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We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.

Secretory phospholipase A2 (PLA2) consists of several 14-kDa isoforms with extensive homology, which makes it difficult to identify a specific isoform. In this study, we have developed and characterized monoclonal antibodies (MAbs) directed specifically against human group V sPLA2 (hVPLA2) derived from cultured hybridomas. These hybridomas were produced from the fusion of BALB/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant hVPLA2. Three hybridomas secreting MAbs, MCL-3G1, MCL-2A5, and MCL-1B7, were selected and subcloned on the basis of their specificity to recognize hVPLA2 using solid-phase enzyme-linked immunoadsorbent assay (ELISA). The purified MAbs demonstrated a common pattern of immunoreactivity to hVPLA2, but not to human group IIa isoform (hIIaPLA2). Isotype analysis indicates that these hybridomas are of the IgG1 type. Under reducing conditions, MCL-3G1 sensitively detected hVPLA2 and demonstrated no cross-reactivity to either hIIaPLA2 or group IV cytosolic PLA2. Although specific for hVPLA2, a relatively modest signal was recognized with MCL-1B7 and MCL-2A5. These newly developed MAbs allow for determination of tissue distribution and cell-specific functions of hVPLA2.

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.

An important early step in atherosclerosis is binding of apolipoprotein B-100 (apoB-100)–containing lipoproteins (VLDL, IDL, and LDL) to the proteoglycan component of the extracellular matrix of the arterial intima.1,2 When oxidative agents or enzymes attack the proteoglycan-bound lipoproteins, they become oxidatively modified, and when various intimal proteases or lipases hydrolyze them, they become nonoxidatively modified. Whereas oxidation of proteoglycan-bound apoB-100–containing particles triggers their release and subsequent uptake by macrophages, the nonoxidative modifications do the opposite: they enhance the strength of particle binding to proteoglycans.3 In addition, such nonoxidatively modified apoB-100–containing lipoprotein particles tend to aggregate and fuse, which allows progressive extracellular accumulation of lipoprotein lipids, a key phenomenon in atherogenesis. The nonoxidative modifications can also lead to intracellular lipid accumulation and so induce foam cell formation, a hallmark of early atherogenesis.4 Thus, local modification of apoB-100–containing lipoproteins leads to both extra- and intracellular accumulation of lipids in the arterial intima. See page 600 In general, the nonoxidative modifications are caused by enzymes that are hydrolytic in nature, either proteolytic or lipolytic. Indeed, the arterial intima contains many different types of hydrolytic enzymes capable of lipoprotein modification. Unfortunately, however, it is currently not known which hydrolytic enzymes of the human arterial intima are actually responsible for turning the intimal lipoproteins into atherogenic particles. Among the lipolytic enzymes potentially involved in such atherogenic conversion of lipoproteins are several members of the secretory phospholipase A2 (sPLA2) family. The hydrolytic action of these enzymes on phosphatidylcholine, the major glycerophospholipid in lipoprotein particles, leads to the generation of lysophosphatidylcholine (lysoPC) and a free fatty acid (FFA). In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Bostrom et al5 have examined in vivo the role of Group V sPLA2 (sPLA2-V) in the context of atherogenesis. …

By means of glycyrrhizin (GL)-affinity and Mono S column chromatographies (HPLC), at least four GL-binding proteins (p25, p17, p15-1 and p15-2) in the two Superdex fractions (P-II and P-III fractions) from Habu snake venom were selectively purified. By determination of their N-terminal partial amino acid sequences, a metalloprotease (p25) and three GL-binding phospholipases A2 (gbPLA2s) [PA2Y (p17), PA21 (p15-1) and PA2B (p15-2)] were identified. PA2B (lysine-49 PLA2) was found to be the most sensitive to GL because (i) it strongly bound to a GL-affinity column; and (ii) its enzyme activity was selectively inhibited by low dose (ID50 = approx. 1.5 microM) of GL, but not by GA. Furthermore, these three gbPLA2s were phosphorylated by casein kinase II (CK-II) in vitro and GL inhibited the CK-II-mediated stimulation of their enzyme activities in vitro.

Group IIA secreted phospholipase A2 (group IIA sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA2s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA2s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2.The bactericidal efficiency of groups V and IIA sPLA2s was shown to be dependent upon the presence of calcium ions and to a less extent Mg2+ ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA2s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA2s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

In this study, we examined the effects of Prostaglandin E1 and tea polysaccharides (TP) on serum estrogen and FSH levels, myocardium sPLA2-V positive levels, and sPLA2-V protein expression in the rats fed on hypercholesterolemic diet. Hyperlipidemic rats were treated with Prostaglandin E1 and TP. Serum estrogen and FSH levels were significantly enhanced by Prostaglandin E1 and TP, whereas myocardium sPLA2-V positive rate and protein expression levels were decreased compared to the HCD group. Our results suggest that Prostaglandin E1 and TP exert strong heart-protective effects and therefore can be used to reduce the risk of heart disorders.

Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5−/−) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5−/− mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5−/− mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5−/− mice. The increased FENa+ observed in Pla2g5−/− mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5−/− mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity.

High mobility group box 1 (HMGB1) mediates proinflammatory responses in inflammatory diseases. Lycopene found in tomatoes and tomato products has anti-oxidant, anti-cancer and antiinflammatory effects. The potential anti-inflammatory roles of lycopene in HMGB1-mediated proinflammatory responses in both primary human umbilical vein endothelial cells (HUVECs) and animal were investigated. The anti-inflammatory effects of lycopene were determined including permeability, monocyte adhesion and migration, and activation of proinflammatory proteins and HMGB1 receptors on HMGB1 activated HUVECs. In the in vivo model, the anti-inflammatory effect of lycopene was assessed by monitoring vascular permeability and migration of leukocytes to the peritoneal cavity of mice injected with lycopene. Lycopene inhibited lipopolysaccharide (LPS)-mediated release of HMGB1, expression of HMGB1-mediated tumor necrosis factor (TNF)-secretory phospholipase A2 (sPLA2)-IIA, and HMGB1-mediated pro-inflammatory signaling responses in endothelial cells. It did this through down-regulation of cell surface expression of cell adhesion molecules (CAMs), HMGB1 receptors, toll-like receptor (TLR)-2, and -4, and receptors for advanced glycation end products (RAGE). These findings suggest that lycopene promotes barrier integrity, inhibits monocyte adhesion and migration to HMGB1 activating HUVECs by blocking activation of proinflammatory cytokines and expression of CAMs and HMGB1 receptors, thereby showing its usefulness as a therapy for vascular inflammatory diseases.

Secretory phospholipases A2 (sPLA2s) are a diverse family of low molecular mass enzymes (13–18 kDa) that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acids and lysophospholipids. We have previously shown that group X sPLA2 (sPLA2-X) had a strong hydrolyzing activity toward phosphatidylcholine in low-density lipoprotein (LDL) linked to the formation of lipid droplets in the cytoplasm of macrophages. Here, we show that group V sPLA2 (sPLA2-V) can also cause the lipolysis of LDL, but its action differs remarkably from that of sPLA2-X in several respects. Although sPLA2-V released almost the same amount of fatty acids from LDL, it released more linoleic acid and less arachidonic acid than sPLA2-X. In addition, the requirement of Ca2+ for the lipolysis of LDL was about 10-fold higher for sPLA2-V than sPLA2-X. In fact, the release of fatty acids from human serum was hardly detectable upon incubation with sPLA2-V in the presence of sodium citrate, which contrasted with the potent response to sPLA2-X. Moreover, sPLA2-X, but not sPLA2-V, was found to specifically interact with LDL among the serum proteins, as assessed by gel-filtration chromatography as well as sandwich enzyme-immunosorbent assay using anti-sPLA2-X and anti-apoB antibodies. Surface plasmon resonance studies have revealed that sPLA2-X can bind to LDL with high-affinity (Kd = 3.1 nM) in the presence of Ca2+. Selective interaction of sPLA2-X with LDL might be involved in the efficient hydrolysis of cell surface or intracellular phospholipids during foam cell formation.

The endothelial protein C receptor (EPCR) plays an important role in cardiovascular disease by binding protein C/activated protein C (APC). EPCR structure contains a hydrophobic groove filled with an unknown phospholipid needed to perform its function. It has not been established whether lipid exchange takes place in EPCR as a regulatory mechanism of its activity. Our objective was to identify this phospholipid and to explore the possibility of lipid exchange as a regulatory mechanism of EPCR activity driven by the endothelially expressed secretory group V phospholipase A2 (sPLA2-V). We identified phosphatidylcholine (PCh) as the major phospholipid bound to human soluble EPCR (sEPCR). PCh in EPCR could be exchanged for lysophosphatidylcholine (lysoPCh) and platelet activating factor (PAF). Remarkably, lysoPCh and PAF impaired the protein C binding ability of sEPCR. Inhibition of sPLA2-V, responsible for lysoPCh and PAF generation, improved APC binding to endothelial cells. EPCR-dependent protein C activation and APC antiapoptotic effect were thus significantly enhanced. In contrast, endothelial cell supplementation with sPLA2-V inhibited both APC generation and its antiapoptotic effects. We conclude that APC generation and function can be modulated by changes in phospholipid occupancy of its endothelial cell receptor.

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.

