Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.

Changes induced by PAF-acether in diacyl and ether phospholipids from guinea-pig alveolar macrophages.

The release and the mobilization of arachidonic acid from guinea-pig alveolar macrophages labeled with [1-14C]arachidonic acid for short (1 h) and long (18 h) periods and stimulated with PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied. After short labeling periods arachidonic acid was primarily incorporated into alkylacyl- and diacylglycerophosphocholine (alkylacylGPC, diacylGPC) and glycerophosphoinositol (GPI), whereas after long labeling periods arachidonic acid was mainly incorporated into alkenylacylglycerophosphoethanolamine (alkenylacylGPE). In macrophages labeled for 1 h, PAF-acether (1 microM) induced a significant decrease in the amount of arachidonic acid esterified into diacyl- and alkylacylGPC and GPI, as well as a significant increase of arachidonate transferred into alkenylacylGPE. No significant decrease in arachidonate esterified in GPC fractions and in GPI was induced by PAF-acether in macrophages labeled for 18 h, whereas the increased transfer of the fatty acid into alkenylacylGPE was still measurable. This study shows that PAF-acether induces the release and the mobilization of newly incorporated arachidonic acid in alveolar macrophages. When cells are labeled for long periods and the majority of arachidonic acid is retained in ether-linked phospholipids, no PAF-acether-induced release of arachidonate was obtained, whereas its transfer was maintained.

Increase of bradykinin-stimulated arachidonic acid release in a delta F508 cystic fibrosis epithelial cell line.

Modification of chloride conductance by bradykinin in epithelial cells has been attributed to an activation of protein kinase A resulting from adenylcyclase stimulation by arachidonic acid cyclooxygenase products. The results presented here compare tracheal epithelial cell lines from one control and two cystic fibrosis patients which were immortalized by transfection with the SV40 large T oncogene. The three cell lines presented the same arachidonic acid content, turnover and mobilisation under basal conditions. Bradykinin stimulated the release of arachidonic acid and the synthesis of cyclooxygenase derivatives (mainly PGE2). The cell line from the cystic fibrosis patient bearing a phenylalanine 508 deletion, which is the major form of the disease, showed a higher bradykinin-induced arachidonic acid release than either control cells or cells from a patient presenting a minor form of the disease. This higher sensitivity suggests a dysregulation of phospholipase A2 stimulation in cystic fibrosis cells and was confirmed on non-immortalized tracheal epithelial cells in primary culture and on skin fibroblasts from patients bearing the same mutation. This defect is associated with a potentiation of cholera toxin pretreatment on cAMP content of delta F508 cell line. The impaired control of arachidonic acid release cannot be attributed to an increased number of bradykinin binding sites, since this increase was similar in the two cystic fibrosis cell lines.

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes.

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.

Insulin-like growth factors counteract the effect of interleukin 1 beta on type II phospholipase A2 expression and arachidonic acid release by rabbit articular chondrocytes.

Interleukin 1 beta was found to stimulate arachidonic acid release, and the synthesis and secretion of type II phospholipase A2 by rabbit articular chondrocytes in vitro. Interleukin 1 beta had no effect on the level of cytosolic phospholipase A2 mRNA. Insulin-like growth factors, which help stabilize the cartilage matrix, reduced the effect of interleukin 1 beta on type II phospholipase A2 activity and mRNA level, and decreased the Interleukin 1 beta-stimulated arachidonic acid release to the basal values. This suggests that type II phospholipase A2 plays a key role in arachidonic acid release from rabbit articular chondrocytes and that insulin-like growth factors counteract the effect of interleukin 1 beta. They may therefore be considered as potential antiinflammatory agents.

Antigenic relatedness between phospholipases A2 from Naja naja venom and from mammalian cells.

Polyclonal antiserum was prepared against phospholipase A2 from Naja naja and used to prepare a purified antibody. It cross-reacted with the antigen, and with intracellular mammalian PLA2. This antibody was immunoreactive and inhibited the PLA2 activity of Naja naja and of guinea pig alveolar macrophages or rat lymphocytes. By immunoblotting, this antiserum revealed one band of PLA2 from Naja naja (14 kDa) and 3 bands for guinea pig alveolar macrophages and rat lymphocytes (30, 45 kDa and a minor band of 14 kDa). These results show an antigenic relatedness between an extracellular PLA2 and membrane-bound PLA2 from two different mammalian species and cell types.

Mobilization of arachidonic acid from diacyl and ether-linked phospholipids in FMLP stimulated alveolar macrophages.

Exposure of [1 14C]AA labeled guinea-pig alveolar macrophages to FMLP for 15 min induced an extensive mobilization of AA from phospholipids. PC and PI mainly contributed to the AA release, and labeled PE remained unchanged. Analysis of ether-linked phospholipids showed a significant breakdown of labeled diacyl and alkyl-acyl PC and an increase in labeled alkenyl-acyl PE.

Phospholipase and prophospholipase activities in bronchoalveolar lavage fluid in severe acute pulmonary disease with or without ARDS.

