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The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX(-/-)) model and found that survival after infection was significantly greater in GX(-/-) mice than in GX(+/+) mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX(-/-) mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX(-/-) mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.

Background— Pharmacological inhibition of cyclooxygenase-2 increases the risk of myocardial infarction (MI) and stroke. Microsomal prostaglandin (PG) E 2 synthase-1 (mPGES-1), encoded by the Ptges gene, functions downstream from cyclooxygenase-2 in the inducible PGE 2 biosynthetic pathway. We caused acute MI in Ptges +/+ and Ptges −/− mice to define the role of mPGES-1 in cardiac ischemic injury. Methods and Results— Twenty-eight days after MI, Ptges −/− mice develop more left ventricular (LV) dilation, have worse LV systolic and diastolic function, and have higher LV end-diastolic pressure than Ptges +/+ mice but have similar pulmonary wet-to-dry weight ratios, cardiac mass, infarct size, and mortality. The length-to-width ratio of individual cardiomyocytes is significantly greater in Ptges −/− than Ptges +/+ mice after MI, a finding consistent with eccentric cardiomyocyte hypertrophy in Ptges −/− mice. Expression of atrial natriuretic peptide, brain natriuretic peptide, and α- and β-myosin heavy chain, markers of ventricular hypertrophy, is higher in the LV of Ptges −/− than Ptges +/+ mice after MI. Ptges +/+ mice express cyclooxygenase-2 and mPGES-1 protein in inflammatory cells adjacent to the infarct after MI but do not express these proteins in cardiomyocytes. Ptges −/− mice express cyclooxygenase-2 in inflammatory cells adjacent to the infarct and do not express mPGES-1 in any cells in the heart. Levels of PGE 2 but not PGD 2 , thromboxane A 2 , PGI 2 , or PGF 2α are higher in the infarct and LV remote from the infarct after MI in Ptges +/+ than Ptges −/− mice. Conclusions— In Ptges +/+ mice, mPGES-1 in inflammatory cells catalyzes PGE 2 biosynthesis in the LV after MI. Deletion of mPGES-1 leads to eccentric cardiac myocyte hypertrophy, LV dilation, and impaired LV contractile function after acute MI.

The role of a cytosolic phospholipase A2-α (cPLA2-α) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA2-α activity was blocked with the specific cPLA2-α inhibitor, Pyrrolidine-1 (human cells), or by cPLA2 -α gene disruption (mice). cPLA2-α inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcγII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA2-α inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B4. Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA2-α(–/–) mice compared with wild type littermates. These studies identify a novel role for cPLA2-α in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A2 (PLA2) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA2 (GV and GX sPLA2) mRNA and contain GV and GX sPLA2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA2s are undetectable. GV sPLA2 is a component of both azurophilic and specific granules, whereas GX sPLA2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA2 and most, if not all, of the PLA2 activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA2. GV sPLA2 does not contribute to fMLP-stimulated leukotriene B4 production but may support the anti-bacterial properties of the neutrophil, because 10–100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA2-α (group IV PLA2α), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B4 in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.

Sulfasalazine is widely used in rheumatoid arthritis and inflammatory bowel diseases. The mechanisms of its activity have not been elucidated. In leukocytes, sulfasalazine and its analogue, CL 42A, inhibited the formation of leukotrienes and possibly of the second messenger compounds at the level of phospholipase C. Partial inhibition of interleukin-lbeta (IL-1beta), IL-6 and tumor necrosis factor-alpha (TNF-alpha) was also found. Since the synthesis of eicosanoids is induced by phospholipase A2 and since secretory phospholipase A2 (sPLA2) is proinflammatory, we investigated the impact of sulfasalazine and related compounds on mRNA, protein synthesis, and release of sPLA2 from osteoblasts. Sulfasalazine and CL 42A markedly inhibited extracellular release of sPLA2. The impact of sulfasalazine was evident at 50 microM (P < 0.001) and maximal at 400 microM, and that of CL 42A at 10 microM (P < 0.001) and 200 microM, respectively. Split products of sulfasalazine, 5-aminosalicylic acid (400 microM) and sulfapyridine (400 microM), had no impact. The effect of sulfasalazine and CL 42A was evident regardless of whether the cells were stimulated with IL-1beta/TNF-alpha, lipopolysaccharide/forskolin, or dibutyryl-cAMP. Sulfasalazine and CL 42A did not alter the level of sPLA2 mRNA. Exposure of stimulated fetal rat calvaria osteoblasts (FRCO) to sulfasalazine did not show accumulation of the intracellular sPLA2 protein as tested by western blot; however, enzymatic activity of PLA2 in disrupted cells was definitely increased. Thus, the impact is on the post-transcriptional release of sPLA2 rather than on the synthesis. There was also an increase in the extracellular release of prostaglandin E2 from FRCO exposed to sulfasalazine or to CL 42A. In contrast, sulfasalazine had no effect on the extracellular release of gelatinase from the cells or on mRNA of cytosolic PLA2 or cyclooxygenase 2. We conclude that the anti-inflammatory activity of sulfasalazine may be related, in part, to the selective inhibition of the extracellular release of proinflammatory sPLA2.

