Werner Pignat

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.

PDGF suppresses the activation of group II phospholipase A2 gene expression by interleukin I and forskolin in mesangial cells

Treatment of rat mesangial cells with interleukin 1β (IL‐1β) and forskolin greatly enhanced the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2 as detected by PLA2 activity measurements and immunoprecipitation of culture media of [35S]methionine‐labelled mesangial cells. PDGF—BB dose‐dependently suppressed the IL‐1β‐ and forskolin‐induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. In contrast, PDGF—AA had no inhibitory effect. The tyrosine kinase inhibitor genistein dose‐dependently antagonized the inhibitory effect of PDGF‐BB on IL‐1β‐stimulated PLA2 secretion, thus suggesting that tyrosine phosphorylation may be required for PDGF‐BB inhibition of PLA2 gene expression in mesangial cells.

Pyrrolidine dithiocarbamate differentially affects cytokine- and cAMP-induced expression of group II phospholipase A2 in rat renal mesangial cells

Renal mesangial cells express group II phospholipase A2 in response to two principal classes activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) and agents that elevate cellular levels of cAMP such as forskolin, an activator of adenylate cyclase. Using pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor NFκB, we determined its role in cytokine — and cAMP — triggered group II PLA2 expression. Micromolar amounts of PDTC suppress the IL‐1β‐ and TNF α‐dependent, but not the forskolin‐stimulated group II PLA2 activity in mesangial cells. Furthermore, PDTC inhibited the increase of group II PLA2 mRNA steady state levels in response to IL‐1β and TNFα, while only marginally affecting forskolin‐induced PLA2 mRNA level. Our data suggest that NFκB activation is an essential component of the cytokine signalling pathway responsible for group II PLA2 gene regulation and that cAMP triggers a separate signalling cascade not involving NFκB. These observations may provide a basis to study the underlying mechanisms involved in the regulation of group II PLA2 gene expression.

Suppression by cyclosporin A of interleukin 1β-induced expression of group II phospholipase A2 in rat renal mesangial cells

We investigated whether cyclosporin A, a potent immunosuppressive drug, affects group II phospholipase A2 (PLA2; EC 3.1.1.4) induction in rat renal mesangial cells. Previously we showed that the expression of group II PLA2 in rat renal mesangial cells is triggered by exposure of the cells to inflammatory cytokines such as interleukin 1β (IL‐1β) or tumour necrosis factor α and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL‐1β for 24 h induced PLA2 activity secreted into cell culture supernatants by about 16 fold. Incubation of mesangial cells with cyclosporin A inhibited IL‐1β‐induced PLA2 section in a dose‐dependent fashion, with an IC50 value of 4.3 μM. Cyclosporin A did not directly inhibit enzymatic activity of PLA2. Immunoprecipitation of radioactively labelled PLA2 protein from mesangial cell supernatants revealed that the inhibition of PLA2 activity is due to a suppression of PLA2 protein levels. This effect was preceded by a reduction of PLA2 mRNA steady state levels, as demonstrated by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. In order to evaluate whether cyclosporin A would affect the transcriptional activity of the PLA2 gene, we performed nuclear run on transcription experiments and provided evidence that the transcription rate of the PLA2 gene is reduced by cyclosporin A. Previously we found that the nuclear transcription factor κB (NFκB) is an essential component of the IL‐1β‐dependent upregulation of PLA2 gene transcription. By electrophoretic mobility shift analysis, we demonstrated that cyclosporin A diminishes the formation of NFκB DNA‐binding complexes, thus suggesting that this transcription factor is a target for cyclosporin A‐mediated repression of PLA2 gene transcription. The data presented in this study strongly suggest that the cellular mechanism involved in the IL1β ‐ dependent transcriptional upregulation of the PLA2 gene in mesangial cells is a target for the action of cyclosporin A.

Cytokine regulation of group II phospholipase A2 expression in glomerular mesangial cells.

SummaryPhospholipase A2 (PLA2) is believed to play an essential role in inflammatory processes by releasing arachidonic acid from membrane phospholipids for synthesis of important lipid mediators, such as prostaglandins, leukotrienes and platelet activating factor.We have used glomerular mesangial cells as a model system to study the regulation of PLA2 under inflammatory conditions. Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) and tumour necrosis factor α (TNFα), as well as agents that increase cellular cAMP levels have been found to increase Group II PLA2 gene expression in a time- and dose-dependent manner.In all cases cytokine-induced synthesis of PLA2 occurred in parallel with cytokine-stimulated prostaglandin (PG) E2 synthesis. Three important classes of compounds that potently antagonise the stimulatory effect of IL-1, TNFα and cAMP on Group II PLA2 expression in mesangial cells have been identified, namely, glucocorticoids, transforming growth factors (TGF) type-β and platelet-derived growth factor (PDGF). Those agents may act sequentially to protect the kidney from damage resulting from cytokine-stimulated mediator release and the subsequent inflammatory reactions.

