Comparison of group I and II soluble phospholipases A2 activities on phagocytic functions of human polymorphonuclear and mononuclear phagocytes

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

Biochemical characterization of soluble phospholipase A2 from rheumatoid synovial fluid

RadiolabeledE. coli, Phosphatidylethanolamine (PE) and Phosphatidylcholine (PC), were used to characterize the phospholipase A₂ (PLA₂) activity in synovial fluid (SF) from rheumatoid arthritis (RA) patients. Cell-free fractions of SF contain a PLA₂ enzyme that preferentially releases [¹⁴C]oleic acid fromE. coli, requires calcium and is optimally active at neutral pH. Purified PE, but not PC is also readily degraded by the soluble enzyme. A cell-associated PLA₂ present in sonicates of SF mononuclear cells and neutrophils preferentially releases [³H]AA fromE. coli. These studies suggest the presence of at least two different enzymes with activity of PLA₂ in rheumatoid SF.     

Selective inhibition of group II phospholipase A2 by quercetin

The influence of quercetin, chlorpromazine, aristolochic acid, and indomethacin on group I phospholipase A₂ (PLA₂) from porcine pancreas and on group II PLA₂ fromVipera russelli was compared. Quercetin and chlorpromazine were found to inhibit PLA₂ activity in lower concentrations (< 100M), while aristolochic acid and indomethacin were inhibitory only in higher concentrations (> 100M). The order of potency againstVipera PLA₂ was: quercetin >chlorpromazin aristolochic acid > indomethacin, while the order of potency against pancreatic PLA₂ was: chlorpromazine > aristolochic acid > indomethacin> quercetin. Thus, quercetin was a potent inhibitor towards group II PLA₂ (IC₅₀=2M), but a very weak inhibitor against group I PLA₂, with maximum 30% inhibition. Aristolochic acid and indomethacin were three to four times more potent towards group II PLA₂ than towards group I PLA₂, while chlorpromazine was equally potent towards the two PLA₂ types. Quercetin and chlorpromazine were also tested against two PLA₂ fractions purified from the plasma of septic shock patients; chlorpromazine was then equally potent towards the two PLA₂ fractions, whereas quercetin was a potent inhibitor of only one of the two PLA₂ fractions (IC₅₀=4M). Together, these results indicate that (1) different PLA₂ inhibitors have different potency depending on which type of PLA₂ they are used against, (2) quercetin selectively inhibits group II PLA₂ and may therefore be used to discriminate between different PLA₂ forms in biological materials, and (3) both PLA₂ of group I and group II are present in septic shock plasma.     

Enzymatic activity and immunoreactivity of extracellular phospholipase A2 in inflammatory synovial fluids

Synovial fluid PLA₂ concentration was measured by an ELISA technique using monoclonal antibodies raised against human recombinant synovial-type group II phospholipase A₂. This ELISA was specific for synovial-type PLA₂ and did not detect pancreatic (group I) PLA₂. In all synovial fluids examined, including rheumatoid, osteoarthritic, psoriatic, and gouty fluids, synovial fluid PLA₂ enzyme activity significantly correlated with PLA₂ immunoreactivity (P<0.001). Within the limits of the ELISA technique, there was no evidence for the presence of specific or nonspecific modulation of PLA₂ activity by either putative PLA₂ activating or inhibitory proteins.     

Edema-inducing activity of phospholipase A2 purified from human synovial fluid and inhibition by aristolochic acid

A neutral-active, Ca²⁺-dependent phospholipase A₂ (PLA₂) purified 11,000-fold from human synovial fluid (HSF) induced edema when injected into the mouse foot pad. The edema produced by HSF-PLA₂ was dose-dependent and was positively correlated with the dose-dependent in vitro expression of PLA₂ activity. Maximum edema was achieved within 45 min after the injection and persisted for atleast 6 h. Aristolochic acid [8-methoxy-6-nitrophenanthro(3,4-d)-1,3-dioxole-5-carboxylic acid], a major chemical component derived from various species ofAristolochia plant, produced a dose-dependent inhibition of in vitro phospholipid hydrolysis by HSF-PLA₂, porcine pancreatic PLA₂, snake venom (Naja naja) PLA₂, and PLA₂ isolated from human platelet. The sensitivity of these PLA₂s to inhibition by aristolochic acid varied markedly: HSF-PLA₂>N.naja PLA₂>human platelet PLA₂>porcine pancreatic PLA₂. The inhibition of HSF-PLA₂ by aristolochic acid was independent of substrate concentration (18–144M and Ca²⁺ concentration (0.1–4.0 mM). These observations indicate that inhibition of HSF-PLA₂ by aristolochic acid may result from direct interaction with the enzyme. When aristolochic acid was mixed with HSF-PLA₂ and then injected into the mouse foot pad, edema was inhibited in a dose-dependent manner and was positively correlated with in vitro inhibition of PLA₂ activity. Alkylation of HSF-PLA₂ withp-bromophenacyl bromide concomitantly inhibited both enzyme and edema-inducing activity. These results clearly demonstrate that the neutral-active, Ca²⁺-dependent PLA₂ isolated from human synovial fluid is proinflammatory and that catalytic activity is positively correlated with in vivo proinflammatory effects.     