The role of Group X secreted phospholipase A2 (GX-sPLA2) during influenza infection has not been previously investigated. We examined the role of (Reviewer 2 Minor Comment 2) GX-sPLA2 during H1N1 pandemic influenza infection in a GX-sPLA2 gene targeted mouse (GX−/−) model and found that survival after infection was significantly greater in GX−/− mice than in GX+/+ mice. Downstream products of GX-sPLA2 activity, PGD2, PGE2, LTB4, cysteinyl leukotrienes and Lipoxin A4 were significantly lower in GX−/− mice BAL fluid. Lung microarray analysis identified an earlier and more robust induction of T and B cell associated genes in GX−/− mice. Based on the central role of sPLA2 enzymes as key initiators of inflammatory processes, we propose that activation of GX-sPLA2 during H1N1pdm infection is an early step of pulmonary inflammation and its (Reviewer 2 Minor Comment 2) inhibition increases adaptive immunity and improves survival. Our findings suggest that GX-sPLA2 may be a potential therapeutic target during influenza.

The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase.

Endotoxin or cytokine-induced expression of a secretory non-pancreatic phospholipase A 2 (sPLA 2 ) has been implicated in the pathogenesis of multisystem organ dysfunction or failure in patients with septic shock. Circulating sPLA 2 levels increase as much as 1000-fold during the course of septic shock. However, the mode of regulation of the activity of this lipolytic enzyme is unknown, since circulating inhibitors have not been identified. We investigated the potential inhibitory activity of the acute phase reactant, C-reactive protein (CRP), a phospholipid-binding protein whose expression increases as much as 1000-fold during severe infections. Serum CRP and sPLA 2 profiles were highly concordant in patients with septic shock. In studies in vitro, human CRP inhibited hydrolysis of PC-lyso-PC (2:1) multilamellar liposomes by human recombinant sPLA 2 in a dose-dependent manner, with an apparent IC 50 of 50 μg/ml CRP and maximal inhibition at 100 μg/ml. Inhibition of sPLA 2 activity by CRP was substrate concentration-dependent, and increasing substrate concentrations reversed the inhibitory effect of CRP using the PC-lyso-PC system. Preincubation of CRP with phosphorylcholine led to a concentration-dependent loss of CRP-induced inhibition of substrate hydrolysis. These observations are consistent with a substrate-depletion model of inhibition of sPLA 2 activity by CRP.

Background— Microsomal prostaglandin E 2 synthase-1 (mPGES-1), encoded by the Ptges gene, catalyzes prostaglandin E 2 biosynthesis and is expressed by leukocytes, cardiac myocytes, and cardiac fibroblasts. Ptges −/− mice develop more left ventricle (LV) dilation, worse LV contractile function, and higher LV end-diastolic pressure than Ptges +/+ mice after myocardial infarction. In this study, we define the role of mPGES-1 in bone marrow–derived leukocytes in the recovery of LV function after coronary ligation. Methods and Results— Cardiac structure and function in Ptges +/+ mice with Ptges +/+ bone marrow ( BM +/+ ) and Ptges +/+ mice with Ptges −/− BM ( BM −/− ) were assessed by morphometric analysis, echocardiography, and invasive hemodynamics before and 7 and 28 days after myocardial infarction. Prostaglandin levels and prostaglandin biosynthetic enzyme gene expression were measured by liquid chromatography–tandem mass spectrometry and real-time polymerase chain reaction, immunoblotting, immunohistochemistry, and immunofluorescence microscopy, respectively. After myocardial infarction, BM −/− mice had more LV dilation, worse LV systolic and diastolic function, higher LV end-diastolic pressure, more cardiomyocyte hypertrophy, and higher mortality but similar infarct size and pulmonary edema compared with BM +/+ mice. BM −/− mice also had higher levels of COX-1 protein and more leukocytes in the infarct, but not the viable LV, than BM +/+ mice. Levels of prostaglandin E 2 were higher in the infarct and viable myocardium of BM −/− mice than in BM +/+ mice. Conclusions— Lack of mPGES-1 in bone marrow–derived leukocytes negatively regulates COX-1 expression, prostaglandin E 2 biosynthesis, and inflammation in the infarct and leads to impaired LV function, adverse LV remodeling, and decreased survival after acute myocardial infarction.

