Gustaf Brunius

Phenytoin potentiates interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts

1 The effect of phenytoin (PHT) on prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts stimulated by interleukin‐1 (IL‐1α, IL‐1β) or by tumour necrosis factor α (TNFα) was studied. 2 IL‐1α (1.5–6.0 ng ml−1) and IL‐1β (30–300 pg ml−1), dose‐dependently, stimulated PGE2 formation, in 24 h cultures, with IL‐β being the most potent agonist. 3 PHT (2.5–20 μg ml−1) did not induce PGE2 formation itself but potentiated IL‐1α‐ and IL‐1β‐induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentrations of both IL‐1 and PHT. 4 IL‐1β (0.1–1.0 ng ml−1) induced release of [3H]‐arachidonic acid ([3H]‐AA) from prelabelled fibroblasts that was potentiated by PHT (20 μg ml−1). 5 TNF‐α (≥0.01 μg ml−1) significantly stimulated the biosynthesis of PGE2 by a process that was potentiated by PHT. 6 Addition of exogenous arachidonic acid (AA) (≥ 1 μm) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by PHT (20 μg ml−1). 7 The results indicate that treatment with PHT results in upregulation of prostaglandin biosynthesis in gingival fibroblasts challenged with IL‐1 or TNFα, at least partly due to enhanced level of phospholipase A2 activity.

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts

Recombinant human interleukin-1β (IL-1β) and bradykinin (BK) synergistically stimulate prostaglandin E2 (PGE2) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1β per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1β, caused a rapid, transient rise of intracellular Ca2+ concentration ([Ca2+]i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1β did not change the effect of BK on [Ca2+]i. Ionomycin and A 23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1β on PGE2 formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1β on PGE2 formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE2. In IL-1β treated cells, the enhancement of PGE2 formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1β. These data show that i) the synergistic interaction between IL-1β and BK on PGE2 formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca2+]i; ii) the signal transducing mechanism by which BK interacts with IL-1β, however, may be linked to a BK induced stimulation of [Ca2+]i and/or protein kinase C; iii) the mechanism involved in the action of IL-1β may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1β and tumor necrosis factor α

Abstract The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1β and TNFα on the production of IL-8. The stimulatory effect of BK on the IL-1β- or TNFα-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1β and TNFα on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1β and TNFα increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1β as well as the IL-1β-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1β- and TNFα-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.

The phenytoin metabolite p-HPPH upregulates prostaglandin biosynthesis in human gingival fibroblasts challenged to interleukin-1

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.