Inhibition of human non-pancreatic phospholipases A2 by retinoids and flavonoids. Mechanism of action

The interaction of retinoids and flavonoids with phospholipases A₂ (PLA₂) was studied to assess the mechanism of inhibition. Retinoids, such as retinal, retinol, retinoic acid and retinol acetate, and flavonoids, such as quercetin, rutin, morin and sciadopitysin, inhibit Ca²⁺-dependent PLA₂ activity of human synovial fluid (HSF)in vitro in a dose-dependent fashion; ID₅₀ s ranged from 2–8 M. Retinal inhibited neutral active Ca²⁺-dependent PLA₂s from human platelets, human plasma, human polymorphonuclear leukocytes andNaja mossambica mossambica venom in a dose-dependent manner while quercetin inhibits extracellular PLA₂ activities of human plasma, HSF andN. m. mossambica venon in a dose-dependent manner but not PLA₂ activity derived from human platelets and polymorphomonuclear leukocytes.Inhibition of PLA₂ activity by both flavonoid and retinoids were independent of Ca²⁺ or Na⁺. Increasing substrate concentration (9–144 nmols) relieved the inhibition of HSF-PLA₂ activity by quercetin indicating probable interaction with the substrate. The inhibition by retinal is independent of substrate concentration suggesting that inhibition by retinal is probably due to direct interaction with the enzyme. both retinal and quercetin quenched the relative fluorescent intensity ofN. m. mossambica PLA₂ and in a dose-dependent manner in the same concentration range at which they inhibitin vitro PLA₂ activity. Retinal and quercetin shift the thermotropic phase transition of distearoylphosphatidylethanolamine (DSPE) liposomes. Both compounds broadened the transition peak, shifted theT _m to lower temperature, and decreased enthalpy significantly. These findings indicate that inhibition of non-pancreatic human PLA₂s by retinoids and flavonoids can be mediated by interaction with enzyme and/or substrate.     

Flavonoids as Phospholipase A2 Inhibitors: Importance of Their Structure for Selective Inhibition of Group II Phospholipase A2

The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A₂ (PLA₂-I) from porcine pancreas and Naja naja, and on group II phospholipase A₂ (PLA₂-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA₂-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA₂-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA₂-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA₂ purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA₂-II from synovial fluid but was only a weak inhibitor of human PLA₂-I from pancreatic juice, suggesting that rutin is a selective PLA₂-II inhibitor. A number of structurally similar flavonoids were tested for their ability to inhibit PLA₂-II from Crotalus atrox and, for comparison, PLA₂-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA₂ activity, and that the hydroxyl groups in 3- and 4-position are required for selective inhibition of PLA₂-II.     

Antiinflammatory action of thielocin A1β, a group II phospholipase A2 specific inhibitor,in rat carrageenan-induced pleurisy

Extracellular phospholipase A₂ (PLA₂) activity was detected in exudate from rat carrageenan-induced pleurisy using [³H]oleic acid-labeledEscherichia coli as substrate. Both exudate volume and PLA₂ activity increased up to 24 h after carrageenin injection. Specific absorption of this activity by anti-group II PLA₂ (PLA₂-II) antibody indicated that the PLA₂ activity in the pleural exudate was PLA₂-II. Thielocin Al, a novel type of PLA₂ inhibitor from fungi, inhibited this PLA₂-II activity in a dose-dependent manner (IC₅₀=0.32M). Thielocin A1 correspondingly reduced both exudate volume and PLA₂-II activity in the exudate in a dose-dependent manner when coinjected with carrageenan. The exudate volume was also significantly decreased when indomethacin, a cyclooxygenase inhibitor, or dexamethasone, a steroidal antiinflammatory drug, was coinjected with carrageenan. However, neither indomethacin nor dexamethasone could significantly attenuate the PLA₂-II activity in exudate In addition, indomethacin and dexamethasone significantly reduced the levels of PGE₂ in the exudate. However, thielocin A1 had no effect on the PGE₂ content in the exudate. These results suggest that thielocin A1 shows antiinflammatory activity due to inhibition of PLA₂-II and offer evidence for the significance of PLA₂1I in the propagation of inflammatory processes.     

