Triclosan reduces prostaglandin biosynthesis in human gingival fibroblasts challenged with interleukin-1 in vitro

Abstract The effect of the toothpaste ingredient triclosan (2,4,4′-trichloro-2′-hydroxyldiphenyl ether) on the prostaglandins biosynthesis in human gingival fibroblasts challenged with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) was studied in vitro. When gingival fibroblasts were treated simultaneously with triciosan and IL-1β, the stimulatory effect of IL-1β on prostaglandin E₂ (PGE₂) and PGI₂ formation was reduced in a dose-dependent manner by triclosan. Triclosan also reduced the PGE: formation induced by TNFα. Furthermore, the capacity of IL-1β to induce release of [³H] arachidonic acid from prelabelled gingival fibroblasts was reduced in the presence of triclosan. Addition of exogenous unlabelled arachidonic acid (AA) to the cells resulted in enhanced PGE₂ formation which was reduced by triclosan. The upregulation of the metabolism of AA to PGE₂ induced by IL-lβ, was markedly reduced in the presence of triclosan. The study indicates that the stimulatory effect of IL-1β on prostanoid formation (PGE₂, PGI₂) in human gingival fibroblasts was diminished in the presence of triciosan partly at the level of phospholipase A₂ and partly at the level of cyclooxygenase. The present data that triclosan. in vitro, inhibits the production of inflammatory mediators such as prostaglandins suggests that this can be an aspect of its clinical effect on gingivitis, in addition to its antibacterial effect.     

Production of Rantes/CCL5 in human gingival fibroblasts challenged with tumor necrosis factor alpha

Chemokines are small-secreted proteins that stimulate the directional migration of leukocytes and thereby mediate the inflammatory process. The present study investigates the capacity of human gingival fibroblasts to produce the beta chemokine Rantes/CCL5. In situ hybridization, immunohistochemistry and ELISA were used to measure the induction of Rantes/CCL5 at the mRNA and protein levels, both in unstimulated gingival fibroblasts as well as in fibroblasts treated with the proinflammatory cytokines tumor necrosis factor (TNF)alpha or interleukin (IL)-1beta. TNFalpha in different concentrations (0.1-10 ng/ml) induced Rantes/CCL5 mRNA expression and protein production in 24-h cultures of human gingival fibroblasts. The expression of Rantes/CCL5-mRNA and protein production, induced by TNFalpha, was evident at 6 h and thereafter increased continuously during the study period (24 h). IL-1beta (3-300 pg/ml) also enhanced the production of Rantes/CCL5 in gingival fibroblasts. The amount of Rantes/CCL5 induced by IL-1beta (300 pg/ml), however, was less than that induced by TNFalpha (10 ng/ml). The study suggests that human gingival fibroblasts, by producing the chemokine Rantes/CCL5, participate in the regulation of the host response during the inflammatory process in the periodontal tissue.     

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.     

Enhanced prostaglandin biosynthesis in human gingival fibroblasts isolated from patients treated with phenytoin

Prostaglandin E2 (PGE2) formation was studied in human gingival fibroblasts derived from three epileptic patients before and after 9 months of phenytoin (PHT) therapy. Interleukin 1 (IL-1 alpha; 0.3-6.0 ng/ml), (IL-1 beta; 10-1000 pg/ml) and tumour necrosis factor (TNF alpha; 0.01-0.1 microgram/ml) dose-dependently stimulated the formation of PGE2 in 24 h cultures. In fibroblasts, derived after 9 months of PHT therapy, IL-1 alpha, IL-1 beta and TNF alpha induced a significantly higher formation of PGE2 compared to that in fibroblasts derived before PHT therapy. IL-1 beta induced a significantly higher release also of 3H-arachidonic acid (3H-AA) from prelabelled PHT fibroblasts compared to that in prelabelled gingival fibroblasts isolated before the drug therapy. Addition of exogenous AA caused a spontaneous increase of PGE2 formation in PHT fibroblasts compared to that in fibroblasts isolated before the PHT treatment. The results indicate that PHT medication results in an upregulation of prostanoid formation in gingival fibroblasts partly due to an increased phospholipase A2 activity and partly due to an increased cyclooxygenase activity.     

