Immunology 1992 77 408-415

Leukotriene A4 modulates generation of leukotriene B4 and sulphidopeptide leukotrienes by human neutrophils R. A. HILGER & W. KONIG Lehrstuhlfur Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Universitat Bochum, Germany

Acceptedfor publication 19 May 1992

SUMMARY We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23 187 or sodium fluoride (NaF). The Ca ionophore A23 187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation ofcells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23 187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response.

INTRODUCTION Leukotrienes are involved in inflammation, immediate type hypersensitivity and respiratory disorders and they are well known for their immune modulatory activity."2 The action of 5lipoxygenase on arachidonate leads to a hydroperoxy intermediate, which is then dehydrated to the allylic epoxide leukotriene A4 (LTA4). LTA4 is stereospecifically converted by anhydrolase to LTB4 or by enzymatic transformation into an isomer of 5,6dihydroxyeicosatetraenoic acid (DiHETE). Spontaneous hydrolysis leads to the biologically inactive stereoisomers of LTB4, 6-trans-LTB4 and 12-epi, 6-trans-LTB4.3 Conjugation of LTA4 with glutathione, leading to LTC4 formation, is catalysed by specific particulate LTC4 synthase4 or various unspecific, mainly cytosolic glutathione-S-transfer-

LTC4 and its metabolites, LTD4 and LTE4, are potent constrictors of vascular and bronchial7 8 smooth muscle. LTC4 and LTD4 also induce plasma leakage from the micro vasculature.9 LTB4 is a potent chemotactic factor for polymorphonuclear leucocytes (PMN)'° and induces neutrophil degranulation, superoxide generation" and leucocyte adherence to vascular endothelium.9 Because ofits chemical instability LTA4 has often been considered solely as an intracellular precursor for leukotriene generation. However, LTA4 can be released and extracellulary stabilized by albumin'2 and then transformed via a transcellular metabolism into LTB4 or LTC4.'3-17 Platelets and human endothelial cells from umbilical veins'8 9 are able to convert extracellular leukotriene A4 into LTC4. Erythrocytes,'4 human airway epithelial cells,20 T and B lymphocytes2' convert LTA4 via a 5-hydrolase into LTB4. Lymphocytes are not able to synthesize LTC4 or LTB4 directly from endogenous or exogenous arachidonic acid. As has been shown extracellular LTA4 provokes neutrophil aggregation, degranulation and stimulates the mobilization of free cytosolic calcium.22'23 Recently, it has become evident that LTA4 is also an important modulator of various effector cells. In this regard LTA4 up-regulates CD23 expression on human B cells and monocytes at suboptimal concentrations of interleukin-4 (IL-4)24 and also increases the cytokine (IL-3, IL-8)-induced leukotriene formation in cells from atopic donors.25 Thus, within an inflammatory environment LTA4 may be present either alone or in combination with other mediators. Therefore, it is of particular interest to study also the modula-

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Abbreviations: DiHETE, dihydroxyeicosatetraenoic acid; 5S,12SDiHETE, 5S, 12S-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid; GSH, glutathione; FMLP, n-formyl-methionyl-leucyl-phenylalanine; HETE, hydroxyeicosatetraenoic acids; LO, lipoxygenase; LT, leukotrienes; LTA4-Me, LTA4 methylester; MK-886 (L-663,536)=3-[1-(4chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimetyl propanoic acid; NaF, sodium fluoride; PMN, polymorphonuclear granulocytes; RP-HPLC, reverse-phase high-performance liquid chromatography. Correspondence: Professor W. Konig, Medizinische Mikrobiologie und Immunologie, Arbeitsgruppe fur Infektabwehrmechanismen, Ruhr-Universitat Bochum, Postfach 102188, Universititsstrate 150, 4630 Bochum 1, Germany.

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Leukotriene A4 modulation of leukotriene B4 and sulphidopeptide leukotrienes tory role of LTA4 on inflammatory effector cells which are

subsequently activated. The Ca ionophore A23187 and sodium fluoride (NaF), which differs in the mode of cellular activation26

used as stimuli. While Ca ionophore A23187 directly elevates intracellular Ca2+ by bypassing membraneous signal transduction, NaF interacts with receptor associated G-proteins and activates the subsequent signal transduction elements. Thus, the purpose of this study was to analyse the effects of LTA4 on leukotriene generation and metabolism from polymorphonuclear leucocytes which were stimulated differently. were

