Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source

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: Leukotriene B4 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 B4 (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 p m o l / 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 p m o l / 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 concentration. 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.

Introduction Abbreviations: PC, cholin~:-linkedphosphoacylglycerols; PE, ethanolamine-linked phosphoacylglycerols; Pl, inositol-linked phosphoacylglycerols; PS, serine-linked phosphoacylglycerols; NL, neutral iipids; TG, triacylglycerols; PAF, l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor); lyso-PAF, 1-O-alkyi-2-1ysosn-glycero-3-phosphocholinc; LTB4; leukotriene B4, (5(S),6Z,8E,10E, 12( R),14Z)-5,12-dihydroxyeicosatetraenoic acid; HPLC, high-performance liquid chromatography; TLC, thin-layerchromatography; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RIA, radioimmunoassay. Correspondence: B. Chabannes, Laboratoire d'Immuno-Ailergologie Respiratoire, INSERM U.205, H6pital Sainte Eug~nie, CHU LyonSud, 1 av. Georges Clemenceau, 69230 Saint Genis Laval, France.

Arac|fidonic acid and its metabolites (leukotrienes, prostanoids), produced by inflammatory cells such as neutrophils, play an important role in inflammatory processes [1]. Leukotriene B4 (LTB4) is among the most potent inflammatory mediators generated by human neutrophils when stimulated with the calcium ionophore 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

48

leukocyte aggregation and activity [9,10], oxygen radical production [11,12], granule exocytosis and to increase cytosolic-free calcium and calcium-phospholipid-dependent protein kinase C activity [13]. T~c concentration of free arachidonic acid in cells under normal conditions is low and this acid is present mainly in ester form at the sn-2 position of phosphoacylglycerols of resting mammalian cells [14]. Its metabolism through 5-1ipoxygenase in neutrophils first involves the action of various lipases that release the fatty acid and make it available to metabolizing enzymes [15]. Walsh et al. [16] provided evidence that the release of arachidonic acid from endogenous stores in human neutrophils occurs by the action of phospholipase A 2. Other studies have revealed that the arachidonic acid is released from two major sources, choline-linked phosphoacylglycerols (PC) and ethanolamine-linked phosphoacylglycerols (PE), which contain 197o and 687o of total cellular arachidonate content and undergo degradation during cell activation by the ionophore A23187 [17-20]. More recent investigations have specified that 66~ of PC-associated and 717o of PE-associated arachidonate are found at the sn-2 position of 1-O-alkyl-2-acyl- and l-O-alkyl-l'-enyl-2-acyl-subclasses, respectively [17]. Finally, it has been shown that phospholipase A 2 acts preferentially upon 1-O-alkyl-2acyl-glycerophospholipids having arachidonic acid as the 2-acyl substituent, thus releasing arachidonate and 1-O-alkyl-2-1yso-sn-glycero-3-phosphocholine (lysoPAF) [18,21,22]. in spite of these investigations, the exact source of the arachidonic acid oxidative cascade remains unknown and its determination is of major interest. The present study was undertaken to examine the effect of exogenous arachidonic acid on LTB4 production by neutrophils from allergic and healthy subjects stimulated with low concentration of the calcium ionophon~ A23187, and to determine the arachidonate source for LTB,s synthesis. Materials and Methods

Materials All solvents (analytical grade) were from Carlo Erba Farmitalia. [l-~4C]arachidonic acid (55 mCi/mmol) and the LTB4 radioimmunoassay kit were obtained from Amersham France. Standard lipids used in thin-layer chromatography, LTB,s, the calcium ionophore A23187, essentially fatty acid-free bovine serum albumin and phospholipase C (Bacillus cereus) were purchased from Sigma. St. Louis MO, U.S.A.

Subjects Fifteen atopic volunteers (nine females and six males, mean age 31 years) were studied. All had a positive allergic rhinitis with positive allergic skin tests and

elevated total IgE and specific IgE levels. They did not take medicine for at least 2 months before the blood punction. Allergic patients have been examined at the hospital during the season of polinization in our region. They were symptomatic, but their symptoms of rhinitis being recent, they were not treated at the time of blood collection. Eleven healthy volunteers with negative personal and family history of atopy and no clinical evidence of atopic or other major diseases served as controls (six females and five males, mean age 30 years). They did not take any drug for at least 2 months.