International audience; Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.

Synovial fluid PLA2 concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant “synovial-type” group II phospholipase A2. This ELISA was specific for synovial-type PLA2 and did not detect pancreatic (group I) PLA2. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA2 enzyme activity significantly correlated with PLA2 immunoreactivity (P

Phospholipase A2 (PLA2) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA2 was noted 3 h after EN challenge (mean 828 +/- 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 +/- 2442 units/ml), and was still evident at 48 h (mean 763 +/- 366 units/ml). In contrast, TNF levels were maximal (mean 712 +/- 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 +/- 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA2 (r = 0.82, P0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA2 response. EN challenge did not affect serum pancreatic PLA2 concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA2 completely abolished PLA2 activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA2 and that the magnitude of this response seems to be related to the prior generation of TNF.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Massive elevations of serum phospholipase A2 activity have been documented in patients with septic shock. Serum PLA2 activity correlated to the degree and duration of circulatory collapse, while purified native PLA2 reproduced hypotension in experimental animals. In a prospective study of patients with septic shock, we have determined the relationship of PLA2 enzyme activity to PLA2 immunoreactivity using radiolabelled E. coli phospholipid substrate and an ELISA specific for group II human nonpancreatic PLA2. In all patients, there was a clear concordance of the two assays. Maximal PLA2 concentration was increased a mean of 554-fold over normal levels. We found no evidence to support the presence of activating or inhibitory proteins. These data confirm that the observed increase in serum PLA2 activity in septic shock is due to intravascular release of group II nonpancreatic PLA2.

Inflammatory processes are characterised by increased levels of extracellular phospholipase A2 (PLA2) and cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF). IL-1, TNF and PLA2 share a number of proinflammatory, arthritogenic effects. The sequential induction, first of the cytokines followed by PLA2, suggests that these cytokines may regulate synthesis and secretion of PLA2. To test this postulate, foetal rat calvarial bone-forming cells (FRCC) were treated with recombinant human IL-1 and TNF and extracellular PLA2 release was quantitated. Both IL-1 and TNF induced the de novo synthesis of PLA2 in a concentration-dependent manner. Continuous exposure of FRCC in primary culture to IL-1 (50 units/ml) over 15 days resulted in as much as 100-fold increase in PLA2 secretion. IL-1 (50 units/ml) added to post-confluent cultures for a 48-h pulse increased PLA2 activity 9.4-fold. The combination of IL-1 (50 units/ml) and TNF (500 units/ml) was synergistic with an observed increase in extracellular PLA2 secretion of 146-fold following a 48-h pulse. Interleukin-6, alone or in combination with IL-1 or TNF, did not further enhance PLA2 synthesis or secretion. Cytokine-induced synthesis of PLA2 was inhibited 80% by 10 μM cycloheximide but not by dexamethasone over the range of 10 −6 to 10 −8 M. FRCC-derived PLA2 was neutral-active with a pH optimum of 6–7.5 and was calcium-dependent with optimal activity in the presence of 2–7 mM calcium. It had absolute 2-acyl specificity using micellar phosphatidylcholine. These data show that bone-forming cells secrete a soluble PLA2 in response to cytokine stimulatino and are, therefore, potential sources of proinflammatory synovial fluid PLA2. Since PLA2 reproduces many of the biologic activities of IL-1 and TNF, and since PLA2 is induced by IL-1 and TNF, these data suggest that PLA2 may serve as a common distal effector of these cytokines.

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.