MATERIALS AND METHODS Materials The reagents used were from the following sources. Ficoll 400 was obtained from Pharmacia (Uppsala, Sweden); Macrodex (6%, w/v) was from Knoll (Ludwigshafen, Germany); sodium metrizoate solution (75%, w/v) from Nyegaard (Oslo, Norway); Zymosan A, FMLP, the Ca ionophore A23 187, cytochalasin B, and heparin were obtained from Sigma (Munich, Germany); acetonitrile [high-performance liquid chromatography (HPLC) grade] was purchased from Baker Chemicals (Gross-Gerau, Germany); methanol, tetrahydrofurane, EDTA, dipotassium hydrogenphosphate, and phosphoric acid were from Riedel de Haen (Seelze, Germany); human serum albumin (HSA) was from Behring (Ludwigshafen, Germany). Synthetic leukotrienes LTB4, LTC4, LTD4 and LTE4 as well as the wo-oxidated products 20-OH- and 20-COOH-LTB4, the monohydroxylated eicosatetraenoic acids (5-HETE, 12-HETE, 15-HETE) and the 5-lipoxygenase inhibitor MK-886 were generous gifts from A. W. Ford-Hutchinson (Merck Frost, Pointe-Claire/Dorval, Quebec, Canada). LTA4 methyl ester was synthesized in a modified way according to Rokach et al.27 [3H]LTA4 methyl ester (14,15-3H(N)-LTA4, methyl ester, 0-74-2-22 TBq/mmol) was purchased from New England Nuclear (NEN, Dreieich, Germany). LTA4 lithium salt (LTA4Li), was prepared as follows: LTA4 methyl ester (100 jg) was dissolved in tetrahydrofurane (93 pl). This solution was degassed with argon and then treated with 7 yl of 1 M aqueous lithium hydroxide. The solution was allowed to stand at 0° for 48 hr. Aliquots of the solution were evaporated with a gentle stream of argon and the residue was resuspended in phosphate-buffered saline (PBS) containing 0 5% (w/v) HSA for further experiments. All other chemicals were from Merck (Darmstadt, Germany).

Buffers Unless stated otherwise the medium used for washing the cells and for mediator release was a modified Dulbecco PBS (referred to as PBS) buffer consisting of 137 mm NaCl, 8 mm Na2HPO4, 2-7 mm KH2PO4 and 2-7 mm KC1 (pH 7-4) and HSA (0 5%).

Preparation of cells PMN were obtained from peripheral blood of healthy donors. Platelets were separated by isolation of platelet-rich plasma (PRP). PRP was obtained by centrifugation of peripheral blood supplemented with PBS containing 1-5% EDTA (10% v/v) at 200 g (25 min at 20°). After centrifugation of the PRP at 1285 g (20 min at 200) the pellet obtained after the first centrifugation was resuspended in the platelet-free plasma. Cells were purified on a Ficoll-metrizoate density gradient. The human neutrophils (PMN) were separated from the erythrocytes by dextran

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sedimentation. The remaining erythrocytes were lysed by exposing the cells to hypotonic conditions. This method results in more than 97% PMN.28 Contamination of the neutrophil preparations with eosinophils was detected by staining as described previously.29 The neutrophil preparations contained 1-3% eosinophils and less than 1% peripheral blood lymphocytes. Cell viability of PMN was assessed microscopically by Trypan blue exclusion analysis. The PMN were suspended to a final concentration of 2 x I07 cells/ml PBS/HSA. Leukotriene A4 conversion The concentration of cells was 2 x 107 PMN/ml PBS/HSA buffer. The cell suspensions (500 p1) were incubated with LTA4 (1 jMg) in the presence of calcium (1 mM), magnesium (0 5 mM) and HSA (0-5% w/v). Incubation proceeded for various times at 370. The reaction was terminated by the addition of 3 ml of methanol/acetonitrile (50:50, v/v). Stimulation of cells Cells (I x 107/500 p1) were preincubated for 10 min with I pg LTA4, or various stimuli (Ca ionophore A23 187, NaF) as well as PBS/HSA buffer at 370 in the presence of calcium (1 mM) and magnesium (0-5 mM). Subsequently, a second stimulus was added as indicated above (Ca ionophore A23 187, NaF, LTA4). The incubation time proceeded for an additional 10 min. The cells were also co-incubated with LTA4 (1 jg) and the various stimuli (Ca ionophore A23187, NaF) for 20 min at 370 in the presence of calcium (1 mM) and magnesium (0-5 mM). In the experiments 3H-labelled LTA4 was used, LTA4 (1 jg/I x 107 PMN) was labelled with [3H]LTA4 (0-05 pCi/l x 107 cells). All buffer controls contained equivalent amounts of the LTA4 vehicle. Stimulations were terminated after the addition of 3 ml methanol/acetonitrile (50:50, v/v).