Group V secretory phospholipase A(2) (sPLA(2)-V) is highly expressed in the heart. This study examined (i) the role of sPLA(2)-V in myocardial ischaemia-reperfusion (I/R) injury and (ii) the cooperative action of sPLA(2)-V and cytosolic PLA(2) (cPLA(2)) in myocardial I/R injury, using sPLA(2)-V knockout (sPLA(2)V(-/-)) mice.Myocardial I/R injury was created by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. The sPLA(2)V(-/-) mice had a 44% decrease in myocardial infarct size, a preservation of echocardiographic LV function (%fractional shortening: 40 ± 3.5 vs. 21 ± 4.6, respectively), and lower content of leucotriene B(4) (LTB(4)) and thromboxane B(2) (TXB(2)) (40 and 37% lower, respectively) in the ischaemic myocardium after I/R compared with wild-type (WT) mice. Intraperitoneal administration of AACOCF3 or MAFP, inhibitors of cPLA(2) activity, decreased myocardial infarct size and myocardial content of LTB(4) and TXB(2) in both genotyped mice. The decrease in myocardial infarct size and content of LTB(4) and TXB(2) after cPLA(2) inhibitor administration was greater in WT mice than in sPLA(2)V(-/-) mice. I/R increased phosphorylation of extracellular signal-related kinase 1/2, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in the ischaemic myocardium in association with cPLA(2) phosphorylation. The I/R-induced increase in the phosphorylation of p38 and cPLA(2) was less in sPLA(2)-V(-/-) mice than in WT mice. Pretreatment with the p38 inhibitor SB202190 suppressed an increase in cPLA(2) phosphorylation after I/R in WT mice.sPLA(2)-V plays an important role in the pathogenesis of myocardial I/R injury partly in concert with the activation of cPLA(2).

We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by D. farinae, and whose function is also regulated by group V sPLA2.

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A2 (sPLA2-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA2-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased > 60-fold and sPLA2-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA2-V–LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA2-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.

LDL particles that enter the arterial intima become exposed to proteolytic and lipolytic modifications. The extracellular hydrolases potentially involved in LDL modification include proteolytic enzymes, such as chymase, cathepsin S, and plasmin, and phospholipolytic enzymes, such as secretory phospholipases A2 (sPLA2-IIa and sPLA2-V) and secretory acid sphingomyelinase (sSMase). Here, LDL was first proteolyzed and then subjected to lipolysis, after which the effects of combined proteolysis and lipolysis on LDL fusion and on binding to human aortic proteoglycans (PG) were studied. Chymase and cathepsin S led to more extensive proteolysis and release of peptide fragments from LDL than did plasmin. sPLA2-IIa was not able to hydrolyze unmodified LDL, and even preproteolysis of LDL particles failed to enhance lipolysis by this enzyme. However, preproteolysis with chymase and cathepsin S accelerated lipolysis by sPLA2-V and sSMase, which resulted in enhanced fusion and proteoglycan binding of the preproteolyzed LDL particles. Taken together, the results revealed that proteolysis sensitizes the LDL particles to hydrolysis by sPLA2-V and sSMase. By promoting fusion and binding of LDL to human aortic proteoglycans, the combination of proteolysis and phospholipolysis of LDL particles potentially enhances extracellular accumulation of LDL-derived lipids during atherogenesis.

Some isoforms of secretory phospholipase A(2) (sPLA(2)) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA(2) isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6-48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA(2) were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.

Secreted phospholipases A(2) (sPLA(2)s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA(2) isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA(2) (sPLA(2)-V). Furthermore, it has recently been shown that sPLA(2)-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA(2)-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA(2)-V null mice (sPLA(2)-V(-/-)) and control wild-type (WT) littermates. We observed that LPS (1 microg/ml)-mediated leukocyte emigration in sPLA(2)-V(-/-) was attenuated by 52% and 86% upon 6 and 12 h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA(2) inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA(2)-V(-/-) mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA(2)-V(-/-) mice as compared to control WT mice. Together, our data demonstrate the role of sPLA(2)-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA(2)-V in the development of inflammatory innate immune responses.

Phospholipase A(2) (PLA(2)) hydrolyzes the sn-2 position of cell membrane phospholipids to release fatty acids and lysophospholipids. We have previously reported that group V secretory PLA(2) (sPLA(2)) translocates from the Golgi and recycling endosomes of mouse peritoneal macrophages to newly formed phagosomes and regulates the phagocytosis of zymosan, suggesting a role in innate immunity. Here we report that in macrophages lacking group V sPLA(2), phagosome maturation was reduced 50-60% at early time points while the binding of zymosan was unimpaired. The ability of group V sPLA(2) to regulate phagocytosis extended to phagocytosis of IgG- and complement-opsonized sheep RBC. Moreover, macrophages lacking group V sPLA(2) had delays in phagocytosis, phagosome maturation, and killing of Candida albicans. Cytokine production and eicosanoid generation were not impaired by the lack of group V sPLA(2). Furthermore, in a model of systemic candidiasis, mice lacking group V sPLA(2) had an increased fungal burden in the kidney, liver, and spleen at day 7 postinfection and increased mortality. Thus, group V sPLA(2) regulates phagocytosis through major phagocytic receptors and contributes to the innate immune response against C. albicans by regulating phagocytosis and killing through a mechanism that is likely dependent on phagolysosome fusion.

