa T, Nagahara T, Matsuda S, Kawaguchi H, Matsuo T, Murakami S, Kurihara H. Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor‐α‐stimulated human gingival epithelial cells. J Periodont Res 2012; 47: 55–61. © 2011 John Wiley & Sons A/S Background and Objective: As epithelial cells function as a mechanical barrier, the permeability of the gingival epithelial cell layer indicates a defensive capability against invasion by periodontal pathogens. We have reported the expression of claudin‐1 and E‐cadherin, key regulators of permeability, in the gingival junctional epithelium. Irsogladine maleate (IM) is a medication for gastric ulcers and also regulates Aggregatibacter actinomycetemcomitans‐stimuated chemokine secretion and E‐cadherin expression in gingival epithelium. In this study, we have further investigated the effects of IM on the barrier functions of gingival epithelial cells under inflammatory conditions. Material and methods: We examined the permeability, and the expression of claudin‐1 and E‐cadherin, in human gingival epithelial cells (HGECs) stimulated with tumor necrosis factor (TNF)‐α, with or without IM. Results: TNF‐α increased the permeability of HGECs, and IM abolished the increase. TNF‐α reduced the expression of E‐cadherin in HGECs, and IM reversed the reduction. In addition, immunofluorescence staining showed that TNF‐α disrupted claudin‐1 expression in HGECs, and IM reversed this effect. Conclusion: The results suggest that IM reverses the TNF‐α‐induced disruption of the gingival epithelial barrier by regulating E‐cadherin and claudin‐1.     

Light‐Emitting Diode Irradiation Promotes Donor Site Wound Healing of the Free Gingival Graft

Background: This study aims to evaluate the effect of light‐emitting diode (LED) light irradiation on the donor wound site of the free gingival graft. Methods: Rat gingival fibroblasts were chosen to assess the cellular activities and in vitro wound healing with 0 to 20 J/cm² LED light irradiation. Seventy‐two Sprague‐Dawley rats received daily 0, 10 (low‐dose [LD]), or 20 (high‐dose [HD]) J/cm² LED light irradiation on the opened palatal wound and were euthanized after 4 to 28 days; the healing pattern was assessed by histology, histochemistry for collagen deposition, and immunohistochemistry for tumor necrosis factor (TNF)‐α infiltration. The wound mRNA levels of heme oxygenase‐1 (HO‐1), TNF‐α, the receptor for advanced glycation end products, vascular endothelial growth factor, periostin, Type I collagen, and fibronectin were also evaluated. Results: Cellular viability and wound closure were significantly promoted, and cytotoxicity was inhibited significantly using 5 J/cm² LED light irradiation in vitro. The wound closure, reepithelialization, and collagen deposition were accelerated, and sequestrum formation and inflammatory cell and TNF‐α infiltration were significantly reduced in the LD group. HO‐1 and TNF‐α were significantly upregulated in the HD group, and most of the repair‐associated genes were significantly upregulated in both the LD and HD groups at day 7. Persistent RAGE upregulation was noted in both the LD and HD groups until day 14. Conclusion: LED light irradiation at 660 nm accelerated palatal wound healing, potentially via reducing reactive oxygen species production, facilitating angiogenesis, and promoting provisional matrix and wound reorganization.     

Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts

Song H, Zhao H, Qu Y, Sun Q, Zhang F, Du Z, Liang W, Qi Y, Yang P. Carbon monoxide releasing molecule‐3 inhibits concurrent tumor necrosis factor‐α‐ and interleukin‐1β‐induced expression of adhesion molecules on human gingival fibroblasts. J Periodont Res 2011; 46: 48–57. © 2010 John Wiley & Sons A/S Background and Objective: Carbon monoxide releasing molecule‐3 (CORM‐3) is a newly reported compound that has shown anti‐inflammatory effects in a number of cells. In this study, we aimed to investigate the influence of CORM‐3 on concurrent tumor necrosis factor‐α (TNF‐α)‐ and interleukin (IL)‐1β‐induced expression of adhesion molecules on human gingival fibroblasts (HGF). Material and Methods: HGF were cultured from the explants of normal gingival tissues. Cells were costimulated with TNF‐α and IL‐1β in the presence or absence of CORM‐3 for different periods of time. The expression of adhesion molecules, nuclear factor‐kappaB (NF‐κB) and phosphorylated p38 was studied using western blotting. RT‐PCR was applied to check the expression of the adhesion molecules at the mRNA level. The activity of NF‐κB was analysed using a reporter gene assay. Results: CORM‐3 inhibited the up‐regulation of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule in HGF after costimulation with TNF‐α and IL‐1β, which resulted in the decreased adhesion of peripheral blood mononuclear cells to these cells. Sustained activation of the NF‐κB pathway by costimulation with TNF‐α and IL‐1β was suppressed by CORM‐3, which was reflected by a reduced NF‐κB response element‐dependent luciferase activity and decreased nuclear NF‐κB‐p65 expression. CORM‐3 inhibited MAPK p38 phosphorylation in response to stimulation with proinflammatory cytokines. Conclusion: The results of this study bode well for the application of CORM‐3 as an anti‐inflammatory agent to inhibit NF‐κB activity and to suppress the expression of adhesion molecules on HGF, which suggests a promising potential for CORM‐3 in the treatment of inflammatory periodontal disease.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell‐surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram‐negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), and tumor necrosis factor‐α (TNF‐α) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer‐membrane proteins from P. gingivalis ATCC 53977. Outer‐membrane protein from P. gingivalis enhanced the production of IL‐6 and IL‐8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL‐8 production activity of polysaccharide from P. gingivalis was higher than that of other cell‐surface components. The levels of IL‐6 and IL‐8 released from the P. gingivalis lipopolysaccharide‐treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer‐membrane protein or lipopolysaccharide inhibited the IL‐6 and IL‐8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer‐membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