Inhibition of 5-lipoxygenase PMN (1 x 107/500 p1 PBS/HSA) were preincubated for 10 min with MK-886 (1 pM) in the presence of calcium (1 mM) and

magnesium (0-5 mM). Stimulation was carried out as already described. Preparation of membrane fractions Isolation of the membrane fractions was carried out as previously described by Pelz et al.30 Stimulated and unstimulated neutrophils were resuspended in Tris buffer (0 05 M, pH 7-5) supplemented with sucrose (0-25 M). EDTA (I mM), EGTA (1 mM), dithiothreitol (1 mM) and leupeptin (100 pg/ml) and cell disruption was carried out by sonication in three periods over 10 seconds (energy output 40 W, sonifier 250 W; Branson, Danbury, CT). Light microscopy revealed a complete cell breakage. The granules, nuclei and unbroken cells were removed by centrifugation at 10,000 g for 20 min (J2-2 1, rotor JA-20; Beckman, Palo Alto, CA). The crude membrane fractions were collected from the supernatant fraction by centrifugation at 100,000 g for 60 min (Beckman centrifuge L8-70, rotor SW60TI). The protein content was assayed according to the method described previously.29

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Determination of GTPase activity GTPase activity was measured as previously shown by Matsumoto et al.3I The membrane fraction (10 ,ig protein) was incubated in 20 mm Tris buffer (pH 7-5) containing 150 mM NaCl, 5 mm MgCl2, 0 1 mm EGTA, 1-14 mM ATP, 0 5 mM App(NH)p, 0 25 mm ouabain and 0 5 ,UM [32P]y-GTP. After incubation for 60 min at room temperature in the absence or presence of the respective stimuli, the reaction was stopped by addition of activated charcoal in 20 mm phosphate buffer (pH 7 5). The samples were then centrifuged at 9600 g for 8 min and the 32p phosphate content of the supernatant fraction was assessed. Determination of marker enzymes The release of lactate dehydrogenase (LDH) as a marker enzyme for cell viability was determined as previously described.29 The extracellular release of LDH was calculated as percentage of the total enzyme activity available after sonication of unstimulated cells (1 x 107). Insignificant amounts (less than 6%) were detected (data not shown). Analysis of leukotriene release The supernatants of incubated cells were deproteinized by addition of 3 ml of methanol/acetonitrile (50:50, v/v), overlaid with nitrogen, and frozen at 70° for 12 hr. After centrifugation at 1000 g for 15 min, the supernatants were evaporated to dryness in a freeze dryer, suspended in 600 pl of methanol/water (30: 70, v/v), overlaid with nitrogen, and stored at 20° for 2 hr. The samples were centrifuged (9700 g for 2 min at room temperature), and 200 yl were then applied to reverse-phase (RP) HPLC. HPLC was performed on reverse-phase columns (4 x 250 mm) packed with Nucleosil (C18) 5-,m particles (Macherey and Nagel, Duren, Germany) and the automatic sample injection module WISP 7 1OB (Waters Associates, Eschborn, Germany). The column temperature was maintained at 400. The absorbance of the column effluent was monitored by using a variable ultraviolet (UV) detector adjusted to 280 nm (detection of DiHETE) and sulphidopeptide leukotrienes) or to 235 nm [detection of monohydroxyeicosatetraenoic acids (HETE)]. In addition, to exclude a relevant contamination with platelets, the samples were monitored at 301 nm for the detection of lipoxins. The peak areas were calculated using a chromatography data system (series 3000; Nelson Analytical, Mannheim, Germany). The solvent system was a mixture of phosphate buffer (17 mM K2HPO4 containing 0 05% EDTA), acetonitrile, and methanol (50:30:20, v/v for the detection of DiHETE; 30:40:30, v/v for the detection of HETE) which was adjusted to pH 5-0 with phosphoric acid. The flow rate was maintained at 1 ml/min. All solvents were degassed before use and were constantly stirred during HPLC analysis. Identification and quantitation of leukotrienes were performed as described previously.32

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Figure 1. Conversion of LTA4 by human neutrophils. Enhancing as well suppressive effects in the presence and absence of Ca ionophore A23 187 (5 pM). The generated leukotrienes were analysed by RP-HPLC. Mean values + SD (n =5) are shown. (a) PMN (1 x 107) were treated with LTA4 (1 Pg) for 10 min and subsequently for a further 10 min with PBS (a) or with Ca ionophore A23 187 (5 ,M) (/3). The decreased LTB4 generation was significant (**P< 001) to the Ca ionophore control (y). (b) PMN were stimulated with Ca ionophore A23 187 (5 gM) for 10 min and subsequently incubated for a further 10 min with PBS (6) or LTA4 (1 pg) (£). The increased LTB4 generation was significant (*P