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

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

Crevicular fluid endothelin-1 levels in periodontal health and disease

Background and Objective:  Endothelin-1 is a 21-amino-acid peptide with multifunctional regulation. Initial research indicated that endothelin-1 levels in the gingival crevicular fluid from patients with chronic periodontitis were higher than those in the gingival crevicular fluid from healthy subjects. The aim of the present study was to assess the relationship between the clinical parameters and the concentrations of endothelin-1 within the gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after the treatment of periodontitis sites.
Material and Methods:  A total of 60 subjects were divided into three groups – healthy (group I), gingivitis (group II) and chronic periodontitis (group III) – based on gingival index, pocket probing depth and clinical attachment loss. A fourth group consisted of 20 subjects from group III, 6–8 wk after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for endothelin-1 using an enzymatic immunometric assay.
Results:  Endothelin-1 was not detected in any sample from any of the study groups.
Conclusion:  The results showed that all the gingival crevicular fluid samples were negative for the endothelin-1 molecule. Therefore, endothelin-1 cannot be considered as a potential biomarker of periodontal disease progression.     

牙龈卟啉单胞菌上调钙结合蛋白1促进牙龈上皮细胞增殖

目的 探究钙结合蛋白1在牙龈卟啉单胞菌(P.gingivalis)影响牙龈上皮细胞增殖和凋亡中的作用.方法 P.gingivalis感染CA9-22细胞,在感染24 h后,采用实时荧光定量聚合酶链反应、免疫印迹法和免疫荧光法检测钙结合蛋白1(CALB1)的表达.通过RNA干扰法抑制CALB1表达,BrdU分析检测细胞增...     

Significance of thrombomodulin release from gingival epithelial cells in periodontitis patients

Background and Objective:  Thrombomodulin, a cell transmembrane glycoprotein, binds to thrombin and converts it from a procoagulant protease to an anticoagulant enzyme that activates protein C. Thrombomodulin is very important in regulating the function of thrombin. Elevated soluble thrombomodulin is present in the gingival crevicular fluid of subjects with periodontitis. The objective of the present study was to investigate the mechanisms about the elevated soluble thrombomodulin in gingival crevicular fluid.
Material and methods:  Gingival sections from six patients with chronic periodontitis and from three periodontally healthy subjects were immunostained for thrombomodulin detection. Thrombomodulin levels were investigated in the gingival crevicular fluid of 11 subjects with chronic periodontitis. The effects of neutrophil enzymes on thrombomodulin release and on thrombomodulin in the gingival crevicular fluid were examined by an enzyme-linked immunosorbent assay or by Western blotting.
Results:  The expression of gingival epithelial thrombomodulin was lost or decrease near infiltrating neutrophils. Thrombomodulin was rapidly released from gingival epithelial cells by neutrophil enzymes, and gingival crevicular fluid with periodontitis included the proteolytic cleavage thrombomodulin using immunoblotting analysis. The thrombomodulin release was not caused by rapid cell damage, on lactate dehydrogenase assay. There were significant differences in thrombomodulin content between gingival crevicular fluid samples from healthy and diseased sites, regardless of the degree of probing depth.
Conclusion:  Neutrophil enzymes induced rapid thrombomodulin release from the membrane surface of gingival epithelial cells. This might explain the thrombomodulin increase in gingival crevicular fluid with local diseased gingiva. Elevation of thrombomodulin in gingival crevicular fluid may be a potential marker of epithelial cell membrane injury.     

