Aspects of interleukin-8 gene expression by gingival and dermal fibroblasts stimulated with interleukin-1beta or tumour necrosis factor alpha

Fibroblast-derived interleukin (IL)-8 is thought to have an important role in the orchestration of immuno-participant cells infiltrating the skin and gingiva in response to continuously recurring bacterial infection. Therefore, the IL-8 gene expression should be under tight regulatory control and it might be temporally and spatially limited in inflammatory tissue. The purpose of this study was to examine the aspect of the IL-8 gene expression by fibroblasts stimulated with pro-inflammatory cytokines, IL-1beta and TNF-alpha. In situ hybridisation revealed that fibroblasts did not express IL-8 mRNA whereas keratinocytes and endothelial cells did in IL-1beta- or TNF-alpha-injected mice skin. However, cultured mouse dermal fibroblasts expressed not only IL-8 but also IL-1beta mRNA without stimulation by exogenous IL-1beta and TNF-alpha, and the expression was not enhanced by the exogenous cytokines. A similar result was obtained in late-passage human gingival fibroblasts. These results suggest that fibroblasts remain insensitive to IL-1beta and TNF-alpha so as to induce the IL-8 gene expression in non-inflammatory mice skin. Mouse dermal and late-passage human gingival fibroblasts in vitro are likely to be altered in phenotype into IL-8-producing cells along with the production of IL-1beta. In skin inflammation and periodontal diseases, fibroblasts may express the IL-8 gene even without an exogenous cytokine, IL-1beta or TNF-alpha, during their proliferation similar to the situation in our culture system.     

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.     

Differential regulation of interleukin-6 and inducible cyclooxygenase gene expression by cytokines through prostaglandin-dependent and -independent mechanisms in human dental pulp fibroblasts

Increased levels of interleukin-1 (IL)-1, tumor necrosis factor-alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) have been detected in inflamed pulp tissue. To gain further insight into the molecular pathogenesis of pulpitis, we investigated the effects of IL-1alpha or TNF-alpha and PGE2, either alone or in combination on IL-6 and cyclooxygenase (COX)-2 messenger RNA (mRNA) production in cultured human dental pulp (HDP) fibroblasts. Exposure of HDP fibroblasts to IL-1alpha or TNF-alpha resulted in elevated levels of IL-6 (approximately 3.4 to approximately 10.4-fold) and COX-2 (approximately 5 to approximately 6.2-fold) mRNA. Simultaneous addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 to the cultures significantly reduced the cytokine-induced IL-6 mRNA synthesis ranging from 45% to 65%. However, indomethacin enhanced the cytokine-stimulated IL-6 mRNA synthesis by approximately 1.7 to approximately 3.4-fold. This action could be reversed by exogenous PGE2. In contrast, PGE2 or indomethacin failed to modify the stimulatory effect of IL-1alpha or TNF-alpha on COX-2 gene expression. Because excessive levels of IL-6 and prostaglandins have been connected with the pathogenesis of several inflammatory diseases, our results suggest the involvement of HDP fibroblasts in the development of pulpitis via producing IL-6 and COX-2. Furthermore, expression of IL-6 and COX-2 genes in this cell seems to be differentially regulated by cytokines through prostaglandin-dependent and -independent pathways.     