Preparation of human neutrophiis Neutrophils were isolated from peripheral venous blood immediately after drawing into citrate. Briefly, the blood was sedimented into 4.57o (w/v) dextran solution (Serva, Heidelberg, F.R.G.) and ncutrophils were isolated by FicoU-Paque (Pharmacia France) density centrifugation [23]. Residual erythrocytes were lysed by hypotonic treatment. Neutrophils were washed twice in a phosphate-buffered saline solution (PBS) without calcium and magnesium (pH 7.4), and cell concentration was adjusted to 4. l0 T neutrophils/ml. More than 957o of the cells were polymorphonuclear neutrophils and cell viability was greater than 957o as judged by the trypan blue exclusion method. The eosinophil levels in whole blood was always below than 37o: 2.6 + 0.9 (n = 15) and 2.3 + 10.57O (n = 13 ) in neutrophils from allergic and healthy subjects, respectively.

Incorporation of [1-14C]arachidonic acid into neutrophii

tipids

Neutrophii lipids were labeled with [1-14C]arachidonic acid according to the method of Bokoch et al. [24]. The standard incubation mixture in 1 ml PBS without calcium and magnesium (pH 7.4) contained 0.25 /~Ci (4.5 nmol) [l-14C]arachidonic acid (55 mCi/mmol) dissolved in 50 /~l of 50 mM Na2CO a. After 60 min incubation at 37°C, the reaction was terminated by the addition of 1 ml ice-cold PBS containing fatty acid-free BSA (1 mg/ml). The cells were removed fiom the supernatant fluid by centrifugation at 225 x g for 10 min, then washed twice with PBS containing fatty acidfree BSA. Neutrophils was resuspended at the concentration of 2.10 7 cells/ml into PBS without BSA. The reaction mixture was then incubated at 37°C for an additional 10 min to ensure that a maximal amount of free labeled arachidonate has been esterified into complex lipids. Cell viability was controlled after these operations and revealed 957o of viable cells.

Extraction and chromatography of iipids Lipids were extracted from cell pellets by the procedure of Bligh and Dyer [25]. Solvents were removed with a stream of nitrogen and the lipids resuspended in diethyl ether/methanol (9:1, v/v). The phospholipids

49 were separated by TLC on silica gel plates LK5 (Whatman), pre-impregnated with 0.8% boric acid in 50% ethanol. The elution was done with chloroform/metha n o l / ammonium hydroxyde/water (60:37.5 : 1 : 3, v/v). The neutral lipids were resolved on silica gel 60G plates (Merck) developed with hexane/diethyi ether/ acetic acid (50:45:4, v/v). The appiopriate standards were employed in these chromatographic systems. The radioactivity incorporated in each lipid band, expressed as a percentage of the total radioactivity spotted on the plate, was determined with a radioscanner (Berthold, Wildbad, F.R.G.) composed of an automatic TLC linear analyser coupled with a chromatographic data system LB 511.

Separation of different species of choline- and ethanolamine-linked phosphoacylglycerols The corresponding bands of PC and PE obtained by TLC was scraped off, extracted three times with chloroform/ methanol/water (5 : 5 : 1, v/v), and extracts were dried under nitrogen. The choline-linked and ethanolamine-linked phosphoacylglycerols were separated into diacyl-, alkyl-acyl- and alkenyl-acyl-subclasses by the procedure of Takamura et al. [26]. Briefly, the extracts were hydrolyzed with phospholipase C (from Bacillus cereus) in 10 mM tris-HCl buffer (pH 7.5) containing 30 mM boric acid and diethyl ether. The hydrolysis was carried out at 30-35°C for 12 h. The liberated 1,2-diradyl-glycerols were extracted and then derivatized with 3,5-dinitrobenzoylchloride at 60°C for 1 h. The dinitrobenzoyl derivatives were extracted three times with hexane and separated by TLC on Silica gel 60G plates (Merck) developed with toluene/hexane/diethyl ether (50 : 45 : 4, v/v). Then, the radioactivity along the plates was monitored with the radioscanner.

