Colocalization of cytosolic phospholipase A,, 5 ... - Wiley Online Library

Eur. J. Biochem. 238, 250-258 (1996) 0 FEBS 1996

Colocalization of cytosolic phospholipase A,, 5-lipoxygenase, and 5-lipoxygenase-activatingprotein at the nuclear membrane of A23187-stimulated human neutrophils Marc POULIOT’, Patrick P. McDONALD’, