Down‐regulation of interleukin‐1α‐induced matrix metalloproteinase‐13 expression via EP1 receptors by prostaglandin E2 in human periodontal ligament cells

In the present study, we investigated the effect of prostaglandin (PG) E₂ on matrix metalloproteinase (MMP)‐13 production in human periodontal ligament cells stimulated with interleukin (IL)‐1α. IL‐1α enhanced both MMP‐13 and PGE₂ production. Indomethacin, a nonselective cyclooxygenase inhibitor, and NS‐398, a specific cyclooxygenase‐2 (COX‐2) inhibitor, significantly enhanced IL‐1α‐induced MMP‐13 production in periodontal ligament cells, although both the agents completely inhibited IL‐1α‐induced PGE₂ production. Exogenous PGE₂ reduced IL‐1α‐induced MMP‐13 mRNA and protein production in a dose‐dependent manner. 17‐phenyl‐ω‐trinor PGE₂, a selective EP₁ receptor agonist, mimicked the inhibitory effect of PGE₂ on IL‐1α‐induced MMP‐13 mRNA and protein production. On the basis of these data, we suggest that COX‐2‐dependent PGE₂ down‐regulates IL‐1α‐elicited MMP‐13 production via EP₁ receptors in human periodontal ligament cells. PGE₂ may be involved in the regulation of destruction of extracellular matrix components in periodontal lesions.     

Assessment of the involvement of the macrophage migration inhibitory factor–glucocorticoid regulatory dyad in the expression of matrix metalloproteinase‐2 during periodontitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and counter‐regulator of endogenous glucocorticoids (GCs). It is implicated in acute and chronic inflammatory diseases. This study investigated the role of the MIF–GC regulatory dyad in the expression and release of matrix metalloproteinase‐2 (MMP‐2) during periodontitis, in vivo and in vitro. In a Mif‐knockout (KO) mouse model of ligature‐induced periodontitis, gingival tissues and blood were collected and analysed for levels of interleukin‐6 (IL‐6), MIF, MMP‐2, and corticosterone. In addition, human gingival fibroblasts (HGFs) were tested for production of IL‐6 and MMP‐2 after stimulation with hydrocortisone (HC), MIF, tumour necrosis factor‐alpha (TNF‐α), or Fusobacterium nucleatum, a pathogen known to elicit immune responses during periodontitis. Wild‐type (WT) mice showed a local and systemic increase of MIF levels during inflammation, which was confirmed by increased local IL‐6 concentrations. Systemic GC levels were reduced in WT and Mif‐KO mice during inflammation, with overall lower concentrations in Mif‐KO mice. In vivo and in vitro, MMP‐2 production was not dependent on MIF or inflammatory stimuli, but was inhibited by HC. Therefore, MIF does not appear to stimulate expression of MMP‐2 in the gingival tissues, whereas GC upregulates MIF and downregulates MMP‐2. Our findings further suggest that MIF may regulate systemic GC levels.     

The Effects of a Novel Botanical Agent on Lipopolysaccharide‐Induced Alveolar Bone Loss in Rats

Background: The development of host‐modulatory agents with low risk of adverse effects has been needed to treat periodontitis, a chronic inflammatory disease. A botanical mixture of extracts from two natural substances, Panax notoginseng and Rehmannia glutinosa Libosch, was developed as a novel botanical agent synthesized with anti‐inflammatory effect. The aim of this study is to evaluate the effects of the botanical mixture on the release of inflammatory cytokines and its inhibitory effect on lipopolysaccharide (LPS)‐induced alveolar bone loss (ABL) in a rat model. Methods: Cytotoxicity was assessed by 3‐(4,5‐dimethylthiazol‐2yl)‐5(3‐carboxymethoxyphenol)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium assay using human gingival fibroblast (hGF) and human periodontal ligament (hPDL) cells. Human acute monocytic leukemia cell line and hGF cells were cultured to assay tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, respectively. Microcomputed tomography analysis and immunofluoresence analysis were performed to evaluate the efficacy of the botanical mixture to inhibit the destruction of alveolar bone and connective tissue in a rat model. Results: The botanical mixture is cytotoxic at concentrations exceeding 2.5 mg/mL (P <0.05). Based on the results from cytotoxicity assay, it can be determined that the pharmacologic ranges of the botanical mixture to be used in all subsequent in vitro and in vivo experiments. The botanical mixture reduced the release of TNF‐α and IL‐6 from human monocytic cells and hGF cells in a dose‐dependent manner (P <0.05). The administration of the botanical mixture significantly reduced the alveolar bone loss in a rat model (P <0.05). In groups treated with the botanical mixture, matrix metalloproteinase (MMP)‐9 was detected along the alveolar bone crest (ABC), but not around the gingival connective tissue, while in the group with LPS‐induced ABL, pronounced expression of MMP‐9 around the ABC, periodontal ligament, and gingival connective tissue was found. Conclusions: The botanical mixture showed a potential adjunctive effect in the treatment of periodontitis. However, the present findings are obtained in vitro and in a rat model, so further clinical study is needed for its clinical application.     