Effect of interleukin-17 on the expression of chemokines in gingival epithelial cells

The role of interleukin (IL)-17 in cellular communication in inflammation has been well described, and a positive correlation between the severity of periodontitis and the level of IL-17 was reported. Although epithelial cells are a major target of IL-17, little is known about the effect of IL-17 on the production of chemokines by human gingival epithelial cells (HGECs). We evaluated the effects of IL-17 on the expression of CXCL8 and CCL2 by HGECs using quantitative real-time PCR and ELISA. In addition, the role of the nuclear factor (NF)-κB signalling pathway in the IL-17-mediated expression of chemokines was assessed using a specific inhibitor. Stimulation with IL-17 up-regulated the expression of CXCL8 mRNA but not of CCL2 mRNA in HGECs, whereas tumour necrosis factor-α (TNF-α) elevated the expression of mRNA for both chemokines. Stimulation with IL-17 up-regulated the secretion of CXCL8 protein, but not the secretion of CCL2 protein. The effect of IL-17 on CXCL8 production was suppressed using an anti-IL-17R Ig, suggesting a role for a specific receptor-ligand interaction. Inhibition of the NF-κB signalling pathway demonstrated that NF-κB activation is required for the CXCL8 expression in HGECs. In conclusion, IL-17 is involved in the regulation of the innate immune response in HGECs by inducing CXCL8 production.     

Cytotoxic effects of short-chain carboxylic acids on human gingival epithelial cells

Previous studies showed that foods that are retained on the dentition can accumulate high levels of short-chain carboxylic acids (acetic, formic, lactic and propionic). Since gingival epithelium is the first periodontal tissue to be challenged by oral factors, a study was undertaken to determine whether short-chain carboxylic acids can affect epithelial cells in vitro. Immortalized human oral epithelial cells were grown in supplemented keratinocyte growth medium at 37°C, and the effects of short-chain carboxylic acids were determined with tetrazolium-based and trypan blue exclusion assays. Low concentrations of short-chain carboxylic acids inhibited the growth of human oral epithelial cells, while higher concentrations led to cell death. The effects of short-chain carboxylic acids on the cells were dose-dependent and varied among the individual acids (propionate >formate >lactate >acetate). Growth inhibition was partly reversible and growth resumed after removal of the acids. However, the time needed for recovery of the cells increased with short-chain carboxylic acids concentration, consistent with progressively greater damage to the cells at higher short-chain carboxylic acids concentrations. The observed effects of short-chain carboxylic acids on gingival cells in vitro supported our hypothesis that short-chain carboxylic acids can damage the integrity of gingival epithelium in situ.     

牙龈上皮细胞的抗菌防御作用

牙龈上皮细胞不仅具有屏障作用,还具有先天免疫抗菌功能,如牙龈上皮细胞表面受体的信号传递、分泌抗菌多肽、蛋白酶和细胞因子等。下面就Toll样受体、抗菌多肽或蛋白、细胞因子或趋化因子、蛋白酶等与牙龈上皮细胞关系的研究进展作一综述,旨在加强对牙龈上皮细胞防御机制的认识。     

Neutrophil-mediated damage to human gingival epithelial cells

Polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of inflammatory gingivitis and periodontitis. To further study the role of PMNs in mediating gingival injury, we cocultured these cells in vitro with monolayers of human gingival epithelial cells. Scanning electron microscopy revealed that the epithelial cells were homogeneous and SDS-PAGE/immunoblot analysis identified the presence of keratins K3, K13 and the K6/16 pair which authenticated the oral origin of the cells. Injury to the gingival cells was determined by scanning electron microscopy and measurement of cell detachment and cytolysis. Unstimulated PMNs produced minimal lysis or detachment, but PMNs stimulated by phorbol myristate acetate produced marked epithelial cell detachment without lysis, which was time- and PMN-dose-dependent. Supernatants of activated PMNs were similarly effective, indicating that the mediator was a stable soluble substance. Elastase and cathepsin G, two neutral proteases of PMN origin, produced time- and concentration-dependent detachment of gingival epithelial cells, suggesting that these enzymes may mediate this form of injury. In other studies, gingival epithelial cells were exposed to PMN myeloperoxidase (MPO), chloride and glucose plus glucose oxidase (GO) as a hydrogen peroxide (H2O2) generating system. The toxic oxygen species produced by this system caused lysis of the epithelial targets which was dependent on the duration of incubation and the concentrations of MPO and GO. Azide, an inhibitor of MPO, and catalase, a scavenger of H2O2, inhibited the lytic activity of this system. Scanning electron micrographs of gingival epithelial cells cocultured with activated PMNs showed lifting of the cells from the plating surface, while target cells attacked by the MPO system revealed extensive damage of cell membranes. These studies indicate that activated PMNs cause nonlytic detachment injury to gingival epithelial cells which may be mediated by digestion of their extracellular matrix by granule neutral proteases. Furthermore, PMN MPO is capable of generating toxic oxygen species which can lyse these epithelial cells. Collectively, these actions could have profound adverse effects on the function and integrity of the gingival epithelium.     