Cell activation Two series of experiments were developed, the first one with unlabeled neutrophiis and the second one with labeled cells. Unlabeled neutrophils (4.107 cells/ml) were diluted with an equal volume of PBS (pH 7.4) supplemented with CaCI 2 (8 mM) and MgCI 2 (2 mM) and incubated for 5 min at 37°C. Then unlabeled or labeled arachidonic acid or PBS (assay without arachidonic acid) and the calcium ionophore A23187 were successively added, and the incubation prolonged for another 5 min at 37°C. 1 ml of the final incubation mixture contained 1 0 7 neutrophils, 2 mM CaCI z, 0.5 mM MgCI z, 0 to 10 /LM unlabeled or labeled arachidonic acid and 0.15 pM ionophore. Arachidonic acid and ionophore solutions in PBS were prepared from stock solutions in ethanol and DMSO, respectively. The final ethanol and DMSO concentrations never exceeded 0.2% and 0.01%, respectively. The reaction was terminated by the addition of 1 vol. of ice-cold

methanol and centrifugation at 1500 × g for 10 min. LTB4 was assayed in the supernatant. With labeled neutrophils, the same protocol was used, but only unlabeled arachidonic acid was incubated with the cells.

Purification and assay of

LTB 4

LTB4 was purified from each supernatant by reversed-phase high-performance liquid chromatography (HPLC) using a LiChrospher 100 RP 18 (5 #M) column (220 × 4 ram) (Merck) and a Kontron HPLC system. The column was isocratically eluted with acetonitrile/ methanol/water/acetic acid (300 : 250 : 400 : 4, v/v), adjusted to pH 5.6 with NH4OH, at the flow rate of 0.8 ml/min. The eluent was monitored at 270 nm. Routinely, 0.1 ml aliquot of supernatant was injected directly without prior extraction or fractionation. When LTB4 was detectable by this chromatographic system, it was quantitated as compared to authentic standard run under the same conditions. When the LTB4 peak was below the detection limit, LTB4 was assayed by radioimmunoassay (RIA). Comparing HPLC and RIA on five assays with neutrophils from allergics stimulated by 0.15 ~M ionophore provided similar results (HPLC: 15.9 + 2.1 and RIA: 14.9 _+ 1.1 pmol LTB4/5 min per 2- 106 neutrophils). In labeled experiments, fractions of the effluent were collected every minute and total LTB4 peak radioactivity was meas~lred by scintillation counting in order to determine the amount of labeled arachidonate converted into LTB4.

Data and statistical analysis LTB4 levels are presented as pmol generated after 5 min stimulation of 2 . 1 0 6 neutrophils. Individual values obtained with neutrophils from each donor are given. Mean + S.E. for separate experiments is also indicated. The amount of radioactivity incorporated into each lipid class separated by TLC is expressed as a percentage of total radioactivity spotted on the plate. Mean + S.E. for separate experiments is indicated. The differences between means of two groups were compared in using the Wilcoxon's non-parametric rank sum test. When the populations could be coupled, the non-parametric paired Wilcoxon's test was also used. Results

Effect of exogenous unlabeled arachidonic acid on LTB 4 generation by neutrophils from allergic and healthy subjects Neutrophils stimulated by 0.15 #M calcium ionophore A23187 produced LTB4 plus two diastereoisomers, the 5(S),12(R)-6-trans-LTB4 and 5(S),12(S)6-trans-LTB4. 20-Hydroxy-LTB4, one oxidized metabolite, was also detectable. A typical separation of these

