Effects of areca nut extracts on the functions of human neutrophils in vitro

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.     

Macrophage tolerance response to Aggregatibacter actinomycetemcomitans lipopolysaccharide induces differential regulation of tumor necrosis factor-alpha, interleukin-1 beta and matrix metalloproteinase 9 secretion

Background and Objective: The lipopolysaccharide of Aggregatibacter actinomycetemcomitans, a potent stimulator of the immune system, induces the secretion of inflammatory mediators that modulate periodontal tissue destruction. In this study, we investigated the tolerance response of human macrophages to stimulation with A. actinomycetemcomitans lipopolysaccharide. Material and Methods: U937 monocytes were differentiated into adherent macrophage‐like cells by treatment with phorbol myristic acid. Macrophage‐like cells were then pretreated for 24 h with either 0.01 or 0.1 μg/mL LPS A. actinomycetemcomitans. Culture medium supernatants were removed and cells were restimulated with LPS at 1 μg/mL. Cell‐free supernatants were collected after 24 h of stimulation and analyzed by ELISA for TNF‐α, IL‐1β, IL‐6, IL‐8, PGE₂ and MMP‐9. Results: Phorbol myristic acid‐differentiated U937 macrophages treated with low doses of lipopolysaccharide developed tolerance to subsequent lipopolysaccharide treatments, resulting in significantly reduced secretion of tumor necrosis factor‐α. However, this tolerance response was associated with increased secretion of interleukin‐1β and matrix metalloproteinase 9, whereas the secretion of interleukin‐6, interleukin‐8 and prostaglandin E₂ was unaffected. Phosphatidylinositol‐3′‐kinase inhibitors added during the tolerance‐induction period markedly attenuated the increase in interleukin‐1β secretion but had no effect on tumor necrosis factor‐α. Conclusion: This study showed that A. actinomycetemcomitans lipopolysaccharide can induce a tolerance response in macrophages that alters the secretion of two important inflammatory mediators as well as of the tissue‐degrading enzyme matrix metalloproteinase‐9. This phenomenon may play a role in modulating the host inflammatory response and the progression of periodontitis.     

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.     

A trend of increase in periodontal interleukin-6 expression across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases

Background and Objective: Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study was to determine the difference in the periodontal expression of MMP‐1, MMP‐8, interleukin‐6, tumor necrosis factor‐α and interleukin‐1β between diabetic and nondiabetic patients. Material and Methods: Periodontal tissue specimens were collected from nine nondiabetic patients without periodontal disease (group 1), from 11 nondiabetic patients with periodontal disease (group 2) and from seven diabetic patients with periodontal disease (group 3). The expression of MMP‐1, MMP‐8, interleukin‐6, tumor necrosis factor‐α and interleukin‐1β was quantified using real‐time polymerase chain reaction. Results: The nonparametric Kruskal–Wallis test showed that the difference in interleukin‐6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal–Wallis nonparametric linear‐by‐linear association test showed a statistically significant trend of increase in the expression of interleukin‐6 from group 1 to group 2 to group 3 (p = 0.02) and a suggestion of such a trend for MMP‐1 (p = 0.05). No increase in MMP‐8 expression was observed in patients in group 3 compared to patients in groups 1 and 2. Although the average expression levels of MMP‐1, interleukin‐1β and tumor necrosis factor‐α were increased from group 1 to group 3, the differences were not statistically significant. Conclusion: A trend of increased interleukin‐6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.     

Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-alpha and interleukin-6 secretion, and CCL25 gene expression, in mouse primary gingival cell lines: interleukin-6-driven activation of CCL2

Background and Objective: Porphyromonas gingivalis infection is strongly associated with periodontitis. Although P. gingivalis is known to elicit a strong inflammatory response, details of that remain fragmentary. To understand the local response to P. gingivalis, primary cell lines derived from mouse gingival tissues were exposed to P. gingivalis or Escherichia coli lipopolysaccharide, and the production of interleukin‐6 and tumor necrosis factor‐α was measured. CCL25 gene expression was measured by real‐time polymerase chain reaction. Cells stimulated with combinations of interleukin‐6, soluble interleukin‐6 receptor and/or soluble gp130 were assayed for CCL2 and tumor necrosis factor‐α secretion. Material and Methods: Primary cell lines were generated from mouse gingival tissues. Enzyme‐linked immunosorbent assays were used to determine cytokine levels, and real‐time polymerase chain reaction was used to quantify CCL25 gene expression. Results: Exposure to P. gingivalis lipopolysaccharide but not to E. coli lipopolysaccharide resulted in significantly elevated levels of both interleukin‐6 and tumor necrosis factor‐α, and stimulation with P. gingivalis lipopolysaccharide also upregulated CCL25 gene expression. In one of three experiments, interleukin‐6 induced CCL2 secretion, whereas interleukin‐6 plus soluble interleukin‐6 receptor induced CCL2 secretion in all three experiments, suggesting that both direct interleukin‐6 signaling and interleukin‐6 trans‐signaling may be involved. However, because soluble gp130 did not inhibit trans‐signaling, and because direct stimulation of gingival cells with soluble gp130 resulted in CCL2 secretion, the possibility exists that soluble gp130 forms binary complexes with soluble interleukin‐6 receptor that promote direct interleukin‐6 stimulation. Conclusion: These findings define a pathway in which exposure of gingival cells to P. gingivalis induces the release of interleukin‐6 and tumor necrosis factor‐α; interleukin‐6, in turn, induces CCL2 secretion.     