Effects and interactions of tumour necrosis factor α and bradykinin on interleukin-1 production in gingival fibroblasts

Effects of and interactions between tumour necrosis factor α (TNFα) and bradykinin (BK) on production of interleukin-1 (IL-lα, IL-lβ) in human gingival fibroblasts were studied. The cytokine TNFα induced production of cell-associated IL-lα and IL-1β in gingival fibroblasts, with IL-lβ being most abundant. Addition of BK, in the presence of TNFα, for 1 h and 6 h, respectively, synergistically enhanced the TNFα induced IL-lβ production, whereas BK alone did not induce 1L-1 production. Similar to BK, two phorbol esters, phorbol 12,13 dibutyrate (PDBu) and phorbol 12-myristate-13-acetate (PMA) which are known to stimulate protein kinase C (PKC), synergistically enhanced the TNFα induced IL-lβ production in the gingival fibroblasts. On the contrary, a phorbol ester which does not activate protein kinase C, 13-phorbolacetate (13-PA), did not potentiate the TNFα induced IL-lβ production. Similar to BK, the phorbol esters (PMA, PDBu, 13-PA) alone did not induce IL-1β production in the gingival fibroblasts. The results indicate that TNFα induces production of cell-associated IL-1 in gingival fibroblasts, which can be upregulated by a PKC dependent pathway.     

Cigarette smoke condensate affects the collagen-degrading ability of human gingival fibroblasts

Background and Objective: Cigarette smoke condensate, the particulate matter of cigarette smoke, is composed of thousands of chemicals, including nicotine. Cigarette smoking is a risk factor for periodontal disease. This study investigated the influence of cigarette smoke condensate on the collagen‐degrading ability of human gingival fibroblasts and its mechanism. Material and Methods: Human gingival fibroblasts were exposed for 72 h to various concentrations of total particulate matter cigarette smoke condensate. Cell proliferation and cytotoxicity were evaluated using water‐soluble tetrazolium‐1 and lactate dehydrogenase, respectively. The collagen‐degrading ability of human gingival fibroblasts was evaluated in collagen‐coated six‐well plates. Conditioned media and membrane extracts were collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Results: Cell proliferation decreased and cytotoxicity increased in human gingival fibroblasts with increasing concentrations of cigarette smoke condensate. Cell proliferation decreased by more than 50% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 200 μg/mL, and cytotoxicity increased to more than 30% (p < 0.05) when the concentrations of total particulate matter cigarette smoke condensate were above 400 μg/mL. Cigarette smoke condensate increased the collagen‐degrading ability of human gingival fibroblasts, especially at a concentration of 100 μg/mL (1.5‐fold increase, p < 0.05) compared with the control. Cigarette smoke condensate increased the production of proMMP‐1, proMMP‐2, MMP‐14 and TIMP‐1, and decreased the production of TIMP‐2, in conditioned media. Furthermore, compared with the control group, cigarette smoke condensate increased the production of MMP‐2, MMP‐14 and TIMP‐2 in membrane extracts, especially at concentrations of 50–100 μg/mL. Conclusion: Cigarette smoke condensate affects human gingival fibroblast proliferation and is toxic at total particulate matter cigarette smoke condensate concentrations of ≥ 400 μg/mL. Cigarette smoke condensate can increase the collagen‐degrading ability of human gingival fibroblasts by altering the production and localization of MMPs and TIMPs.     

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.     

Effect of phenytoin on interleukin-1β production in human gingival fibroblasts challenged to tumor necrosis factor αin vitro

Effects and interaction of tumor necrosis factor α (TNFα) and the antiepileptic drug phenytoin (PHT) on interleukin-1β (IL-1β) production as well as on prostaglandin E₂ (PGE₂) formation were studied in gingival fibroblasts in vitro. TNFα, in contrast to PHT, dose-dependently stimulated the production of cell-associated IL-1β. The stimulatory effect of TNFα on IL-1β production was accompanied by enhanced PGE₂ formation. When PHT and TNFα were added simultaneously, the drug potentiated the stimulatory effect of TNFα on both IL-Iβ production and PGE₂ formation. The major PHT metabolite, p-HPPH, did not affect IL-1β production, either alone or in combination with TNFα. The production of IL-1β induced by TNFα and the combination of TNFα and PHT was further enhanced in the presence of the prostaglandin endoperoxide (PGH) synthase inhibitors, indomethacin and flurbiprofen. The PHT-mediated enhancement of TNFα-induced IL-1β production and PGE₂ formation in gingival fibroblasts may be an important link in the pathogenesis of gingival overgrowth induced by PHT.     