Objective Hydrolysis of LDL by phospholipase A 2 (PLA 2 ) generates free fatty acids (FFAs) and lysophospholipids (lysoPCs). Binding of the PLA 2 -modified LDL to proteoglycans, and their uptake by macrophages are increased. Since the extracellular pH is locally decreased in advanced atherosclerotic plaques, we examined the effects of acidic pH on PLA 2 -induced LDL modification and its proatherogenic consequences. Results LDL particles were avidly hydrolyzed by sPLA 2 -V at pH range 7.5–5.5. With decreasing pH, the ability of albumin to sequester the formed FFAs and lysoPCs from the sPLA 2 -V-modified LDL particles decreased, and, as a consequence, more of the hydrolytic products accumulated in the particles. At acidic pH, the sPLA 2 -V-modified LDL particles had higher binding strength to human aortic proteoglycans, and their uptake by human monocyte-derived macrophages and ensuing foam cell formation were enhanced. Conclusions The present data show that the proatherogenic effects exerted by sPLA 2 -V-induced lipolysis of LDL are enhanced with decreasing pH and suggest that sPLA 2 -V is particularly atherogenic in advanced atherosclerotic lesions, in which local acidic conditions prevail.

El pdf del artículo es la versión pre-print., Macrophages can be activated through TLRs for a variety of innate immune responses. In contrast with the wealth of data existing on TLR-dependent gene expression and resultant cytokine production, very little is known on the mechanisms governing TLR-mediated arachidonic acid (AA) mobilization and subsequent eicosanoid production. We have previously reported the involvement of both cytosolic group IVA phospholipase A2 (cPLA2) and secreted group V phospholipase A2 (sPLA2-V) in regulating the AA mobilization response of macrophages exposed to bacterial LPS, a TLR4 agonist. In the present study, we have used multiple TLR agonists to define the role of various PLA2s in macrophage AA release via TLRs. Activation of P388D1 and RAW2647.1 macrophage-like cells via TLR1/2, TLR2, TLR3, TLR4, TLR6/2, and TLR7, but not TLR5 or TLR9, resulted in AA mobilization that appears to involve the activation of both cPLA2 and sPLA2 but not of calcium-independent phospholipase A2. Furthermore, inhibition of sPLA2-V by RNA interference or by two cell-permeable compounds, namely scalaradial and manoalide, resulted in a marked reduction of the phosphorylation of ERK1/2 and cPLA2 via TLR1/2, TLR2, TLR3, and TLR4, leading to attenuated AA mobilization. Collectively, the results suggest a model whereby sPLA2-V contributes to the macrophage AA mobilization response via various TLRs by amplifying cPLA2 activation through the ERK1/2 phosphorylation cascade. Copyright © 2009 by The American Association of Immunologists, Inc., This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055 and BFU2007-67154).

Objective— In vitro data indicate that human LDL modified by Group V secretory phospholipase A 2 (GV sPLA 2 ) is proatherogenic. Consistent with this, gain and loss of function studies demonstrated that GV sPLA 2 promotes atherosclerosis in LDLR −/− mice. The current study investigates whether GV sPLA 2 promotes atherosclerotic processes in apoE −/− mice. Methods and Results— LDL ( d =1.019 to 1.063) from apoE −/− and LDLR −/− mice fed chow or Western diet were hydrolyzed by GV sPLA 2 . Phosphatidylcholine on LDL from LDLR −/− mice fed either a chow or Western diet was hydrolyzed to a greater extent (61.1±0.4% and 45.3±4.6%) than the corresponding fractions from apoE −/− mice (41.7±3.6% and 39.4±1.2%). ApoE −/− LDL induced macrophage foam cell formation in vitro without modification by GV sPLA 2 , whereas hydrolysis of LDLR −/− LDL was a prerequisite for foam cell formation. In contrast to findings in LDLR −/− mice, GV sPLA 2 deficiency did not significantly reduce atherosclerosis in apoE −/− mice, although collagen content was significantly reduced in lesions of apoE −/− mice lacking GV sPLA 2 . Conclusions— The ability of GV sPLA 2 to promote atherosclerotic lipid deposition in apoE −/− and LDLR −/− mice may be related to its ability to increase the atherogenic potential of LDL from these mice as assessed in vitro.

