q Birkha¨user Verlag, Basel, 1998 Inflamm. res. 47, Supplement 1 (1998) S24–S25 1023-3830/98/010S24-02 $ 1.50+0.20/0

Inflammation Research

Role of phospholipase A2 and split lipid products in histamine release from mast cells M. Varsani and F. L. Pearce Department of Chemistry, University College London, 20 Gordon Street, London WC1H 0AJ, UK, Fax +171 380 7463, e-mail: [email protected]

degrees of histamine release from the various cells studied. Representative results are shown in Table 1. Phosphatidylethanolamine (PE), phosphatidylinositol (PI) and their corresponding lyso-derivatives were essentially ineffective in all cases (maximum release < 2%). Lyso-PS produced a small release of histamine from the different cell phenotypes but strikingly augmented immunological secretion from the RPMC. Thus, the releases induced by anti-IgE (300-fold dilution), lyso-PS (1 mM) alone, and the two factors together were 32.3 6 3.7%, 8.2 6 2.8% and 73.9 6 4.5%, respectively. Similar results were also seen with PS (data not shown). None of the other lipids had any such effect in any of the test systems. The potentiating effects of PLA2 in combination with IgE-directed and related ligands in rat mast cells [3] may then be due to the generation of lyso-PS. Lyso-PC produced a marked release of histamine from RPMC, human lung mast cells and human basophils (Table 1). Lyso-PA was rather less active against RPMC, showed modest activity against the basophils and was ineffective against the human lung cells. In all three systems, the secretion induced by lyso-PA was suppressed by metabolic blockade with 2deoxy-D-glucose and antimycin A. However, the releases evoked by lyso-PC were unaffected by these agents and were thus presumably cytolytic. The non-cytotoxic release of histamine induced by PLA2 may then be due to the generation of lyso-PA, which has a signalling function in other systems

Introduction The enzyme phospholipase A2 (PLA2) cleaves fatty acid moieties from the sn-2 position of the glycerol phosphate backbone to yield the lyso-phospholipid and free arachidonic acid [1]. The latter may be further metabolised to generate leukotrienes and prostaglandins. In addition, PLA2 may also be intrinsically involved in stimulus-secretion coupling in the mast cell. The enzyme may be classified phylogenetically into various subtypes [2] and we have recently shown that purified PLA2 from bee and cobra (Naja naja) venom produce a dose-dependent release of histamine from different mast cell phenotypes [3]. In some cases, the release was non-cytotoxic whilst, in others, components of the process were cytolytic. Suboptimal amounts of the enzyme also potentiated immunological histamine release from rat mast cells. To elucidate the possible mechanisms involved in these processes, we have now examined the effects of various membrane L-aphospholipids, together with their corresponding lysoderivatives, on rat peritoneal mast cells (RPMC), human lung mast cells and human basophils. Materials and methods RPMC were recovered by direct lavage and human basophil leukocytes were isolated by dextran sedimentation of peripheral blood [4]. Mast cells from the human lung were obtained by enzymic dissociation of the tissue with the protease collagenase [5]. Cytotoxicity was judged by the effects of the metabolic inhibitors 2-deoxy-D-glucose (5 mM) and antimycin A (1 mM). Histamine release following stimulation with different secretagogues was determined spectrofluorometrically as previously described [6]. All biochemicals were obtained from Sigma (London, UK) except anti-rat IgE (ICN Immunochemicals, Thame, Oxfordshire, UK). All values are given as means 6 SEM for 4 experiments.

Table 1. Histamine release from different mast cell phenotypes treated with various L-a-phospholipids and their corresponding lysoderivatives (1 mM unless otherwise indicated). Secretagogue

Histamine release (%) from RPMC

human lung

human basophils

6.4 6 1.0 5.5 6 2.3 2.6 6 0.5 48.8 6 2.1 80.8 6 2.7 9.9 6 1.8

1.7 6 0.3 0.1 6 0.2 0.3 6 0.6 4.0 6 0.4 81.9 6 3.9 1.2 6 0.7

0.4 6 0.3 0.1 6 0.4 0.2 6 0.1 14.5 6 2.4 69.8 6 5.0 0.9 6 1.2

The L-a-phospholipids (1 mM–1 mM) phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylserine (PS) and their corresponding lyso-derivatives induced varying

PA PC PS (0.1 mM) Lyso-PA Lyso-PC Lyso-PS (0.1 mM)

Correspondence to: F. L. Pearce

All values are given as means 6 SEM for 4 experiments. For abbreviations, see text.

Results and discussion

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Inflamm. res., Supplement 1 (1998) Table 2. Histamine release (%) from different mast cell phenotypes treated with lyso-PA (0.5 mM) and lyso-PC (0.1 mM) in the absence and presence of metabolic inhibitors.

25

Mast cell phenotype

Lyso-PA

RPMC Human lung Human basophils

Lyso-PC

(¹) inhibitors

(+) inhibitors

(¹) inhibitors

(+) inhibitors

33.0 6 5.8 4.0 6 0.4 13.7 6 2.6

5.1 6 2.3 0.2 6 0.1 0.2 6 0.3

15.4 6 4.5 40.8 6 3.7 20.9 6 1.8

27.4 6 5.8 42.4 6 5.3 22.4 6 2.9

All values are given as means 6 SEM for 4 experiments. For experimental details and abbreviations, see text.

[7]. In contrast, the lytic release of the amine induced by PLA2 may be caused by the production of lyso-PC (lyso-lecithin), which is a known surfactant [8]. In conclusion, the present data indicate that the differential effects of PLA2 on various histaminocytes may reflect variations in overall membrane composition leading to the production of one or other lipid metabolite which then induces or augments the secretory response.

[2] Davidson FF, Dennis EA. Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms. J Mol Evol 1990;31:228–38. [3] Varsani M, Pearce FL. Role of phospholipase A2 in mast cell activation. Inflamm Res 1997;46 Suppl 1:S9–10. [4] Hirst SJ, Hayes NA, Burridge J, Pearce FL, Foreman JC. Human basophil degranulation is not triggered by very dilute antiserum against human IgE. Nature 1994;366:525–7. [5] Ali H, Pearce FL. Isolation and properties of cardiac and other mast cells from the rat and the guinea pig. Agents Actions 1985;16:138–40. [6] White JR, Pearce FL. Characteristics of histamine secretion from rat peritoneal mast cells sensitised to the nematode Nippostrongylus brasiliensis. Immunology 1982;46:353–9. [7] Durieux ME, Lynch KR. Signalling properties of lysophosphatidic acid. Trends Pharmacol Sci 1993;14:249–54. [8] Marquardt DL, Walker LL. Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation. J Allergy Clin Immunol 1991;88:721–30.

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

References [1] Dennis EA. Regulation of eicosanoid production: Role of phospholipases and inhibitors. Biotechnology 1987;5:1294–300.

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