Interleukin-1beta induces matrix metalloproteinase-1 expression in cultured human gingival fibroblasts: role of cyclooxygenase-2 and prostaglandin E2

OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrices and are responsible for excessive connective tissue breakdown in inflammatory disorders. We investigated the mechanism of MMP-1 expression in human gingival fibroblasts in response to the stimulation with interleukin-1beta (IL-1beta), and the role of inducible-type cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the regulation of MMP-1 expression. MATERIALS AND METHODS: We stimulated cultured human gingival fibroblasts with r(h)IL-1beta, and examined the expression of MMP-1 mRNA and protein by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of indomethacin, dexamethasone, or cycloheximide (CHX) on the IL-1beta-induced expression of MMP-1 was examined. The expression of MMP-1 in gingival fibroblasts stimulated with PGE2 was also examined. RESULTS: IL-1beta stimulated the expressions of mRNA and protein for MMP-1, in cultured fibroblasts, in time- and concentration-dependent manners. Pretreatment of the cells with indomethacin or dexamethasone inhibited the IL-1beta-induced MMP-1 expression. CHX, a protein synthesis inhibitor, also suppressed the MMP-1 expression. IL-1beta also induced COX-2 expression in gingival fibroblasts, and PGE2, a major COX-2 product, was found to enhance MMP-1 expression. CONCLUSION: The IL-1beta-induced MMP-1 expression in gingival fibroblasts may be mediated, at least in part, by COX-2 and its product PGE2.     

Prostaglandin F2alpha upregulates interleukin-6 production in human gingival fibroblasts

Prostaglandin F2alpha (PGF2alpha) is a bioactive lipid mediator which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2alpha in periodontal lesions are poorly understood. In the present study, we investigated the effect of PGF2alpha on interleukin (IL)-6 production in human gingival fibroblasts (HGF). PGF2alpha stimulated IL-6 production in a time- and concentration-dependent fashion. IL-1beta and tumor necrosis factor alpha (TNFalpha), proinflammatory cytokines, induced IL-6 production in a time-dependent manner, and PGF2alpha synergistically enhanced IL-6 production induced by IL-1beta and TNFalpha. IL-6 mRNA was expressed in PGF2alpha-stimulated HGF, and PGF2alpha increased IL-6 mRNA levels induced by IL-1beta and TNFalpha. Fluprostenol, a selective FP receptor agonist, could mimic PGF2alpha-induced IL-6 production. Since FP receptors are coupled to elevation of intracellular calcium and activation of protein kinase C (PKC), the mechanism of IL-6 production by PGF2alpha was investigated using TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores, and calphostin C, an inhibitor of PKC. TMB-8 significantly suppressed PGF2alpha-induced IL-6 production, whereas calphostin C showed a stimulatory effect on PGF2alpha-induced IL-6 production. From these data, we suggest that PGF2alpha upregulates IL-6 production through FP receptors in HGF, that PGF2alpha synergistically enhances IL-6 production in IL-1beta- and TNFalpha-stimulated HGF, and that PGF2alpha-induced IL-6 production may be dependent on intracellular Ca2+ mobilization and be downregulated by PKC activation. PGF2alpha may be involved in the pathogenesis of periodontal disease by enhancing IL-6 levels in periodontal lesions.     

Interleukin-1β stimulates collagenase production by cultured human periodontal ligament fibroblasts

To examine the effects of interleukin-lβ (IL-1β) on collagenase production by human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) in Culture, collagenase activity in conditioned media was determined using a novel procedure that circumvented interference by enzyme inhibitors. Fibroblasts obtained from five paired periodontal ligament and gingival tissues were cultured for two weeks, and then incubated for a further 72 h in α-MEM supplemented with various concentrations of IL-1β (0 to 1250 pg/ml). The conditioned media from individual cultures were harvested and treated with dithiothreitol to inactivate TIMPs, and then with APMA, to activate the latent collagenase. Collagenase activity was measured fluorometrically using FITC-collagen as a substrate. IL-lβ induced a ∼2.4 to 5.2-fold increase in collagenase activity in PLF compared to a ∼1.4 to 2.2-fold increase in GF. These results are in contrast to previous studies in which collagenase activity was measured in the presence of TIMPs, and indicate that PLF are more sensitive to IL-1β than GF. Since both PLF and GF are present in periodontal lesions, it is possible that collagenase secretion stimulated by exposure to inflammatory cell products such as IL-lβ may participate in the destruction of collagen fibers involved in periodontal attachment.     