Introduction. mPGES functions downstream from COX-2 in the inducible PGE 2 biosynthetic pathway, and can be expressed by cardiac myocytes and inflammatory cells. mPGES gene targeted mice (mPGES −/− ) develop LV dilation and impaired contractile function after MI. The purpose of this study was to determine if mPGES in bone marrow (BM) derived inflammatory cells regulates LV remodelling after MI. Methods. Six week old female mPGES +/+ mice were irradiated with 9.5 Gy and transplanted with BM from male mPGES +/+ or mPGES −/− mice (BM +/+ or BM −/− chimeras, respectively). After 10 weeks, the left coronary artery was ligated. Echocardiography was used to assess LV dimensions and function in vivo . After fixation in situ , LV dimensions and infarct volume was assessed by planimetry. The efficiency of BM transplantation, determined by the ratio of syr to gapdh DNA, measured by real time PCR, was 98%. Results. There was no difference in LV dimensions or function between BM +/+ and BM −/− mice before MI (not shown). Between 7 and 28 days post MI, ejection fraction did not change in BM +/+ mice, but decreased significantly in BM −/− mice (Table ). BM −/− mice also had a higher LV diameter (2.7 ± 0.1 vs. 3.1 ± 0.1 mm, p = 0.0001) and LV volume (14 ± 0.8 vs. 18 ± 1.0 mm 3 , p = 0.025) than BM +/+ mice 28 days after MI. In contrast, there was no difference in the percentage of infarcted LV, heart rate or cardiac output between BM +/+ and BM −/− mice after MI. Survival of BM +/+ vs. BM −/− 28 days after MI was similar (75% vs. 72%, log rank, p = 0.86). Conclusion. mPGES1 in bone marrow cells that localize to the heart after MI is necessary to maintain post infarction LV contractile function, and to prevent adverse LV remodelling. Use of mPGES inhibitors as substitutes for COX-2 inhibitors may be detrimental in patients with coronary artery disease.

Microsomal prostaglandin (PG) E(2) synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE(2), a key proinflammatory mediator. The purpose of this study was to elucidate the regulation of mPGES-1 mRNA expression in cardiomyocytes, define the role of JNK enzymes in this process, and characterize the role of mPGES-1 in cardiomyocyte PGE(2) biosynthesis. In neonatal cardiomyocytes, interleukin-1beta and lipopolysaccharide (LPS) both stimulated mPGES-1 mRNA expression and increased mPGES-1 mRNA stability and protein synthesis but failed to increase mPGES-1 mRNA transcription. Treatment with the JNK1/2 inhibitor, SP600125, abrogated the increases in mPGES-1 mRNA stability, mPGES-1 protein synthesis, and PGE(2) release induced by interleukin-1beta or LPS. mPGES-1 protein synthesis was observed in LPS-stimulated neonatal cardiomyocytes from jnk1(-/-) or jnk2(-/-) mice. In contrast, infection of jnk1(-/-) cardiomyocytes with an adenovirus encoding phosphorylation-resistant JNK2 (ad-JNK2-DN), or of jnk2(-/-) cardiomyocytes with ad-JNK1-DN, significantly decreased LPS-stimulated mPGES-1 protein synthesis. Similarly, co-infection with ad-JNK1-DN and ad-JNK2-DN attenuated LPS-stimulated mPGES-1 protein synthesis in cardiomyocytes from wild type mice. Targeted deletion of the gene encoding mPGES-1 led to a 3.2-fold decrease in LPS-stimulated PGE(2) release by cardiomyocytes in comparison with wild type cells but had no effect on COX-1, COX-2, mPGES-2, or cytosolic PGES mRNA levels. These studies provide direct evidence that mPGES-1 mRNA levels in cardiomyocytes are augmented by stabilization of mPGES-1 mRNA, that JNK1 or JNK2 can participate in the regulation of mPGES-1 protein synthesis in these cells, and that mPGES-1 catalyzes the majority of LPS-induced PGE(2) biosynthesis by cardiomyocytes.

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in delayed prostaglandin biosynthesis. The purpose of this study was to evaluate the role of the MAP kinase kinase 6 (MKK6)–p38 MAPK signaling cascade in the regulation of myocardial COX-2 gene expression, in vitro and in vivo. RT-PCR analysis identified p38α and p38β2 MAPK mRNA in rat cardiac myocytes. Interleukin-1β induced the phosphorylation of p38α and p38β2 MAPK in cardiomyocytes and stimulated RNA polymerase II binding to the COX-2 promoter, COX-2 transcription, COX-2 protein synthesis, and prostaglandin E 2 (PGE 2 ) release. Infecting cardiomyocytes with adenoviruses that encode mutant, phosphorylation-resistant MKK6 or p38β2 MAPK inhibited interleukin-1β–induced p38 MAPK activation, COX-2 gene expression, and PGE 2 release. Treatment with the p38α and p38β2 MAPK inhibitor, SB202190, attenuated interleukin-1β–induced COX-2 transcription and accelerated the degradation of COX-2 mRNA. Cells infected with adenoviruses encoding wild-type or constitutively activated MKK6 or p38β2 MAPK, in the absence of interleukin-1β, exhibited increased cellular p38 MAPK activity, COX-2 mRNA expression, and COX-2 protein synthesis, which was blocked by SB202190. In addition, elevated levels of COX-2 protein were identified in the hearts of transgenic mice with cardiac-restricted expression of wild-type or constitutively activated MKK6, in comparison with nontransgenic littermates. These results provide direct evidence that MKK6 mediated p38 MAPK activation is necessary for interleukin-1β–induced cardiac myocyte COX-2 gene expression and PGE 2 biosynthesis in vitro and is sufficient for COX-2 gene expression by cardiac myocytes in vitro and in vivo.