Involvement of MT1‐MMP and TIMP‐2 in human periodontal disease

Oral Diseases (2010) 16 , 388–395 Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. Methods: MT1‐MMP and TIMP‐2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis‐affected human subjects. MT1‐MMP and TIMP‐2 expression were assessed through Western‐blot of tissue homogenates. The main cell types involved in MT1‐MMP and TIMP‐2 production were evaluated by means of immunohistochemistry. Results: Both MT1‐MMP and TIMP‐2 were significantly increased in periodontitis‐affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1‐MMP and its inhibitor TIMP‐2 was altered in periodontitis‐affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1‐MMP in fibroblasts and macrophages in gingival tissues. MT1‐MMP was detected in cells in close association with the gingival collagen matrix. TIMP‐2 expression was identified in fibroblasts, macrophages and epithelial cells. Conclusions: Our observations show an increased expression of MT1‐MMP and TIMP‐2 in periodontitis‐affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.     

Is There an Interaction Between Polycystic Ovary Syndrome and Gingival Inflammation?

Background: The aim of this study is to evaluate the gingival crevicular fluid (GCF), saliva, and serum concentrations of tumor necrosis factor‐α (TNF‐α), TNF‐α receptor‐1 (TNF‐αR1), TNF‐αR2, and interleukin‐6 (IL‐6) in non‐obese females with polycystic ovary syndrome (PCOS) and either clinically healthy periodontium or gingivitis. Methods: Thirty‐one females with PCOS and healthy periodontium, 30 females with PCOS and gingivitis, and 12 systemically and periodontally healthy females were included in the study. GCF, saliva, and serum samples were collected, and clinical periodontal measurements, body mass index, and Ferriman‐Gallwey score (FGS) were recorded. Sex hormones, cortisol, and insulin levels were measured. TNF‐α, TNF‐αR1, TNF‐αR2, and IL‐6 were determined by enzyme‐linked immunosorbent assay. Kruskal‐Wallis followed by Bonferroni‐corrected post hoc Mann‐Whitney U tests were used to analyze the data. Results: The PCOS + gingivitis group revealed significantly higher GCF, saliva, and serum IL‐6 concentrations than the PCOS + healthy group (P <0.0001). The two PCOS groups exhibited significantly higher saliva TNF‐α concentrations than the control group (P = 0.024 and P = 0.013, respectively). The FGS index was significantly higher in the PCOS + gingivitis group than the PCOS + healthy group (P = 0.030). The PCOS + gingivitis group revealed significantly higher insulin concentration than the PCOS + healthy and control groups (P = 0.014 and P <0.0001, respectively). Serum TNF‐α, TNF‐αRs, and serum, GCF, and salivary IL‐6 levels correlated with the clinical periodontal measurements. Conclusions: PCOS and gingival inflammation appear to act synergistically on the proinflammatory cytokines IL‐6 and TNF‐α. Thus, PCOS may have an impact on gingival inflammation or vice versa. Additional studies are warranted to clarify the possible relationship between PCOS and periodontal disease.     

