Biochimica et Biophysica Acta, 1093 (1991) 47-54 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 A DONIS 016748899100178G 47 BBAMCR 12944 Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of ca.lcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source Bernard Chabannes, Rachid Hosni, Patrick Moli6re, Martine Croset, Yves Pacheco, Max Perrin-Fayolle and Michel Lagarde INSERM U.205, Service de Chimie Biologique, institut National des Sciences Appliqu~es, Villeurbanne (France) and H~pital Sainte Eugdnie, Saint Genis Lava/(France) (Received 19 November 1990) Key words: Arachidonicacid metabolism: LeukotrieneB 4 synthesis; Calcium ionophore A23187; Human neutrophil; Allergy; Ether-linked glycerophospholipid Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 370C with 0.15/zM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 F M). In neutrophils from allergic patients, the leukotriene B 4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 4- 4.2 and 38.1 4- 6.8 pmol/5 min per 2 • 106 cells in the absence and presence of 10 itM arachidonic acid, respectively; P < 0.005; n = 8). The LTB4 level in neutrophiis from healthy subjects was only 0.97 + 0.17 pmol/5 min per 2-106 cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-t4C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentra- tion. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-t4C]arachidonate for I h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyi-, alkenylacy|- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidyicholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB 4 by the 5-1ipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycego-3-phosphocholine. Abbreviations: PC, cholin~:-linkedphosphoacylglycerols; PE, ethanol- amine-linked phosphoacylglycerols; Pl, inositol-linked phosphoacyl- glycerols; PS, serine-linked phosphoacylglycerols; NL, neutral iipids; TG, triacylglycerols; PAF, l-O-alkyl-2-acetyl-sn-glycero-3-phos- phocholine (platelet-activating factor); lyso-PAF, 1-O-alkyi-2-1yso- sn-glycero-3-phosphocholinc; LTB4; leukotriene B 4, (5(S),6Z,8E,10E, 12(R),14Z)-5,12-dihydroxyeicosatetraenoic acid; HPLC, high-perfor- mance liquid chromatography; TLC, thin-layerchromatography; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RIA, radio- immunoassay. Correspondence: B. Chabannes, Laboratoire d'Immuno-Ailergologie Respiratoire, INSERM U.205, H6pital Sainte Eug~nie, CHU Lyon- Sud, 1 av. Georges Clemenceau, 69230 Saint Genis Laval, France. Introduction Arac|fidonic acid and its metabolites (leukotrienes, prostanoids), produced by inflammatory cells such as neutrophils, play an important role in inflammatory processes [1]. Leukotriene B 4 (LTB4) is among the most potent inflammatory mediators generated by human neutrophils when stimulated with the calcium iono- phore A23187 [2-5]. LTB4 causes neutrophils to move to the site of inflammation, to degranulate and to generate active oxygen species [5,6-8]. Arachidonic acid has been demonstrated to induce polymorphonuclear