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Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in inflammation by catalyzing the release of arachidonic acid, a substrate for lipoxygenase and cyclooxygenase enzymes, from membrane phospholipids. In the present study we examined the role of cPLA(2) in inflammatory responses through the use of a specific inhibitor of the enzyme, cPLA(2), arachidonyl trifluoromethyl ketone (AACOCF3). Interestingly, we observed that AACOCF3 is an inhibitor of chronic but not acute inflammatory responses. Specifically, AACOCF3 inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice. Additionally, oral treatment of ovalbumin-sensitized/ovalbumin-challenged BALB/c mice with 20 mg/kg AACOCF3 prevented the development of airway hyper-responsiveness in a model of asthma. Furthermore, AACOCF3 decreased cellular recruitment in the airway lumen and airway inflammation after the ovalbumin challenge. Taken together, these results suggest that a potent and specific chemical inhibitor of cPLA(2) may be useful for the treatment of chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.

Although the expression of the prototypic secretory phospholipase A(2) (sPLA(2)), group IIA (sPLA(2)-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA(2) enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA(2)s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA(2)-V, and to a lesser extent that of sPLA(2)-IID, -IIE, and -IIF, were increased, whereas that of sPLA(2)-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA(2)-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA(2)-IIA inhibitor that turned out to inhibit sPLA(2)-IID, -IIE, -V and -X as well. In contrast, sPLA(2)-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA(2)s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.

In HepG2 cells phosphorothioate modified antisense oligonucleotides against a sequence in the Ca2+ binding domain (AS-Ca2+) of type II sPLA2 mRNA restrained IL-6-induced synthesis of sPLA2 protein, sPLA2 mRNA (northern blot), and abolished IL-6 stimulated PGE2 release. An antisense oligonucleotide corresponding to a sequence in the catalytic domain (AS-Cat) of sPLA2 was less effective. The antisense oligonucleotides did not affect albumin synthesis in HepG2 cells, additionally demonstrating their specificity. The corresponding AS-Ca2+ against a homologous part of the rat sPLA2 mRNA depressed rat carrageenin oedema for 60-70%. Identical suppression was achieved by specific low molecular weight inhibitors of sPLA2. Since cyclo- and 5-lipoxygenase inhibitors exerted similar reductions of carrageenin oedema type II sPLA2 dependent eicosanoid formation seems to be a key cascade in this type of inflammation.

The effects of bakuchiol, a meroterpenoid isolated from the leaves of Psoralea glandulosa L., on phospholipase A2 (PLA2) activity from different sources, human neutrophil responses, zymosan air pouch and topical inflammation in mice, were investigated. This natural product was a weak inhibitor of secretory and intracellular PLA2 but dose-dependently reduced the formation of LTB4 and TXB2 by human neutrophils and platelet microsomes, respectively. In addition, bakuchiol inhibited degranulation in human neutrophils, whereas superoxide generation was not affected. In mice, bakuchiol decreased cell migration, myeloperoxidase activity and eicosanoid levels in the air pouch inflammation induced by zymosan. After topical administration, this compound was effective as an inhibitor of oedema and myeloperoxidase activity in the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear oedema and significantly reduced the PGE2 content and ear oedema in the arachidonic acid-induced response. Bakuchiol is a natural anti-inflammatory agent able to control leukocytic functions such as eicosanoid production, migration and degranulation in the inflammatory site.

We investigated the concentration of immunoreactive pancreatic phospholipase A2 (pan-PLA2) and the catalytic activity of phospholipase A2 (CA-PLA2) in plasma, peritoneal fluid, and pancreas of rats in which acute hemorrhagic pancreatitis was induced by an intraductal injection of sodium taurocholate. The contribution of pancreas to the CA-PLA2 in plasma was studied by removing pancreatic PLA2 by absorbing plasma samples with a polyclonal antibody raised in a rabbit against rat pancreatic PLA2. Sodium taurocholate injected into the pancreatic duct produced hemorrhagic pancreatitis with necrosis and inflammatory cell invasion within 8 hr. Saline injection caused edematous pancreatitis, but sham operation did not alter pancreatic morphology from normal. The concentration of pan-PLA2 increased rapidly in plasma in all animals, but significantly more in sodium taurocholate-injected animals than in saline-injected or sham-operated animals. The level of CA-PLA2 in plasma increased in sodium taurocholate-injected animals only. There was no correlation between pan-PLA2 and CA-PLA2 values in plasma in sodium taurocholate-injected animals. The CA-PLA2 was marginally increased in pancreatic tissue of sodium taurocholate-injected animals compared to that of saline-injected and sham-operated animals at 8 hr. Treatment by the anti-pan-PLA2 antibody effectively removed pan-PLA2 from plasma and peritoneal fluid samples in sodium taurocholate-injected animals. The level of CA-PLA2 in plasma was similar before and after antibody treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study by using the human recombinant non-pancreatic-secreted platelet PLA2 (r-hnps-PLA2) and rabbit polyclonal antibodies directed against either the human (group II) or the porcine enzyme (group I), we have shown a possible involvement of platelet PLA2 in poly-L-arginine (25 kDa)-induced rat paw edema. Local treatment of rats with the anti-platelet-PLA2 antibody (anti-hnps-PLA2) but not with anti-porcine-PLA2 antibody (anti-porc-PLA2) significantly reduced the edema induced by a maximal dose of poly-L-arginine (1 mg/paw). Furthermore when r-hnps-PLA2 (1-10 micrograms) was injected together with a subliminal dose of poly-L-arginine (50 micrograms/paw), a dose-dependent increase in both edema and protein leakage was observed. This effect was selectively inhibited by the anti-hnps-PLA2 (10-100 micrograms/paw) but not anti-porc-PLA2 (10-100 micrograms paw). Thus, platelets seem to be involved in both vascular and cellular components of the inflammatory response by contributing, most likely in the early phase, to the edema formation through secretion of PLA2.

Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A2 (HSF-PLA2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 microM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A2 (PLA2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA2 activity.

Two phospholipase A2 (PLA2) enzymes, TMVPLA2 I and TMVPLA2 II, isolated from Trimeresurus mucrosquamatus venom induced rat hind-paw oedema. Recovered myeloperoxidase activity increased within 1 h and was greatly elevated in the rat paw 3-6 h after subplantar injection of these venom PLA2 enzymes. Methotrexate pretreatment significantly reduced not only the peripheral leucocyte count but also venom PLA2-induced paw oedema. In rat isolated PMN leucocyte suspension, venom PLA2 induced superoxide radical formation. Paw swelling caused by TMVPLA2 I or TMVPLA2 II was only slightly or not, respectively, reduced in the rats pretreated with anti-platelet plasma, which reduced peripheral blood platelet count by greater than 96%, suggesting platelets are not involved. In isolated platelet preparation, TMVPLA2 I induced platelet activation in a concentration-dependent manner, while TMVPLA2 II had no effect. Pretreatment with diphenhydramine/methysergide greatly suppressed the oedematous responses caused by the two venom PLA2 enzymes; the residual responses were significantly further depressed by aspirin. The oedematous responses caused by the enzymes were also suppressed by FPL 55712, BW 755C, dexamethasone, superoxide dismutase/catalase, isoprenaline and terbutaline. However, BN 52021 and L 652731, both platelet aggregating factor antagonists, were not effective on these responses. Thus, in addition to histamine and 5-hydroxytryptamine release by the mast cells in PLA2-induced paw oedema (Wang & Teng 1990), the results of this study indicate minor, but significant, roles for neutrophils and inflammatory mediators including prostaglandins, leukotrienes and superoxide radicals.

The edema-producing activity of NNAVPLA2, an acidic phospholipase A2 (PLA2) enzyme from Naja naja atra venom (NNAV), was less potent than that of TMVPLA2 II, a basic PLA2 from Trimeresurus mucrosquamatus venom (TMV). These edema-forming effects were greatly suppressed by pretreatment of rats with diphenhydramine/methysergide or compound 48/80, which reduced the tissue content of histamine and serotonin. Heparin abolished and suppressed the paw edema caused by protamine and TMVPLA2 II, respectively, but had no effect on the NNAVPLA2-induced response. In isolated rat peritoneal mast cells, both PLA2 concentration dependently induced the release of histamine and beta-glucuronidase. Again, TMVPLA2 II was more potent than NNAVPLA2. This degranulation effect of mast cells caused by TMVPLA2 II and protamine was inhibited by heparin, while that caused by NNAVPLA2 was unaffected. The edema-forming and mast cell degranulation effects were greatly decreased in both PBPB-modified NNAVPLA2 and PBPB-modified TMVPLA2 II, in which the catalytic activity of the enzymes was completely lost. PBPB-modified TMVPLA2 II-induced paw edema was also suppressed by heparin. Furthermore, this edematous response was totally reversed in rat pretreated with aspirin in combination with diphenhydramine and methysergide. These results suggest that the edema-forming effect of PLA2 is probably dependent on the presence of catalytic, positive charge and pharmacological sites on its molecule.

Appreciable phospholipase A2 activity was detected in a hind paw homogenate from rats with adjuvant arthritis or carrageenan-induced edema. The activity was neutralized by treatment with antibody raised against rat platelet secretory phospholipase A2, indicating that 14-kDa group II phospholipase A2 was induced in the hind paw during the process of inflammation. Injection of purified rat platelet phospholipase A2 into the hind paw of rats with adjuvant-induced arthritis resulted in exacerbation of edema in a dose-dependent manner, whereas no effect was observed on either normal rats or animals with carrageenan-induced edema under the same experimental conditions. These results suggest the importance of mammalian group II phospholipase A2 in the pathogenesis of some types of inflammation.

According to the aim of the simultaneous and sequential inhibition of key enzymes of the arachidonic acid cascade, combinations of inhibitors of the phospholipase A2 (PLA2), cyclooxygenase (COX) and of lipoxygenases (LOX) were administered to rats with carrageenin edema and adjuvant arthritis, respectively. While the COX inhibitors diclofenac-Na and acetylsalicylic acid in combination with PLA2 and LOX inhibitors mostly gave overadditive antiinflammatory effects in the carrageenin edema, phenylbutazone in combination at most displayed additivity. Admixture of ascorbic acid led to subadditive effects. In the adjuvant arthritic rat, the combined administration of diclofenac-Na and dexamethasone yielded additive effects, only. The same result is valid for the combination of piroxicam and a LOX inhibitor in this model. Results are discussed with respect to the mode of action of substances as well as to the model peculiarities.