50 four compounds provided from stimulated neutrophils from allergic patients, is shown in Fig. 1. The insert represents the ultraviolet spectrum of the compound eluted as peak 4 which is closely superimposable to the spectrum of authentic LTB4. In the absence of exogenous arachidonic acid, neutrophils from allergic patients stimulated by 0.15/~M calcium ionophore A23187 produce 20 to 40-fold higher LTB4 than cells from healthy subjects (Fig. 2). The !~ormer produce 16.2 ± 4.1 pmol LTB4/5 rain per 2- 106 cells, as measured by ultraviolet absorption peak integration after HPLC, whereas the latter generate 0.97 :l: 0,27 pmol LTB4/5 min per 2.106 cells as assayed by RIA. When neutrophils from allergic patients were activated in the presence of increasing concentrations of exogenous unlabeled arachidonic acid, LTB4 level increased significantly in a dose-dependent manner. In the presence of 10 ~M arachidonic acid, the LTB4 level in neutrophils from allergic patients was 38.1 + 6.8 pmol/5 min per 2.106 cells (P < 0.005 versus LTB4 level in the absence of arachidonic acid; n--8). In contrast, increasing the fatty acid concentrations had no effect on LTB4 level in neutrophils from healthy subjects (1.28 _+

lX10

-2

C

' |1

is

220

i i i i i i

|,

270

i i i

ii

i

iIJ

320

Wavelength ( nm )

05

6C

A 23187 (0.15 klM) f~ o r

40

o ×

E

20 E Q. ~t I-/

J v

2.5

7,5 10 0 2.5 Arachidonic acid ( H M )

Allergic

7.5

10

Healthy

Fig. 2. Effect of exogenous arachidonic acid on LTB4 production by neutrophils from allergic and healthy subjects. Neutrophils (10v cells/ml) were stimulated with the ionophore A 23187 (0.15/tM) in the absence or presence of increasing concentrations of unlabeled arachidonic acid. After 5 rain at 37°C, the reaction was terminated with one volume of methanol, the homogenate was centrifuged and LTB4 was quantitated in the supernatant by integration of ultraviolet absorption peak given by HPLC (neutrophils from allergic patients) or by RIA (neutrophils from healthy subjects). Individual values (pmol/5 rain per 2.106 cells) from separate experiments are plotted.

0.22 pmol LTB4/5 min per 2.106 cells in the presence of 10/~M arachidonic acid) (Fig. 2). In a control experiment without activation by the ionophore, neutrophils from allergic or healthy subjects did not produce any LTB4 in the absence as well as in the presence of increasing concentration of arachidonate.

Incorporation of exogenous [l-14C]arachidonic acid into lipids of neutrophils from allergic and healthy subjects

0

10

20

Retention time

30 (min)

Fig. 1. Purification of LTB4 by HPLC. Colunm: LiChrospher 100 RP 18 (5 Fro; 220x4 nun); mobile phase: acetonitrile/methanol/water/ acetic acid (300:250:400:4, v/v) adjusted to pH 5.6 with NH4OH; flow rate: 0.8 ml/min. Ultraviolet absorbance monitored at 270 nm. Peaks: 1, 20.OH-LTB4; 2, 5(S),12(R).6-trans-LTB4; 3, 5(S),12(S)-6trans-LTB4; 4, LTB4. The inset, represents the ultraviolet-spectrum of compound eluted as peak 4. AUFS = absorption unit at full scale.

60 min after addition of [1-t4C]arachidonate, 80~; of the added radioactivity was cell-associated both in neutrophils from allergic and healthy subjects. From this cell-associated [1-14C]arachidonate, 46~ and 26~; were incorporated in phospholipids of neutrophils from allergic and healthy subjects, respectively (Table I). PI, PC and PE are primarily labeled. However, we found that labeled arachidonate was incorporated to the same extent in PI and PC species of cells from healthy donors, whereas about 2-fold more radioactivity was incorporated in PI compared to PC in neutrophils from allergic patients. Then, PI from allergic subjects incorporated significantly more radioactivity (about 2-fold) than PI from healthy volunteers.

51 TABLE I

Incorporation of [1-14C]arachidonic acid into lipids of neutrophils from allergic and healthy subjects Neutrophils (107 cells/mi) were incubated with [l-14C]arachidonic acid for 60 rain at 37°C, washed twice, and incubated for an additional 10 min. Lipids were extracted from the cell pellets as described in Materials and Methods. The data represent the amount of radioactivity in each lipid species as a percentage of total radioactivity spotted on TLC plates. The values indicated represent the mean + S.E. of seven separate experiments with neutrophils from allergic patients and four separate experiments with cells from healthy subjects, n.d., not determined Lipid fractions