The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.

Of 10 mammalian secreted phospholipase A(2) (sPLA(2)) enzymes identified to date, group V and X sPLA(2)s, which are two potent plasma membrane-acting sPLA(2)s, are capable of preventing host cells from being infected with adenovirus, and this anti-viral action depends on the conversion of phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) in the host cell membrane. Here, we show that human group III sPLA(2), which is structurally more similar to bee venom PLA(2) than to other mammalian sPLA(2)s, also has the capacity to inhibit adenovirus infection into host cells. Mass spectrometry (MS) demonstrated that group III sPLA(2) hydrolyzes particular molecular species of PC to generate LPC in human bronchial epithelial cells. Remarkably, in addition to the catalytically active sPLA(2) domain, the N-terminal, but not C-terminal, domain unique to this enzyme was required for the anti-adenovirus effect. To our knowledge, this is the first demonstration that the biological action of group III sPLA(2) depends on its N-terminal domain. Finally, our MS analysis provided additional and novel evidence that group III, V and X sPLA(2)s target distinct phospholipid molecular species in cellular membranes.

Although surgical resection is the first choice for oral cancer, the development of new anti-cancer drugs is of great interest. The effect of the histone deacetylation inhibitor, sodium butyrate (NaBu) on oral cancer cell (OCC) HSC-3 and HSC-4 proliferation in vitro was investigated. The synthesis of rate-limiting enzymes such as sPLA2 (-IIA, -V, -X) and COX-2 was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, as well as PGE2 by ELISA. NaBu acted in a concentration-dependent manner. Over 3 mM, it inhibited OCC proliferation, due to increased p21 expression and cell cycle arrest in the G2/M-phase. At low concentration (or = 1 mM), NaBu showed no effects or enhanced cell proliferation. NaBu also regulated COX-2 and sPLA2-X expression, and augmented PGE2 synthesis in OCC. These results indicate that NaBu is a novel candidate agent for the treatment of oral cancer. The treatment efficacy must be investigated in additional experiments considering NaBu concentration and tumor cell heterogeneity.

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A2 (sPLA2-V) was required for COX-2 induction, but the nature of the sPLA2-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA2-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA2-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA2-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA2-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel. Copyright © 2007 by The American Association of Immunologists, Inc., This work was supported by the Spanish Ministry of Education and Science (Grants BFU2004-01886/BMC and SAF2004-04676), the Fundación La Caixa (Grant BM05-248-0), and the Spanish Ministry of Health (ISCIII-RETIC RD06).

Animal and human studies suggest that both secretory PLA2 (sPLA2)-V and sPLA2-IIA (encoded, respectively, by the neighbouring PLA2G5 and PLA2G2A genes) contribute to atherogenesis. Elevated plasma sPLA2-IIA predicts coronary heart disease (CHD) risk, but no mass assay for sPLA2-V is available. We previously reported that tagging single nucleotide polymorphism (tSNP) haplotypes of PLA2G2A are strongly associated with sPLA2-IIA mass, but not lipid levels. Here, we use tSNPs of the sPLA2-V gene to investigate the association of PLA2G5 with CHD risk markers. Seven PLA2G5 tSNPs genotypes, explaining >92% of the locus genetic variability, were determined in 519 patients with Type II diabetes (in whom PLA2G2A tSNP data was available), and defined seven common haplotypes (frequencies >5%). PLA2G5 and PLA2G2A tSNPs showed linkage disequilibrium (LD). Compared to the common PLA2G5 haplotype, H1 (frequency 34.9%), haplotypes H2-7 were associated with overall higher plasma LDL (P < 0.00004) and total cholesterol (P < 0.00003) levels yet lower oxLDL/LDL (P = 0.006) and sPLA2-IIA mass (P = 0.04), probably reflecting LD with PLA2G2A. Intronic tSNP (rs11573248), unlikely itself to be functional, distinguished H1 from LDL-raising haplotypes and may mark a functional site. In conclusion, PLA2G5 tSNP haplotypes demonstrate an association with total and LDL cholesterol and oxLDL/LDL, not seen with PLA2G2A, thus confirming distinct functional roles for these two sPLA2s.

Confocal immunofluorescence analysis indicated a relatively high localization of group V secretory phospholipase A(2) (GV) in the nuclei of cultured PC12 and U251 astrocytoma cells. Here, we report the biochemical evidence for the presence of a secretory PLA(2) in the nuclei of neuronal and glial cells from rat brain cortex. Enzymic activity was determined using [(3)H]oleate labelled Escherichia coli membranes in intact nuclei and in their soluble fractions in which the specific activity was significantly more elevated. The treatment of soluble nuclear fractions with inhibitors of cytosolic Ca(2+)-dependent or Ca(2+)-independent phospholipases A(2) was ineffective whereas DTT or Indoxam, a specific inhibitor of all isoforms of sPLA(2), abolished enzyme activity. The enzyme was identified as group V secretory phospholipase A(2) (GV) by Western blot analysis and its nucleoplasmic localization was demonstrated by CLSM.