Butyrate,a bacterial metabolite,induces apoptosis and autophagic cell death in gingival epithelial cells

Tsuda H, Ochiai K, Suzuki N, Otsuka K. Butyrate, a bacterial metabolite, induces apoptosis and autophagic cell death in gingival epithelial cells. J Periodont Res 2010; 45: 626–634.©2010 John Wiley & Sons A/S Background and Objective: Butyrate is produced by some types of anaerobic periodontal bacteria. Millimolar concentrations of butyrate are found in mature dental plaque from periodontitis patients. Although butyrate reportedly has a variety of effects in many mammalian cells, its effect on gingival epithelial cells is not well known. In this study, we investigated the effect of butyrate on gingival epithelial Ca9‐22 cell death. Material and Methods: Death of Ca9‐22 cells was assessed after treating the cells with or without butyrate. A SYTOX Green dye, which exhibits strong green fluorescence once it enters dead cells through ruptured cell membranes, was used for cell death detection. Phosphatidylserine redistribution was measured using fluorescein isothiocyanate‐labeled annexin V. The activity of caspase‐3 was measured as the amount of cleaved substrate peptide. Anti‐apoptotic bcl‐2 mRNA expression was measured using real‐time RT‐PCR. Western blotting and fluoromicroscopic analysis with anti‐microtubule‐associated protein 1 light chain 3 (LC3) antibodies were performed for detection of autophagy. Results: Stimulation with millimolar concentrations of butyrate for 48 h induced Ca9‐22 cell death. The stimulation also caused increased caspase‐3 activity, phosphatidylserine redistribution and bcl‐2 down‐regulation, suggesting butyrate‐induced apoptosis. However, the pan‐caspase inhibitor, Z‐VAD‐FMK, did not inhibit cell death completely. This implies the existence of other types of cell death. In addition, markers of autophagy, namely, the conversion of LC3‐I to LC3‐II and increased LC3 accumulation, were observed. Moreover, inhibition of autophagy by 3‐methyladenine suppressed the butyrate‐induced cell death, suggesting that butyrate could induce cell death through autophagy. Conclusion: These data suggest that butyrate induces apoptosis and autophagic cell death.     

高迁移率族蛋白1在慢性牙周炎病变牙龈组织中的表达

目的 研究慢性牙周炎病变牙龈组织中高迁移率族蛋白1（HMGB1）的表达。方法 提取健康志愿者外周血单核细胞（PBMC）,以1 pg·mL-1的细菌脂多糖（LPS）刺激细胞,24 h后用免疫荧光染色法检测HMGB1的表达,48 h 后用酶联免疫吸附试验检测细胞上清液中HMGB1的表达;分别以50 ng·mL-1肿瘤坏死因子-α（TNF-α）和100 ng? mL-1 HMGB1刺激PBMC,48 h后检测细胞上清液中HMGB1和TNF-α的表达。另外收集健康者和慢性牙周炎患者的牙龈组织和龈沟液,分别检测牙龈组织和龈沟液内HMGB1的表达。结果 LPS刺激PBMC 24 h后,HMGB1自细胞核移出至细胞质中;刺激48 h后,细胞上清液中HMGB1的表达量明显高于对照组（P<0.01）。TNF-α和HMGB1分别刺激 PBMC 48 h后,上清液中HMGB1和TNF-α的表达水平较对照组亦有明显增强（P<0.01）。在慢性牙周炎牙龈组织上皮钉突下方浸润的细胞中,HMGB1自细胞核转移至细胞质和细胞外;其龈沟液内HMGB1的表达量也明显高于健康对照组（P<0.01）。结论 HMGB1可能在牙周炎病理进程中有重要作用。     

