Retinoic acid-inducible gene-I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin-1beta, -6 and -8 expression

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1beta (IL-1beta) induced RIG-I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG-I expression induced by lipopolysaccharide (LPS) or double-stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG-I in the expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG-I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG-I induction by these stimuli was examined. The expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts transfected with RIG-I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG-I in concentration- and time-dependent manners. We also examined the localization of RIG-I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA-induced RIG-I expression. Introduction of RIG-I cDNA into gingival fibroblasts resulted in enhanced expression of IL-1beta, IL-6 and IL-8; moreover, overexpression of RIG-I stimulated with LPS or dsRNA synergistically increased expression of IL-1beta, IL-6 and IL-8. RIG-I may have important roles in the innate immune response in the regulation of IL-1beta, IL-6 and IL-8 expression in gingival fibroblasts in response to LPS and dsRNA.     

Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts

Lu H‐K, Tseng C‐C, Lee Y‐H, Li C‐L, Wang L‐F. Flutamide inhibits nifedipine‐ and interleukin‐1β‐induced collagen overproduction in gingival fibroblasts. J Periodont Res 2010; 45: 451–457. © 2010 John Wiley & Sons A/S Background and Objective: To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin‐1β‐ and nifedipine‐induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined. Material and Methods: Gingival fibroblasts from healthy subjects and patients with dihydropyridine‐induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin‐1β or both. The mRNA expression was examined using real‐time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot. Results: Interleukin‐1β was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen α1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen α1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin‐1β. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue. Conclusion: The data suggest that both nifedipine and interleukin‐1β play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine‐induced overgrowth cells.     

Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes

Gaibani P, Caroli F, Nucci C, Sambri V. Major surface protein complex of Treponema denticola induces the production of tumor necrosis factor α, interleukin‐1β, interleukin‐6 and matrix metalloproteinase 9 by primary human peripheral blood monocytes. J Periodont Res 2010; 45: 361–366. © 2010 John Wiley & Sons A/S Background and Objective: Treponema denticola is a micro‐organism that is involved in the pathogenesis of periodontitis. Major surface protein complex (MSPc), which is expressed on the envelope of this treponeme, plays a key role in the interaction between T. denticola and gingival cells. The peptidoglycan extracted from T. denticola induces the production of a large variety of inflammatory mediators by macrophage‐like cells, suggesting that individual components of T. denticola cells induce the inflammatory response during periodontal disease. This study was designed to demonstrate that MSPc of T. denticola stimulates release of proinflammatory mediators in primary human monocytes. Material and Methods: Primary human monocytes were separated from the blood of healthy donors and incubated for up to 24 h with varying concentrations of MSPc. The production of tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6) and matrix metalloproteinase 9 (MMP‐9) was measured at different time points with commercially available enzyme‐linked immunosorbent assays. Results: T. denticola MSPc induced the synthesis of TNF‐α, IL‐1β, IL‐6 and MMP‐9 in a dose‐ and time‐dependent manner. Similar patterns of TNF‐α, IL‐1β and IL‐6 release were observed when cells were stimulated with 100 and 1000 ng/mL of MSPc. The production of MMP‐9 was significant only when cells were treated with 1000 ng/mL of MSPc. Conclusion: These results indicate that T. denticola MSPc, at concentrations ranging from 100 ng/mL to 1.0 μg/mL, activates a proinflammatory response in primary human monocytes.     

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.     

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

Chotjumlong P, Khongkhunthian S, Ongchai S, Reutrakul V, Krisanaprakornkit S. Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E ₂ synthesis in human gingival fibroblasts. J Periodont Res 2010; 45: 464–470. © 2010 John Wiley & Sons A/S Background and Objective: Oral epithelial cells express three antimicrobial peptide human β‐defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) synthesis in non‐immune cells, such as human gingival fibroblasts. Material and Methods: Cultured fibroblasts were treated with different concentrations of hBD‐1, ‐2, ‐3 or interleukin‐1β, as a positive control, for various times, in the presence or absence of NS‐398, a specific COX‐2 inhibitor. The levels of COX‐1 and COX‐2 mRNA expression were analyzed using RT‐PCR and real‐time PCR. Whole cell lysates were analyzed for COX‐1 and COX‐2 protein expression by western blotting. Cell‐free culture supernatants were assayed for PGE₂ levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Results: Ten and 40 μg/mL of hBD‐3 up‐regulated COX‐2 mRNA and protein expression, consistent with COX‐2 up‐regulation by interleukin‐1β, whereas hBD‐1 and hBD‐2 did not. However, COX‐1 mRNA and protein were constitutively expressed. The time‐course study revealed that hBD‐3 up‐regulated COX‐2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX‐2 up‐regulation, 10 and 40 μg/mL of hBD‐3 significantly increased PGE₂ levels in cell‐free culture supernatants (p < 0.05), and this was inhibited by NS‐398 in a dose‐dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human β‐defensin‐3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.     

Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts

Tamai R, Kiyoura Y, Sugiyama A. Alendronate regulates cytokine production induced by lipid A through nuclear factor‐κB and Smad3 activation in human gingival fibroblasts. J Periodont Res 2011; 46: 13–20. © 2010 John Wiley & Sons A/S Background and Objective: Nitrogen‐containing bisphosphonates (NBPs) are widely used as anti‐bone‐resorptive drugs. However, use of NBPs results in inflammatory side‐effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. Methods: The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)‐1β, IL‐6, IL‐8 and monocyte chemoattractant protein‐1 (MCP‐1) in culture supernatants were measured by ELISA. We also examined nuclear factor‐κB (NF‐κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. Results: Pretreatment of PBMCs with alendronate promoted lipid A‐induced production of IL‐1β and IL‐6, but decreased lipid A‐induced IL‐8 and MCP‐1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A‐induced production of IL‐6 and IL‐8, and increased NF‐κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate‐augmented IL‐6 and IL‐8 production by human gingival fibroblasts but up‐regulated alendronate‐decreased IL‐8 production by PBMCs. Conclusion: These results suggest that alendronate‐mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF‐κB and Smad3 activity.     

Peroxisome proliferator‐activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide‐induced activation of matrix metalloproteinase‐2 by downregulating NADPH oxidase 4 in human gingival fibroblasts

We investigated the roles of peroxisome proliferator‐activated receptor δ (PPARδ) in Porphyromonas gingivalis‐derived lipopolysaccharide (Pg‐LPS)‐induced activation of matrix metalloproteinase 2 (MMP‐2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg‐LPS‐induced activation of MMP‐2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP‐2 activity, suggesting a mechanism in which Nox4‐derived ROS modulates MMP‐2 activity. Furthermore, c‐Jun N‐terminal kinase and p38, but not extracellular signal‐regulated kinase, mediated PPARδ‐dependent inhibition of MMP‐2 activity in HGFs treated with Pg‐LPS. Concomitantly, PPARδ‐mediated inhibition of MMP‐2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg‐LPS. These results indicate that PPARδ‐mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS‐dependent regulation of MMP‐2 activity.     

Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease

Da? A, F?rat ET, Kadiro?lu AK, Kale E, Y?lmaz ME. Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease. J Periodont Res 2010; 45: 445–450. © 2010 John Wiley & Sons A/S Background and Objective: The prevalence of chronic renal disease in industrialized countries is increasing, and chronic renal disease and periodontitis can have significant, reciprocal effects. The aim of this study was to evaluate the associations between specific clinical parameters and the levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) in the gingival crevicular fluid of hemodialysis (HD) patients with periodontal disease. Material and Methods: Forty‐three HD patients and 43 systemically healthy subjects were enrolled in this study. Plaque index (PI), gingival index (GI) and pocket depth were used to determine periodontal status. Venous blood samples were obtained from each patient in the morning before the dialysis session and analyzed to determine the levels of inflammatory, biochemical and hematological parameters. Gingival crevicular fluid was collected from all subjects, and the levels of TNF‐α and IL‐8 were determined in the gingival crevicular fluid samples. Results: The following results were obtained from HD patients and controls: TNF‐α (pg/mL), 31.40 ± 1.46 and 3.06 ± 0.15 (p < 0.001); IL‐8 (pg/mL), 90.98 ± 94.03 and 35.05 ± 16.86 (p < 0.001); PI, 1.69 ± 1.02 and 0.04 ± 0.02 (p < 0.001); GI, 0.82 ± 0.06 and 0.04 ± 0.02 (p < 0.001); and pocket depth, 2.23 ± 0.63 and 1.51 ± 0.05 (p < 0.001), respectively. In addition, there were positive correlations between TNF‐α and PI (r = 0.642, p < 0.001), between TNF‐α and GI (r = 0.565, p < 0.001), between TNF‐α and pocket depth (r = 0.522, p < 0.001), between IL‐8 and PI (r = 0.402, p = 0.002), between IL‐8 and GI (r = 0.396, p = 0.002), and between IL‐8 and pocket depth (r = 0.326, p = 0.012). There were negative correlations between albumin and PI (r = ?0.491, p < 0.001), albumin and GI (r = ?0.406, p < 0.001), albumin and pocket depth (r = ?0.464, p < 0.001) and albumin and CRP (r = ?0.467, p = 0.002) and between the gingival crevicular fluid levels of TNF‐α and IL‐8, TNF‐α and hemoglobin (r = ?0.745, p < 0.001; r = ?0.285, p < 0.05) (respectively). Conclusion: The levels of TNF‐α and IL‐8 in gingival crevicular fluid were significantly higher in HD patients than in controls. There were strong, positive correlations between clinical periodontal parameters and the levels of inflammatory cytokines in gingival crevicular fluid from the HD patients.