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts

Background and Objective: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. Material and Methods: Fibroblasts were stimulated for 48 h with 10^?4 or 10^?9 m Substance P; untreated fibroblasts served as controls. The expression of MMP‐1, ‐2, ‐3, ‐7 and ‐11 and of TIMP‐1 and ‐2 was evaluated using real‐time polymerase chain reaction and western blotting. Results: There was a significant, concentration‐dependent stimulatory effect of Substance P on MMP‐1, ‐2, ‐3 and ‐7 and TIMP‐2 gene expression (p < 0.05), and a probable effect on MMP‐11 (p = 0.06). At the higher concentration (10^?4 m Substance P), MMP‐1, ‐2, ‐3, ‐7 and ‐11 and TIMP‐2 showed the greatest up‐regulation; at the lower concentration (10^?9 m Substance P), MMP‐1, ‐3 and ‐7 and TIMP‐2 exhibited diminished up‐regulation, with MMP‐2 and ‐11 showing down‐regulation (p < 0.05). Expression of TIMP‐1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up‐regulated MMP‐1, ‐2, ‐3 and ‐11 and TIMP‐2. MMP‐1, ‐3 and ‐11 and TIMP‐2 showed greater up‐regulation at the higher Substance P concentration and diminished up‐regulation at the lower concentration. MMP‐2 was up‐regulated to a similar degree at both Substance P concentrations. Conclusion: In gingival fibroblast cells, Substance P at the higher concentration (10^?4 m ) induced greater up‐regulation of MMP‐1, ‐3 and ‐11 and TIMP‐2 expression, but at the lower concentration (10^?9 m ) induced diminished up‐regulation, which may represent a mechanism for modulating periodontal breakdown.     

Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines

BACKGROUND AND OBJECTIVE: Cyclosporin-induced gingival overgrowth arises from an alteration in collagen homeostasis and is enhanced by inflammatory changes in the gingival tissues. The aim of this study was to investigate the interaction among interleukin-1, oncostatin M, cyclosporin and nifedipine in promoting the up-regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase by gingival fibroblasts. MATERIAL AND METHODS: Fibroblast cultures (n = 5) were obtained from healthy controls and from patients with cyclosporin-induced gingival overgrowth, and cells were harvested between the fourth and ninth passages. Cells were stimulated with interleukin-1 and oncostatin M, alone or in combination, and with different concentrations of cyclosporin (0-2000 ng/mL) and nifedipine (0-200 ng/mL). MMP-1 and tissue inhibitor of metalloproteinase-1 production was determined using an enzyme-linked immunosorbent assay technique. A CyQuant cell proliferation assay was used to determine the DNA concentration in the sample. RESULTS: Fibroblasts obtained from patients with cyclosporin-induced gingival overgrowth produced significantly lower levels of MMP-1 than control fibroblasts (p < 0.001); tissue inhibitor of metalloproteinase-1 levels were significantly lower (p < 0.05), and the ratio of MMP-1 to tissue inhibitor of metalloproteinase-1 was reduced, in the conditioned medium of patients with cyclosporin-induced gingival overgrowth compared with controls. Interleukin-1 and oncostatin M produced a significant increase in the up-regulation of MMP-1, which was reversed when cyclosporin and nifedipine were added to the cell cultures (p < 0.05). CONCLUSION: Pro-inflammatory cytokines significantly up-regulate MMP-1 in cultured gingival fibroblasts. Up-regulation is attenuated by both cyclosporin and nifedipine. The interaction may account for the synergism between inflammation and cyclosporin-induced gingival overgrowth.     

Prostaglandin E2 potentiates interleukin-1β induced interleukin-6 production by human gingival fibroblasts

Abstract Increased levels of cytokines and prostanoids have been detected in inflamed gingival tissue and may play an important role in periodontal pathogenesis. Recent studies suggest that monocytic products, such as interleukin (IL)-1β, could stimulate IL-6 production by human gingival fibroblasts (HGF). In this context, the production of local cytokines and inflammatory mediators could regulate the secretory capacity of resident gingival fibroblasts. Therefore, the purpose of this study was to determine if PGE₂ induced by IL-1β could potentiate the IL-6 response by HGF. Utilizing an ELISA, it was determined that maximal IL-6 occurred when HGF were stimulated with 0.10–10 nM IL-1β. These concentrations of IL-1β also induced a small, but significant increase in PGE₂ production by HGF. Interestingly, the combination of ILγβ and PGE₂ induced a synergistic rise in IL-6 production by HGF. Moreover, inclusion of indomethacin caused a 20% reduction in IL-6 production and totally eliminated PGE₂ production. These findings provide additional rationale for the clinical use of NSAIDs in the management of periodontal disease due to their ability to attenuate production of both PGE₂, and IL-6. These results suggest the endogenous PGE₂ induced by IL-1β plays an important regulatory role in IL 6 production by HGF. Moreover, they support the concept that elevated PGE₂ induced during inflammation can regulate HGF secretory function.