Percentage of recovered radioactivity healthy

Pl PC PE PS Free fatty acid Triacylglycerols

11.9 + 1.7 11.8 -I-0.9 2.8 + 0.4 n.d. 1.3 + 1.3 70.7 + 0.2

allergic P < 0.05

P < 0.05

26.5 + 1.5 14.4+ 1.2 4.4 + 1.3 0.5 + 0.5 1.1 + 0.2 53.1 + 2.1

The remaining cell-associated label was incorporated in neutral lipids co-migrating with triacylglycerols (TG). The radioactivity incorporated in TG was significantly higher in neutrophils from healthy than from allergic donors. There was no significant changes in the level of label incorporated in PC and PE between both cells populations.

Metabolism of arachidonic acid in labeled neutrophiis When labeled neutrophils from allergic patients were stimulated by the ionophore (0.15/~M) in the absence or presence of unlabeled arachidonic acid, LTB4, its two isomers and the 20-hydroxy metabolite were produced in quantities high enough to be measured by HPLC. In the absence of exogenous arachidonic acid, LTB4 level after 5 min stimulation by the ionophore (0.15 pM) was 23.8 + 6.4 pmol/5 min per 2- 106 neutrophils, whereas it was 53.4 + 7.8 in the presence of 10/~M unlabeled arachidonate (P < 0.05) (means + S.E. from seven separate experiments). Surprisingly, LTB4 and the other 5-1ipoxygenase metabolites were totally unlabeled. According to our early observations (Fig. 2), the stimulation of neutrophils from healthy subjects did not produce LTB4 or other metabolites detectable by HPLC and no radioactivity was detectable in the effluent. Arachidonic acid metabolism through 5-1ipoxygenase first involves the action of phospholipase A 2 that releases the acid from the esterified arachidonic acid pool. This lipase is activated during stimulation by various stimuli, i.e., calcium ionophore A23187. However, although all lipid classes of neutrophils used were labeled (Table I), LTB4 produced was unlabeled. We then attempted to determine the source of arachidonic acid used in the biosynthesis of unlabeled LTB4.

Distribution of [l-14C]arachidonic acid in choline- and ethanolamine-linked phosphoacylglycerol subclasses During the stimulation of neutrophils by the calcium ionophore A23187, activated phospholipase A 2 released arachidonic acid and lyso-PAF further acetylated to produce PAF-acether. 1-O-alkyl-l'-enyl-2-arachidonoylsn-glycero-3-phosphoethanolamine may provide arachidonate [17] together with 1-O-alkyl-2-arachidonoyl-snglycero-3-phosphocholine [18,21], the latter providing the lyso-PAF as well [18,21]. Since it is assumed that the choline- and ethanolamine-linked pools are the main sources of arachidonic acid for the biosynthesis of arachidonate oxygenated metabolites, we have studied the arachidonate labeling of PC and PE subclasses after hydrolysis of the PC and PE pools with phospholipase C and derivatization into dinitrobenzoyl derivatives (see Methods). The results are summarized in Table II. 1,2-Diacyl- and 1-O-alkenyl-2-acyl-subclasses of PC, and 1,2-diacyl-, 1-O-alkyl-2-acyl- and 1-O-alkyl-l'-enyl2-acyl-subclasses of PE were labeled in both neutrophils from allergic and healthy subjects. In contrast [1-~4C]arachidonate was not incorporated in the 1-O-alkyl-2-acyl-subclass of PC. No significant difference could be observed between both neutrophil populations. Effect of exogenous [l-14C]arachidonic acid on LTB 4 production The results depicted in Table III show that in the absence of exogenous arachidonic acid, HPLC measurable LTB4 was generated after stimulation of unlabeled

TABLE !!

