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.     

Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-1β release from rat periosteal and human gingival fibroblasts in vitro

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease-digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum-free medium with 0.1, 1.0 and 10.0 micrograms/ml LPS. The levels of prostaglandin E2 (PGE2) and interleukin-1 beta (IL-1 beta) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE2 (24.5 +/- 1.5 (SD) ng/ml) and IL-1 beta (0.34 +/- 0.29 ng/ml). LPS stimulated statistically significant dose-related increases in PGE2 and IL-1 beta at the concentrations of LPS tested. At 10.0 micrograms/ml, LPS-stimulated fibroblasts produced 363.5 +/- 40.3 ng/ml PGE2 and 1.81 +/- 0.1 ng/ml IL-1 beta in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE2 and IL-1 beta release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.     

Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures

Oral keratinocytes are the first cells in contact with tobacco components and are capable of producing various inflammatory mediators, including PGE2 and IL-1. The purpose of this study was to examine PGE2 and IL-1 concentrations in nicotine-exposed oral keratinocyte cultures. Gingival keratinocyte cultures were established from healthy gingival tissues obtained from 7 subjects. Cultures were divided into 4 groups exposed to serum free medium (control), 0.1 μ m , 10 μ m or 1 mM nicotine for 4, 24 or 48 h. Using enzyme-linked immunosorbent assays, PGE₂ and IL-1α were quantified in culture supernatants; IL-1 α and β were also measured in lysed cells. A repeated measures analysis of variance was used to identify significant differences over time and treatment. Nicotine exposure did not significantly alter PGE2 levels at any given time period; however, PGE ₂ quantities declined significantly (p=0.0001) over time. At both 24 and 48 h, IL-1 α concentrations in lysates from 1 mM nicotine-exposed cells were significantly (p < 0.01) greater than those for all other treatments. Interleukin-1α quantities also declined significantly (p=0.037) over time in the cultures. Interleukin-1β concentrations were elevated, albeit not significantly, in the 1 mM treated cells at 24 and 48 h. Cell viability, mass and counts were not affected by nicotine treatment; these parameters increased significantly (p < 0.005) over time. In summary, nicotine treatment significantly increased IL-1 a concentrations in cultured keratinocytes; however, PGE₂ synthesis was not altered. Elevated IL-1 production by keratinocytes may have implications in tobacco-induced lesions, given the central role IL-1 plays in tissue response to injury.     

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.     

Nicotine effects on PGE2 and IL-1β release by LPS-treated human monocytes

Cigarette smoking is a major risk factor in the development and further progression of periodontitis. However, little is known regarding the pathogenesis of smoking-related periodontal diseases. The purpose of this study was to examine the effects of nicotine, alone and in combination with lipopolysaccharide (LPS), on monocyte secretion of bone-resorbing factors, PGE₂ and IL-1β. Peripheral blood monocytes (PBM) were isolated by counterflow centrifugal elutriation from 15 healthy, non-smoking donors. PBM were incubated for 24 h in RPMI 1640 containing nicotine (0, 50 μg/ml, I μg/ml, 10 μg/ml and 100 μg/ml) with or without 10 μg/ml Porphyromonas gingivalis LPS or Escherichia coli LPS. Culture supernatants were assayed for PGE₂ and IL-1β by ELISA. None of the nicotine preparations resulted in significant PBM secretion of PGE₂ and IL-1β above that of unstimulated cultures. However, PGE₂ release was potentiated 1.7-fold by the combination of P. gingivalis LPS and 10 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.05, one-way ANOVA). Prostaglandin E₃ release also was potentiated 3.5-fold by P. gingivalis LPS and 100 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.00001, one-way ANOVA) and 3.1-fold by E. coli LPS and 100 μg/ml nicotine relative to E. coli LPS alone (p<0.00001, I. one-way ANOVA). IL-1β secretion was lower for either LPS plus 100 μg/ml nicotine relative to LPS alone, although not significantly. These data demonstrate upregulation of LPS-mediated monocyte secretion of PGE₂ by nicotine and suggest a potential role for nicotine in periodontal disease pathogenesis.     

Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-1β release from rat periosteal and human gingival fibroblasts in vitro

Lipopolysaccharide (LPS) was extracted from Porphyromonas gingivalis (W83) by the Westphal procedure, nuclease-digested and ultracentrifuged. Fibroblasts were obtained from human gingival tissue and rat periosteum, grown to confluence then stimulated in serum-free medium with 0.1, 1.0 and 10.0μg/ml LPS. The levels of prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) released were measured after 2, 4 and 6 d by specific radioimmunoassays. Unstimulated gingival fibroblasts produced low levels of PGE₂ (24.5 ± 1.5 (SD) ng/ml) and IL-1β (0.34 ± 0.29 ng/ml). LPS stimulated statistically significant dose-related increases hi PGE₂ and IL-1β at the concentrations of LPS tested. At 10.0μg/ml, LPS-stimulated fibroblasts produced 363.5 ± 40.3 ng/ml PGE, and 1.81±0.1 ng/ml IL-1β in 6 d. These results demonstrate that LPS from P. gingivalis is capable of stimulating PGE, and IL-1β release from fibroblasts. This would appear to be an additional mechanism by which LPS can induce tissue breakdown in periodontal disease.     

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.     

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.     

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.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.     

Prostaglandin F2 alpha upregulates intercellular adhesion molecule-1 expression in human gingival fibroblasts

Prostaglandin F2 alpha (PGF2 alpha) is a bioactive lipid mediator, which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2 alpha in the disease are not well understood. In the present study, we investigated the effect of PGF2 alpha on intercellular adhesion molecule-1 (ICAM-1) expression in human gingival fibroblasts (HGF) and the effect of PGF2 alpha on ICAM-1 expression elicited by proinflammatory cytokines, interferon-gamma (IFN-gamma) and tumour necrosis factor alpha (TNF alpha) in the cells. PGF2 alpha-stimulated HGF expressed ICAM-1 expression in a time- and dose-dependent manner. IFN-gamma-elicited ICAM-1 expression was synergistically increased by PGF2 alpha, whereas TNF alpha-induced ICAM-1 expression was slightly inhibited by PGF2 alpha. Fluprostenol, a selective FP receptor agonist, could mimic PGF2 alpha-induced effect on ICAM-1 expression. Furthermore, signal transduction for the regulation of ICAM-1 by PGF2 alpha was investigated using N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), a calcium calmodulin antagonist, and 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C (PKC). W-7 and H-7, remarkably, suppressed PGF2 alpha-induced ICAM-1 expression and synergistic increase of ICAM-1 expression by combination of PGF2 alpha and IFN-gamma, while IFN-gamma-elicited ICAM-1 expression was only partially inhibited by W-7 and H-7. From these data, we suggest that PGF2 alpha upregulates ICAM-1 expression in HGF and synergistically enhances IFN-gamma-induced ICAM-1 expression through FP receptor by calcium calmodulin-dependent and PKC-dependent pathways. PGF2 alpha may be involved in the pathology of periodontal disease by upregulating ICAM-1 expression in HGF.     

Osteocalcin, prostaglandin E2 and alkaline phosphatase in gingival crevicular fluid: their relations to periodontal status

Abstract. Prostaglandin E₂ (PGE₂) and alkaline phosphatase (ALP) have often been measured in gingival crevicular fluid (GCF) as possible indicators of gingival inflammation and bone metabolism. Osteocalcin (OC), a major component of bone matrix, is mainly produced by osteoblasts, and could also be considered as a marker of bone turnover. The aims of this preliminary study were lo examine if OC was present in GCF and to assess the relationships of OC, PGE₂, and ALP in GCF to periodontal conditions. GCF samples were collected with durapore strips from 34 healthy, 12 gingivitis and 118 periodontitis sites in 17 subjects. ELISA techniques were used for the determinations of OC and PGE₂. ALP was measured spectrophotometrically by using p-nitrophenyl phosphate as substrate. Total amounts and concentrations of PGE₂ and ALP were significantly higher in periodontitis as compared to healthy and gingivitis sites, and were significantly and positively correlated with probing depth (PD) and gingival index (GI). OC was present in GCF from both healthy and diseased sites with mean concentrations more than ten times greater than normal serum levels. Total OC amounts from strips soaked with GCF from periodontitis sites were significantly higher than those Found in healthy and gingivitis sites. When the data were expressed as concentrations, OC showed significantly positive correlations with GI, but not with PD. However, total amounts of OC significantly correlated with both clinical parameters. OC, PGE₂ and ALP were found to have significantly positive correlations with each other, both when expressed as total amounts and concentrations. These data suggest that a significant amount of OC present in GCF is produced locally by periodontal tissues. Total OC amounts in GCF, as well as PGE₂, and ALP levels, can be considered as potential markers of periodontitis. However, in order to better define the capacity of such mediators for diagnosis, additional longitudinal studies are needed.     