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.     

Cimetidine Reduces Interleukin‐6, Matrix Metalloproteinases‐1 and ‐9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease

Background: Histamine seems to act, via H₂ receptor, on inflammatory processes by stimulating interleukin (IL)‐6 and matrix metalloproteinase (MMP) release. As cimetidine is an H₂ receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL‐6, MMP‐1, and MMP‐9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL‐6 on MMPs, the relationship between IL‐6/MMP‐1 and IL‐6/MMP‐9 immunoexpression was evaluated. Methods: Forty‐six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the alveolar process surface and number of IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05). Results: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP‐positive osteoclasts and IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL‐6/MMP‐1 and IL‐6/MMP‐9 was detected in PDSG and PDCimG. Conclusion: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL‐6, MMP‐1, and MMP‐9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL‐6 immunolabeling in the inflamed gingival mucosa.     

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.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

MAPKs, activator protein-1 and nuclear factor-κB mediate production of interleukin-1β-stimulated cytokines, prostaglandin E₂ and MMP-1 in human periodontal ligament cells

Murayama R, Kobayashi M, Takeshita A, Yasui T, Yamamoto M. MAPKs, activator protein‐1 and nuclear factor‐κB mediate production of interleukin‐1β‐stimulated cytokines, prostaglandin E ₂ and MMP‐1 in human periodontal ligament cells. J Periodont Res 2011; 46: 568–575. © 2011 John Wiley & Sons A/S Background and Objective: Determination of the interleukin‐1 (IL‐1) signaling cascades that lead to the production of various inflammatory mediators and catabolic factors may clarify attractive targets for therapeutic intervention for periodontitis. We comprehensively assessed the involvement of MAPKs, activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in IL‐1β‐induced production of interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), prostaglandin E₂ (PGE₂) and MMP‐1 in human periodontal ligament cells. Material and Methods: Human periodontal ligament cells were pretreated with an inhibitor for each of the MAPKs or NF‐κB and subsequently treated with IL‐1β. Following treatment, phosphorylation of three types of MAPK (ERK, p38 MAPK and c‐Jun N‐terminal kinase), IκB kinase (IKK) α/β/γ and IκB‐α, as well as the DNA binding activity of AP‐1 and NF‐κB and the production of IL‐6, IL‐8, PGE₂ and MMP‐1, were determined by western blotting, a gel mobility shift assay and ELISA, respectively. Results: The three MAPKs, simultaneously activated by IL‐1β, mediated the subsequent DNA binding of AP‐1 at various magnitudes, while IKKα/β/γ, IκB‐α and NF‐κB were also involved in the IL‐1 signaling cascade. Furthermore, IL‐1β stimulated the production of IL‐6, IL‐8, PGE₂ and MMP‐1 via activation of the three MAPKs and NF‐κB, because inhibitors of these significantly suppressed the IL‐1β‐stimulated production of these factors. Conclusion: Our results strongly suggest that MAPK, AP‐1 and NF‐κB mediate the IL‐1β‐stimulated synthesis of IL‐6, IL‐8, PGE₂ and MMP‐1 in human periodontal ligament cells. Therefore, inhibition of activation of MAPK, AP‐1 and/or NF‐κB may lead to therapeutic effects on progression of periodontitis.     

Cytokine expression profile in the bone‐anchored hearing system: 12‐week results from a prospective randomized,controlled study

Objective

To study the effect of implanting the percutaneous bone‐anchored hearing system (BAHS) itself and inflammation of the peri‐abutment skin warrant clarification. In this study, we aimed to acquire further insight into the immune responses related to BAHS surgery and peri‐implant skin inflammation.

Materials and Methods

During surgery and 12 weeks post‐implantation, skin biopsies were obtained. If applicable, additional biopsies were taken during cases of inflammation. The mRNA expression of IL‐1β, IL‐6, IL‐8, TNFα, IL‐17, IL‐10, TGF‐ß, MIP‐1α, MMP‐9, TIMP‐1, COL1α1, VEGF‐A, FGF‐2 TLR‐2, and TLR‐4 was quantified using qRT‐PCR.