白细胞介素-1β对遗传性牙龈纤维瘤和正常牙龈上皮细胞β-防御素的影响

目的:比较白细胞介素1β(IL-1β)刺激体外培养的正常牙龈、遗传性牙龈纤维瘤(HGF)上皮细胞β-防御素(HBD)表达的差异,探讨该差异与遗传性牙龈纤维瘤发病机制的可能相关性。方法:体外培养3例遗传性牙龈纤维瘤病人和6例正常人牙龈上皮细胞,经0,0.01,0.1,1,10,100 ng/mL的IL-1β分别刺激12、24、36、48 h,提取细胞总RNA,逆转录后,采用HBD-1、2、3特异性引物经PCR扩增,以β-肌动蛋白为内参,计算HBD-1、2、3与内参扩增产物相对值,对HBD-1、2、3进行半定量分析,采用SPSS软件单向方差统计分析法分析结果。结果:HBD-1 mRNA在正常牙龈和遗传性牙龈纤维瘤上皮细胞中呈固有表达,不受IL-1β刺激的影响。IL-1β可上调两种牙龈上皮细胞HBD-2、3的表达,在浓度为0.1～10 ng/mL时,作用时间大于12 h,受刺激组与未受刺激组差异有显著性(P<0.01)。相同浓度IL-1β作用不同时间时,正常牙龈上皮细胞中HBD-2、3的表达水平显著高于遗传性牙龈纤维瘤上皮细胞中表达水平(P<0.05,P<0.01)。且随作用时间延长差异更加显著。结论:遗传性牙龈纤维瘤病变组织和正常牙龈组织中β-防御素的表达有差异,这种差异可能与遗传性牙龈纤维瘤上皮细胞的分化及发病机制相关。     

牙龈卟啉单胞菌膜泡对牙龈上皮细胞MMPs基因表达的影响

目的:探讨牙龈卟啉单胞菌膜泡对牙龈上皮细胞基质金属蛋白酶(MMPs)基因表达的影响,揭示牙龈卟啉单胞菌在牙周炎中的致病作用.方法:以Real-time RT-PCR法检测牙龈卟啉单胞菌膜泡刺激下牙龈上皮细胞MMP-1和MMP-3的mRNA表达水平.结果:牙龈卟啉单胞菌膜泡显著地上调MMP-1和MMP-3 mRNA表达水平.结论:牙龈卟啉单胞菌诱导牙龈上皮细胞发生细胞炎症反应,可能是牙周炎发生、发展的重要因素.     

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目的    探讨精氨酸-甘氨酸-天冬氨酸（RGD）肽修饰纯钛表面对人牙龈成纤维细胞（human gingival fibroblasts,HGF）和上皮细胞（human gingival epithelial cells,HGE）初期黏附行为的影响。方法    应用羰基二咪唑（1,1′-carbonyldiimidazole,CDI）活化法将含RGD的短肽共价连接到纯钛表面,免疫荧光法检测钛表面RGD肽。评价RGD接枝与未接枝纯钛表面对HGF和HGE初期黏附的影响。结果    RGD肽可以通过CDI活化方法接枝到纯钛表面,HGF和HGE在RGD接枝钛表面黏附和增殖的细胞数量显著高于未接枝钛表面,差异有统计学意义（P < 0.01）。结论    生物活性肽RGD接枝纯钛表面可有效促进HGF和HGE在其表面的黏附。     