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.

We previously showed that group V secretory phospholipase A(2) (sPLA(2)V) is inhibited by sphingomyelin (SM), but activated by ceramide. Here, we investigated the effect of sphingolipid structure on the activity and acyl specificity of sPLA(2)V. Degradation of HDL SM to ceramide, but not to ceramide phosphate, stimulated the activity by 6-fold, with the release of all unsaturated fatty acids being affected equally. Ceramide-enrichment of HDL similarly stimulated the release of unsaturated fatty acids. Incorporation of SM into phosphatidylcholine (PC) liposomes preferentially inhibited the hydrolysis of 16:0-20:4 PC. Conversely, SMase C treatment or ceramide incorporation resulted in preferential stimulation of hydrolysis of 16:0-20:4 PC. The presence of a long chain acyl group in ceramide was essential for the activation, and long chain diacylglycerols were also effective. However, ceramide phosphate was inhibitory. These studies show that SM and ceramide in the membranes and lipoproteins not only regulate the activity of phospholipases, but also the release of arachidonate, the precursor of eicosanoids.

Phospholipase A2s (PLA2) are a class of enzymes, which catalyze the hydrolysis of membrane phospholipids at the sn-2 position to release fatty acids and lysophospholipids. When the fatty acid is arachidonic acid (AA), a complementary metabolism leads to pro-inflammatory mediators collectively known as eicosanoids. Thus, inhibiting PLA2 activity remains a prime target for the development of new drugs for the treatment of inflammation-related diseases. More than one type of PLA2s plays a major role in inflammatory disease conditions. In the present study, quantitative structure-activity relationship (QSAR) study was performed for a series of 48 Me-indoxam derivatives as human group V PLA, (hVPLA2) inhibitors, using molecular operating environment (MOE) software. The hVPLA2 is a secretory PLA2 (sPLA2), involved in eicosanoid formation in inflammatory cells such as macrophages and mast cells. These studies have come out with three good predictive models (r = 0.82-0.84), which are cross-validated (rcv = 0.68-0.70) by leave-out-one method (Loo). The positive correlation of spatial descriptor Pmiz with inhibitory activity shows that proper orientation of the substitution at R position towards Z-axis is necessary to facilitate the possible interactions of the indole core with active site residues of the PLA2 enzyme. The negative contribution of b_rotN (atom and bond count-type descriptor) suggests that increasing flexibility conferred by the R substitution is detrimental for the activity. In addition to the hVPLA2 inhibitory activity is found to be highly influenced by molecular size, energy and polarity of the Me-indoxam derivatives.

Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.

Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.

Group V sPLA(2) is unique among the family of secretory sPLA(2) enzymes in being able to bind to cell membranes through both interfacial-binding and through binding to proteoglycan. The function of group V sPLA(2) as an enzyme and its cross-talk with cPLA(2)alpha in initiating eicosanoid generation is well documented. Evidence, though, is emerging on the ability of this molecule to act as a regulator of several intracellular and extracellular pathways independently of its ability to provide arachidonic acid for eicosanoid generation, acting within the cell or as a secreted enzyme. In this article we will provide an overview of the properties of the enzyme and how they relate to our current understanding of its function.

Clinical observations strongly support an association of circulating levels of secretory phospholipases A 2 (sPLA 2 ) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA 2 -IIA and sPLA 2 -V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.

sPLA2 (secretory phospholipase A2) enzymes have been implicated in various biological events, yet their precise physiological functions remain largely unresolved. In the present study we show that group V and X sPLA2s, which are two potent plasma membrane-acting sPLA2s, are capable of preventing host cells from being infected with an adenovirus. Bronchial epithelial cells and lung fibroblasts pre-expressing group V and X sPLA2s showed marked resistance to adenovirus-mediated gene delivery in a manner dependent on their catalytic activity. Although adenovirus particles were insensitive to recombinant group V and X sPLA2s, direct addition of these enzymes to 293A cells suppressed both number and size of adenovirus plaque formation. Group V and X sPLA2s retarded the entry of adenovirus into endosomes. Moreover, adenoviral infection was suppressed by LPC (lysophosphatidylcholine), a membrane-hydrolytic product of these sPLA2s. Thus hydrolysis of the plasma membrane by these sPLA2s may eventually lead to the protection of host cells from adenovirus entry. Given that group V and X sPLA2s are expressed in human airway epithelium and macrophages and that the expression of endogenous group V sPLA2 is upregulated by virus-related stimuli in these cells, our present results raise the possibility that group V and X sPLA2s may play a role in innate immunity against adenoviral infection in the respiratory tract.