Results

Thirty‐five patients agreed to the surgery and 12‐week biopsy. Twenty‐two patients had mRNA of sufficient quality for analysis. Ten were fitted with a BAHS using the minimally invasive Ponto surgery technique. Twelve were fitted with a BAHS using the linear incision technique with soft‐tissue preservation. Five biopsies were obtained during episodes of inflammation. The post‐implantation mRNA expression of IL‐1β (P = .002), IL‐8 (P = .003), MMP9 (P = .005), TIMP‐1 (P = .002), and COL1α1 (P < .001) was significantly up‐regulated. IL‐6 (P = .009) and FGF‐2 (P = .004) mRNA expression was significantly down‐regulated after implantation. Within patients, no difference between post‐implantation mRNA expression (at 12 weeks) and when inflammation was observed. Between patients, the expression of IL‐1β (P = .015) and IL‐17 (P = .02) was higher during cases of inflammation compared with patients who had no inflammation at 12‐week follow‐up.

Conclusions

As part of a randomized, prospective, clinical trial, the present study reports the molecular profile of selected cytokines in the soft tissue around BAHS. Within the limit of this study, the results showed that 12 weeks after BAHS implantation the gene expression of some inflammatory cytokines (IL‐8 and IL‐1β) is still relatively high compared with the baseline, steady‐state, expression. The up‐regulation of anabolic (COL1α1) and tissue‐remodeling (MMP‐9 and TIMP1) genes indicates an ongoing remodeling process after 12 weeks of implantation. The results suggest that IL‐1β, IL‐17, and TNF‐α may be interesting markers associated with inflammation.     

Correlation between periodontal disease, inflammatory alterations and pre-eclampsia

Politano GT, Passini R, Nomura ML, Velloso L, Morari J, Couto E. Correlation between periodontal disease, inflammatory alterations and pre‐eclampsia. J Periodont Res 2011; 46: 505–511. © 2011 John Wiley & Sons A/S Background and Objective: Several studies have hypothesized that periodontal disease may increase the risk of pre‐eclampsia. The correlation between the two diseases would probably be based on hypertension‐related cytokine release in the local periodontal environment. The aim of this study was to evaluate the association between periodontal disease and pre‐eclampsia, and the correlation of the two conditions with interleukin‐6 (IL‐6) and tumor necrosis factor‐α(TNFα) mRNA expression. Material and Methods: A case–control analysis of 116 pregnant women, 58 with pre‐eclampsia (cases) and 58 normotensive pregnant women (controls) was performed. In addition to collection of socio‐demographic data and periodontal evaluation, peripheral blood samples were collected for laboratory analysis of IL‐6 and TNFα mRNA expression by real‐time PCR. Results: There was an association between periodontitis and pre‐eclampsia (adjusted odds ratio 3.73; 95% confidence interval 1.32–10.58). Increased TNFα mRNA expression was observed in pre‐eclamptic women; however, there was no correlation between periodontitis and systemic cytokine expression. In the case group, systemic cytokine mRNA levels were similar in pregnant women with and without periodontitis (means ± SD): 0.73 ± 0.24 vs. 0.82 ± 0.38 for TNFα and 1.31 ± 1.49 vs. 1.09 ± 0.74 for IL‐6, respectively. Conclusion: Periodontitis was clinically related to pre‐eclampsia; however, the supposed mechanism that correlates the two diseases, i.e. a systemic inflammatory process involving cytokines TNFα and IL‐6 in the presence of periodontal disease, could not be confirmed in this study.     

Stimulation of epithelial cell matrix metalloproteinase (MMP‐2, ‐9, ‐13) and interleukin‐8 secretion by fusobacteria

Background/aims: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin‐8 (IL‐8) secretion upon exposure to fusobacteria. Methods: Eight different oral and non‐oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP‐13), gelatinase A (MMP‐2), gelatinase B (MMP‐9), and IL‐8 secretion by epithelial cells. Results: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP‐9 and MMP‐13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP‐2 secretion. F. nucleatum and F. necrophorum also increased IL‐8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL‐8 production, suggesting that their expression is differently regulated. Conclusion: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP‐9, MMP‐13, and IL‐8 from epithelial cells.     

Periodontitis and Endothelial Dysfunction: Periodontal Clinical Parameters and Levels of Salivary Markers Interleukin‐1β, Tumor Necrosis Factor‐α, Matrix Metalloproteinase‐2, Tissue Inhibitor of Metalloproteinases‐2 Complex,and Nitric Oxide