Time-dependent cytokine production in the croton oil-induced mouse ear oedema and inhibition by prednisolone

The croton oil-induced mouse ear oedema is a well established model for investigating anti-inflammatory effects of nonsteroidal and steroidal compounds [1]. Corticosteroids are highly effective inhibitors of this irritant-induced local inflammation [2]. They are still the most potent inhibitors of cytokine biosynthesis and are widely used as reference compounds for discovering and profiling novel inhibitors. This study was undertaken to investigate whether measurable amounts of IL-113 are produced during croton oil-induced skin inflammation and whether prednisolone is able to inhibit IL-1/3 production in this model. The kinetics of IL-I~ production, leukocyte infiltration (myeloperoxidase activity, MPO) and oedema development were compared.

Tetranactin inhibits interleukin 1β and cAMP induction of group II phospholipase A2 in rat renal mesangial cells

Renal mesangial cells express secretory phospholipase A2 in response to two principal classes of activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines, such as interleukin 1 beta, and agents that elevate cellular levels of cAMP. Treatment of mesangial cells with tetranactin, a cyclic antibiotic produced by Streptomyces aureus with a molecular structure similar to cyclosporin A inhibits interleukin 1 beta- and cAMP-dependent group II phospholipase A2 secretion in a dose-dependent manner with IC50 values of 43 and 33 nM, respectively. However, tetranactin does not directly inhibit group II phospholipase A2 activity. Western blot analyses of mesangial cell supernatants reveal that the inhibition of phospholipase A2 activity is due to suppression of phospholipase A2 protein synthesis. This effect is preceded by the reduction of phospholipase A2 mRNA steady-state levels as shown by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. Thus, tetranactin is a potent inhibitor of group II phospholipase A2 expression in cytokine- and cAMP-stimulated mesangial cells and represents a new class of group II phospholipase A2 inhibitors with IC50 values in the low nanomolar range. This compound may be useful in the therapy of diseases associated with increased group II phospholipase A2 secretion.

Platelet-derived growth factor and fibroblast growth factor differentially regulate interleukin 1β- and cAMP-induced group II phospholipase A2 expression in rat renal mesangial cells

Expression of group II phospholipase A2 (PLA2; EC 3.1.1.4) in rat renal mesangial cells is triggered in response to two principal classes of activating signals. These two groups of activators comprise inflammatory cytokines such as interleukin 1beta (IL-1beta) or tumor necrosis factor alpha and agents that elevate cellular levels of cyclic AMP (cAMP) such as forskolin, an activator of adenylate cyclase. Treatment of mesangial cells with IL-1beta or forskolin for 24 h induces group II PLA2 activity secreted into cell culture supernatants by about 15-fold and 11-fold, respectively. Platelet-derived growth factor (PDGF)-BB potently inhibits secretion of IL-1beta- and forskolin-induced group II PLA2 activity. By Western and Northern blot analyses, we demonstrate that this is due to a reduction of PLA2 protein levels and the corresponding PLA2 mRNA steady-state levels. Basic fibroblast growth factor (bFGF) virtually does not inhibit IL-1beta-stimulated group II PLA2 activity, but markedly inhibits forskolin-induced expression of group II PLA2 activity. These effects are caused by changes in the corresponding PLA2 protein and PLA2 mRNA steady-state levels. Inhibition of protein kinase C (PKC) by the potent and selective PKC inhibitor calphostin C converted the inhibitory action of PDGF into a bFGF-type of response thus suggesting that PKC is a major effector in PDGF-induced inhibition of IL-1beta-stimulated group II sPLA2 expression. In summary, our data suggest that PDGF and bFGF differentially modulate in a stimulus-specific manner the expression of group II PLA2 in mesangial cells.

The influence of anti-rheumatic drugs on hepatic mRNA levels of acute-phase proteins in rats with adjuvant arthritis.

Using specific cDNA probes, we have investigated drug-induced changes in hepatic mRNA levels of the major acute-phase proteins (APP) fibrinogen, α2-macroglobulin (α2-MG), albumin and α1-acid glycoprotein (α1-AGP) in male Lewis rats with adjuvant arthritis. Test compounds were given orally from day 0 to 20 and hepatic mRNA analysis was performed at day 21. Prednisolone (1,3, 10 mg/kg), Cyclosporine A (1, 3, 10 mg/kg) and cyclophosphamide (3 mg/kg) dose-dependently normalized hepatic mRNA levels of all four APP. Equipotent anti-inflammatory doses of indomethacin (0.3, 1 mg/kg) significantly downregulated α2-MG mRNA levels but were much less effective in influencing albumin and α1-AGP mRNA levels and even slightly increased hepatic fibrinogen mRNA levels. These results suggest that cytokine overproduction, which is thought to be responsible for the acute-phase response in rats with adjuvant arthritis, can be effectively downregulated by immunosuppressive drugs, but is distinctly less affected by the cyclooxygenase inhibitor indomethacin.