Phospholipase A2 Type Research Articles

Inhibition of Secretory Phospholipase A2 Activity Attenuates Acute Cardiogenic Pulmonary Edema Induced by Isoproterenol Infusion in Mice After Myocardial Infarction

Several types of secretory phospholipase A2 (sPLA2) are expressed in lung tissue, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA2 activity attenuates acute cardiogenic pulmonary edema in mice. Acute cardiogenic pulmonary edema was induced in C57BL/6J male mice by an increase in heart rate with continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/h) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg LY374388, a prodrug of LY329722 that inhibits sPLA2 activity, or vehicle was administered. The ISP infusion after myocardial infarction induced interstitial and alveolar edema on lung histology. Furthermore, it increased the lung-to-body weight ratio, pulmonary vascular permeability evaluated by the Evans blue extravasation method, lung activity of sPLA2, and lung content of thromboxane A2 and leukotriene B4. These changes were significantly attenuated by LY374388 treatment. In Kaplan-Meier analysis, the survival rate during the ISP infusion after myocardial infarction was significantly higher in LY374388- than in vehicle-treated mice. Similar results were obtained with another inhibitor of sPLA2 activity, para-bromophenacyl bromide. In conclusion, inhibition of sPLA2 activity suppressed acute cardiogenic pulmonary edema.

Secretory phospholipase A2 type III enhances α-secretase-dependent amyloid precursor protein processing through alterations in membrane fluidity

In the non-amyloidogenic pathway, amyloid precursor protein (APP) is cleaved by alpha-secretases to produce alpha-secretase-cleaved soluble APP (sAPP(alpha)) with neuroprotective and neurotrophic properties; therefore, enhancing the non-amyloidogenic pathway has been suggested as a potential pharmacological approach for the treatment of Alzheimer's disease. Here, we demonstrate the effects of type III secretory phospholipase A(2) (sPLA(2)-III) on sAPP(alpha) secretion. Exposing differentiated neuronal cells (SH-SY5Y cells and primary rat neurons) to sPLA(2)-III for 24 h increased sAPP(alpha) secretion and decreased levels of Abeta(1-42) in SH-SY5Y cells, and these changes were accompanied by increased membrane fluidity. We further tested whether sPLA(2)-III-enhanced sAPP(alpha) release is due in part to the production of its hydrolyzed products, including arachidonic acid (AA), palmitic acid (PA), and lysophosphatidylcholine (LPC). Addition of AA but neither PA nor LPC mimicked sPLA(2)-III-induced increases in sAPP(alpha) secretion and membrane fluidity. Treatment with sPLA(2)-III and AA increased accumulation of APP at the cell surface but did not alter total expressions of APP, alpha-secretases, and beta-site APP cleaving enzyme. Taken together, these results support the hypothesis that sPLA(2)-III enhances sAPP(alpha) secretion through its action to increase membrane fluidity and recruitment of APP at the cell surface.

Staphylococcus aureus Enriched in Ordered Lipids Present Resistance Towards the Antibacterial Agent sPLA2-IIA: An Unusual Mechanism to Survive

Bacterial membranes present solid-ordered/liquid-disordered (so/ld) cooperative melting event close to physiological temperature. The cellular advantage of this thermotropic melting event is yet to be determined. We show that this thermal behavior provides resistance towards a membrane active antibacterial agent. Phospholipase A2 type IIA (sPLA2-IIA) is a hydrolytic enzyme which presents antibacterial properties towards Gram positive bacteria. The enzyme has higher activity in ld phase compared to so phase in anionic membranes. We show that the lipid phase behavior of Staphylococcus aureus (S.aureus) membranes as measured by FTIR modulates sPLA2-IIA by inducing a sharp drop in activity below the melting temperature of the membrane (centered at 15.3°C). The effects of sPLA2-IIA treatment on cell viability are also investigated. While above the main melting event viability drops to 20% of the initial CFU after treatment, below the main melting event cell viability only drops to 60% under the same treatment. This strongly suggests that cells in the solid-ordered phase are better adapted to survive the enzymatic insult. These results led us to explore if a subpopulation of S.aureus enriched in ordered lipids can be selected after repeated treatment with sPLA2-IIA at 37°C. After selecting for resistance at 37°C we measured growth curves, membrane order, and cell viability as a function of treatment temperature. The results suggest that at 37°C there is a bacterial subpopulation with increased membrane rigidity that insures survival of the colony to an insult by sPLA2-IIA. This subpopulation also presents a longer latency period which can be explained by the increased presence in ordered lipids, which are known to inhibit cell division. Even if the growth conditions are not optimal, the presence of this subpopulation ensures survival from the antibacterial insult.

