The effects of tumour necrosis factor‐α on bone cells involved in periodontal alveolar bone loss; osteoclasts,osteoblasts and osteocytes

Periodontitis is the most common bone loss pathology in adults and if left untreated is responsible for premature tooth loss. Cytokines, such as tumour necrosis factor‐α (TNFα), involved in the chronic inflammatory response within the periodontal gingiva, significantly influence the normal bone remodelling processes. In this review, the effects of TNFα on bone metabolism in periodontitis are evaluated in relation to its direct and indirect actions on bone cells including osteoclasts, osteoblasts and osteocytes. Evidence published to date suggests a potent catabolic role for TNFα through the stimulation of osteoclastic bone resorption as well as the suppression of osteoblastic bone formation and osteocytic survival. However, the extent and timing of TNFα exposure in vitro and in vivo greatly influences its effect on skeletal cells, with contradictory anabolic activity observed with TNFα in a number of studies. None the less, it is evident that managing the chronic inflammatory response in addition to the deregulated bone metabolism is required to improve periodontal and inflammatory bone loss treatments???????????????????????????????????????.     

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.     

Locally administered interferon-γ accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation

Ayon Haro ER, Ukai T, Yokoyama M, Kishimoto T, Yoshinaga Y, Hara Y. Locally administered interferon‐γ accelerates lipopolysaccharide‐induced osteoclastogenesis independent of immunohistological RANKL upregulation. J Periodont Res 2011; 46: 361–373. © 2011 John Wiley & Sons A/S Background and Objective: Interferon‐γ (IFN‐γ) potently inhibits RANKL‐induced osteoclastogenesis in vitro. In contrast, previous studies have shown that an increase in IFN‐γ expression is correlated with an increase in lipopolysaccharide (LPS)‐induced bone loss in vivo. However, it is not clear whether local IFN‐γ accelerates osteoclastogenesis or not in vivo. Therefore, the aim of this study was to clarify the role of local IFN‐γ in LPS‐induced osteoclastogenesis. Material and Methods: We induced bone loss in calvaria by injecting LPS. One group of mice received an IFN‐γ injection together with LPS injection, while another group received IFN‐γ 2 d after LPS injection. Bone resorption was observed histologically. Next, we stimulated murine bone marrow macrophages with macrophage‐colony stimulating factor and RANKL in vitro. We added different doses of IFN‐γ and/or LPS at 0 or 48 h time points. Cells were stained with tartrate‐resistant acid phosphatase at 72 h. Results: Local administration of IFN‐γ together with LPS injection did not affect osteoclast formation. However, IFN‐γ injected after LPS injection accelerated osteoclast formation. Also, we confirmed that IFN‐γ added at 0 h inhibited RANKL‐induced osteoclastogenesis in vitro. However, inhibition by IFN‐γ added at 48 h was reduced compared with that by IFN‐γ added at 0 h. Interestingly, IFN‐γ together with a low concentration of LPS accelerated osteoclast formation when both were added at 48 h compared with no addition of IFN‐γ. Conclusion: The results suggest that local IFN‐γ accelerates osteoclastogenesis in certain conditions of LPS‐induced inflammatory bone loss.     

Epithelial Cells Secrete Interferon‐γ Which Suppresses Expression of Receptor Activator of Nuclear Factor Kappa‐B Ligand in Human Mandibular Osteoblast‐Like Cells

Background: Prostaglandin (PG)E₂ accumulates in inflamed periodontal tissue and induces receptor activator of nuclear factor kappa‐B ligand (RANKL)‐RANK‐osteoprotegerin (OPG) signaling associated with bone resorption. Although oral epithelial cells maintain tissue homeostasis, the role of these cells in RANKL regulation remains unknown. Methods: To mimic an inflamed condition, RANKL upregulation in human mandibular osteoblast‐like cells (HMOBs) were stimulated with PGE₂. Effect of recombinant human interferon (IFN)‐γ or epithelial‐derived IFN‐γ in constitutively released or Porphyromonas gingivalis lipopolysaccharide (PgLPS)‐stimulated epithelial supernatant was investigated in HMOBs. Some HMOBs were pretreated with an anti‐IFN‐γ antibody before PGE₂ stimulation. THP‐1 human monocytes and HMOBs were cocultured in a transwell system to investigate RANKL‐driven THP‐1 osteoclastic activity. Results: PGE₂ significantly increased RANKL messenger RNA (mRNA) and protein in HMOBs in a dose‐dependent manner, while OPG protein remained similar to baseline. Epithelial cells constitutively released IFN‐γ, which was substantially increased by PgLPS. HMOBs treated with epithelial supernatant or recombinant IFN‐γ, concurrently with PGE₂ stimulation, reduced RANKL, but not OPG, expression. In contrast, anti‐IFN‐γ antibody reversed the effect of epithelial mediators on RANKL expression. When cocultured with THP‐1, RANKL released by PGE₂‐stimulated HMOBs is adequate to drive THP‐1 differentiation as osteoclastogenic gene expression and bone resorption pit are increased. However, recombinant IFN‐γ, or IFN‐γ derived from oral epithelial cells, suppressed RANKL expression at both the mRNA and protein level, resulting in decreased THP‐1‐derived osteoclastic activity. Conclusion: Oral epithelial cells interact with HMOBs by releasing IFN‐γ to regulate RANKL expression and contribute to osteoclastogenesis.     

