Gilbert Bereziat

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.

Essential Fatty Acids Interconversion in the Human Fetal Liver

In order to study the role played by the fetoplacental unit in providing the human fetus with arachidonic acid, delta 5- and delta 6-desaturase activities were studied in microsomes from human fetal liver and placenta after 18 and 22 weeks of gestation. We evidenced for the first time delta 5- and delta 6-desaturase activities in fetal liver microsomes. As in adult liver, delta 6-desaturation is the rate-limiting step of arachidonic acid synthesis. No activity was found in the placenta. Arachidonic acid concentrations were higher in fetal serum than in maternal serum while the opposite was observed for linoleic acid. The fetal liver microsomal content in arachidonic acid was low. Taken together the data suggest that arachidonic acid is supplied to the fetus through a preferential transfer across the placenta.

Parenteral nutrition providing a restricted amount of linoleic acid in severely burned patients: a randomised double-blind study of an olive oil-based lipid emulsion v . medium/long-chain triacylglycerols

It has been claimed that lipid emulsions with a restricted linoleic acid content can improve the safety of total parenteral nutrition (TPN). The tolerability of TPN and its effects on the metabolism of fatty acids were assessed in this prospective, double-blind, randomised study comparing an olive/soyabean oil long-chain triacylglycerol (LCT) with a medium-chain triacylglycerol (MCT)/LCT; 50:50 (w) based lipid emulsion in two groups (O and M, respectively; eleven per group) of severely burned patients. After resuscitation (48-72 h), patients received TPN providing 147 kJ/kg per d (35 kcal/kg per d) with fat (1.3 g/kg per d) for 6 d Plasma fatty acids, laboratory parameters including liver function tests, and plasma cytokines were assessed before and after TPN. Adverse events encountered during TPN and the clinical outcomes of patients within the subsequent 6 months were recorded. With both lipid emulsions, the conversion of linoleic acid in its higher derivatives (di-homo-gamma-linolenic acid) improved and essential fatty acid deficiency did not appear. Abnormalities of liver function tests occurred more frequently in the M (nine) than in the O (three) group (P = 0.04, Suissa-Shuster test). Seven patients (four from group O and three from group M) died as a consequence of severe sepsis 3-37 d after completion of the 6 d TPN period. When compared with the surviving patients, those who died were older (P = 0.01) and hyperglycaemic at baseline (P < 0.001), and their plasma IL-6 levels continued to increase (P < 0.04). Although fatty acid metabolism and TPN tolerability were similar with both lipid emulsions, the preservation of liver function noted with the use of the olive oil-based lipid emulsions deserves confirmation.

Differentiation regulates interleukin-1beta-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase.

Chondrocyte dedifferentiation has been noted in osteoarthritic cartilage, but the contribution of this phenomenon is poorly understood. Interleukin (IL)-1beta, the major pro-inflammatory cytokine found in osteoarthritic synovial fluid, induces the dedifferentiation of cultured articular chondrocytes, whereas E-series prostaglandins (PGE) are capable of inducing cell differentiation. Since PGE(2) synthesis is up-regulated by IL-1beta, we addressed the question of whether the state of chondrocyte differentiation may influence the production of IL-1-induced PGE(2) by modulating cyclooxygenase (COX)-2 expression. Immortalized human articular chondrocytes, (tsT/AC62) cultured in monolayer after passage through alginate matrix (alg+) produced 5-fold greater amounts of PGE(2) than continuous monolayer cultures (alg-) after stimulation with IL-1beta. Moreover, IL-1beta induced COX-2 expression at 0.01 ng/ml in (alg+) cells, whereas a 100-fold higher dose of cytokine was necessary for stimulation in (alg-) cells. SB203580, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor, completely abolished the IL-1beta-induced COX-2 mRNA. Overexpression of p38 MAPK induces a COX-2 reporter, whereas overexpression of dominant negative p38 MAPK represses IL-1beta-induced promoter expression. Interestingly, IL-1beta-induced p38 MAPK activity was greatly enhanced in (alg+) compared with (alg-) cells. Our results suggest that differentiated articular chondrocytes are highly responsive to IL-1beta and that p38 MAPK mediates this response by inducing COX-2 gene expression.