Phospholipase A2 inhibitors as potential therapeutic agents for the treatment of inflammatory diseases

Importance of the field: The various phospholipase A2 (PLA2) types have been implicated in diverse kinds of lipid signaling and inflammatory diseases. Rheumatoid arthritis, lung inflammation, neurological disorders, such as multiple sclerosis, cardiovascular diseases, including atherosclerosis, and cancer are included among the diseases where PLA2 enzymes are involved. Thus, there is a great interest in developing potent and selective PLA2 inhibitors and some of them have entered clinical trials.Areas covered in this review: This review article discusses the role of each PLA2 class in inflammatory diseases and the advances in the development of inhibitors presented in patent literature from January 2004 to May 2009.What the reader will gain: PLA2s cluster in four main types: secreted sPLA2, cytosolic cPLA2, Ca2+-independent iPLA2 and lipoprotein-associated LpPLA2. Each of those types has been implicated in diverse kinds of inflammatory diseases. Readers will rapidly gain an overview of the various PLA2 inhibitors reported in the patent literature in the past 5 years. Furthermore, the readers will learn the difficulties related to the development of PLA2 inhibitors as new drugs and also the different companies and research groups that are the main players in the field.Take home message: Although the role of each PLA2 is not yet distinct in different diseases, the development and future use of different PLA2 inhibitors to treat human disease seems very promising.

The antibacterial activity of phospholipase A2 type IIA is regulated by the cooperative lipid chain melting behavior in Staphylococcus aureus

As one of its primary physiological functions, sPLA(2)-IIA appears to act as an antibacterial agent. In particular, sPLA(2)-IIA shows high activity towards Gram-positive bacteria such as Staphylococcus aureus (S. aureus). This antibacterial activity results from the preference of the enzyme towards membranes enriched in anionic lipids, which is a common feature of bacterial membranes. An intriguing aspect observed in a variety of bacterial membranes is the presence of a broad but cooperative lipid chain melting event where the lipids in the membrane transition from a solid-ordered (so) into a liquid-disordered (ld) state close to physiological temperatures. It is known that the enzyme is sensitive to the level of lipid packing, which changes sharply between the so and the ld states. Therefore, it would be expected that the enzyme activity is regulated by the bacterial membrane thermotropic behavior. We determine by FTIR the thermotropic lipid chain melting behavior of S. aureus and find that the activity of sPLA(2)-IIA drops sharply in the so state. The activity of the enzyme is also evaluated in terms of its effects on cell viability, showing that cell survival increases when the bacterial membrane is in the so state during enzyme exposure. These results point to a mechanism by which bacteria can develop increased resistance towards antibacterial agents that act on the membrane through a cooperative increase in the order of the lipid chains. These results show that the physical behavior of the bacterial membrane can play an important role in regulating physiological function in an in vivo system.

25‐hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR

In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.

Peroxiredoxin VI in Complex With a Transition Inhibitor

Peroxiredoxin VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs. Its antioxidant properties are due to its ability to reduce hydroperoxides found in lung surfactants and thus prevent the toxicity associated with hyperoxia. Prdx6 is a bifunctional protein that contains two distinct active sites. One active site catalyzes a phospholipase A2 (PLA2) type hydrolysis of phospholipids, and the second active site catalyzes the reduction of lipid hydroperoxides typical of 1‐cys peroxiredoxins. The structure of Prdx6 has been solved; however, there is no structural evidence to elucidate its catalytic mechanism. This study is designed to solve the structure of Prdx6 in complex with MJ33, a PLA2 transition‐state inhibitor, in an effort to understand the mechanism and conformational change Prdx6 undergoes during its catalytic cycle. Here we report that Prdx6 has been successfully purified to homogeneity. Our crystallization screens have produced protein crystals that have diffracted to a resolution of 2.8Å. This research is supported by the Chemistry Alumni Scholars.

Phospholipase A2 structure/function, mechanism, and signaling

Tremendous advances in understanding the structure and function of the superfamily of phospholipase A2 (PLA2) enzymes has occurred in the twenty-first century. The superfamily includes 15 groups comprising four main types including the secreted sPLA2, cytosolic cPLA2, calcium-independent iPLA2, and platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated (Lp)PLA2. We review herein our current understanding of the structure and interaction with substrate phospholipids, which resides in membranes for a representative of each of these main types of PLA2. We will also briefly review the development of inhibitors of these enzymes and their roles in lipid signaling.