古曲抑菌素A对脂多糖诱导人牙髓细胞炎症反应的影响

目的研究组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）对脂多糖（LPS）诱导人牙髓细胞（hDPC）炎症反应的影响。 方法采用酶组织块法体外分离培养hPDC,按处理因素不同分为空白对照组、LPS组（1 μg/ml）、TSA（25或50 nmol/L预处理）+LPS（1 μg/ml）组,实时荧光定量聚合酶链反应（PCR）和ELISA检测促炎因子白细胞介素（IL）-6、IL-8 mRNA及蛋白表达量,Western blot检测NF-κB信号通路关键信号蛋白IKKα/β、p65及IκB-α磷酸化程度的变化。IL-6、IL-8 mRNA及蛋白表达采用方差分析,NF-κB信号通路Western blot结果采用独立样本t检验。 结果TSA（25 nmol/L）预处理能显著降低LPS刺激引起的IL-6、IL-8 mRNA与蛋白表达（F_{IL-6 mRNA}= 22.538,P_{IL-6 mRNA}= 0.002;F_{IL-8 mRNA}= 20.253,P_{IL-8 mRNA}= 0.002;F_IL-6蛋白= 9.327,P_IL-6蛋白= 0.007;F_IL-8蛋白= 9.6894,P_IL-8蛋白= 0.011）;LPS刺激能激活NF-κB信号通路中关键蛋白p-IKKα/β、p-p65和p-IκB-α的表达,而TSA预处理可降低IKKα/β（t_{30 min}= 6.437,P_{30 min}= 0.003;t_{60 min}= 6.386,P_{60 min}= 0.003;t_{120 min}= 4.368,P_{120 min}= 0.012）和IκB-α磷酸化程度（t_{15 min}= 3.822,P_{15 min}= 0.019;t_{30 min}= 4.467,P_{30 min}= 0.011）。 结论TSA可显著抑制LPS刺激下hDPC促炎因子的分泌,同时降低IKKα/β和IκB-α磷酸化程度,提示TSA可能通过降低NF-κB信号通路活性抑制牙髓炎症发展。     

microRNA-223在牙龈卟啉单胞菌诱导牙龈成纤维细胞炎症调控的研究

目的 探讨microRNA-223在牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharides, P.g-LPS)诱导牙龈成纤维细胞(gingival fibroblasts,GFs)炎症过程中对相关炎症因子表达水平的调控作用。 方法 采用慢病毒转染、干扰GFs中的microRNA-223的表达,在最适P.g-LPS刺激浓度(800 μg/L)分别刺激过表达、抑制以及正常表达microRNA-223的GFs,采用实时聚合酶联反应（Real-time quantitative polymerase chain reaction,qPCR）检测TNF-α、IL-1β、IL-6的mRNA表达水平变化,酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测其蛋白水平的变化。 结果 LPS刺激GFs产生炎症反应时,细胞内microRNA-223以及相关促炎因子TNF-α、IL-1β、IL-6的mRNA和蛋白表达水平较正常细胞中的表达量明显上调。当细胞内microRNA-223上调时,促炎因子的mRNA和蛋白表达水平也会上调(P<0.001);当细胞内microRNA-223下调时,促炎因子TNF-α、IL-1β的蛋白水平会显著下降(P<0.001)。 结论 当GFs受P.gingivalis-LPS刺激发生炎症时,microRNA-223的表达量增多,上调促炎因子TNF-α、IL-1β、IL-6,进一步加重组织细胞的炎症。     

interleukin-1β and tumor necrosis factor-α stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament

The present study demonstrates that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1β and TNF-α induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-γ. The peak expression of the MCP-1 gene by IL-1β or TNF-α was observed at 3 h after initiation of their treatment. Furthermore, IL-1β in combination with TNF-α synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1β also stimulated synergistically MCP-1 protein expression in combination with TNF-α. These results suggest that IL-1β and TNF-α may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.     

Influence of HEMA on LPS- and LTA-stimulated IL-6 release from human dental pulp cells