Hypertension favors the inflammatory process in rats with experimentally induced periodontitis

Bonato CF, do‐Amaral CCF, Belini L, Salzedas LMP, Oliveira SHP. Hypertension favors the inflammatory process in rats with experimentally induced periodontitis. J Periodont Res 2012; 47: 783–792. © 2012 John Wiley & Sons A/S Background and Objective: Cardiovascular diseases are significantly correlated with chronic periodontitis. The aim of this study was to evaluate bone‐loss level, neutrophil migration, CXCL2/CINC‐2α, CXCL5/LIX, CCL20/MIP‐3α and tumor necrosis factor‐α (TNF‐α) production, inducible nitric oxide synthase (iNOS) expression and C‐reactive protein (CRP) release in spontaneously hypertensive rats (SHRs) and normotensive (WTK) rats after experimental induction of periodontal disease. Material and Methods: Periodontitis was induced by placement of silk yarn ligatures around the first molar counterparts. The levels of CRP, CCL20/MIP‐3α and CXCL5/LIX were evaluated in the peripheral blood, and bone‐loss level, neutrophil recruitment, the production of myeloperoxidase, CXCL2, CXCL5, CCL20 and TNF‐α, and the expression of iNOS were evaluated in the gingival tissue. Histological sections were taken to evaluate and measure bone resorption and neutrophil recruitment in the furcation region. Results: Rats with periodontitis had alveolar bone resorption. SHRs with periodontitis showed marked bone loss and increased neutrophil infiltration in comparison with WTK rats. SHRs with periodontitis showed increased levels of TNF‐α and CXCL2, and a slight tendency for increased levels of CXCL5, in the gingival tissue but no increase in the level of CCL20. In SHRs, even without periodontitis, the levels of TNF‐α, CXCL2, CXCL5 and CCL20 showed a slight tendency to increase. In the WTK rats, TNF‐α, CXCL2 and CXCL5 levels were increased with periodontitis, but the level of CCL20 was not. iNOS was expressed in the gingival tissue of WTK rats and SHRs with periodontitis; however, SHRs appeared to express a higher level of iNOS than did WKT rats. The CRP level was elevated in both types of rats with periodontitis; however, the CRP level was higher in SHRs with periodontitis than in WTK rats with periodontitis. Conclusion: In SHRs, the hypertensive condition per se seems to favor the inflammatory processes that become potentiated with periodontitis, when compared with WKT rats.     

Transforming growth factor‐β‐regulated fractalkine as a marker of erosive bone invasion in oral squamous cell carcinoma

Patients with oral squamous cell carcinoma (OSCC) bone invasion are surgically treated with bone resection, which results in severe physical and psychological damage. Here, we investigated the potential of fractalkine (CX3CL1), which is regulated by transforming growth factor (TGF‐β), as a novel biomarker for correct prediction and early detection of OSCC‐associated bone invasion. TGF‐β knockdown and treatment with a TGF‐β‐neutralizing antibody decreased the level of fractalkine in the culture media of HSC‐2 and YD10B OSCC cells. Treatment with a fractalkine‐neutralizing antibody reduced TGF‐β‐stimulated invasion by HSC‐2 and YD10B cells. Fractalkine treatment increased the viability, invasion, and uPA secretion of both OSCC cell lines. Furthermore, OSCC cell bone invasion was assessed following subcutaneous inoculation of wild‐type or TGF‐β knockdown OSCC cells in mouse calvaria. TGF‐β knockdown prevented erosive bone invasion, reduced the number of osteoclasts at the tumor‐bone interface, and downregulated fractalkine expression in mouse tumor tissues. Our results indicate that the production of fractalkine is stimulated by TGF‐β and mediates TGF‐β‐induced cell invasion in several OSCC cell lines showing an erosive pattern of bone invasion. Fractalkine may be a useful predictive marker and therapeutic target for OSCC‐induced bone destruction.     

Immunization by Arg‐gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon‐γ production

We previously demonstrated that a Porphyromonas gingivalis rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice (Yonezawa et al. Infect Immun 2001: 69: 2858–2864). In the present study, reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine‐immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of interleukin (IL)‐2, IL‐4, IL‐5 and IL‐12 was elevated; however, interferon (IFN)‐γ was lower in the serum of the DNA vaccine‐immunized mice than in the serum of nonimmunized mice. The regulation of IFN‐γ production elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice.