ABSTRACT
Prostaglandins, especially prostaglandin E2 (PGE2), play a crucial role in the pathogenesis of periodontal disease. We have previously reported that inflammatory mediators interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) increase the production of PGE2 in human gingival fibroblasts. In this study, we investigated the effect of cell-to-cell interactions between gingival fibroblasts and lymphocytes on PGE2 production by using co-culture technique. Cell-to-cell contact between gingival fibroblasts and lymphocytes synergistically enhanced the production of PGE2 in co-cultures. In contrast to lymphocytes, the cyclooxygenase-2 (COX-2) mRNA expression in gingival fibroblasts was strongly enhanced following cell contact between gingival fibroblasts and lymphocytes. The level of COX-1 mRNA expression, however, was not affected either in gingival fibroblasts or in lymphocytes by the interactions between fibroblasts and lymphocytes. The study demonstrates that cell contact between gingival fibroblasts and lymphocytes strongly stimulates PGE2 production partly due to enhanced COX-2 mRNA expression in gingival fibroblasts. The cell-to-cell contact between gingival fibroblasts and lymphocytes should be considered as an important regulatory aspect for the enhancement of PGE2 in periodontal disease.
Subject(s)
Fibroblasts/metabolism , Gingiva/metabolism , Isoenzymes/genetics , Lymphocytes/physiology , Peroxidases/genetics , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , Cell Communication , Cell Count , Cell Culture Techniques , Coculture Techniques , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/antagonists & inhibitors , Dinoprostone/biosynthesis , Fibroblasts/physiology , Gene Expression Regulation, Enzymologic , Gingiva/cytology , Humans , Isoenzymes/antagonists & inhibitors , Membrane Proteins , Nitrobenzenes/pharmacology , Periodontal Diseases/enzymology , Periodontal Diseases/metabolism , Peroxidases/antagonists & inhibitors , Statistics as Topic , Sulfonamides/pharmacology , Up-RegulationABSTRACT
The in vitro effect of phenytoin (PHT) on the production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in human gingival fibroblasts, challenged with or without interleukin-1beta (IL-1beta), was studied. PHT (20 microg/ml) alone increased the mRNA level for both IL-6 and IL-8, as well as synergistically enhancing the production of IL-6 and IL-8, at both transcriptional and translational level in fibroblasts challenged with IL-1beta (30 pg/ml). The stimulatory effect of PHT on IL-1beta-induced IL-6 production was strongly reduced by the specific cyclooxygenase-2 inhibitor NS-398 (1 microM). The anti-inflammatory drug, dexamethasone (1 microM), abolished the production of both IL-6 and IL-8 in gingival fibroblasts challenged with PHT in the presence or absence of IL-1beta. The ability of PHT, alone as well as in combination with IL-1, to upregulate the production of IL-6 and IL-8 in human gingival fibroblasts may contribute to enhanced recruitment and activation of inflammatory cells. This effect of PHT may thereby give a prerequisite for the establishment of an interaction between cytokines and connective tissue cells in the periodontal tissue, which is suggested to lead to gingival overgrowth.
Subject(s)
Anticonvulsants/pharmacology , Gingiva/drug effects , Interleukin-1/pharmacology , Interleukins/biosynthesis , Phenytoin/pharmacology , Adolescent , Cells, Cultured , Child , Child, Preschool , Drug Synergism , Enzyme-Linked Immunosorbent Assay , Fibroblasts/drug effects , Fibroblasts/metabolism , Gingiva/cytology , Gingiva/metabolism , Humans , In Situ Hybridization , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , RNA, Messenger/analysis , Stimulation, Chemical , Up-RegulationABSTRACT
The production of interleukin 6 (IL-6) was studied in human gingival fibroblasts challenged with bradykinin (BK), in the presence or absence of either tumour necrosis factor alpha (TNF-alpha) or interleukin 1 (IL-1). The inflammatory mediator BK as well as the cytokines TNF-alpha and IL-beta dose dependently stimulated IL-6 production in gingival fibroblasts. When the cells were treated simultaneously with BK and either IL-1 beta or TNF-alpha, the inflammatory mediator BK synergistically upregulated IL-6 production in a dose-dependent manner. The BK B1 receptor agonist des-arg9-BK as well as the BK B2 receptor agonist Lys-BK also induced IL-6 production and synergistically enhanced the effect of IL-1 and TNF-alpha on the production of IL-6. The upregulation of IL-6 production induced by BK was abolished by the anti-inflammatory agent dexamethasone (DEX) and the phospholipase A2 (PLA2) inhibitor 4-bromphenacyl bromide (BPB). Treatment of the cells with the cyclooxygenase (COX) inhibitor flurbiprofen resulted in a minor reduction of the stimulatory effect of BK. The results show that BK together with IL-1 or TNF-alpha act in concert to enhance IL-6 production and that the synergy was obtained by both the BK B1 and the BK B2 receptor agonist. The study indicates that the synergy between BK and IL-1 or TNF-alpha on IL-6 production is mediated partly at the level of PLA2 and partly at the level of COX. The synergism between the pro-inflammatory mediator BK and the cytokines IL-1 or TNF-alpha indicates that gingival fibroblasts, by producing cytokines, affect the local immune response in the connective tissue and thereby play a role in the pathogenesis of periodontal disease.