Cholesterol Content And Domain Formation As Regulators Of PLA2-IIA Activity In Anionic Membranes

Phospholipase A2 type IIA (PLA2-IIA) hydrolyses the sn-2 position of glycerolipids to produce free fatty acids and lysolipids. The enzyme presents a strong affinity towards membranes enriched in anionic lipids, and lipid-packing is known to influence the ability of PLA2-IIA to extract lipids from the membrane. This leads to an interrelation between membrane structure, and enzyme activity. In this study we evaluate the activity of PLA2-IIA on unilamellar vesicles composed of either 1- palmitoyl-2-oleoyl phosphatidylglycerol (POPG) or dimyristoyl - phosphatidylglycerol (DMPG), in combination with cholesterol (Chol) and sphyngomyelin (SM). We expose the vesicles to PLA2-IIA and monitor the activity at 37°C, where both POPG and DMPG are in the liquid-disordered phase in their pure form. For DMPG/Chol or POPG/Chol the results show that adding cholesterol alone inhibits PLA2-IIA activity. However, this effect is more accentuated in DMPG vesicles compared to POPG vesicles. We attribute this difference to a closer interaction of cholesterol with the saturated acyl chains of DMPG, leading to tighter lipid packing and a reduced hydrolysis rate. In the second part of the study, for POPG and DMPG samples in which we included cholesterol and sphyngomyelin in equal molarities, we detect high hydrolytic activity in a wider range of compositions for a given Chol/PG ratio compared to samples without SM. We propose that a strong affinity between SM and cholesterol, related to liquid-ordered domain formation, leads to a depletion of cholesterol from the PG-rich regions, maintaining a high PLA2-IIA hydrolysis rate. In the third part of our study, we focus on changes in rigidity of the membranes after exposure to PLA2-IIA based on Laurdan General Polarization measurements. We find a general increase in rigidity following the hydrolytic burst in POPG binary and ternary systems.

Secretory Phospholipase A2 Type III Enhances α-secretase-dependent Amyloid Precursor Protein Processing by its Effect on Membrane Fluidity and Endocytosis

Owing to the non-amyloidogenic pathway of amyloid precursor protein (APP) processing to produce neuroprotective and neurotrophic α-secretase-cleaved soluble APP (sAPPα), and preclude the amyloidogenic pathway which produces neurotoxic amyloid-β peptide (Aβ), increasing α-secretase activity and sAPPα levels have been suggested as pharmacological approaches for treatment of Alzheimer's disease (AD). In this study, we demonstrated that cytokines including TNFα, IL-1β and IFNγ stimulated immortalized astrocytes (DITNC cells) to secrete secretory phospholipase A2 type III (sPLA2-III) into culture medium. When this conditioned medium was applied to differentiated human neuroblastoma (SH-SY5Y cells), it enhanced sAPPα secretion from cells. To further demonstrate the effect of sPLA2-III on sAPPα secretion, SH-SY5Y cells were exposed to sPLA2-III from bee venom, which is homologous to mammalian sPLA2-IIIs, and hydrolyzed products of sPLA2 including arachidonic acid (AA), lysophosphatidylcholine (LPC) and palmitic acid (PA). We found that either sPLA2-III or AA, but not LPC and PA, increased membrane fluidity, increased the localization of APP at the cell surface without altering the total APP expression in cells, and enhanced sAPPα secretion in SH-SY5Y cells. In addition, neither sPLA2-III nor AA altered the expressions of α-secretases including ADAM 9, 10, and 17. APP has the motif which can target to clathrin-coated pits. We demonstrated that monodansylcadaverine (MDC), clathrin-mediated endocytosis inhibitor, can increase sAPPα secretion from SH-SY5Y cells by reducing APP internalization. Based on these results, sPLA2-III-enhanced sAPPα secretion is suggested to be a consequence in part, due to increased membrane fluidity, and reduced APP internalization through clathrin-mediated endocytosis. Further study will focus on the effect of sPLA2-III on clathrin-mediated endocytosis.

Secretory phospholipase A2 type IIA: a regulator of immune function in atherosclerosis?

