European Journal of Clinical Investigation (1991) 21, 631-637

Leukotriene B4 levels from stimulated neutrophils from healthy and allergic subjects: effect of platelets and exogenous arachidonic acid R. HOSNI, B. CHABANNES, Y. PACHECO, P. MOLIERE, M. GROSCLAUDE, M. PERRIN FAYOLLE & M. LAGARDE*, INSERM U 205, Laboratoire d'Immuno-allergologie Respiratoire, Hapita1 Sainte-

Eugtnie, Saint Genis Laval, France, and * INSERM U 205, Laboratoire de Chimie Biologique, Institut National des Sciences Appliqukes, Villeurbanne, France Received 5 July 1990 and in revised form 13 March 1991

Abstract. Leukotriene B4 (LTB4) levels were measured in peripheral blood neutrophils from allergic and healthy donors after stimulation by calcium ionophore A 23187. This level was higher in neutrophils from allergic subjects than in neutrophils from healthy subjects in the presence as well as in the absence of exogenous arachidonic acid. Platelets from allergics increased LTB4 levels from neutrophils from allergics but not levels in those from healthy donors. Moreover, platelets from healthy subjects reduced LTB4 in neutrophils from both groups. These results suggest that biochemical differences exist in neutrophils and platelets from allergics which contribute to changes in arachidonic acid metabolism via the 5-lipoxygenase pathway. In addition, they support the concept that platelets may play an important role in the regulation of neutrophil LTB4 levels, possibly by affecting the 5lipoxygenase activity during the course of allergic inflammatory reactions. Keywords. Allergy, arachidonic acid, asthma, human neutrophils, human platelets, leukotriene B4. Introduction There is increasing evidence that clinical manifestations of atopic inflammatory diseases depend on a Abbreviations: PBS, phosphate buffered saline; LTB4, leukotriene B4 or 5S, 12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoicacid; 12-HPETE, 12S-hydroperoxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid; 12-HETE. 12S-hydroxy-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid; (5S-12R)-6-truns-LTB4, 5S,12R-dihydroxy-6,8,10,14(E,E,E,Z)-eicosatetraenoic acid; (5S-12S)-6-trans-LTB4, 5S,12Sdihydroxy-6,8,10,14-(E,E,E,Z)-eicosatetraenoic acid; 5s-12sDHETE, 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid; 20-OH-LTB4,5S,12R,20-trihydroxy-6,8,10, I4-(Z,E,E,Z)-eicosatetraenoic acid; PGB2, prostaglandin B2; RP-HPLC, reverse phase-high performance liquid chromatography. Portions of this work were presented at the XIIIth International Congress of Allergology and Clinical Immunology, Montreux, Switzerland, October 16-21, 1988, and were published in abstract form (Allergy Proceedings 1988; 9: 438). Correspondence: Dr B. Chabannes, Laboratoire d'Immunoallergologie Respiratoire, INSERM U 205, HBpital Sainte-Eugtnie BBt SF, 1 Avenue Georges Cltmenceau, 69230 Saint Genis Laval, France.

complex interaction between a variety of cells successively stimulated by numerous mediators. Histological, biochemical and physiological studies demonstrate that neutrophils [1,2] and platelets [3-51 play a supporting role in atopic inflammatory and hypersensitivity reactions. Natural factors influencing the neutrophi1 activity are still unknown. Recent attention has focused upon the role of leukotriene B4 (LTB4), a product of arachidonic acid through the 5-lipoxygenase pathway. LTB4 stereospecifically activates neutrophils. It is a complete secretagogue in these cells [6,7] and a potent stimulator of neutrophil chemotaxis [8,9]. In addition, LTB4 increases leucocyte adherence to the vascular endothelium [8,10,113and transendothelial migration of neutrophils [12]. This leukotriene B4 can play an important role in the elaboration and amplification of inflammatory response by neutrophils [13]. Some differences in physiological and biochemical behaviour of neutrophils and platelets have been observed when allergic and non-allergic subjects were compared. In neutrophils from atopic patients stimulated with the ionophore A 23187, the formation of some arachidonate 5-lipoxygenase products of arachidonic acid is modified [ 14,151 and the respiratory burst is higher than in cells from healthy donors [16]. In platelets from asthmatic patients, some physiological functions are also altered [ 17,181. It has been reported that a cellular cooperation between neutrophils and platelets may promote the LTB4 production in human neutrophils [ 19-2 11 in a way that 12-HPETE produced by arachidonate 12-lipoxygenase in platelets may stimulate LTB4 synthesis [20]. These observations could mean that platelets might be implicated in the initiation and/or amplification of allergic inflammatory diseases and hypersensitivity reactions, but the part played by platelets is still largely unexplored. Although LTB4 is not the end-product of the nonpeptidic leukotriene pathway, the present work focused on its level because of its biological activity. We measured LTB4 levels in stimulated peripheral blood neutrophils from healthy subjects and allergic patients, in the presence and absence of platelets and/ or arachidonic acid. 63 1