Background: Periodontitis has been associated with a greater risk for atherosclerotic cardiovascular disease (ACD). Endothelial dysfunction (ED) is a parameter of early ACD, and its association with periodontitis has rarely been investigated to date. The objective of this study is to evaluate the association between periodontitis and ED by means of periodontal clinical parameters and salivary markers interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, nitric oxide (NO), and matrix metalloproteinase (MMP)‐2 and tissue inhibitor of metalloproteinases (TIMP)‐2 complex. Methods: Forty‐seven individuals were divided into two groups: 1) 24 individuals with chronic periodontitis (CP); and 2) 23 individuals without CP. Periodontal examinations of bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL) were performed. ED was evaluated by means of flow‐mediated dilatation (FMD) of the brachial artery. Salivary concentrations of IL‐1β, TNF‐α, and MMP‐2/TIMP‐2 complex were assessed using enzyme‐linked immunosorbent assay. NO determination was based on the reaction of Griess. Results: Individuals with CP presented higher occurrence of ED than individuals without CP (P = 0.03 after reactive hyperemia; P = 0.05 after sublingual nitrate). A significant association among the production of MMP‐2/TIMP‐2 complex with the presence of CP (P = 0.008) and periodontal parameters PD, CAL, and BOP was identified. Concentration of salivary markers IL‐1β, TNF‐α, and NO was similar in individuals with and without CP. A significant positive correlation between NO and ED was also identified. Conclusions: Periodontitis was positively associated with ED, expressed by a smaller percentage of FMD of the brachial artery and higher salivary levels of MMP‐2/TIMP‐2 complex. Additionally, salivary levels of NO were significantly associated with better functioning of the vascular endothelium.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.     

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.     

Short‐Term Effects of Non‐Surgical Periodontal Treatment on the Gingival Crevicular Fluid Cytokine Profiles in Sites With Induced Periodontal Defects: A Study on Dogs With and Without Streptozotocin‐Induced Diabetes

Background: The aim of this study is to assess the short‐term effects of non‐surgical periodontal therapy (NSPT) on the gingival crevicular fluid (GCF) cytokine profile in sites with standardized periodontal bony defects in beagle dogs with and without diabetes. Methods: Four beagle dogs with streptozotocin (STZ)‐induced diabetes and four healthy dogs were included. Fasting blood glucose levels were measured using a glucometer. In all animals, a 3‐walled bony defect was created on the mesial surface of the second premolar and first molar in all quadrants. After 12 weeks, all animals underwent weekly NSPT for 3 weeks. Baseline and post‐NSPT GCF samples were collected, and levels of interleukin (IL)‐1, IL‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α were measured using enzyme‐linked immunosorbent assay. Statistical analyses were performed using a software program, and P values <0.05 were considered statistically significant. Results: Mean fasting blood glucose levels were significantly higher in dogs with induced diabetes than those without diabetes (P <0.01). At baseline, mean IL‐6 (P <0.01) and IL‐8 (P <0.05) levels were higher in dogs with diabetes than those without diabetes. A significant reduction in levels of IL‐1, IL‐1β, IL‐6, IL‐8, and TNF‐α was noted in dogs without diabetes 1 week after NSPT. However, this significant reduction (P <0.05) only appeared 2 weeks after NSPT in dogs with diabetes. Conclusions: NSPT reduces GCF levels of proinflammatory cytokines in dogs with and without STZ‐induced diabetes; however, chronic hyperglycemia seems to retard the effect of NSPT on GCF cytokine concentration.     

Tumor Necrosis Factor‐α and Interleukin (IL)‐1β, IL‐6, and IL‐8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells,Delaying Wound Healing

Background: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, IL‐6, and IL‐8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. Methods: GFs and ECs were seeded in 96‐well plates (1 × 10⁴ cells/well) in plain culture medium (Dulbecco’s modified Eagle’s medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF‐α (100 ng/mL); 2) IL‐1β (1 ng/mL); 3) IL‐6 (10 ng/mL); and 4) IL‐8 (10 ng/mL). All cytokines were diluted in serum‐free DMEM. Control cultures were exposed only to serum‐free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme‐linked immunosorbent assay). Data were analyzed by Kruskal–Wallis and Mann–Whitney U tests (α = 0.05). Results: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL‐1β. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL‐6 and IL‐8 significantly increased synthesis of TNF‐α and IL‐1β. Conclusions: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.     

Role of MyD88‐dependent and MyD88‐independent signaling in Porphyromonas gingivalis‐elicited macrophage foam cell formation

Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. Toll‐like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and TRIF during macrophage foam cell formation in response to P. gingivalis. In the presence of human low‐density lipoprotein (LDL) mouse bone‐marrow‐derived macrophages (BMφ) cultured with P. gingivalis responded with significant reduction in tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). The BMφ stained strongly with oil red O, regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P. gingivalis stimulated foam cell formation in a similar way to live bacteria. The BMφ from MyD88‐knockout and Lps2 mice revealed a significant role for MyD88, and a minor role for TRIF in P. gingivalis‐elicited foam cell formation. Porphyromonas gingivalis‐elicited TNF‐α and IL‐6 were affected by MyD88 ablation and to a lesser extent by TRIF status. These data indicate that LDL affects the TNF‐α and IL‐6 response of macrophages to P. gingivalis challenge and that MyD88 and TRIF play important roles in P. gingivalis‐elicited foam cell formation.     

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.