Soluble phospholipase A2 type IIA (sPLA2-IIA) is an acute-phase reactant that is markedly increased in inflammatory disorders including cardiovascular disease. Inflammatory cytokines such as interleukins IL-6 and IL-1β, interferon (IFN)-γ, and tumour necrosis factor (TNF)-α increase its expression in vascular smooth muscle cells and hepatocytes, two cell types largely responsible for its elevations in plasma. sPLA2-IIA is also highly expressed in atherosclerotic lesions and associated with smooth muscle cells, macrophages, and glycoaminoglycans,1 where it exerts proatherogenic effects in part by modifying low-and high-density lipoproteins (LDL and HDL). It hydrolyses the sn -2 ester bond in the glyceroacyl phospholipids present in both LDL and HDL and cell membranes, releasing fatty acids and lysophospholipids. sPLA2-IIA can also exert effects that are independent of its hydrolytic functions. It elevates IL-6 production by macrophages2 and contributes to elevations in cyclooxygenase-2 expression by mast cells:3 two cell types with significant roles in atherosclerosis. Thus, sPLA2-IIA is thought to promote atherosclerosis by hydrolysing lipoproteins and regulating macrophage and mast cell responses via non-hydrolytic mechanisms. In this issue of Cardiovascular Research , Ibeas et al. 4 provide additional new and somewhat unexpected insights as to how … *Corresponding author. Tel: +61 3 8532 1190; fax: 61 3 8532 1100. E-mail address : alex.bobik{at}bakeridi.edu.au

Abstract 2617: Inhibition of Secretory Phospholipase A 2 Activity Attenuates Acute Cardiogenic Pulmonary Congestion Induced by Isoproterenol Infusion in Mice with Post-Myocardial Infarction

Several types of secretory phospholipase A 2 (sPLA 2 ) are expressed in airway epithelial cells and alveolar inflammatory cells, yielding various eicosanoids that might cause pulmonary edema. This study examined whether inhibition of sPLA 2 activity attenuates acute cardiogenic pulmonary congestion in mice. Acute cardiogenic pulmonary congestion in 10-week-old male C57BL/6J mice was induced by continuous intravenous infusion of isoproterenol (ISP) (10 mg/kg/hr) at 2 weeks after the creation of myocardial infarction by left coronary artery ligation. Just before ISP infusion, a single intraperitoneal injection of 100 mg/kg of LY374388 (LY), a prodrug of LY329722 that is a specific inhibitor of activity of all sPLA 2 , or vehicle was administered. Arterial systolic pressure, heart rate, and LV end-diastolic pressure were comparably increased after ISP in both LY-mice and vehicle-mice. Also, lung expression levels of mRNA and protein of group V and X sPLA 2 were comparably increased from baseline by 3 ~ 5-fold in both groups of mice. sPLA 2 activity in lung tissue after ISP was increased from baseline by 5-fold in vehicle-mice, while the activity was not detected in LY-mice. In isolated neutrophils, incubation with LY329722 inhibited the respiratory burst and migration in response to fMLP or C5a by 40~50%. LY significantly reduced the lung-to-body weight ratio after ISP (71% of vehicle-mice) and attenuated alveolar neutrophil accumulation based on the lung histology (56% of vehicle-mice). Using the Evans Blue extravasation method, pulmonary vascular permeability was suppressed in LY-mice (Evans Blue content in lung; 66% of vehicle-mice). The lung content of thromboxane A 2 and leukotriene B 4 was increased in both groups, but was lower in LY-mice (71% and 64% of vehicle-mice, respectively). In Kaplan-Meyer analysis, the survival rate during the 12hr ISP infusion was higher in LY-mice than vehicle-mice (p < 0.01, n = 15 mice per group). Similar results were obtained with another inhibitor of sPLA 2 activity, para -bromophenacyl bromide. Inhibition of sPLA 2 activity suppressed acute cardiogenic pulmonary edema, and sPLA 2 inhibition may have therapeutic value in acute cardiogenic pulmonary congestion.

Phospholipase A2 Biochemistry

The phospholipase A(2) (PLA(2)) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. There are five main types of PLA(2): the secreted sPLA(2)'s, the cytosolic cPLA(2)'s, the Ca(2+)independent iPLA(2)'s, the PAF acetylhydrolases, and the lysosomal PLA(2)'s. This review focuses on the superfamily of PLA(2) enzymes, and then uses three specific examples of these enzymes to examine the differing biochemistry of the three main types of these enzymes. These three examples are the GIA cobra venom PLA(2), the GIVA cytosolic cPLA(2), and the GVIA Ca(2+)-independent iPLA(2).