Distribution of [l-14C]arachidonate in phosphatidylcholine and phosphatidylethanolamine subclasses of neutrophils from allergic and healthy subjects Choline-linked (PC) and ethanolamine-linked (PE) glycerophospholipids were separated by TLC, elnted from the plates, hydrolyzed by phospholipase C (Bacillus cereus) and derivatized into dinitrobenzoyl derivatives. The diradyi derivatives were then separated by TLC. The spots visualized by exposition to iodine were scraped off and counted by scintillation counting. The amount of radioactivity in each lipid subclass is expressed as a percentage of total radioactivity spotted on the plates. The data represent the mean+S.E, of seven separate experiments with neutrophils from allergic patients and four with cells from healthy subjects. *: PE fractions were pooled before hydrolysis and derivatization Phospholipid classes

Phosphol,pid subclasses

Healthy

Allergic

PC

diacyl-GPC alkyl-acyl-GPC alkenyl-acyl-GPC

64.6 + 4.4 0 35.4 + 4.4

56.4 + 2.5 0 43.6 ± 3.4

PE

diacyl-GPE alkyi-acyi-GPE aikenyl-acyl-GPE

19.3 * 34.8 * 45.9 *

35.4 :!:4.7 23.2 + 4.5 41.5 + 6.6

52 Discussion

TABLE !11

Effect of exogenous [I-14C]arachidonic acid on LTB4 production by unlabeled neutrophilsfrom allergic subjects Neutrophils (10 Tcells/ml) were stimulated by the ionophore A23187 (0.15 pM) for 5 rain at 37°C, in the absence or presence of increasing concentrations of [l-14C]arachidonic acid. LTB4 was purified by RPHPLC and the effluent recorded at 270 nm. Fractions of the effluent were collected every minutes and total radioactivity of LTB4 peak measured by scintillation counting [1-14C]AA (/~M)

Total LTB4 a

Labeled LTB4 b

Unlabeled LTB4 c

0 2,$ 10.0

30.2 + 8.6 39.0 + 9.8 $8.4+8.2

11.6 + 4.8 43,64-5.2

27.4 4- 6.6 14.84-4.9

a Total LTB4 measured by peak integration. t, Amount of labeled LTI~ determined from specific activity of arachidonic acid. c Unlabeled LTI~ calculated by substraction of b from a. The results (pmol/5 min per 2.106 cells) represent the mean 4-S.E. of seven separate experiments.

neutrophils from allergic patients with 0.15/tM calcium ionophore A23187 (30.2:1:8.6 pmol/5 min per 2-106 cells). This LTB4 must be produced from an endogenous pool of arachidonic acid. In the presence of increasing concentrations of exogenous labeled arachidonic acid (2.5 and 10 /zM), the LTB4 level was also increased (58.4 + 8.2 pmol/5 rain per 2.106 cells at 10 ~tM). These results confirm the data shown in Fig. 2. Scintillation counting of LTB4 fractions collected after HPLC revealed that LTB4 produced in the presence of exogenous labeled fatty acid was labeled and that the total activity incorporated increased with the exogenous labeled arachidonate concentration. With neutrophils from healthy subjects, essentially no HPLC detectable LTB4 was formed and no radioactivity was detectable in the effluent. The amount of labeled LTB4 may be calculated from the specific activity of exogenous labeled arachidonic acid and the total radioactivity of LTB4. The difference between total LTB4 produced and labeled LTB4 is likely the part of LTB4 generated from the unlabeled endogenous arachidonate pool. As reported in Table Ill, the amount of labeled LTB4 increases and the part of unlabeled LTB4 decreases with increasing concentrations of exogenous labeled arachidonic acid. At 2.5 and 10/~M, about 30~ and 75~, respectively, of total LTB4 produced by neutrophils from allergic patients was [1t4C]arachidonate-derived, whereas about 70% and 25%, respectively, of LTB4 should be synthesized from the substrate released from the endogenous unlabeled site. Thus, it appears that exogenous arachidonic acid is easily used by 5-1ipoxygenase pathway and inhibits the LTB4 formation from the endogenous source of arachidonic acid, in neutrophils from allergic patients.

Leukotriene B4 production in neutrophils is generaily studied during cell stimulation by different agents including the calcium ionophore A23187 used at micromolar concentrations from 2 to 10 /~M. Under these conditions, large quantities of LTB4 are produced, easily detectable by HPLC and measurable by ultraviolet spectrophotometry. In preliminary investigations, we have compared LTB4 production by human peripheral blood neutrophils from allergic and healthy subjects challenged with 1.5/~M calcium ionophore A23187. We have shown that after 5 min stimulation at 37°C, LTB4 was significantly higher in neutrophils from allergic patients than from healthy donors (unpublished data). The present data establish that a low concentration of calcium ionophore A23187 such as 0.15/~M strongly stimulates neutrophils from allergic patients. The LTB4 level was easily detectable and assayable by HPLC after 5 min challenge at 37°C. In contrast, the LTB4 level in neutrophils from healthy subjects was always very low, undetectable by HPLC and measurable only by RIA. Moreover, increasing concentrations of exogenous arachidonic acid had no effect on the LTB4 level in neutrophils from healthy donors, whereas it increased this level in neutrophils from patients with allergic rhinitis. In addition, when neutrophiis from healthy volunteers were challenged in the presence of increasing exogenous [1-t4C]arachidonate, labeled LTB4 was never detectable in the incubation medium, whereas the stimulation of neutrophils from allergic subjects resulted in an increased production of labeled LTB4 under the same conditions. The latter increase was accompanied by a progressive decrease of unlabeled LTB4 produced from arachidonic acid released from an endogenous store of the cell. 5-Hydroxyeicosatetraenoic acid (5-HETE) also generated via the 5-1ipoxygenase pathway after cell stimulation was detectable only in neutrophils from allergic patients. We focused on LTB4 levels because of its biological activity and also because part of 5-HETE generated is known to be reesterified into membrane phospholipids [27]. It is widely accepted that in resting mammalian cells the free arachidonate concentration is very low and that the fatty acid is almost exclusively located under ester form at the sn-2 position of glycerol in glycero-phospholipids [14]. Esterified arachidonic acid is released by activation of phospholipase A e in stimulated cells, i.e., with the calcium ionophore A23187 [16]. In human neutrophils, it has been shown that phospholipase A 2 acts preferentially on 1-O-alkyl-2-arachidonoyl-sn-2glycerophospholipids then releasing arachidonic acid and lyso-PAF [17,21], the released arachidonate acting as a substrate for eicosanoids biosynthesis [14,28]. Indeed, in human neutrophils, choline-linked (PC) and

53 ethanolamine-linked (PE) glycerophospholipids contain 19% and 68% of total cellular arachidonate [17]. Moreover, 66% of PC- and 71% of PE-arachidonate are located in 1-O-alkyl-2-acyl-glycerophosphocholine and 1- O-alkyl-1 '-enyl-2-acyl-glycerophosphoethanolamine subclasses, respectively [17]. [1-14C]arachidonate was rapidly incorporated at similar extent in neutrophils from allergic and healthy subjects. According to others [17,18,24], labeled arachidonate is primarily incorporated into PI and PC and to a lesser extent into PE, while the radioactivity incorporated into PS is very low. The stimulation of [14C]arachidonate labeled neutrophils from allergic and healthy subjects by 0.15 pM calcium ionophore A23187 resulted in the formation of unlabeled LTB4. Numerous investigators have found that labeled LTB4 is synthesized in labeled neutrophils challenged with high concentrations (higher than 2 pM) of calcium ionophore A23187 [28,29]. Indeed we found that labeled neutrophils from allergic and healthy donors stimulated by 1.5 #M ionophore, in the absence of exogenous arachidonic acid, released labeled LTB4 (data not shown). Therefore, in neutrophils stimulated by 0.15 /~M ionophore, arachidonate substrate for LTB4 production must be released from an endogenous pool not labeled after cell incubation with [1-14C]arachidonic acid. Examination of different subclasses of PE and PC revealed that only 1-O-alkyl-2-acylglycerophosphocholine was unlabeled, suggesting that arachidonic acid contained in this species might be the source of the substrate for the 5-1ipoxygenase pathway in neutrophils from allergic patients stimulated with low concentration of ionophore in the absence of exogenous arachidonate. The suggestion that 1-O-alkyl-2-acyl-GPC is the main source of LTB4 endorses that Chilton [30]. Our results show that LTB4 levels in neutrophiis from allergic patients are higher than those in neutrophils from healthy subjects in response to 0.15 #M ionophore A23187. Our results concerning normal neutrophils are consistent with those of Biilah and coworkers [29], showing that LTB4 and 5-HETE are TLCor HPLC-detectable only when neutrophils from healthy donors are stimulated by ionophore A23187 at concentrations higher than 0.3 #M. The present experiments have been carried out with neutrophils from allergic patients during the period of exposure to various pollen allergens in our region (March to October). Out of this period, the LTB4 level after cell stimulation was low or undetectable (unpublished observations). The increased production of LTB4 by peripheral blood neutrophils from allergic patients could then indicate intrinsic cell abnormalities and/or extrinsic neutrophil stimulation ('priming') in allergic diseases. Among the intrinsic abnormalities, 5-1ipoxygenase [31] and phospholipase A 2 hyperactivity [32] have been reported. Recent observations have shown that neutrophils may be 'primed' by various mediators