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Patients and methods Patients Nineteen atopic volunteers, 8 females and 11 males (mean age: 30 years; age range: 13 to 50 years) were studied. All had a positive atopic allergic disease (allergic rhinitis and asthma), positive allergic skin tests, elevated IgE and specific IgE levels. Asthmatic patients were treated with beta-2-mimetic drugs alone or in association with theophylline. Steroid antiinflammatory drugs were always excluded. However, the treatment was stopped 48 h before the venipuncture. Patients with allergic rhinitis did not take any medicine for two months before the blood punction. Allergic patients were studied either in the critical season or in the free interval.

Healthy subjects Fifteen healthy volunteers with negative personal and family history of atopy and no clinical evidence of atopic or other major diseases, served as controls (7 females and 8 males; mean age: 30 years; age range: 23 to 39 years). They claimed to have not taken any drugs for at least two months. Cell preparation All subjects were fasted for at least 12 h before venipuncture. Venous blood (50 ml) was drawn, collected into polypropylene tubes containing 5 ml of 3.8% sodium citrate solution, and was immediately centrifuged at 120 g for 20 min at room temperature. The upper phase (platelet-rich plasma) and lower phase (remaining blood cells) were removed and treated separately. Platelet-rich layers were diluted with 4 volumes of Tyrode's solution, without sodium bicarbonate (Sigma Chemical Co., St Louis, MO, USA), and centrifuged at 1500 g for 10 min. The resulting pellets were washed twice and suspended into the same solution at the concentration of about 48 x lo7 platelets per ml. Cells were counted with Unopett (Becton-Dickinson Co., Rutherford, NJ, USA). Neutrophils were purified by the method of Boyum [22]. Briefly, the remaining blood cells (lower phase) were mixed with a 4.5% Dextran (PM 100000200 000) solution (Serva Feinbiochemica GMBH, Heidelberg, Germany) at a lower phase/Dextran solution ratio of 5: 1 and sedimented for 30 min at 37°C. The upper neutrophil-rich fraction (6 ml) was diluted twice with phosphate buffered saline solution (PBS), containing (mmol 1-'): NaCl 136.75, KC1 2.68, Na2HP04 8.04 and KH2P04 1.47 (pH 7.4), layered onto 5 ml Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) and centrifuged at 600 g for 20 min. The erythrocytes in the neutrophil-containing pellets were lysed by vortexing for 30 s with sterile water. Then 0.15 mol I-' NaCl (10 ml) was added and the mixture centrifuged in the same conditions. The resulting neutrophil pellets were washed twice with PBS supplemented with CaC12 (8 mmol I-') and MgC12 (2 mmol

1-I) and cell concentration was adjusted to 4 x lo7cells per ml. An aliquot of the final neutrophil preparation was stained with May-Grunwald Giemsa (Farmitalia Carlo-Erba, Romilly sur Andelle, France) for differential cell counting. The neutrophil count and viability were greater than 96%. Neutrophil contamination by platelets measured by phase contrast microscopy (Leitz, Wetzlar, Germany) was about 0.5-1 platelet per neutrophil, depending on the preparation, in neutrophils from both allergic and healthy subjects. Eosinophi1 content was always below 5%. Incubations In 3 ml polypropylene tubes, 50 pl neutrophil suspension (2 x lo6 cells) were mixed with 50 pl platelet suspension (24 x lo6 cells) or 50 pl Tyrode's solution (assay without platelets) and incubated for 5 min at 37°C. Then 50 pl ionophore A 23187 (Sigma Chemical Co.) (final concentration 1.5 pmol 1-I) and 50 p1 arachidonic acid (Sigma Chemical Co.) (final concentration lOpmoll-') in PBS or 50 pl PBS (assay without arachidonic acid) were added, and the mixture was incubated for another 5 min at 37°C. The final concentrations of CaClz and MgC12 in the incubation medium were 2 mmol I-' and 0.5 mmol I-', respectively. The incubation was terminated by the addition of 0.2 ml methanol containing 60 pmol of prostaglandin B2, cooling at 4°C and acidification to pH 3.5 by 10 pl of 0.3 moll-' H3P04.After centrifugation at 1500g and 4°C for 10 min, the supernatant was stored at - 20°C until the analysis. PuriJication and assay of leukotriene B4 LTB4 was purified and assayed by reversed phase high performance liquid chromatography (RP-HPLC). A Kontron HPLC system (Miinchen, Germany) consisting of a 420 solvent delivery pump, a 430 UV absorbance detector and a 450 data system was used. Separation of eicosatetraenoic acids was achieved on a reversed phase Spherisorb ODs-2 C IS Microbore column (length: 22 cm, internal diameter: 0.4 cm, particle size: 5 pm) (Interchim, MontluCon, France). The column was isocratically eluted with the mobile phase acetonitrile-methanol-water-acetic acid (300:250:400:4 by vol) adjusted to pH 4.5 with NH40H and at the flow rate of 0.8 ml per min. All solvents were HPLC grade (Farmitalia Carlo-Erba). The eluent was monitored at 270 nm. Routinely, 0.1 ml aliquot of supernatant was injected directly without prior extraction or fractionation. Four metabolites of arachidonic acid were detected and identified by comparison with authentic standards. They were 5S, 12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (leukotriene B4, LTBd), 5S, 12R-dihydroxy6,8,10,14-(E,E,E,Z)-eicosatetraenoicacid [(5S- 12R)6-trans-LTB41 (Sigma Chemical Co.), 5S, 12sdihydroxyd,8,10,14-(E,E,E,Z)-eicosatetraenoic acid [(5S,12S)-6-trans-LTB4], 5S, 12S-dihydroxy-6,8,10,14(E,Z,E,Z)-eicosatetraenoicacid (5S,I2S-DHETE) and