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.     

Modulation of phagocytosis, chemotaxis, and adhesion of neutrophils by areca nut extracts

BACKGROUND: A higher prevalence of periodontal diseases among areca chewers than non-areca chewers has been demonstrated. Neutrophils, representing the first line of the host defense mechanism against microbial infection, play important roles in maintaining periodontal health. This study determined the possible effects of areca nut on phagocytosis, chemotaxis, and adhesion of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE) and fresh and tender areca nut with husk (tANE) were examined for their effects on neutrophil phagocytosis using flow cytometry and confocal laser scanning microscopy. The effects of rANE and tANE on chemotaxis and adhesion of neutrophils to human aortic endothelial cells were examined using fluorescence-labeled neutrophils. RESULTS: Both rANE and tANE inhibited the phagocytic activity of neutrophils in a dose-dependent manner. The levels of internalized fluorescent bacteria in neutrophils decreased after ANE treatment. However, exposure of neutrophils to rANE and tANE stimulated the chemotaxis activity of neutrophils to N-formyl-Met-Leu-Phe (fMLP) and enhanced adhesion of neutrophils to human aortic endothelial cells in a dose-dependent manner. Moreover, treatment of neutrophils with rANE was more effective than incubation with tANE. CONCLUSIONS: Components of areca nut inhibited phagocytosis activity of neutrophils but enhanced chemotaxis and adhesion of neutrophils. Alterations in functions of neutrophils may lead to signs of clinical diseases associated with areca chewing. The components in ANEs that are responsible for these observations remain to be elucidated.     

Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells

Wang C‐C, Chen T‐Y, Wu H‐Y, Liu T‐Y, Jan T‐R. Areca nut extracts suppress the differentiation and functionality of human monocyte‐derived dendritic cells. J Periodont Res 2012; 47: 198–203. © 2011 John Wiley & Sons A/S Background and Objective: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. Material and Methods: Human peripheral blood monocytes were cultured in the presence of granulocyte–monocyte colony‐stimulating factor and interleukin‐4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS) was examined. Results: Monocytes cultured in granulocyte–monocyte colony‐stimulating factor and interleukin‐4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)‐DR, CD11c and the co‐stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA‐DR and CD11c, but markedly attenuated the proportion of CD40‐positive cells and the mean fluorescence intensity of CD86. The expression of co‐stimulatory molecules in LPS‐activated dendritic cells was not affected, whereas the mRNA expression of interleukin‐12 induced by LPS was markedly suppressed by ANE treatment in a concentration‐dependent manner. Conclusion: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte‐derived dendritic cells was attenuated in the presence of ANE.     

Activation of interleukin 1β gene transcription by human cytomegalovirus: molecular mechanisms and relevance to periodontitis

In recent years, studies have demonstrated an association between human cytomegalovirus (HCMV) and destructive periodontal disease. It has been shown that reactivation of HCMV in periodontitis lesions may be related to progressing periodontal disease. Several possible mechanisms by which HCMV exerts periodontopathic potential have been previously proposed. These are reviewed and include the upregulation of bone resorptive cytokines such as interleukin‐1beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) by active HCMV infection at the periodontitis site. This review focuses on the molecular basis of IL‐1β gene activation by HCMV immediate early (IE) gene products. A novel hypothesis is also described whereby HCMV plays a significant role in the pathogenesis of periodontal disease by the ability of its IE proteins to strongly transactivate IL‐1β gene expression. More studies are needed to further explore this hypothesis and clarify the association between HCMV and periodontitis.     

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.     

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.     