(granulocyte and macrophage colony stimulating factor, GM-CSF, and other cytokines). Such a cytokine stimulation has been proposed to explain the increased LTB4 production, after neutrophil stimulation with calcium ionophore or chemotactic peptides [33,34], and neutrophil respiratory burst observed in allergic diseases [35]. In summary, our results show that neutrophils from allergic patients are much more sensitive to calcium ionophore A23187 stimulation than those from healthy subjects and produce higher quantities of LTBa when challenged by low ionophore concentrations (0.15 #M). Moreover, in contrast to neutrophils from healthy subjects, neutrophils from allergies seem to incorporate directly exogenous arachidonate into LTB4. In addition, LTB4 might be produced from endogenous arachidonic acid released essentially from the 1-O-aikyi-2-arachidonoylglycerophosphocholine subclass. This suggests that arachidonic acid release under neutrophil stimulation could be different according to the concentration of ionophore A23187, the highest concentration releasing the fatty acid from various classes of phospholipids, leading to labeled LTB4 from labeled neutrophils [17]. Acknowledgement This work was supported by the 'Institut National de la Sant6 et de la Recherche M6dicale'. References 1 Holgate, S.T. (1988) Postgr. Med. J. 64, 82-95. 2 Borgeat, P. and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA 76 2148-2152. 3 Borgeat, P., Fruteau de Laclos, B. and Maclouf, J. (1983) Biochem. Pharmacol. 32, 381-387. 4 Borgeat, P. (1989) Can. J. Physiol. Pharmacol. 67, 936-942. 5 Ford-Hutchinson, A. (1990) Crit. Rev. lmmunol. 10, 1-12. 6 Samuelsson, B. (1983) Science 220, 568-575. 7 Lewis, R.A. and Austen, K.F. (1984) J. Clin. Invest. 73, 889-897. 8 Smith, R.J., lden, S.S. and Bowman, B.J. (1984) Inflammation 8, 365-384. 90'Fiaherty, J.T., Showell, H.J., Becker, E.L. and Ward, P.A. (1979) Prostaglandins 17, 915-927. 10 Shasby, D.M.., Shasby, S.S. and Peach, M.J. (1985) J. Appl. Physiol. 59, 47-55. 11 Badwey, J.A., Curnutte, J.T. and Karnovsky, M.J. (1981) J. Biol. Chem. 256, 12640-12643. 12 Badwey, J.A., Curnutte, J.T., Robinson, J.M., Berde, C.B., Kamovsky, M.J. and Karnovsky, M.L. (1984) J. Biol. Chem. 259, 7870-7877. 13 Smith, R.J., Sam, L.M., Justen. J.M., Leach, K.L. and Epps, D.E. (1987) Br. J. Pharmacol. 91,641-649. 14 Irvine, R.F. (1982) Biochem. J. 204, 3-16. 15 Needleman, P. (1978) Biochem. Pharmacoi. 27, 1515-1519. 16 Walsh, C.E., Dechatelet, L.R., Chilton, F.H., Wykle, R.L. and Waite, M. (1983) Biochim. Biophys. Acta 750, 32-40. 17 Chilton, F.H. and Connell, T.R. (1988) J. Biol. Chem. 263, 52605265. 18 Swendsen, C.L., Ellis, J.M., Chilton, F.H., O'Flaherty, J.T. and Wykle, R.L. (1983) Biochem. Biophys. Res. Commun. 113, 72-79.

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