Fritz Märki

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.

Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2

Effects of the lanthionine-containing peptide antibiotics duramycin, duramycin B, duramycin C and cinnamycin on the activity of phospholipase A2 from six different sources were studied, and their mode of action was investigated. The four antibiotics inhibited potently all tested phospholipases A2, with IC50 values of around 1 microM, using phosphatidylethanolamine or [1-14C]oleate-labelled Escherichia coli, whose phospholipids are rich in phosphatidylethanolamine, as substrates. No inhibition was observed when the substrate was phosphatidylcholine. Binding of the antibiotics to the lipid fraction of E. coli could be demonstrated by co-sedimentation with whole, but not with lipid-depleted E. coli. In addition, preincubation of duramycin B with vesicles of phosphatidylethanolamine, but not those of phosphatidylcholine, prevented the inhibition of phospholipase A2 activity. The interaction of duramycin B and C, but not that of the biologically inactive compounds actagardine and the duramycin B trisulphoxide, with phosphatidylethanolamine was demonstrated using circular dichroism studies. On the other hand, no interaction of duramycin B with phosphatidylcholine could be demonstrated. A strict correlation between the physico-chemical interaction of the studied lantibiotics, demonstrated by circular dichroism spectroscopy, and their inhibition of phospholipase A2 was observed. These results suggest that lanthionine-containing peptide antibiotics inhibit phospholipase A2 indirectly by specifically sequestering the substrate phosphatidylethanolamine. This mode of action is analogous to the one described for the protein lipocortin.

Interleukin-1β, tumor necrosis factor and forskolin stimulate the synthesis and secretion of group II phospholipase A2 in rat mesangial cells

Abstract Treatment of rat glomerular mesangial cells with interleukin-1β, tumor necrosis factor or forskolin resulted in the release of phospholipase A2 activity in the culture medium. Essentially all of this phospholipase A2 activity was bound to immobilized monoclonal antibodies raised against rat liver mitochondrial 14 kDa group II phospholipase A2. Gelfiltration confirmed the absence of higher molecular weight phospholipases A2 in the culture medium. Immunoblot experiments showed the virtual absence of this 14 kDa group II phospholipase A2 in unstimulated mesangial cells. The time-dependent increase of phospholipase A2 activity in both cells and culture medium upon stimulation with interleukin-1β plus forskolin is accompanied with elevated 14 kDa phospholipase A2 protein levels. These results indicate that the increased phospholipase A2 activity upon treatment of mesangial cells with these stimulators is due to increased synthesis of group II phospholipase A2. Over 85 % of this newly synthesized phospholipase A2 appears to be secreted from the cells.

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.

Studies on the acyl-chain selectivity of cellular phospholipases A2

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A2 are discussed.

Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet.

Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved.

Cytokine-and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.

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.

Transforming growth factors type-β and dexamethasone attenuate group II phospholipase A2 gene expression by interleukin-1 and forskolin in rat mesangial cells

Treatment of rat mesangial cells with interleukin‐1β(IL‐1β) and forskolin induced, in a synergistic fashion,the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2. In contrast, interleukin‐6 did not increase PLA2 mRNA levels of PLA2 activity. Transforming growth factor (TGF) β1, TGFβ2 and TGFβ3 equipotently attenuated the IL‐1β‐ and forskolin‐induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. The glucocorticoid dexamethasone only partially suppressed the IL‐1β‐ and forskolin‐induced elevation of PLA2 mRNA, but totally inhibited PLA2 synthesis and secretion.

‘Antiflammins’: Two nonapeptide fragments of uteroglobin and lipocortin I have no phospholipase A2 ‐inhibitory and anti‐inflammatory activity

The ‘antiflammin’ nonapeptides P1 and P2 [(1988) Nature 335, 726‐730] were synthesized and tested for inhibition of phospholipase A2 and release of prostaglandin E2, and leukotriene C4 in stimulated cells in vitro, and in vivo for anti‐inflammatory activity in rats with carrageenan‐induced paw oedema. Porcine pancreatic phospholipase A2, was not inhibited at concentrations of 0.5–50 μM. Prostaglandin E2, and leukotriene C4 release by mouse macrophages stimulated with zymosan or ATP was not affected up to a concentration of 10 μm, nor was prostaglandin release by interleukin 1β‐stimulated mesangial cells and angiotensin II‐stimulated smooth muscle cells. Both peptides exhibited no anti‐inflammatory activity in carrageenan‐induced rat paw oedema after topical (250 μg/paw) or systemic administration (1 or 4 s.c.). These results do not support the claim of potent phospholipase A2‐inhibitory and anti‐imflammatory activity of the ‘antiflammins’ P1 and P2 [1].