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.

Acute inflammatory responses to invading bacteria such as Staphylococcus aureus include mobilization of polymorphonuclear leukocytes (PMN) and extracellular group IIA phospholipase A2 (gIIA-PLA2). Although accumulating coincidentally, the in vitro anti-staphylococcal activities of PMN and gIIA-PLA2 have thus far been studied separately. We now show that degradation of S. aureus phospholipids during and after phagocytosis by human PMN requires the presence of extracellular gIIA-PLA2. The concentration of extracellular gIIA-PLA2 required to produce bacterial digestion was reduced 10-fold by PMN. The effects of added gIIA-PLA2 were greater when present before phagocytosis but even apparent when added after S. aureus were ingested by PMN. Related group V and X PLA2, which are present within PMN granules, do not contribute to bacterial phospholipid degradation during and after phagocytosis even when added at concentrations 30-fold higher than that needed for action of the gIIA-PLA2. The action of added gIIA-PLA2 required catalytically active gIIA-PLA2 and, in PMN, a functional NADPH oxidase but not myeloperoxidase. These findings reveal a novel collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Secretory phospholipase A2 is associated with ischaemic injury in the human heart, but the distribution of type V secretory phospholipase A2 (sPLA2-V) remains unknown. The significance of sPLA2-V in myocardial infarction was investigated histopathologically.Sequential changes in the localization of sPLA2-V and its mRNA in myocardial tissues obtained from 30 autopsied hearts were examined by immunohistochemistry and in situ hybridization and compared with those of fibronectin, vascular endothelial growth factor (VEGF), interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, and cyclooxygenase-2 (COX-2).No expression of sPLA2-V was observed in normal heart, but it was promptly expressed in wavy myofibres positive for fibronectin just after the onset of infarction. sPLA2-V was subsequently expressed in ischaemic cardiomyocytes around the lesion. The expression decreased at the granulation tissue and disappeared at the chronic stage with scar formation. The distribution of the signal for sPLA2-V mRNA paralleled that of the protein. Ischaemic myocytes around the lesion expressed VEGF, IL-1beta, TNF-alpha and COX-2 at all stages.sPLA2-V production in myocardium is limited to the acute phase of infarction. sPLA2-V may play a dual role, acting both to remove degraded cell-membrane through cooperative activity with COX-2 in necrotic areas and to attack ischaemic myocytes around the lesion via degradation of membrane phospholipids.

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA2s were compared at a concentration of 1 microg/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA2 were up to 20 times more reactive than group IIA sPLA2. Group X sPLA2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA2s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA2, as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.

Background and Aim: Type V secretory phospholipase A 2 (sPLA 2 -V) is a key enzyme in the arachidonate cascade. However, the distribution of SPLA 2 -V in human liver has not yet been investigated. In this study, the significance of sPLA 2 -V expression in human hepatocytes damaged by liver disease was investigated. Methods: Samples of liver tissue from patients with chronic hepatitis B and C, hepatitis virus-related liver cirrhosis, and congestive hepatocyte injury were immunostained with antibodies against sPLA 2 -V, cyclooxygenase (COX)-2, hepatitis viral antigens, transforming growth factor (TGF)-β 1 , interleukin (IL)-1β and tumor necrosis factor (TNF)-a. Results: In chronic hepatitis patients, sPLA 2 -V-positive hepatocytes were scattered in the liver lobules, while cyclooxygenase-2, IL-1β, and TNF-a were diffusely expressed. Hepatocytes around necroinflammatory lesions were strongly positive for sPLA 2 -V. Some sPLA 2 -V-positive hepatocytes were also positive for viral antigens. TGF-β 1 was expressed only in fibrotic lesions. The pattern of distribution of these proteins in liver cirrhosis patients was similar to that in chronic hepatitis patients, but sPLA 2 -V expression tended to be more intense than in chronic hepatitis. In the congestive liver, sPLA 2 -V, COX-2, and the two cytokines were diffusely expressed in surviving hepatocytes. Conclusions: sPLA 2 -V expression in hepatocytes is induced by viral infection, fibrosis, and circulatory disturbance. Immunostaining using sPLA 2 -V antibody is useful for the detection of injured hepatocytes in patients with liver diseases.

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.