Contribution of group II phospholipase A2 to arachidonic acid release and prostaglandin synthesis by mesangial cells

Interleukin 1 (IL-1) and tumour necrosis factor (TNF) have been found to increase group II phospholipase A₂ (PLA₂) synthesis and secretion by mesangial cells. In all cases 85%–90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PGE₂) synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA₂ attenuates IL-1 and TNF-stimulated PGE₂ production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA₂, potently blocks IL-1- and TNF-stimulated PGE₂ synthesis in intact mesangial cells with 1C₅₀s of 1.3 and 1.0 M, respectively.     

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246–254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726–730 [1988]). P2 (0.05 M) was also reported to inhibit porcine pancreatic phospholipase A₂ (PLA₂). The effect of synthetic P2 (98% pure) on PLA₂ was evaluated in two assay systems. Using porcine pancreatic PLA₂ and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005–50 M) had no effect on PLA₂ activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA₂ as the enzyme and [¹⁴C]-oleate-labelledE. coli substrate, P2 (0.005–50 M) had no significant effect on PLA₂ activity. A reported PLA₂ inhibitor, manoalide, was a potent inhibitor of PLA₂ in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA₂ inhibitory activity.     

Structure-activity relationships leading to WAY-121,520, a tris aryl-type,indomethacin-based,phospholipase A2 (PLA2)/leukotriene biosynthesis inhibitor

We were intrigued by reports of the inhibition of phospholipase A₂ (PLA₂) by indomethacin. In order to increase the potency of the indomethacin system as an inhibitor of PLA₂, it was decided to make more lipophilic analogs. Indeed, covalent attachment of a quinoline ring to the methoxy substituent of indomethacin affords WAY-122,220 which is almost an order of magnitude more potent than indomethacin in inhibiting human synovial fluid PLA₂ (IC₅₀=15 and 145 μM, respectively). TheN−p-chloro-benzyl analog of this compound, WAY-121,520, was an even more potent inhibitor of PLA₂ (IC₅₀=4 μM). Structural analyses and molecular modeling suggest that these compounds may inhibit PLA₂ by mimicking arachidonic acid. WAY-121,520 is also a potent leukotriene biosynthesis inhibitor both in the rat PMN and mouse macrophage assays (IC₅₀=10 and 4 nM, respectively), possibly acting via a 5-LO (5-lipoxygenase) translocation inhibition mechanism. The multiple actions of WAY-121,520 may contribute to its favorable anti-inflammatory profile.

     

Contribution of group II phospholipase A2 to arachidonic acid release and prostaglandin synthesis by mesangial cells

Interleukin 1 (IL-1) and tumour necrosis factor (TNF)α have been found to increase group II phospholipase A₂ (PLA₂) synthesis and secretion by mesangial cells. In all cases 85%–90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PGE₂) synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA₂ attenuates IL-1β and TNFα-stimulated PGE₂ production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA₂, potently blocks IL-1β- and TNFα-stimulated PGE₂ synthesis in intact mesangial cells with 1C₅₀s of 1.3 and 1.0 μM, respectively.     

Phospholipase A2 (PLA2) activity in undifferentiated U937 cells

PLA₂ activity has been described in U937 cells. The present study characterized PLA₂ activity in these undifferentiated cells. Cells were grown in suspension culture, harvested by centrifugation, and washed and homogenized in a neutral buffer containing standard proteinase inhibitors. A low speed supernatant was fractionated either by acid extraction or by sucrose density gradient centrifugation. PLA₂ activity was measured using either L--1-palmitoyl-2-arachidonoyl [1-¹⁴C]-phosphatidylcholine or heat-inactivated [³H]oleic acid-labeledE. coli as substrates. Substrate-specific PLA₂ activity was found in the acid-extracted and in the 25% sucrose fractions. Standard inhibitors were investigated with these PLA₂ activities. Our results suggest undifferentiated U937 cells contain three distinct PLA₂ activities. This is the first indication that more than one PLA₂ activity is present in undifferentiated U937 cells.     

Pharmacological characterization of WAY-121,520: A potent anti-inflammatory indomethacin-based inhibitor of 5-lipoxygenase (5-LO)/phospholipase A2 (PLA2)

WAY-121,520 inhibited human synovial fluid PLA₂ (HSF-PLA₂) (IC₅₀=4 M) using arachidonic acid-labeledE. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC₄, IC₅₀=4nM) and rat PMN (LTB₄, IC₅₀=10 nM) and an ability to antagonize LTD₄ binding to isolated guinea-pig trachea (pK _B=6.0).In vivo anti-inflammatory activity was noted in murine TPA-induced (ED₅₀=91 g/ear) and arachidonic acid-induced (66% inhibition at 400 g/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA₂ with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA₂ and LTD₄ antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.     