Subject(s)
Bradykinin/pharmacology , Fibroblasts/drug effects , Gingiva/drug effects , Interleukin-1/pharmacology , Interleukin-6/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Acetophenones/pharmacology , Adolescent , Child , Dexamethasone/pharmacology , Drug Synergism , Fibroblasts/metabolism , Flurbiprofen/pharmacology , Gingiva/cytology , Humans , Receptors, Bradykinin/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Effects of and interactions between interleukin-1 beta (IL-1 beta) and phenytoin (PHT) on alpha 1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E2 (PGE2) formation were studied. IL-1 beta (300 pg/ ml) reduced the steady-state level of alpha 1 (I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 micrograms/ml) reduced the level of alpha 1 (I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1 beta, PHT potentiated the inhibitory effect of IL-1 beta on alpha 1 (I) procollagen mRNA level that was accompanied by an increased PGE2 formation. Preincubation with indomethacin (10(-6) M) partially reduced the inhibitory effect of IL-1 beta as well as of IL-1 beta in combination with PHT on the mRNA level of alpha 1 (I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE2 (> or = 10 nM) dose-dependently reduced steady-state level of alpha 1 (I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of alpha 1 (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 alpha 1 (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.
Subject(s)
Anticonvulsants/pharmacology , Fibroblasts/drug effects , Gingiva/drug effects , Interleukin-1/pharmacology , Phenytoin/pharmacology , Procollagen/antagonists & inhibitors , RNA, Messenger/antagonists & inhibitors , Blotting, Northern , Cells, Cultured , Child , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/administration & dosage , Dinoprostone/biosynthesis , Dinoprostone/genetics , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Gingiva/cytology , Gingiva/metabolism , Humans , Indomethacin/pharmacology , Nucleic Acid Hybridization , Procollagen/analysis , Procollagen/genetics , Prostaglandin-Endoperoxide Synthases/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , RNA, Messenger/analysis , RNA, Messenger/geneticsABSTRACT
The effects of and interactions between the major phenytoin (PHT) metabolite 5-parahydroxyphenyl-5-phenylhydantoin (p-HPPH) and interleukin-1 (IL-1 alpha, IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on prostaglandin biosynthesis in human gingival fibroblasts were studied. IL-1 alpha, IL-1 beta and TNF alpha, dose-dependently, stimulated PGE2 formation in gingival fibroblasts. The metabolite, p-HPPH (1.2-2.4 micrograms/ml), did not induce PGE2 formation itself but potentiated IL-1 alpha and IL1 beta induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentration of both IL-1 and p-HPPH. The metabolite also stimulated IL-1 induced formation of 6-Keto PGF1 alpha, the stable breakdown product of PGI2, in a dose dependent manner. IL-1 beta induces release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts, which was potentiated by p-HPPH (> or = 1.2 micrograms/ml). TNF alpha (> or = 1 ng/ml) significantly stimulated the biosynthesis of PGE2 by a process that was also potentiated by p-HPPH. Addition of exogenous, unlabelled AA (10 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by p-HPPH (1.6 micrograms/ml). The results indicate that treatment with p-HPPH results in upregulation of prostaglandin synthesis in gingival fibroblasts challenged to IL-1 or TNF alpha at the level of phospholipase A2.
Subject(s)
Gingiva/drug effects , Interleukin-1/pharmacology , Phenytoin/analogs & derivatives , Prostaglandins/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Cells, Cultured , Child , Drug Synergism , Female , Fibroblasts/metabolism , Gingiva/cytology , Gingiva/metabolism , Humans , Male , Phenytoin/pharmacology , Up-Regulation/drug effectsABSTRACT
The aim of the study was to determine the effect of bradykinin (BK) on the level of cytoplasmic-free Ca2+, [Ca2+]i, in human gingival fibroblasts and its relation to BK-induced prostanoid formation. BK, but not des-Arg9-BK, induced a significant rapid (within seconds) and transient increase in [Ca2+]i, that was not dependent on extracellular Ca2+. The stimulatory effect of BK was seen in concentrations at or above 10(-8) M, with the most pronounced effect at 10(-6) M. D-Arg0-Hyp3-Thi5,8-DPhe7-BK, a BK B2 receptor antagonist, but not des-Arg9-Leu8-BK, a BK B1 receptor antagonist, blocked BK-induced rise in [Ca2+]i. The BK B2 receptor antagonist also significantly reduced BK-induced PGE2 formation. When extracellular Ca2+ in the incubation medium was depleted, either by addition of EGTA or by omission of Ca2+ addition, BK still caused a significant stimulation of PGE2 formation. The calcium ionophores A23187 and ionomycin, similar to BK, caused a burst of PGE2 formation. The two phorbol esters phorbol 12,13-dibutyrate and 4-beta-phorbol-didecanoate positively amplified calcium ionophore A23187-induced PGE2 formation. The results indicate that BK-induced PGE2 formation in gingival fibroblasts is coupled to an increase in [Ca2+]i mediated by the BK B2 receptor, and which is independent of extracellular Ca2+.
Subject(s)
Bradykinin/pharmacology , Calcium/metabolism , Dinoprostone/biosynthesis , Gingiva/metabolism , Receptors, Neurotransmitter/drug effects , Bradykinin/analogs & derivatives , Calcium/physiology , Cells, Cultured , Child , Cytoplasm/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Gingiva/cytology , Gingiva/drug effects , Humans , Receptors, Bradykinin , Receptors, Neurotransmitter/physiology , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Dinoprostone/biosynthesis , Fibroblasts/metabolism , Gingiva/metabolism , Gingival Hyperplasia/metabolism , Phenytoin/adverse effects , 6-Ketoprostaglandin F1 alpha/biosynthesis , Arachidonic Acids/pharmacokinetics , Arachidonic Acids/pharmacology , Cells, Cultured , Child , Dose-Response Relationship, Drug , Epilepsy/drug therapy , Fibroblasts/drug effects , Gingiva/drug effects , Gingiva/pathology , Gingival Hyperplasia/chemically induced , Gingival Hyperplasia/pathology , Gingivitis/chemically induced , Gingivitis/metabolism , Gingivitis/pathology , Humans , Interleukin-1/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Up-RegulationABSTRACT
Human gingival fibroblasts derived from 2 patients before and 9 months after the start of phenytoin (PHT) therapy were studied with respect to the effect of epidermal growth factor (EGF) on the incorporation of 3H-thymidine into DNA, binding of EGF to its cell-surface receptor, internalization of EGF-receptor-ligand complexes and, finally, with respect to EGF receptor mRNA levels. In fibroblasts derived from the patient who developed gingival overgrowth during the PHT medication (responder) as well as in the fibroblasts derived from the patient where gingival overgrowth did not develop (non-responder), the affinity of the EGF receptor for EGF was not significantly changed. In the non-responder patient the internalization of EGF receptor ligand was decreased, whereas it was increased in the fibroblasts derived from the responder patient after PHT therapy. The steady-state level of EGF-r mRNA increased significantly (p less than 0.001) in the cultured fibroblasts derived from the non-responder but decreased (p less than 0.05) in the responder patient following PHT therapy. Ligand affinity cross-linking studies revealed one major component of EGF receptor with a molecular weight of 170 KDa in fibroblasts from the non-responder as well as from the responder. The study indicates that PHT medication results in a down-regulation of EGF receptor metabolism in fibroblasts derived from a responder patient, whereas in the non-responder patient EGF receptor metabolism is up-regulated.