Interleukin-1α produced in human gingival fibroblasts induces several activities related to the progression of periodontitis by direct contact

Previous observations suggest that interleukin-1 (IL-1) may play an important role in the progression of periodontitis. In the present study, we investigated whether a cell-associated IL-1α (CAIL-lα) produced in human gingival fibroblasts (HGF) induces biological activities related to the progression of periodontitis. HGF were treated with recombinant human IL-1β (rhIL-1β) for 12 h. After that, the cell layers of HGF were washed 3 times with fresh medium and were then fixed with 1% paraformaldehyde. The fixed cell layers of HGF were used for assays for bone resorbing activity, prostaglandin E₂ (PGE₂) production and collagenase activity. Fixed cell layers of HGF treated with rhIL-1β enhanced not only calcium release from BALB/c mouse calvaria but also PGE₂ production and collagenase activity in HGF and human periodontal ligament fibroblasts (HPLF) cultured on the fixed cell layers. These activities were neutralized by treatment with monoclonal mouse anti-human IL-1α antibody, but monoclonal mouse antihuman IL-1β antibody showed no effects on these activities. The induction of these activities by fixed cell layers of HGF required direct contact between the fixed cell layers and the calvaria, HGF, or HPLF. These results suggest that CAIL-1α produced in HGF treated with rhIL-1β induces bone resorbing activity, PGE₂ production and collagenase activity in the target cells by direct contact; CAIL-lα may play an important role in the progression of periodontitis.     

Prostaglandin E2 levels in blood plasma and in crevicular fluid of advanced periodontitis patients before and after surgical therapy

AIM: The aim of the study was to determine PGE2 levels in venous blood plasma (VBP), gingival blood plasma (GBP) and gingival crevicular fluid (GCF) in advanced periodontal patients before and after 6 months surgical treatment.PATIENTS AND METHODS: There were 12 patients aged 28-45 years and seven healthy subjects as controls included into the study. Clinical parameters such as PLI, GI, BI, PD, AL and bone height % from radiographs were recorded before and after treatment. PGE2 levels were determined by radioimmunoassay (125 I RIA Kit, NEN(R)).RESULTS: Before treatment the mean VBP, GBP and GCF PGE2 levels in periodontal patients were higher than healthy controls (P < 0.001, Student's t-test). Six months after treatment the mean levels of PGE2 in VBP, GBP and GCF were significantly reduced in patients with improvement of clinical and roentgenological parameters (P < 0.001, Student's t-test). CONCLUSIONS: It can be concluded that PGE2 is involved in the pathogenesis of periodontal disease. The inflamed periodontal tissues may produce significant amount of PGE2 and the degree of inflammation might be determined by the ratios of PGE2. The lower levels of PGE2 in blood plasma and in gingival crevicular fluid after treatment are signs of improvement of periodontal disease.     

Interleukin-1β+3953 allele 2: association with disease status in adult periodontitis

Abstract. Adult periodontitis is a complex multifactorial disease whose etiology is not well defined. The pro-inflammatory and bone resorptive properties of interleukin-1 beta (IL-1β) strongly suggest a role for this cytokine in the pathogenesis of periodontal disease. In the study reported here, the frequency of IL-1β⁺³⁹⁵³ genotypes including allele 2 of the IL-1β⁺³⁹⁵³ restriction fragment length bi-allelic polymorphism was significantly increased in patients with advanced adult periodontitis compared to those with early and moderate disease. Furthermore, allele 2 was associated with increased production of IL-1β by activated peripheral blood polymorphonuclear cells of patients with advanced disease, although this increase failed to reach statistical significance. Finally, the data obtained revealed significant linkage disequilibrium between allele 2 of the IL-1β⁺³⁹⁵³ polymorphism and allele 2 of the bi-allelic IL-1α⁻⁸⁸⁹ polymorphism in both patients and orally healthy controls. These findings provide new insight into the possible role of IL-1α and β gene polymorphisms in the susceptibility to adult periodontitis.