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography

Summary Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA₂-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA₂ activity was found without prior trypsin activation (67×10³ U/min/100 µl). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1–2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic fluid collections could account for pseudocyst formation after an acute pancreatitis attack.     

Influence of plasma proteins on activity of proinflammatory enzyme phospholipase A2

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

Characterization of extracellular phospholipase A2 (PLA2) activity in fluid and peritoneal cells from casein-treated rats

Extracellular phospholipase A₂ activity (PLA₂) found in the fluid and cells of the peritoneal cavity of rats injected with casein is described. PLA₂ activities from both the fluid and cells require Ca²⁺ and have pH optima of 7. Acid-extraction increased PLA₂ activity in the polymorphonuclear leukocyte (PMN) homogenates 20-fold but not the PLA₂ activity in the extracellular fluid. Acid extraction also increased the sensitivity of the PLA₂ activities to standard inhibitors. Since the PLA₂ activities described in this model have characteristics similar to other inflammatory PLA₂s, including human synovial fluid PLA₂, casein stimulation should prove useful for testing potential inhibitors.     

Phosphonate-phospholipid analogues inhibit human phospholipase A2

A phosphonate-containing phospholipid (PL) analogue (Compound 1) designed as a transition-state inhibitor competively inhibits non-human extracellular PLA₂ at a mole fraction of 0.003 in the kinetic scooting mode (Jain et al., Biochem. 284135 (1989)). To further profile the activity of Compound 1, we examined its activity with purified human enzyme and in whole cell systems. Compound 1 effectively inhibited a 14 kDa human PLA₂ purified from joint synovial fluid of patients with rheumatoid arthritis using³H-AA labeledE. coli as substrate (IC₅₀=1.7M) and a high MW PLA₂ (110 kDa) isolated from the cytosol of a human monocytic cell line, U-937, which selectively hydrolyzes AA-containing PL (IC₅₀=165M). It failed to reduce A23187-induced PGE₂ or LTC₄ production by human adherent monocytes or LTB₄ release from human neutrophils which may be due, in part, to poor membrane partitioning.     

Pharmacologic modulation of D-49 phospholipase A2-induced paw edema in the mouse

Paw edema was produced in CD-1 mice by the injection of 0.3 g of snake venom PLA₂ (A.p. piscivorus D-49) into the hind paw. Edema peaked at 10 min, remained elevated until 60 min, and then declined slowly. The PLA₂ inhibitors, luffariellolide and aristolochic acid, reduced the edema but only when coinjected with the PLA₂. The histamine/serotonin antagonists were the most effective drug class against PLA₂-induced paw edema. The PAF antagonists, CV-6202 (iv) and kadsurenone (coinjected) reduced the PLA₂-induced edema, whereas high doses of the corticosteroids, dexamethasone and hydrocortisone, were also effective. NSAIDs only partially inhibited the paw edema. The LO/CO inhibitors yielded varying activities, with only BW755C and NDGA inhibiting the edema. These results suggest that PLA₂ induces paw edema in the mouse via the action of several classes of inflammatory mediators.     

Modulation of phospholipase A2 activity in human synovial fluid by cations

Cell-free, Ca²⁺ dependent phospholipase A₂ activity (PLA₂) was measured in human synovial fluid of patients with various kinds of arthritis using [1–¹⁴C] oleate-labelled autoclaved Escherichia coli as substrate. PLA₂ activity at pH 7.0 and with 5 mM added Ca²⁺ was stimulated and then inhibited in a dose-dependent fashion by NaCl; maximal stimulation of 8.8 fold was found at 150 mM Na⁺. Similar effects were obtained with K⁺ Li⁺ and Ru⁺. In the absence of added Na⁺, PLA₂ activity was maximal with 25 mM Ca²⁺ (145 nmols/hr/mg), but in the presence of 150 mM Na⁺, activity was maximal with 4 mM Ca²⁺ (415 nmols/hr/mg). PLA₂ activity was optimal between pH 6.5–8.0 in presence of 150 mM Na⁺¹ and 4 mM Ca²⁺. There was no significant difference between PLA₂ activity in synovial fluids from rhematoid and other types of arthritis. Neutral active, Ca²⁺-dependent PLA₂ activity in acid extracts of human platelets, plasma, polymorphonuclear leukocytes and synovial fluid varied in response to added Na⁺. In presence of 150 mM added Na⁺ and 5 mM PLA₂ activity in human synovial fluid was inhibited by all multivalent cations tested. In the absence of Na⁺, Cu²⁺ and Mg²⁺ stimulated PLA₂ activity in a dose dependent fashion; whereas, Fe²⁺, Fe³⁺ and Al³⁺ were inhibitory. The extent of stimulation by Mg²⁺ was inversely related to the concentration of added Ca²⁺.