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

Multifactorial interaction among lipoproteins, vascular wall cells, and inflammatory mediators has been recognized as the basis of atherogenesis. In the arterial wall high-density lipoprotein (HDL) and human secretory phospholipase A(2) (sPLA(2)) colocalize with vascular smooth muscle cells and concentrate in the atherosclerotic lesions. It has been shown that gr IIA sPLA(2) hydrolyzes lipoproteins, altering their structure and releasing active agents such as lyso-phosphatidylcholine (PtdCho) and free fatty acids. We investigated the impact of normal HDL(3) (NHDL(3)), acute phase HDL(3) (APHDL(3)), and low-density lipoprotein (LDL), both unhydrolyzed and sPLA(2)-hydrolyzed, and some products of hydrolysis, such as lyso-PtdCho, oleic and linoleic acid, on [(3)H] thymidine incorporation by DNA of cultured human vascular smooth muscle cells (VSMC). NHDL(3) markedly enhanced mitogenic activity of VSMC in a dose- and time-dependent manner. Doubling of thymidine incorporation was usually achieved by 40 microg/ml of NHDL(3) after 4 hours of incubation. APHDL(3) had invariably a stronger inducing effect on the mitogenic activity than NHDL(3); 40 microg/ml more than tripled [(3)H] thymidine incorporation after 4 hours of incubation. NHDL(3) preincubated with human apo serum amyloid A apolipoprotein-induced higher mitogenic activity in VSMC than NHDL(3) alone. Hydrolysis of NHDL(3), APHDL(3), or LDL by gr IIA sPLA(2) markedly enhanced mitogenic activity of VSMC as compared with unhydrolyzed lipoproteins. sPLA(2) concentrations that can be found in atherosclerotic vascular walls markedly enhanced lipoprotein-induced mitogenic activity of VSMC. sPLA(2) per se did not affect thymidine incorporation and VSMC did not release sPLA(2) into the medium. There was no evidence for hydrolysis of the wall of VSMC by gr IIA sPLA(2). The presence of the products of hydrolysis of lipoproteins such as oleic and linoleic acids and lyso-PtdCho or their combinations with NHDL(3) explains in part markedly enhanced mitogenic activity of VSMC. It is conceivable that sPLA(2,) which is known to colocalize with lipoproteins in the vascular wall in the domain of VSMC, is capable of induction of the mitogenic activity in these cells in vivo and should be considered as a proatherogenic enzyme.

Rationnel La microsomal prostaglandin E2 synthase-1 (mPGES-1), regulee par le gene Ptges, est en aval de la COX-2 dans la voie de synthese de la PGE2. En post infacrtus du myocarde (IDM) mPGES-1 est exclusivement exprimee par les leucocytes lies a la reponse inflammatoire. L’objectif de ce travail etait d’une part d’identifier les varietes de leucocytes issus de la moelle osseuse (MO) et migrant dans le myocarde en post IDM exprimant mPGES-1, et d’autre part de rechercher un effet de l’expression de mPGES-1 sur le remodelage ventriculaire gauche (VG) post IDM. Methode l’expression de l’ARNm codant pour mPGES-1 a ete mesuree en RT PCR dans les monocytes circulants, les macrophages/ cellules dendritiques, et les monocytes Ly-6Chi et LY-6Clo isoles dans la zone infarcie du myocarde de souris Ptges+/+. Un model murin chimerique a ete realise en irradiant des souris Ptges+/+ avant de les transplanter avec de la MO de souris Ptges+/+ ou Ptges-/-. Apres 10 semaines, un IDM etait provoque par ligature de la coronaire gauche. La fonction VG etait evaluee en echocardiographie et par catheterisme cardiaque. Les dimensions du VG et la taille d’IDM etaient mesurees en planimetrie. Resultats mPGES-1 etait majoritairement exprime par les monocytes LY-6Clo 14 a 28 jours apres l’IDM. Avant l’IDM, il n’y avait pas de difference concernant les dimensions et la fonction du VG entre les souris chimeriques ayant une MO Ptges+/+ ou Ptges-/-. Vingt huit jours apres l’IDM, les souris chimeriques MO Ptges-/- presentaient des differences significatives en comparaison aux souris MO Ptges+/+: un diametre et un volume VG plus importants, une fraction de raccourcissement et un debit cardiaque plus alteree et des parametres hemodynamiques plus pejoratifs. Il n’y avait pas de difference entre les deux groupes concernant la taille d’IDM, l’epaisseur du myocarde, et la survie. Conclusion l’ARNm de mPGES-1 est majoritairement exprime dans les monocytes LY-6Clo dans le myocarde infarcis. mPGES-1 semble jouer un role dans la reponse inflammatoire des leucocytes issus de la MO en modifiant le remodelage et la fonction VG en post IDM.