Hepatocytes produce tumor necrosis factor-α and interleukin-6 in response to Porphyromonas gingivalis

Takano M, Sugano N, Mochizuki S, Koshi RN, Narukawa TS, Sawamoto Y, Ito K. Hepatocytes produce tumor necrosis factor‐α and interleukin‐6 in response to Porphyromonas gingivalis. J Periodont Res; 2012; 47: 89–94. © 2011 John Wiley & Sons A/S Background and Objective: The liver plays a major role in clearing systemic bacterial infections. In addition, inflammatory cytokines produced in the liver play a critical role in systemic cytokine levels. The aim of this study was to investigate the production of tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) by hepatocytes in response to periodontal pathogens. Material and Methods: The mouse hepatic carcinoma cell line Hepa‐1.6 and the mouse macrophage‐like cell line RAW 264 were co‐cultured in Transwell insert plates. Cells were stimulated with bacterial extracts prepared from Porphyromonas gingivalis and the induction of TNF‐α and IL‐6 was measured using real‐time PCR and ELISA. Results: After stimulation with bacteria, the induction of TNF‐α and IL‐6 was observed in RAW 264 cells and Hepa‐1.6 cells. Significant reduction of TNF‐α mRNA expression in Hepa‐1.6 cells was observed after treatment with antibody to TNF‐α. Conclusion: The results obtained in the present study show that P. gingivalis extract induces TNF‐α and IL‐6 in an in vitro liver model and that macrophage‐derived TNF‐α mediates the induction of TNF‐α in hepatocytes.     

Tumor Necrosis Factor‐α Induces Matrix Metalloproteinases‐3, ‐10, and ‐13 in Human Periodontal Ligament Cells

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)‐α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus. Methods: Cultured PDL cells were stimulated with TNF‐α and analyzed with an MMP antibody array. Real‐time polymerase chain reaction (PCR), enzyme‐linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP‐3, ‐10, and ‐13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP‐3, ‐10, and ‐13 was observed after blocking the TNFR with an antagonist. Results from real‐time PCR, ELISA, and western blot were analyzed by paired t test. Results: The antibody array showed that the protein most strongly upregulated by TNF‐α stimulation was MMP‐3, followed by MMP‐13 and MMP‐10. The TNF‐α receptor blocker specifically inhibited expression of MMP‐3 and ‐13. In addition, TNF‐α increased levels of MMP mRNAs in MMP‐3, ‐13, and ‐10 (in decreasing order). However, ELISAs showed that MMP‐13 was the most upregulated protein, followed by MMP‐10 and MMP‐3. Western blotting indicated that TNF‐α increased MMP‐3 and ‐13 levels but had no significant effect on the level of MMP‐10, and zymography showed that TNF‐α increased the activities of all forms of MMP‐3 and ‐13, but MMP‐10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1. Conclusions: TNF‐α (10 ng/mL) upregulates levels of MMP‐3, ‐10, and ‐13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.     

Epidermal growth factor stimulates urokinase-type plasminogen activator expression in human gingival fibroblasts. Possible modulation by genistein and curcumin

BACKGROUND: Regulation of the extracellular matrix turnover is a crucial process in wound healing and the progress of periodontal disease. It has been proposed that urokinase-type plasminogen activator (uPA), under the control of growth factors or cytokines, provides the proteolytic potential to the accomplishment of these cellular events. Epidermal growth factor (EGF) is one of the growth factors that has been shown to be active in uPA regulation. METHODS: In this study, we have assessed the effect of EGF on uPA expression in primary cultures of human gingival fibroblasts. We also studied the signaling pathways involved in this process and the role of the dietary phytoestrogens curcumin and genistein as potential modulators of this response. RESULTS: Human gingival fibroblasts expressed a basal uPA activity, which was inhibited by genistein, but not by curcumin. After treatment with 10 ng/ml EGF, uPA production was strongly stimulated. Exposure to genistein and curcumin inhibited EGF-stimulated urokinase production, although only genistein showed a statistically significant inhibitory response. Using more specific inhibitors, we found that the mitogen-activated extracellular kinase and c-Jun N-terminal kinase (JNK) inhibitors PD98059 and SP600125 also blocked the EGF-dependent stimulatory effect. On the other hand, SB203580, inhibitor of the p38 member of mitogen-activated protein kinase family, did not alter this response. In accordance to these findings, EGF stimulated a potent activation of JNK and a mild activation of extracellular signal-regulated kinases 1/2. Finally, EGF stimulated the phosphorylation of its receptor and tyrphostin (AG1478), curcumin and genistein were able to inhibit this stimulatory effect. CONCLUSIONS: These results indicate that EGF constitutes a strong stimuli on uPA expression in human gingival fibroblasts. Our data also shows that EGF-stimulated uPA production involves the activation of the extracellular signal-regulated kinases 1/2 and JNK signaling pathways and might be modulated by the natural phytoestrogens curcumin and genistein.     

Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.