Pro-inflammatory Secretory Phospholipase A2 Type IIA Binds to Integrins αvβ3 and α4β1 and Induces Proliferation of Monocytic Cells in an Integrin-dependent Manner

Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin alphavbeta3 at a high affinity (K(D)=2 x 10(-7) M). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to alpha4beta1. We showed that sPLA2-IIA competed with VCAM-1 for binding to alpha4beta1, and bound to a site close to those for VCAM-1 and CS-1 in the alpha4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins alphavbeta3 and alpha4beta1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrin-sPLA2-IIA interaction is a novel therapeutic target.

Secreted phospholipase A2 type IIA as a mediator connecting innate and adaptive immunity: new role in atherosclerosis

Human atherosclerotic plaques express markers of macrophage/dendritic cells as well as high levels of inflammatory proteins such as secreted phospholipase A(2) type IIA (sPLA(2)-IIA). To understand the cellular changes associated with the progress of atherosclerosis, we evaluated the role of sPLA(2)-IIA in mediating monocyte recruitment and differentiation into antigen-presenting cells. The effect of sPLA(2)-IIA on monocyte differentiation was evaluated in human THP-1 cells, a cellular line widely used as a model for monocyte-macrophage differentiation. Changes in functional processes, morphology and expression of antigens, characteristic of differentiated cells, were monitored over a 1-3 day period. sPLA(2)-IIA inhibited CD14 expression in a time- and concentration-dependent manner and upregulated dendritic cell-specific ICAM-3 grabbing non-integrin levels at the cell surface, findings that were the same for human monocytes. In addition, sPLA(2)-IIA-differentiated cells showed a dendritic cell phenotype characterized by the generation of fine dendritic protrusions and an increase in surface markers such as CD40, CD83, CD54, CD61, and CD62L. Furthermore, cell adhesion, migration, endocytic activity, and allogeneic T cell proliferation capacity were markedly increased after sPLA(2)-IIA treatment. sPLA(2)-IIA induces the differentiation of mononuclear cells and increases their adhesive and migratory capabilities, which suggests a novel function for sPLA(2)-IIA as a mediator connecting innate and adaptive immunity. These findings may provide insight into the immuno-inflammatory processes occurring in atherosclerosis, helping us to understand the cellular changes associated with the development of atherosclerosis.

Proteins Associated With the Early Intrauterine Equine Conceptus

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

Release of arachidonic acid induced by tumor necrosis factor-α in the presence of caspase inhibition: Evidence for a cytosolic phospholipase A2α-independent pathway

Stimulation of L929 cells with tumor necrosis factor-α (TNFα) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFα-treated L929 cells, its role in the TNFα-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A2 (PLA2). To find out the mechanisms underlying the TNFα-induced release of AA, we investigated the relationship between TNFα stimulation and PLA2 regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFα and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking α type of cytosolic PLA2 (cPLA2α)). Stimulation with TNFα/zVAD also caused the release of AA from L929-cPLA2α-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA2α) completely inhibited the TNFα-induced release of AA, but only partially inhibited the TNFα/zVAD-induced response in L929 cells. The TNFα/zVAD-induced release of AA from C12 and L929-cPLA2α-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA2 activity, decreased the amount of AA released by TNFα/zVAD. TNFα/zVAD appears to stimulate release of AA from C12 cells in a cPLA2α-independent, BHA-sensitive manner. The possible roles of secretory PLA2 and reactive oxygen species from different pools in the release of AA and cell death were discussed.

Endothelial lipase is associated with inflammation in humans

The aim of this study was to investigate the extent to which inflammation is linked with plasma endothelial lipase (EL) concentrations among healthy sedentary men. Plasma C-reactive protein (CRP) concentrations were measured with a highly sensitive commercial immunoassay, plasma interleukin-6 (IL-6) concentrations were measured using a commercial ELISA, and plasma secretory phospholipase A(2) type IIA (sPLA(2)-IIA) concentrations were measured using a commercial assay in a sample of 74 moderately obese men (mean body mass index, 29.8 +/- 5.2 kg/m(2)). Plasma EL concentrations were positively correlated with various indices of obesity, fasting plasma insulin, and plasma CRP, IL-6, and sPLA(2)-IIA concentrations. Multiple regression analyses revealed that plasma CRP concentrations explained 14.5% (P = 0.0008) of the variance in EL concentrations. When entered into the model, LPL activity accounted for 16.1% (P < 0.0001) and plasma CRP concentrations accounted for 20.9% (P < 0.0001) of the variance in EL concentrations. The combined impact of visceral adipose tissue (VAT) and of an inflammation score on EL concentrations was investigated. Among subjects with high or low VAT, those having a high inflammation score based on plasma CRP, IL-6, and sPLA(2)-IIA concentrations had increased plasma EL concentrations (P = 0.0005). In conclusion, our data reveal a strong association between proinflammatory cytokines and plasma EL concentrations among healthy people with low or high VAT levels.