Secretory group II (sPLA2) and cytosolic (cPLA2) phospholipases A2 and cyclooxygenase-2 (Cox-2) play a pivotal role in release of proinflammatory eicosanoids. Excessive activity of sPLA2 per se can also propagate inflammation. Endogenous control of the above enzymes has not been completely elucidated. We investigated the combined impact of promoting cytokines and inhibitory peptide growth factors on the expression of mRNA of the above enzymes, on protein content and extracellular release of sPLA2 and on PGE2 production in osteoblasts (FRCO). The synthesis and release of sPLA2 were enhanced by about 20-fold by 0.5 ng/ml IL-1beta or by 50 ng/ml of TNFalpha. Coaddition of both cytokines resulted in synergistic 150-fold increase in the release of sPLA2 implying the existence of two paths of induction. IL-1beta and TNFalpha markedly enhanced the transcription of sPLA2 mRNA. Kinetic study showed that IL-1/TNF initiated sPLA2 release after 12 h, reaching maximum at 48 h. IL-1alpha was a weak stimulator of sPLA2 release, whereas IL-6, IL-8, IGF, IFN-gamma, growth hormone, insulin and GM-CSF were not stimulatory. Peptide growth hormones TGFbeta, PDGF-BB, EGF and bFGF markedly inhibited the extracellular release of sPLA2. TGFbeta and PDGF-BB significantly reduced the level of sPLA2 mRNA, thus acting upon transcription whereas EGF and bFGF were not inhibitory, acting rather upon the translational or posttranslational steps. IL-1/TNF and growth factors had no significant effect on cPLA2 mRNA expression. Cox-2 mRNA expression was markedly enhanced by IL-1/TNF and suppressed by all growth factors tested. Cytokines enhanced the extracellular release of PGE2 and further enhancement was induced by growth factors with the exception of TGFbeta. Cycloheximide abolished completely the release of sPLA2 and markedly reduced the release of PGE2 from cytokine-stimulated FRCO, regardless of whether growth factors were present or not. NS-398, a specific inhibitor of Cox-2 abolished almost completely the release of PGE2 from cytokine-stimulated cells, regardless of the presence of growth factors. Thus, different signalling mechanisms are involved in the impact of growth factors on mRNA expression of sPLA2, cPLA2 and Cox-2. The differences between the impact on FRCO sPLA2 and that reported in other cells, imply that endogenous control of arachidonic acid cascade is cell-specific.

SUMMARY The early response to inflammation is characterized by the synthesis of a variety of proteins under cytokine and glucocorticoid control. During episodes of infection or inflammation, a secretory phospholipase A2 (sPLA2) appears in the circulation along with a variety of acute-phase proteins (APP), suggesting possible common regulatory elements amongst sPLA2 and APP. Using the human hepatoma line, HepG2, regulation of sPLA2 expression was examined in relation to synthesis of HP and ACH. The patterns of induction of sPLA2, HP and ACH were distinct for each of IL-1, tumour necrosis factor (TNF) and IL-6, oncostatin M, IL-11 and leukaemia inhibitory factor. Dexamethasone had an enhancing effect on IL-6-induced expression of HP and ACH, but inhibited sPLA2 expression by 50%. Both 8-bromo-cAMP and dibutyryl cAMP increased sPLA2 expression (48.8-fold and 64.2-fold, respectively), whereas KT5720, an inhibitor of protein kinase A, down-regulated cytokine-induced sPLA2 synthesis by 51%. These data show that a panel of cytokines induced varying patterns of up-regulation of sPLA2, ACH and HP. Although dexamethasone potentiated IL-6-induced APP expression in HepG2 cells, it suppressed sPLA2 expression in a dose-dependent manner. In several respects, sPLA2 regulation is similar to that of HP and ACH, but a notable difference is the reciprocal effect of glucocorticoids on sPLA2 expression compared with that of ACH and HP.