Bronchoalveolar lavages were performed in 21 patients undergoing mechanical ventilation: Group I: coma due to sedative overdose (11 cases), without pulmonary impairment, serving as control group; Group II: severe acute pulmonary disease without ARDS (5 cases); Group III: patients with ARDS (5 cases). In the recovered fluid we measured: total proteins (P) and phospholipids (PL), phospholipasic (PLase) and prophospholipasic (PPLase) activities. In ARDS group, considerable increase of P, (p less than 0.001), and a doubling of PL (p less than 0.02) was found. Total PLase activity was present in all three groups, with a higher mean level in Group III (p less than 0.01). PPLase activities were low or undetectable in four patients of this group. PLase/PL ratio was increased in acute respiratory insufficiency, with or without ARDS, suggesting an increase of surfactant catabolism. The decrease of this ratio when pulmonary function improved, or its increase after deterioration suggest that it is related to changes of surfactant.

Interaction between PAF-acether and drugs that stimulate cyclic AMP in guinea-pig alveolar macrophages.

The PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine)-induced arachidonate release from alveolar macrophages was significantly reduced by prostaglandin E2 (PGE2) and by the beta-adrenoceptor agonist salbutamol. In addition, PAF-acether markedly reduced the increase in intracellular cyclic AMP (cAMP) concentrations induced by PGE2 and salbutamol. Our data indicate an inverse relationship between intracellular cAMP levels and free arachidonate availability in alveolar macrophages treated with PAF-acether. A rise in intracellular cAMP therefore represents an important alternative route for controlling the effects of PAF-acether and the resulting inflammatory alterations in the respiratory system.

Decreased phosphatidyl choline content in bronchoalveolar lavage fluids of children with bronchopulmonary dysplasia: a preliminary investigation.

Bronchoalveolar lavage (BAL) was performed on 12 infants who had recovered from neonatal acute respiratory failure and on 12 patients with bronchopulmonary dysplasia (BPD) in order to evaluate the concentration of phosphatidyl choline (PC) in BAL fluid. These two groups were similar at birth (mean birth weight: 1,980 and 1,750 g, respectively; mean gestational age: 33.4 and 32.1 weeks respectively). Mechanical ventilation based on oxygen requirement lasted longer in the group with BPD. BAL was performed at the end of the first year of life (at 8.5 and 10.3 months, respectively) and the results were compared to control values (from infants of the same age without neonatal disease). Whereas the protein concentration in BAL fluid was similar in the two groups, a dramatic decrease of the BAL PC was found in BPD: The mean values of BAL-PC over protein ratio were 0.9 in the group without pulmonary sequelae and 0.3 in the group with BPD. These preliminary results suggest an impairment of the pulmonary surfactant metabolism in this chronic lung disease following neonatal acute respiratory failure.

Effects of oxygen-derived free radicals on type II pneumocytes in primary culture.

After a brief review of oxygen-derived free radicals formation, cellular antioxidant equipment, and in vivo oxidant lung injury, the model of type II pneumocytes in primary culture is described with its characteristics and limitations. This model has been used to study either the in vivo or the in vitro effects of free radicals: the resistance of type II cells isolated from animals exposed to oxidants may be explained by their higher level of anti-oxidant enzymes. In contrast, type II cells isolated from control animals do not develop a resistance to the toxic effects on an in vitro exposure to free radicals. This discrepancy might be the result of a possible interaction of alveolar macrophages or fibroblasts with type II cells, which remains to be explored.

[Coupling of seven transmembrane domains receptors cytosolic PLA2: role of G proteins and protein kinases]. / Couplage des récepteurs à sept segments transmembranaires/PLA2 cytosolique: rôle des protéines G et des proteines kinases.

Cytosolic phospholipase A2 is a constitutive and ubiquitous enzyme. Although its role in the early production of lipid mediators is well established, the mechanisms leading to its activation and its place in the signal transduction pathways triggered by G-protein-coupled receptors is still unclear. Two main mechanisms, have been involved in its activation: its translocation by the increase of intracellular calcium allowing access to its phospholipidic substrate, and its phosphorylation by serine/threonine protein kinases such as protein kinase C or MAP kinases. However these two mechanisms do not fully explain the activation of cytosolic phospholipase A2. The irreversible association to membranes observed after receptor stimulation suggests that a still unknown anchoring mechanism might be involved. The elucidation of this anchoring might take place in the overall synthesis of distinct lipid mediators pools able to play an intracellular role of second messengers or an extracellular role of autocrine/paracrine mediators.

Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages. Differential receptor/G-protein interaction assessed by pertussis and cholera toxins.