LEUKOTRIENE B4 LEVELS IN HEALTHY AND ALLERGIC NEUTROPHILS

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Retention time (min) Figure 1. Separation of LTB4 from its diastereoisomersby RP-HPLC. Column: Spherisorb ODS-2 CIS(22 x 0.4cm;5 pm particle size); mobile phase: methanol-acetonitrile-water-acetic acid (250: 300:400:4 by vol); pH: 4.5; flow rate: 0.8 ml min-'. UV absorbance monitored at 270 nm. AUFS=absorbance units at full scale. (a) Assay with additional platelets. (b) Assay without additional platelets. Peaks: 1= 20-OH-LTB4; 2 = PGB2; 3 = (5S,12R)-6-trans-LTB4; 4 =(5S,12S)-6-trans-LTB4; 5 = LTB4; 6=SS,IZS-DHETE. The insert of the figure shows the UV spectrum of peak 5 recorded directly on the effluent.

5S,12R,20-trihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (20-OH-LTB4) (Cayman Chemical Company, Ann Arbor, MI, USA). The ultraviolet spectrum of LTB4 was recorded with a photodiode array multispectral analyser (1 040A HPLC-detection-system, Hewlett Packard, San Diego, CA, USA). Quantitation of LTB4 was done with PGB2 used as an internal standard. Data and statistical analysis LTB4 levels are presented as pmol present after 5 min stimulation of 2 x lo6 neutrophils. Individual values obtained with neutrophils from each donor are given. MeanfSEM for each population is also indicated. The differences between means of each group were compared using the unpaired Kruskal and Wallis nonparametric rank sum test. When populations could be coupled, the non-parametric paired Wilcoxon's test was used. Results

Isolation and assay of LTB4 by HPLC (Fig. I) When neutrophils were stimulated with the ionophore

A 23 187 with or without arachidonic acid, LTB4 could be detected with two diastereoisomers: (5S,12R)-6trans-LTB4 and (5S,12S)-6-trans-LTBd. In the presence of additional platelets, a third isomer (5S,12S)DHETE was also released. We have improved the mobile phase which includes methanol, acetonitrile, water and acetic acid (250:300:400:4 by vol), and leads to the complete separation of these four substances by RP-HPLC. Figure 1 represents two typical chromatograms obtained after stimulation of neutrophils alone (Fig. lb) or in the presence of additional platelets (Fig. la). Peaks 1,2, 3,4, 5 and 6 co-migrate with authentic 20OH-LTB4, PGB2, (5S,12R)-6-trans-LTB4, (5S,12S)-6trans-LTB4, LTB4 and (SS,12S)-DHETE, respectively. In an attempt to verify the identity of the compound eluted at peak 5, we have recorded the UV spectrum directly in the effluent with a multispectral analyser. The insert of Fig. 1 shows that this compound has a typical UV spectrum of a conjugated triene [23] and that this spectrum is superimposable to that of LTB4 (maximal absorbance at 270 nm with two shoulders at 260 and 280 nm). Moreover, the ratio of absorbances measured at 270 nm and 280 nm (1.26 in this case) is

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