硝苯地平对人牙龈上皮细胞bcl-2基因表达的影响

目的:体外观察硝苯地平(nifedipine,NIF)对人牙龈上皮细胞(human gingival epithelial cells,HGECs)bcl-2基因转录水平的调节,探讨NIF诱导的药物性牙龈增生(drug-induced gingival overgrowth,DGO)与凋亡抑制基因bcl-2的相关性.方法:采用牙周手术切除的健康牙龈组织.用酶消化法分离培养HGECs;免疫组织化学方法对培养细胞进行细胞鉴定;实时定量PCR技术检测不同浓度NIF(1 μg/ml、2 μg/ml和3 μg/ml)刺激下HGECs中bcl-2 mRNA水平,以0 μg/ml NIF为空白对照.采用SPSS 11.0软件包对所得数据进行单因素方差分析.结果:酶消化法获得的HGECs在体外培养中生长状态良好;免疫组织化学显示,HGECs抗角蛋白染色阳性,抗波形蛋白染色阴性;NIF处理24h后的HGECs bcl-2 mRNA水平随NIF浓度的增高而上升,3 μg/ml浓度组与空白对照组有显著差异(P＜0.05);NIF处理48h后.2 μg/ml、3 μg/ml浓度组HGECs bcl-2 mRNA水平与空白对照组差异明显(P＜0.05).结论:NIF调节体外培养的HGECs中bcl-2基因转录的水平.     

Adherence of Porphyromonas gingivalis to gingival epithelial cells: modulation of bacterial protein expression

The protein profiles of Porphyromonas gingivalis (ATCC 33277 and W83) bound to KB gingival epithelial cells were analyzed by SDS-PAGE and immunoblotting. We found that a 51-kDa component was formed in bacteria that adhered to the KB cells, whereas 26- to 29-kDa bands were less intensive, in contrast to the protein profile of free bacteria. P. gingivalis ATCC 33277 incubated with protease-treated KB cells retained the profile of free bacteria. These results demonstrate the specificity of bacterial recognition of eukaryotic membrane components.     

Porphyromonas gingivalis gingipains mediate the shedding of syndecan-1 from the surface of gingival epithelial cells

Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan-1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan-1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan-1 detected in the culture medium increased significantly in a time-dependent manner. However, neither a heat-inactivated supernatant nor a supernatant from a gingipain-deficient mutant had a significant effect on syndecan-1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.     

Porphyromonas gingivalis gingipains mediate the shedding of syndecan‐1 from the surface of gingival epithelial cells

Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan‐1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan‐1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan‐1 detected in the culture medium increased significantly in a time‐dependent manner. However, neither a heat‐inactivated supernatant nor a supernatant from a gingipain‐deficient mutant had a significant effect on syndecan‐1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.     

Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22

Suzuki K, Sakiyama Y, Usui M, Obama T, Kato R, Itabe H, Yamamoto M. Oxidized low‐density lipoprotein increases interleukin‐8 production in human gingival epithelial cell line Ca9‐22. J Periodont Res 2010; 45: 488–495. © 2010 John Wiley & Sons A/S Background and Objective: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low‐density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. Material and Methods: Cells of the human gingival epithelial cell line Ca9‐22 were cultured in media containing OxLDL, and the amounts of interleukin‐8 (IL‐8) and prostaglandin E₂ (PGE₂) produced were measured using ELISAs. Results: Production of IL‐8 by Ca9‐22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE₂ by Ca9‐22 cells was enhanced by co‐incubation with OxLDL and interleukin‐1β (IL‐1β). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL‐induced IL‐8 and PGE₂ production in the presence of IL‐1β. The p³⁸ MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL‐induced IL‐8 production. Among oxidized lipids and chemically modified LDL, 7‐ketocholesterol enhanced IL‐8 production. Conclusion: This is the first report to show that OxLDL enhances IL‐8 production in epithelial cells.