The involvement of guanine-nucleotide-binding regulatory proteins (G proteins) in the regulation of arachidonic-acid release induced by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or platelet-activating factor (PAF) was examined in guinea-pig alveolar macrophages. We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet-Leu-Phe or PAF) and of GTP (or GTP[S]). Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic-acid release and of the labeling of a 40-kDa protein in membranes incubated with [32P]NAD and pertussis toxin. fMet-Leu-Phe, but not PAF, allows the ADP-ribosylation of a 40-KDa protein by cholera toxin in the presence of Mg2+. This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin. The 40-kDa protein ADP-ribosylated seems to be alpha i1 and/or alpha i2. Stimulation of GTPase activity by fMet-Leu-Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin. Prior treatment of alveolar macrophages with cholera toxin, which ADP-ribosylates Gs, inhibits PAF-stimulated and fMet-Leu-Phe-stimulated arachidonic-acid release to the same extent, via a cAMP-protein-kinase-A cascade. The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet-Leu-Phe and PAF is associated with a strong increase of in-vitro [32P]NAD labeling of Gi proteins either by pertussis or by cholera toxin. This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein-phosphatase inhibitor, suggesting the involvement of protein-kinase A in this process. In conclusion, our results demonstrate that fMet-Leu-Phe and PAF receptors interact differently with Gi1/2 proteins in guinea-pig alveolar macrophages. Gi1/2 proteins are a possible target of the cross-regulation of arachidonic-acid release by a Gs-mediated pathway.

The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A.

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5'-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

G proteins as biological targets for anti-allergic drugs?

The inhibiting effect of the H1 antihistamine cetirizine on the release of mediators (LTB4, arachidonic acid and phospholipase A2) was measured in different cells in vitro (human PMN, deltaF508 cells, chinese hamster ovary cells and rabbit chondrocytes) using different agonists (fMLP, NaF, calcium ionophore A 23187, bradykinin, adrenaline and IL-1). It was shown that physiological concentrations of the drug inhibited the release when activation of receptor-coupled G proteins was involved. By contrast, there was no inhibiting effect of cetirizine when the release was induced by a calcium ionophore which bypasses the G proteins coupled to cell membrane receptors.

[Cellular phospholipases A2, structure-function relationship]. / Les phospholipases A2 cellulaires, relations structures-fonction.

Two PLA2 are involved in cell signaling and in phospholipid homeostasis in mammalian cells. The first one is a 14 kDa protein whose cDNA was cloned in 1989. This enzyme exhibits a strong homology with pancreatic PLA2 but is more related to type II PLA2. This PLA2 is secreted by different tissues in response to inflammatory processes. Their main function seems to be the hydrolysis of membranes of altered cells or of bacteria and the stimulation of lipid mediator synthesis. The first cDNA of an another important PLA2 group was cloned in 1991. The protein deduced is a 88 kDa cytoplasmic protein. It is involved in cell signaling by stimulating the production of free fatty acids and of their oxygenated products. These products might in turn either activate transducing proteins or stimulate membrane receptors.

Synergistic effect of interleukin-1 beta and tumor necrosis factor alpha on PGE2 production by articular chondrocytes does not involve PLA2 stimulation.

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL1), cause increased production of prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE2 production, and the activities of cytosolic phospholipase A2 (cPLA2), secreted phospholipase A2 (sPLA2), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA2, sPLA2, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE2 production. This synergy did not involve PLA2 stimulation, as there were no increases in AA release, cPLA2 and sPLA2 activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE2 production in articular chondrocytes. As sPLA2 and cPLA2 do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.

Mechanism of N-formyl-methionyl-leucyl-phenylalanine- and platelet-activating factor-induced arachidonic acid release in guinea pig alveolar macrophages: involvement of a GTP-binding protein and role of protein kinase A and protein kinase C.

Various pharmacological effectors were used to investigate the mechanism of arachidonic acid release by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) in guinea pig alveolar macrophages. The fMLP- and PAF-stimulated arachidonic acid release (i) was mimicked by sodium fluoride and inhibited by Bordetella pertussis toxin, suggesting the participation of a guanine nucleotide-binding protein; ii) was mimicked by A23187 but was insensitive to the calmodulin inhibitor R24571, making the involvement of a calmodulin-dependent pathway unlikely; and (iii) was mimicked by 12-O-tetra-decanoyl phorbol 13 acetate (TPA) and was, like the TPA-stimulated release, markedly decreased when protein kinase C (PKC) had been down-regulated by TPA (65% decrease) or inhibited by sphingosine, a diacylglycerol-competitive PKC inhibitor shown to completely abolish the enzyme activity from alveolar macrophages at 40 microM. Moreover, PAF and fMLP, under conditions where they stimulated arachidonic acid release, promoted an appreciable, albeit transient, translocation of PKC, suggesting a possible involvement of the enzyme in the agonist-stimulated process. However, staurosporine, another PKC inhibitor decreasing PKC activity from alveolar macrophages by 60% at 20 nM, failed to alter fMLP- and PAF-stimulated release. These data lead us to suggest that fMLP- and PAF-stimulated arachidonic acid release is mediated by mechanisms involving either a staurosporine-insensitive PKC isoform or a sphingosine-sensitive coupling between a pertussis toxin-sensitive guanine nucleotide-binding protein and phospholipase A2. Finally, the fMLP- and PAF-stimulated arachidonic acid release was inhibited by cholera toxin and was, like A23187-stimulated release, potentiated by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8), an exclusive protein kinase A inhibitor in alveolar macrophages, suggesting a negative regulation by protein kinase A.