Production of inflammatory mediators and cytokines by human gingival fibroblasts following bacterial challenge

Bacteria can indirectly affect the course of periodontal diseases by activating host cells to produce and release inflammatory mediators and cytokines. These mediators and cytokines, manifest potent proinflammatory and catabolic activity and may play key roles in local amplification of the immune response as well as in periodontal tissue breakdown. This study tested the effect of Actinobacillus actinomycetemecomitans (Aa) and Campylobacter rectus (Cr) challenge on PGE₂, IL-1β, IL-6 and IL-8 production by human gingival fibroblasts (HGF). Contact-inhibited HGF were prepared and formalin-killed bacterial cells ( Aa JP2, ATCC 29523 & 33384 and Cr ATCC 33238) at 10⁶-10⁹ were added to the HGF. Culture supernatants were collected at varying time intervals and analyzed for cytokine and mediator content. All concentrations of Aa JP2 and Cr ATCC 33238 suppressed IL-1β production up to approximately 50% during the initial 3-12-h period. No bacterial concentration tested was able to increase IL-1β production above the maximum basal levels. Both bacterial species stimulated production of IL-6 and IL-8. Aa JP2 did not affect PGE₂ levels significantly, whereas Cr ATCC 33238 was stimulatory only at the highest concentration tested (10⁹). There were no significant differences among the three Aa strains with respect to IL-1β production. However, Aa ATCC 29523 and ATCC 33384 were less capable of stimulating IL-6 secretion and more efficient in stimulating. IL-8 production than Aa JP2. In general. Cr was the most potent enhancer of cyto-kine and mediator production by HGF. In conclusion, Aa and Cr are capable of amplifying the local immune response and promoting periodontal tissue inflammation by stimulating HGF to secrete mainly IL-6 and IL-8.     

In situ hybridisation and immunocytochemical localisation of osteolytic cytokines and adhesion molecules in ameloblastomas

Ameloblastomas produce interleukin-1-like activity that could explain some part of their osteolytic capability. However, the cellular source of this osteolytic activity is unknown. In the present study, cytokines with known inflammatory and osteolytic activity, i.e., interleukin-1 (IL-1), tumour necrosis factor (TNF), and interleukin-6 (IL-6), have been localised by immunocytochemistry and in situ hybridisation. The cellular adhesion receptors ICAM-1, E-selectin and VCAM-1 have also been immunolocalised. Immunocytochemistry demonstrated that all seven specimens showed positive staining for IL-1α and IL-6 with these cytokines being located in the stellate reticulum-like cells and vascular endothelium. Very faint staining for IL-1β was seen in four of seven specimens. No reaction was seen for TNF-α. All specimens demonstrated E-selectin staining in the vascular endothelium and ICAM-1 and VCAM-1 staining in the stellate reticulum-like cells and the endothelium. In situ hybridisation for the cytokines showed the presence of mRNA of both IL-1α and IL-6 in the stellate reticulum-like cells. Faint staining for IL-1β was also seen. No staining was seen for TNF. These findings show that ameloblastomas synthesize two bone-modulating cytokines, IL-1α and IL-6, and that these are synthesized mainly by the stellate reticulum-like cells. These tumours also contain a proportion of activated blood vessels in which endothelial cells express the cellular adhesion receptors ICAM-1, E-selectin and VCAM-1.     

Immunocytochemical localization of inflammatory cytokines and vascular adhesion receptors in radicular cysts

Odontogenic cysts are one of the commonest bone destroying lesions of the maxillofacial skeleton, with the inflammatory radicular cyst being the commonest jaw cyst. Explains of radicular cysts produce an interleukin-1-like activity which could explain the osteolysis seen with these tumours though the cellular source of this osteolytic activity is unknown. In the present study, cytokines with known inflammatory and osteolytic activity: interleukin-1 (IL-1), tumour necrosis factor (TNF), inlerleukin-6 (IL-6). and the chemotactic cytokine interleukin-8 (IL-8) have been localized immunocytochemically in radicular cysts. The cellular adhesion receptors ICAM-1 and ELAM-1 have also been immunolocalized. All specimens showed positive staining for IL-1 (alpha and beta) and IL-6, with these cytokines being located in epithelial and vascular endothelial cells. Only two specimens demonstrated TNF and IL-8 staining, which was located in macrophages. All specimens demonstrated ELAM-1 staining in endothelium and ICAM-1 staining in epithelium, endothelium and mononuclear cells. These findings show that radicular cysts contain two bone-modulating cytokines. IL-1 and IL-6, and that these appear to be synthesized mainly by the epithelial cells. Cysts also contain a proportion of activated blood vessels whose endothelial cells express the cellular adhesion receptors ICAM-1 and ELAM-1.     