Different effects of n-6 and n-3 polyunsaturated fatty acids on the activation of rat smooth muscle cells by interleukin-1β

There is good evidence that the n-3 polyunsaturated fatty acids (PUFAs) in fish oil have antiinflammatory effects and reduce the pathogenesis of atherosclerosis. However, the mechanisms underlying these actions are largely unknown. This study was designed to investigate the effects of membrane incorporation of two major components of fish oil [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)], on rat smooth muscle cells (SMCs) activation induced by interleukin-1β (IL1β). We compared their effects with those of n-6 arachidonic acid (AA). Expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 adhesion molecules involved in SMCs migration was enhanced by AA, whereas EPA and DHA had no similar effects. We established that AA potentiates IL1β-induced expression of the type IIA secreted phospholipase A2 (sPLA2) gene, whereas EPA and DHA reduce this stimulation. EPA and DHA also abolished proinflammatory prostaglandin PGE2 production by inhibiting the IL1β-induced production of cyclooxygenase-2 (COX-2) mRNA. Much interest was then focused on three transcriptional factors implicated in inflammation control and especially in modulating rat sPLA2 and COX-2 gene transcription: nuclear factor-κB, CCAAT/enhancer binding protein β, and E26 transformation-specific-1. electrophoretic mobility shift assay revealed that the binding activity of all three factors was increased by AA and reduced (or not affected) by n-3 PUFA. These results indicate that EPA and DHA act in opposition to AA by modulating various steps of the inflammatory process induced by IL1β, probably by reducing mitogen-activated protein kinase p42/p44 activity.

Transcriptional regulation of inflammatory secreted phospholipases A2

Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene.

Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release.

The coding sequence of type II phospholipase A2 from human placenta was cloned in a bovine papilloma virus-derived eukaryotic expression vector under the control of the metallothionein promoter. Stably transfected C127 mouse fibroblast lines were obtained with this vector. These transfected cells overexpressed a functional 14 kDa phospholipase A2, which was bulky secreted. However, a significant phospholipase A2 activity was measured in cell homogenates. The involvement of this 14 kDa phospholipase A2 in mechanisms related to stimulated arachidonic acid release was investigated. We could parallel the overexpression of phospholipase A2 with an increase in phorbol ester and fluoroaluminate-stimulated arachidonic acid release. Pertussis toxin inhibited this stimulation. These results suggest that the 14 kDa type II phospholipase A2 might contribute to stimulation of arachidonic acid release, and therefore to eicosanoid production.

Induction of Secreted Type IIA Phospholipase A2 Gene Transcription by Interleukin-1β ROLE OF C/EBP FACTORS

Secreted type IIA phospholipase A2, which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1β stimulates the phospholipase A2 [−1614; +20] promoter activity by 6–7-fold and that the [−210; −176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) β and C/EBPδ transcription factors bind to this element as shown by bandshift experiments. Interleukin-1β increased the levels of C/EBPδ mRNA as soon as 2 h and up to 24 h whithout affecting those of C/EBPβ. Higher amounts of C/EBPδ proteins correlate with the stimulation of C/EBPδ mRNA. Mutations or 5′ deletions in the upstream [−247; −210] region reduced by 2-fold the basal and interleukin-1β-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1β. A [−114; −85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1β or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A2 gene by interleukin-1β in chondrocytes absolutely requires C/EBPβ and C/EBPδ factors but does not involve NF-κB.

Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors.

The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.

A 3-month double-blind randomised study comparing an olive oil- with a soyabean oil-based intravenous lipid emulsion in home parenteral nutrition patients.

Intravenous lipid emulsions (ILE) have demonstrated advantages including prevention of essential fatty acid (EFA) deficiency; however, too much EFA can down regulate fatty acid elongation leading to an imbalance of nutritional compounds in plasma and cell membranes. An olive oil-based ILE containing long-chain triacylglycerols (LCT) with a low content (20 %) of PUFA was administered for home parenteral nutrition (HPN) and compared with a conventional soyabean oil-based ILE (PUFA content, 60 %). Thirteen patients (26-92 years) with stable intestinal failure were randomised after a 1-month run-in period with a medium-chain triacylglycerols-LCT-based ILE, to receive 3 months of HPN with either olive oil- (n 6) or soyabean oil-based (n 7) ILE. The nutritional impact and safety of HPN, oral intakes and absorption rates, phospholipid fatty acids in plasma and lymphocyte cell membrane were assessed. The only clinical event reported was one case of pneumonia (soya group). In both groups, 20 : 3n-9:20 : 4n-6 ratios remained within normal ranges (0.03-0.07). There was a significant increase of gamma-linolenic acid (gamma-LA) in plasma and lymphocyte cell membrane (P=0.02) and of oleic acid in plasma (P<0.01) in the olive compared with the soya group. A significant correlation was found between gamma-LA (day 90 - day 0) in plasma and PUFA parenteral intakes (P=0.02), but neither with fat intakes nor with fat absorption rates. In conclusion, plasma and lymphocyte EFA pattern remained in normal ranges without EFA deficiency with both lipid emulsions, despite a lower content of n-3 and n-6 series with the olive oil-based ILE.