Acute inflammation can be considered in terms of a series of checkpoints where each phase of cellular influx, persistence, and clearance is controlled by endogenous stop and go signals. It is becoming increasingly apparent that in addition to initiating the inflammatory response, eicosanoids may also mediate resolution. This suggests there are two phases of arachidonic acid release: one at onset for the generation of proinflammatory eicosanoids and one at resolution for the synthesis of proresolving eicosanoids. What is unclear is the identity of the phospholipase (PLA2) isoforms involved in this biphasic release of arachidonic acid. We show here that type VI iPLA2 drives the onset of acute pleurisy through the synthesis of PGE2, LTB4, PAF, and IL-1beta. However, during resolution there is a switch to a sequential induction of first sPLA2 (types IIa and V) that mediates the release of PAF and lipoxin A4, which, in turn, are responsible for the subsequent induction of type IV cPLA2 that mediates the release of arachidonic acid for the synthesis of proresolving prostaglandins. This study is the first of its kind to address the respective roles of PLA2 isoforms in acute resolving inflammation and to identify type VI iPLA2 as a potentially selective target for the treatment of inflammatory diseases.

Objectives— Secretory phospholipase A 2 (sPLA 2 ) enzymes hydrolyze the sn-2 fatty acyl ester bond of phospholipids to produce a free fatty acid and a lysophospholid. Group V sPLA 2 is expressed in cultured macrophage cells and has high affinity for phosphatidyl choline-containing substrates. The present study assesses the presence of group V sPLA 2 in human and mouse atherosclerotic lesions and its activity toward low-density lipoprotein (LDL) particles. Methods and Results— Group V sPLA 2 was detected in human and mouse atherosclerotic lesions by immunohistochemical staining. Electron microscopic analysis showed that mouse group V sPLA 2 -modified LDL is significantly smaller (mean diameter±SEM=25.3±0.25 nm) than native LDL (mean diameter±SEM=27.7±0.29 nm). Hydrolysis by group V sPLA 2 induced spontaneous particle aggregation; the extent of aggregation was directly proportional to the degree of LDL hydrolysis. Group V sPLA 2 modification of LDL led to enhanced lipid accumulation in cultured mouse peritoneal macrophage cells. Conclusions— Group V sPLA 2 may play an important role in promoting atherosclerotic lesion development by modifying LDL particles in the arterial wall, thereby enhancing particle aggregation, retention, and macrophage uptake.

Conclusions regarding the contribution of low molecular weight secretory phospholipase A2 (sPLA2) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA2 enzymes. To elucidate the role of group V sPLA2, we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C4 and prostaglandin E2 was attenuated approximately 50% in peritoneal macrophages from group V sPLA2-null mice compared with macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA2-null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.

Both secretory and cytosolic phospholipase A2 enzymes have been implicated in the pathogenesis of arthritis in animal models, but the exact expression patterns of the enzymes in diseased human joint tissue are uncertain. We investigated the messenger RNA expression of group IIa, IValpha and V phospholipase A2 and localized the presence of group IIa and IValpha phospholipase A2 at protein levels in articular cartilage from patients with rheumatoid arthritis, osteoarthritis and patients with non-arthritic joints. Both group IIa phospholipase A2 messenger RNA and protein were detected in all samples independent of diagnosis, but were far more prominent in cartilage from rheumatoid arthritis samples. In cartilage with rheumatoid arthritis, the enzyme was detected both within the chondrocytes and in the extracellular matrix, whereas only few osteoarthritic cartilage samples showed positive staining in the matrix. In the cartilage matrix of non-arthritic controls, group IIa phospholipase A2 was totally absent. Messenger RNA for the group IValpha and V phospholipase A2 was, except for one osteoarthritic cartilage sample, exclusively detected in rheumatoid arthritic cartilage. For group IValpha phospholipase A2 this was also confirmed at the protein level. These results suggest that each phospholipase A2 enzyme has distinct roles in both healthy and diseased joint tissue, and that the diversity and amount of enzyme correlate with the grade of inflammation and disease severity.

In macrophages and other major immunoinflammatory cells, two phospholipase A2 (PLA2) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A2 (cPLA2) and a secreted phospholipase A2 (sPLA2). In this study, the molecular mechanism underlying cross-talk between the two PLA2s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA2-dependent manner, and addition of exogenous group V sPLA2 to the activated cells increases the release. This sPLA2 effect is abolished if the cells are pretreated with cPLA2 inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA2. In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA2 activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA2 response to levels found in cells displaying normal cPLA2 activity. Moreover, 12- and 15-HPETE are able to enhance sPLA2 activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA2-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA2 with cPLA2 for a full AA mobilization response.

Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IValpha cytosolic PLA2 (cPLA2alpha) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i). MC+/+, which lack group IIa and V PLA2s, and (ii). MC-/-, which lack groups IIa, V, and IValpha PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC-/-. It has been argued that cPLA2alpha plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IValpha PLA2s were expressed in MC-/- or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2alpha in H2O2-treated MC-/- increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC-/-. When sPLA2 and cPLA2alpha are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N',N'-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2alpha and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2alpha is responsible for AA release.