ObjectiveDental pulp cells interact with immunogenic components such as LPS (lipopolysaccharide) or LTA (lipoteichoic acid) released from microorganisms in carious lesions. In the present investigation, the formation of the pro-inflammatory cytokines TNFα and IL-6 in LPS- or LTA-stimulated cells from the dental pulp interface and pulp fibroblasts was analyzed in the presence of the resin monomer 2-hydroxyethyl methacrylate (HEMA) under varying cellular redox conditions.MethodHuman pulp fibroblasts (HPC) or cells from the dental pulp interface expressing an odontoblast phenotype (hOD-1) were exposed to LTA, LPS or HEMA for 1 h or 24 h. Redox homeostasis was modified by the prooxidant BSO (L-buthionine sulfoximine) or the antioxidant NAC (N-acetyl cysteine). Formation of TNFα or IL-6 was analyzed by ELISA, and cell survival was determined by a crystal violet assay. Statistical analyses were performed using the Mann-Whitney-U-test.ResultsSecretion of TNFα was not detected in LPS- or LTA-stimulated HPC or hOD-1, and IL-6 was not found after a short exposure (1 h). After a 24 h exposure, LPS induced a 3-fold increase in IL-6 formation in HPC, while LTA stimulated IL-6 release about 20-fold. Likewise, LTA was more effective than LPS in hOD-1 stimulating IL-6 levels about 50-fold. HEMA inhibited the LPS- and LTA-induced IL-6 release, and this effect was enhanced by BSO but counteracted by NAC in both cell types. IL-6 release was independent of cell survival rates.ConclusionsThe protective immune response in odontoblasts and pulp fibroblasts is impaired by monomers such as HEMA through the disturbance of the redox homeostasis.     

interleukin-1β and tumor necrosis factor-α stimulate synergistically the expression of monocyte chemoattractant protein-1 in fibroblastic cells derived from human periodontal ligament

The present study demonstrates that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce and synergistically stimulate monocyte chemoattractant protein-1 (MCP-1) expression in fibroblasts from human periodontal ligament. IL-1β and TNF-α induced in a dose-dependent manner the expression of the MCP-1 gene in the fibroblasts from the human periodontal ligament. However, such an inducing effect was not observed with IL-6 and interferon-γ. The peak expression of the MCP-1 gene by IL-1β or TNF-α was observed at 3 h after initiation of their treatment. Furthermore, IL-1β in combination with TNF-α synergistically stimulated the MCP-1 gene expression in the cells. We also observed significant chemotactic activity for human monocytes in conditioned medium of fibroblasts from the human periodontal ligament treated with both cytokines. The stimulated chemotactic activity induced by these cytokines depended on both dose and treatment time. The chemotactic activity in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was neutralized by antiserum specific for MCP-1 protein. The MCP-1 gene product in conditioned medium of IL-1β-treated fibroblasts from the human periodontal ligament was shown to have a molecular mass of 11,000 Da by immunoprecipitation with the specific antiserum, and IL-1β also stimulated synergistically MCP-1 protein expression in combination with TNF-α. These results suggest that IL-1β and TNF-α may contribute to the infiltration of monocytes into inflammatory sites of periodontal ligament tissues via the MCP-1 gene product produced by fibroblasts from the human periodontal ligament.     

人β防御素在牙髓组织中的表达及与炎症因子的关系

目的:探索人β-防御素(humanβ-defensin,HBD)家族成员在人牙髓组织中的表达,以HBD2为代表,探讨HBD在牙髓炎症反应中所受调控及HBD家族成员之间的调节关系.方法:在NCBI GEO Profiles数据库中检索HBD在人牙髓组织中表达的基因芯片信息,并运用反转录聚合酶链式反应(RT-PCR)进行验证.运用4种炎症因子TNF-α、IL-1 α、IL-1β和IL-6不同组合刺激人牙髓细胞,采用实时荧光定量核酸扩增检测法(qPCR)检测相应组分对HBD2表达水平的影响;HBD110预处理及LPS刺激牙髓细胞后,采用qPCR方法检测TNF-α、IL-1α和HBD2的表达水平.采用GraphPad Prism5.01对实验组和对照组的结果进行统计学分析.结果:在NCBI GEO Profiles数据库中检索到27种HBD家族成员在人牙髓组织中表达.TNF-α、IL-1α、IL-1β和IL-6联合作用,可显著提升HBD2的表达水平.HBD110通过调控TNF-α和IL-1α的表达,提高HBD2的表达水平.结论:除已有报道的HBD1、HBD2、HBD3和HBD4外,其他HBD家族成员在人牙髓组织中表达,且HBD2在牙髓组织中受炎症因子及其他HBD家族成员的调控.     