Lipid mediators of inflammation, contribute to airway hyper-reactivity in asthma. Since production of lipid mediators is largely regulated by phospholipase A2 (PLA2), and since PLA2 expression in mesenchymal cells is induced by cytokines and other signals, we examined PLA2 expression by rat tracheobronchial smooth muscle cells (TBSMC). PLA2 expression in TBSMC cultures was markedly increased by tumour-necrosis factor (TNF) alpha (130-fold) and interleukin-1beta (IL-1beta) (7.4-fold). Lipopolysaccharide (LPS;100 ng/ml) resulted in a 51-fold increase in extracellular PLA2 activity. PLA2 expression by LPS-stimulated or cytokine-stimulated cells was downregulated by dexamethasone. Whereas forskolin or dibutyrl cAMP increased PLA2 activity, inhibition of protein kinase A but not tyrosine kinase reduced PLA2 expression. Northern blot analysis showed that TNF alpha and IL-1beta increased both PLA2 and inducible cyclooxygenase (Cox-2) mRNA transcription. Addition of dexamethasone substantially blunted the increase in PLA2 and Cox-2 mRNA. In contrast, the level of Cox-1 mRNA was very low and did not change with the various treatments. Since proinflammatory lipid mediators have been implicated in the pathogenesis of asthma and PLA2 activity regulates generation of these lipid mediators, cytokine-stimulated synthesis and release of PLA2 by airway smooth cells may contribute to the potentiation of airway inflammation in asthma.

We have shown previously that interleukin-1 (IL-1) has biphasic effects on the formation of bone nodules in long-term cultures of fetal rat calvarial (RC) cells (Ellies and Aubin, Cytokine 2:430-437, 1990). To determine the role of arachidonic acid metabolism in this process, we have examined the release of prostaglandin E2 (PGE2) and phospholipase A2 (PLA2) from RC cells under conditions that allowed concomitant analysis of the formation of bone nodules. Recombinant human IL-1 alpha (rhIL-1 alpha) stimulated PGE2 and PLA2 release in a time- and dose-dependent manner. PGE2 release was highest in preconfluent cultures (days 1-6) and was stimulated up to 8.5-fold in response to 50 U/ml of rhIL-1 alpha. In contrast, extracellular PLA2 activity was maximal in postconfluent cultures, with 50 U/ml of rhIL-1 alpha causing a 20-fold increase by day 15. PLA2 release by RC cells was not significantly affected by PGE2, the glucocorticoid dexamethasone, or the cyclooxygenase inhibitor indomethacin. Indomethacin partially blocked the inhibition of bone nodule formation caused by rhIL-1 alpha, and exogenous PGE2 reversed this effect. Addition of group I PLA2 from Naja naja venom to RC cells had no effect on bone nodule development; however, group II PLA2 from Crotalus adamanteus venom inhibited the formation of bone nodules in a dose range similar to that induced by rhIL-1 alpha. These results indicate that PGE2 release does not have a direct temporal correlation with increases in PLA2 activity. In addition, the data show that only part of the inhibition of bone formation seen with rhIL-1 alpha is mediated by PGE2 and suggest that extracellular PLA2 also accounts for part of the inhibition.

Soluble phospholipase A2 (PLA2) purified from rheumatoid synovial fluid (group II) and repurified Naja naja venom PLA2 (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mononuclear (MO) phagocytes. Group II PLA2 reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA2 suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA2 purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group I Naja naja and porcine pancreatic PLA2 had no effect on superoxide generation. Group II but not group I PLA2 reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA2, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA2s may mean that these enzymes exert different influences on cell membrane.

