q Birkha¨user Verlag, Basel, 1997 Inflamm. res. 46, Supplement 1 (1997) S9–S10 1023-3830/97/0100S9-02 $ 1.50+0.20/0

Inflammation Research

Role of phospholipase A2 in mast cell activation M. Varsani and F. L. Pearce Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK

the inhibitors, the enzyme induced a non-cytotoxic release of histamine from murine peritoneal mast cells, human lung mast cells and human basophils. This release may be due to production of arachidonic acid and its further metabolism to 5hydroperoxyeicosatetraenoic acid (5-HPTE), which is thought to modulate secretion [7]. However, the releases from tissue mast cells of the rat and guinea pig were essentially unaffected by metabolic blockade and were presumably cytotoxic. This may be due to generation of lyso-phosphatidylcholine (lysolecithin), which is a known surfactant. Suboptimal amounts (0.1 units/ml) of PLA2 from Naja naja venom potentiated IgE-mediated histamine secretion from rat peritoneal mast cells. Thus, the releases induced by anti-IgE alone (300-fold dilution), PLA2 alone, and the two factors together were 13:1 6 2:3%, 11:1 6 3:4%, and 45:9 6 3:4%, respectively. Similar results were seen with nerve growth factor and concanavalin A (data not shown). However, the combined (32:3 6 4:3%) effects of PLA2 (11:3 6 2:8%) and compound 48/80 (25 ng/ml, 19:7 6 1:3%) were simply additive. The potentiating effect may be due to generation of lyso-phosphatidylserine, which is known to augment histamine release induced by IgE-directed and related ligands [8]. As shown in Table 2, histamine release induced by compound 48/80 and anti-IgE was attenuated by the PLA2 inhibitors p-bromophenacyl bromide (p-BPB, 10 mM) and (p-amylcinnamoyl)anthranilic acid (ACA, 100 mM). Oleoyloxyethylphosphocholine (OEPC, 50 mM), and AACOCF3 (a

Introduction The enzyme phospholipase A2 (PLA2 ) cleaves fatty acid moieties from the sn-2 position of the glycerol phosphate backbone. It predominantly liberates free arachidonate which may be metabolised to generate leukotrienes and prostaglandins. In addition, PLA2 may also be intrinsically involved in stimulus-secretion coupling in the mast cell. Thus some reports, but not others, have claimed that purified preparations of the enzyme may evoke mast cell degranulation [1]. We have therefore now re-examined this problem in light of the recent phylogenetic classification of the enzyme into different subtypes [2]. Materials and methods Rat and mouse peritoneal mast cells were isolated by direct lavage. In some experiments, rat cells were reversibly permeabilised by treatment with ATP [3]. Human basophil leukocytes were isolated by dextran sedimentation of peripheral blood [4]. Mast cells from human and animal tissues were obtained by enzymic dissociation with the protease collagenase [5]. Histamine release was determined spectrofluorometrically as previously described [6]. All biochemicals were obtained from Sigma except anti-rat IgE (ICN Immunochemicals) and the PLA2 inhibitors (Calbiochem). All values are given as means 6 SEM for 4 experiments.

Results and discussion PLA2 from Naja naja (cobra) venom (0.3–10 units/ml) produced a dose-dependent release of histamine from rat peritoneal mast cells, with a maximum secretion of 72:5 6 2.6%. Over the same concentration range, the enzyme from bee venom was much less active (maximum release 19:0 6 0.9%), and the enzymes from Crotalus adamanteus, Crotalus atrox (eastern and western diamondback rattlesnakes, respectively) and bovine pancreas were essentially ineffective (maximum release <10%). PLA2 (10 units/ml) from Naja naja venom induced histamine release from a wide range of mast cell phenotypes and Table 1 shows the responses obtained in the absence and the presence of the metabolic inhibitors 2-deoxy-D-glucose (5 mM) and antimycin A (1 mM). As judged by the effects of

Table 1. Histamine release from different mast cell phenotypes treated with PLA2 from Naja naja venom (10 units/ml) in the absence and presence of metabolic inhibitors.

Correspondence to: F. L. Pearce

All values are given as means 6 SEM for 4 experiments.

Mast cell phenotype

Rat peritoneal Rat lung Rat mesentery Rat skin Guinea pig lung Guinea pig mesentery Guinea pig skin Mouse peritoneal Human lung Human basophils

m

Histamine release (%) ð¹Þ inhibitors

ðþÞ inhibitors

73:5 6 3:7 30:5 6 4:6 33:5 6 3:7 52:7 6 3:2 24:5 6 1:6 12:7 6 0:6 42:2 6 3:2 13:4 6 3:4 29:9 6 9:2 5:6 6 1:7

9:3 6 1:6 27:6 6 3:5 28:6 6 3:4 45:2 6 4:5 18:6 6 2:4 9:3 6 0:9 38:6 6 4:8 1:7 6 0:9 2:4 6 3:1 0:3 6 0:2

Inflamm. res., Supplement 1 (1997)

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Table 2. Inhibition of histamine release from rat peritoneal mast cells stimulated with anti-rat IgE (250-fold dilution) and compound 48/80 (0.2 mg/ml).

Inhibitor

Inhibition (%) of histamine release induced by

p-BPB (10 mM) ACA (100 mM) AACOCF3 (100 mM) OEPC (50 mM)

anti-IgE

compound 48/80

anti-IgE (permeabilised cells)

87:7 6 4:5 75:2 6 4:8 1:8 6 3:6 ¹2:3 6 3:7

98:9 6 3:0 95:4 6 3:6 ND ND

ND ND 87:6 6 2:5 54:3 6 3:1

Inhibitors were pre-incubated (10 min) with the cells or introduced by permeabilisation with ATP [3]. For explanation and abbreviations, see text. All values are given as means 6 SEM for 4 experiments.

trifluoromethylketone analogue of arachidonic acid, 100 mM) were inactive in normal rat mast cells but were effective inhibitors in cells reversibly permeabilised with ATP. This indicates the importance of incorporation of the compounds into the cytosol. In total, the present data indicate that activation of endogenous PLA2 may be an essential step in signal transduction in the mast cell. Inhibition of this enzyme may then provide a novel target for the therapeutic modulation of this cell type.

[2] [3] [4] [5]

Acknowledgement. This work was supported by a grant from the Wellcome Trust.

[6] [7]

References

[8]

[1] Murakami M, Kudo I, Fujimori Y, Suga H, Inoue K. Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-

m

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