Functional TLRs and NODs in human gingival fibroblasts

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-kappaB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.     

Leptin induces IL-6 and IL-8 expression through leptin receptor Ob-Rb in human dental pulp fibroblasts

Objective: Leptin, through binding to its special receptor (Ob-Rb), has potent effects on immunity and inflammation. This study aimed to investigate the expression of leptin receptor Ob-Rb in human dental pulp fibroblasts (HDPFs) and the effects of leptin on the production of proinflammatory cytokines of IL-6 and IL-8 by HDPFs.

Methods: Ob-Rb expression was determined by quantitative real-time PCR (real-time PCR), Western blot and immunofluorescence analyses in cultured HDPFs. Small interfering RNA (siRNA) was transfected into HDPFs to down-regulate the expression of Ob-Rb. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were used to determine the proinflammatory cytokines of IL-6 and IL-8 levels in leptin-stimulated HDPFs. The involved signalling pathways that mediate the leptin-stimulated production of proinflammatory cytokines were investigated using Western blot and specific signalling inhibitor analyses.

Results: The expression levels of Ob-Rb mRNA and protein were detected in HDPFs. Leptin could stimulate mRNA and protein expression of IL-6 and IL-8 in HDPFs in a concentration-dependent and time-dependent manner. Transfection with siRNA targeting Ob-Rb resulted in remarkable reduction of IL-6 and IL-8 expressions by HDPFs. In accordance with the enhanced expression of proinflammatory cytokines, leptin stimulation resulted in rapid phosphorylation of STAT3, p38 MAPK, ERK and Akt in HDPFs. Inhibiting JAK2/STAT3, p38 MAPK or PI3K/Akt substantially decreased leptin-induced IL-6 production, whereas blocking ERK and p38 MAPK substantially suppressed IL-8 production from leptin-stimulated HDPFs.

Conclusions: Leptin may up-regulate IL-6 and IL-8 production through binding with Ob-Rb in HDPFs via the activation of different intracellular signalling pathways.     

Induction of dental pulp fibroblast matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression by interleukin-1alpha and tumor necrosis factor-alpha through a prostaglandin-dependent pathway

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are involved in the degradation of extracellular matrix in many inflammatory diseases. Little is known regarding the expression of these mediators in dental pulp fibroblasts. The effects of proinflammatory cytokines (interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha)) and prostaglandin E2 (PGE2) on pulp fibroblast MMP-1 and TIMP-1 gene expression were investigated. Northern hybridization showed that IL-1alpha and TNF-alpha induced significant MMP-1 gene expression, with only little effect on TIMP-1 gene. Exogenous PGE2, however, upregulated TIMP-1 mRNA synthesis but not MMP-1. Concomitant addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 suppressed MMP-1 mRNA production, compared with the groups treated with IL-1alpha or TNF-alpha alone. In contrast, PGE2 enhanced the upregulatory effects of TIMP-1 mRNA by IL-1alpha or TNF-alpha. Furthermore, cytokine stimulation of MMP-1 and TIMP-1 gene expressions can be enhanced or blocked by indomethacin, respectively, and reversed by exogenous PGE2. These results suggested that cytokine-stimulated MMP-1 and TIMP-1 gene expression in dental pulp fibroblasts was mediated, at least in part, by a prostaglandin-dependent pathway. The differential regulation of IL-1alpha or TNF-alpha-induced MMP-1 and TIMP-1 mRNA synthesis, as well as the direct upregulation of TIMP-1 gene expression by PGE2, also implied that prostaglandin may serve as a protective mechanism from excessive tissue breakdown during pulpitis.