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis

Background and Objective: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. This study was designed to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Material and Methods: Ligature‐induced periodontitis was applied in periodontitis‐susceptible Fischer 344 rats. The animals were either given daily intraperitoneal injections of the nicotinic acetylcholine receptor antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous injections in the neck skin of nicotine (0.8 mg/kg), or treated with the same amount of saline intraperitoneally and nicotine subcutaneously, or treated with mecamylamine and saline. Control animals received intraperitoneal and subcutaneous injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 wk. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally) to induce a robust immune and stress response. Results: Compared with saline/saline‐treated control animals, saline/nicotine‐treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor‐α, transforming growth factor‐1β and interleukin‐10. Mecamylamine pretreatment of nicotine‐treated rats abrogated the increased periodontal bone loss and the LPS‐induced decrease in tumour necrosis factor‐α, but had no significant effects on the levels of transforming growth factor‐1β and interleukin‐10, or the stress hormone corticosterone. Conclusion: The results indicate that nicotine enhances the susceptibility to periodontitis via nicotinic acetylcholine receptors, which may act by suppressing protective immune responses through the cholinergic anti‐inflammatory pathway.     

Hypoxia inducible factor‐1α expression in areca quid chewing‐associated oral squamous cell carcinomas

Oral Diseases (2010) 16 , 696–701 Objectives: Hypoxia inducible factor (HIF)‐1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF‐1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF‐1α expression in normal human oral epithelium and areca quid chewing‐associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF‐1α expression. Methods: Twenty‐five OSCC from areca quid chewing‐associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N‐acetyl‐l ‐cysteine (NAC), AP‐1 inhibitor curcumin, extracellular signal‐regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. Results: Hypoxia inducible factor‐1α expression was significantly higher in OSCC specimens than normal specimen (P < 0.05). Arecoline was found to elevate HIF‐1α expression in a dose‐ and time‐dependent manner (P < 0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline‐induced HIF‐1α expression (P < 0.05). Conclusions: Hypoxia inducible factor‐1α expression is significantly upregulated in areca quid chewing‐associated OSCC and HIF‐1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.     

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.     

Effects of areca nut extract on the apoptosis pathways in human neutrophils

Ho W‐H, Lee Y‐Y, Chang L‐Y, Chen Y‐T, Liu T‐Y, Hung S‐L. Effects of areca nut extract on the apoptosis pathways in human neutrophils. J Periodont Res 2010; 45: 412–420. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. Material and Methods: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. Results: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP‐ribose) polymerase, and of caspase‐3 and caspase‐8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase‐3α/β may be involved in the ANE‐modulated effects of neutrophils. Conclusion: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.     

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.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

Effects of tobacco smoke on the secretion of interleukin‐1β, tumor necrosis factor‐α, and transforming growth factor‐β from peripheral blood mononuclear cells

Alterations of the host response caused by short‐term exposure to high levels of smoke during the act of smoking (acute smoke exposure) as well as long‐term exposure to lower levels of tobacco substances in the bloodstream of smokers (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. In this study, we examined the secretion of three cytokines [interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, and transforming growth factor (TGF)‐β] from mononuclear blood cells from current smokers and non‐smokers exposed to in vitro tobacco smoke (which may be comparable to in vivo acute smoke exposure) and mononuclear blood cells from current smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure). Peripheral blood mononuclear cells were isolated from eight healthy current smokers and eight healthy non‐smokers, plated in culture wells, exposed in vitro for 1–5 min to cigarette smoke in a smoke box system or not exposed (baseline controls), and then incubated without further smoke exposure for another 24 h. Supernatants from each well were then collected and assayed for the concentrations of the three cytokines by enzyme‐linked immunosorbent assay (ELISA). At baseline, mean IL‐1β levels were higher in smokers than in non‐smokers (mean: 10.6 vs. 5.9 pg/ml, anova : P < 0.05). In both smokers and non‐smokers, secreted levels of IL‐1β increased from 0 to 5 min of in vitro smoke exposure (mean: 5.9–9.9 pg/ml, t‐test: P < 0.05 for non‐smokers only) with levels in smokers higher than in non‐smokers (P > 0.05). Mean TNF‐α levels increased from 0 to 2 min of smoke exposure and decreased from 2 to 5 min in smokers and non‐smokers, with higher levels in non‐smokers than smokers at all time‐points (P > 0.05). Mean TGF‐β levels were higher in smokers than in non‐smokers at all time‐points (mean: 180.5 vs. 132.0 pg/ml, P < 0.05 at 5 min only) with no significant alteration of the pattern of secretion with cigarette smoke exposure. These observed alterations in the secretion of cytokines from mononuclear blood cells in smokers, relative to non‐smokers, and with in vitro smoke exposure may play a role in the pathogenesis of periodontal diseases in smokers.