Multiple forms of soluble phospholipase A2 (PLA2) are known to coexist in venoms of individual reptilian species. While similar observations in several mammalian species suggest that this is a common phenomenon, the functional implications are not yet understood. In attempting to devise therapeutic strategies for treatment of inflammatory disorders by inhibition of PLA2, it is imperative to define the various PLA2 species in the relevant compartments. Herein, we report the presence of three PLA2 isotypes in rheumatoid arthritis serum, one pancreatic and two nonpancreatic phospholipases A2. The pancreatic and one of the nonpancreatic forms were optimally active in 7 mM calcium at pH 7.5. The other nonpancreatic form was calcium-independent and optimally active at pH 7.O. Only the calcium-dependent nonpancreatic form was observed in rheumatoid synovial fluid. Of the three serum isotypes, only the calcium-dependent nonpancreatic form correlated with markers of disease activity, such as the joint count and Landsbury index. Therefore, not all soluble or circulating phospholipases A2 are relevant to inflammatory processes. Selective inhibition of the proinflammatory form of PLA2 may prove to have some therapeutic benefit while minimizing the possible adverse effects of this form of intervention.

Circulating phospholipase A2 (PLA2) has been recognized as a mediator of circulatory collapse in experimental endotoxic shock. To assess the role of serum PLA2 in septic shock in man, we determined serum PLA2 profiles in a prospective study in 12 patients with septic shock. During the hypotensive phase of sepsis, serum PLA2 levels were consistently elevated as high as 33,428 U/ml (normal range 115 +/- 12 [SE]; n = 101). In all 12 patients, PLA2 levels correlated directly with the magnitude and duration of circulatory collapse (p less than .001), with a progressive fall of serum PLA2 levels during convalescence. In contrast, serum PLA2 levels in patients with cardiogenic shock secondary to myocardial infarction remained low. In pancreatitis, PLA2 levels paralleled fluctuations of serum amylase and lipase, whereas in septic shock without pancreatic involvement, PLA2 changes were discordant with changes in pancreatic enzymes. As well, septic shock serum PLA2 failed to crossreact by radioimmunoassay with antiserum against human pancreatic PLA2. These data are consistent with an extrapancreatic source of intravascular PLA2 release during sepsis. Since endogenous serum PLA2 levels correlate directly with the magnitude of hypotension in both experimental endotoxic shock and clinical septic shock, and since parenteral administration of purified exogenous PLA2 reproduces hypotension in experimental models, we conclude that high levels of intravascular PLA2 may contribute similarly to the circulatory collapse in septic shock in man.

Soluble phospholipase A2 has been implicated in the pathogenesis of local and systemic inflammatory reactions. Elevated levels of circulating phospholipase A2 (PLA2) correlate with the severity of circulatory collapse and pulmonary dysfunction in gram-negative septic shock. Characterization of septic shock serum PLA2 revealed a calcium-dependent enzyme with absolute 2-acyl specificity with a pH optimum of 7.5. We tested a number of therapeutic agents for their ability to inhibit PLA2 from human septic shock serum. Chloroquine, chlorpromazine, dexamethasone base, dexamethasone sodium phosphate, indomethacin, lidocaine, oleic acid, palmitic acid, promethazine, trans-retinoic acid, rutin and dl-alpha-tocopherol were all studied over the range of 10(-2) to 10(-7) M. All agents, with the sole exception of dexamethasone base, inhibited PLA2 activity at concentrations greater than 10(-3) M. PLA2 inhibition by dexamethasone sodium phosphate was factitious, due to the formation of calcium-phosphate complexes. Of the 11 agents studied, chlorpromazine was the most effective, with an IC50 of 7.5 X 10(-5) M, a membrane concentration achievable within its therapeutic range. Inhibition was non-competitive with an apparent Ki of 5 nM. Since serum PLA2 levels correlate with mortality in both experimental endotoxemia and clinical gram-negative septic shock, and chlorpromazine was previously shown to improve survival in these conditions, we postulate that its therapeutic efficacy resides at least in part in its PLA2-inhibitory activity.

The influence of plasma proteins on quantitation of the proinflammatory enzyme phospholipase A2 (PLA2) from rheumatoid synovial fluid was studied using two different assays. Human and bovine serum albumins increased the rate of PLA2 hydrolysis of membrane-associated phospholipid substrates. In contrast, albumin profoundly inhibited the PLA2 hydrolysis of synthetic phospholipids in micellar dispersion. Other plasma proteins (alpha, beta and gamma-globulins) had minimal effect on PLA2 activity in either assay system. Since the presence of albumin may compromise estimation of PLA2 and of phospholipase-inhibitory proteins, the appropriate selection of assay conditions is obligatory for the accurate quantitation of their respective activities.