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.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1β

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1β (IL-β). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1β, IL-6 and IL-8. IL-1β activation of GF cells showed that IL-1β not only induces the expression of IL-6, IL-8 and TNF-α, but also acts in an autocrine manner on GF cells and induces IL-1βexpression. Furthermore, the continuous presence of IL-1β in GF cell cultures did not down regulate the response of GF cells to IL-1β. Pretreatment of GF cells with IL-lβ resulted in the enhanced synthesis of TNF-α in response to additional IL-lβ. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell‐surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram‐negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), and tumor necrosis factor‐α (TNF‐α) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer‐membrane proteins from P. gingivalis ATCC 53977. Outer‐membrane protein from P. gingivalis enhanced the production of IL‐6 and IL‐8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL‐8 production activity of polysaccharide from P. gingivalis was higher than that of other cell‐surface components. The levels of IL‐6 and IL‐8 released from the P. gingivalis lipopolysaccharide‐treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer‐membrane protein or lipopolysaccharide inhibited the IL‐6 and IL‐8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer‐membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

一氧化碳对炎症环境下人牙龈成纤维细胞黏附分子表达影响的机制研究

目的 研究一氧化碳对炎症环境下人牙龈成纤维细胞（HGF）黏附分子表达影响的分子机制。方法 体外培养HGF,以50 ng•mL-1的TNF-α和10 ng•mL-1的IL-1β刺激加入或不加入500 μmol•L-1一氧化碳释放分子-3（CORM-3）的HGF;分别在刺激10、20 min后收集部分细胞,用Western blot法检测丝裂原激活蛋白激酶（MAPK）通路中细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38的磷酸化水平;在刺激4 h后用Western blot法检测核转录因子-κB（NF-κB）的核内表达;在部分实验中,HGF在TNF-α、IL-1β及CORM-3刺激前以鸟苷酸环化酶特异性抑制剂1H-[1,2,4]噁二唑[4,3-α]喹喔啉-1-酮（ODQ）预处理8 h,刺激24 h后用Western blot法检测细胞间黏附分子-1（ICAM-1）的表达。结果 在TNF-α和IL-1β刺激细胞10 min后,MAPK通路中p38、ERK和JNK的磷酸化水平即有明显升高,CORM-3能够明显抑制p38的磷酸化,而对ERK和JNK的磷酸化水平无明显影响;4 h后,CORM-3可明显抑制NF-κB-p65的核内表达。ODQ不能改变CORM-3对TNF-α和IL-1β共同刺激下ICAM-1表达水平的影响,说明CORM-3的作用并非通过鸟苷酸环化酶系统实现。结论 一氧化碳对炎症环境下HGF黏附分子表达的抑制作用可能是通过对NF-κB的活性抑制和对MAPK p38磷酸化水平的抑制作用来实现的。     

Cyclosporin A and hydroxycyclosporine (M-17) affect the secretory phenotype of human gingival fibroblasts

The responsiveness of human gingival fibroblast populations to cyclosporin A (CsA) and its principal metabolite, hydroxycyclosporine (M17), was evaluated in cell culture. Gingival fibroblasts exhibited a dose-dependent accumulation and bell-shaped distribution of dansylated CsA. A 100-fold excess of non-labeled CsA prevented the accumulation of the fluorescent probe in the fibroblasts. Both CsA (400 ng/ml) and M17 (100 ng/ml) stimulated mean gingival fibroblast cell number to 23.2% and 36.7% above controls, and reduced mean collagen production by 37.7% and 37.4% below controls, respectively; however, neither CsA nor M17 affected mean protean production in comparison to control cultures. Analyses of responses to CsA and M17 by ligand-accumulating and non-accumulating fibroblasts sorted out from the parent cultures did not provide consistent interstrain responses either by cells representing the upper quartile of fluorescence or cells representing the bottom quartiles of fluorescence. These data demonstrate that CsA is accumulated by gingival fibroblasts and that CsA and M17 are potent modulators of gingival fibroblast phenotype.     

lnterleukin-1β and phenytoin reduce α1 (1) procollagen mRNA expression in human gingival fibroblasts

Effects of and interactions between interleukin-1β (IL-1 β) and phenytoin (PHT) on α1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E₂ (PGE₂) formation were studied. IL-1β (300 pg/ ml) reduced the steady-state level of αl(I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 μg/ml) reduced the level of α1(I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1β, PHT potentiated the inhibitory effect of IL-1β on αl(I) procollagen mRNA level that was accompanied by an increased PGE₂ formation. Preincubation with indomethacin (10^-6m) partially reduced the inhibitory effect of IL-1β as well as of IL-1β in combination with PHT on the mRNA level of αl(I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE₂ (≥10 nm) dose-dependently reduced steady-state level of α1(I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of αl(I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces α1(I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.