Interferon γ-Induced Gene Expression of the Novel Secretory Phospholipase A2 Type IID in Human Monocyte-Derived Macrophages is Inhibited by Lipopolysaccharide

Phospholipase A(2) (PLA(2)) is a superfamily of enzymes that may play a major role in airways inflammation. We investigated the effect of interferon-gamma (IFN-gamma) on the gene expression of 19 different PLA(2) types in human monocyte-derived macrophages and nasal epithelial cells (RPMI 2650). The cells were stimulated with IFN-gamma for different lengths of time (up to 48 h), and the mRNA levels of the different PLA(2) types were determined by reverse transcriptase-PCR (RT-PCR) and normalized to those of the house-keeping gene, GAPDH. It appeared that IFN-gamma clearly increased the expression of secretory PLA(2) IID (but not IIA) in macrophages, while both PLA(2) IID and IIA were upregulated in RPMI 2650 cells. Moreover, after 18 h, the mRNA levels of cytosolic PLA(2) IVA were 2-3 times higher in IFN-gamma-stimulated macrophages than controls, while there was no such effect of IFN-gamma in RPMI 2650 cells. Lipopolysaccharide (LPS) augmented the increased gene expression of PLA(2) IVA but decreased both the basal and the IFN-gamma-induced PLA(2) IID mRNA expression in macrophages (but not in RPMI 2650 cells). The NF-kappaB inhibitor Pyrrolidine dithiocarbamate (PDTC) and the phoshatidylinositol 3-kinase (PI3K) inhibitor wortmannin were employed to get an insight into the mechanism behind these observations. Incubation of macrophages with PDTC had no effect on the LPS impairment of PLA(2) IID gene expression, but inhibited the LPS mediated activation of PLA(2) IVA. No significant effect was noted of PDTC on IFN-gamma stimulation, while PI3K had no effect at all on any of the stimuli used. Furthermore, LPS (but not IFN-gamma) increased the mRNA levels of the nuclear factor (NF)-kappaB inhibitors alpha and xi in macrophages, but not in RPMI 2650 cells. These findings indicate that (a) the gene expression of secretory types PLA(2) IID and IIA in response to IFN-gamma is much dependent on cell type, and (b) the regulation of PLA(2) type IID in human macrophages is clearly different from that of PLA(2) type IVA. (c) PLA(2) IVA is probably under control of both NF-kappaB and IFN-gamma-responsive elements (GRE) or IFN-gamma-activating sites (GAS). The possibility that PLA(2) IID is involved in cytokine-mediated inflammation in the nasal mucosa is inferred, as is the potential role of PLA(2) IID in the host defense against LPS-containing bacteria.

Paneth cells: leukocyte-like mediators of innate immunity in the intestine

Paneth cells are secretory intestinal epithelial cells located at the base of the crypts of Lieberkühn in the small intestine. They display prominent cytoplasmic granules, containing antibacterial proteins such as lysozyme, secretory phospholipase A2 type IIA, and alpha-defensins, which are released into the intestinal lumen in response to a range of stimuli. In this, they resemble circulating leukocytes, which also elaborate and secrete lysozyme and alpha-defensins as part of an antibacterial defense function, and the resemblance is sustained at other levels. The cells also strongly and specifically express the NOD2 gene product, one of an emerging family of critical, intracellular mediators of innate immune responses, which is also highly expressed in peripheral blood mononuclear cells, and they express RNA for tumor necrosis factor alpha, a major myelomonocytic cell-derived cytokine, which has a crucial role in the pathogenesis of diseases such as rheumatoid arthritis and Crohn's disease (CD). Thus, these cells, which are derived from the pluripotent intestinal epithelial stem-cell lineage, are sessile, resident host-defense cells, which may share with leukocytes the beneficial function of secreting antimicrobial peptides, as well as the potentially harmful capacity for promoting inflammation and tissue damage. Paneth cells are most abundant in the distal small intestine, which is the region most frequently affected by CD, and there is great interest in the potential role of these cells in this condition. This brief review summarizes current knowledge and speculates on how the study of these fascinating cells might be advanced.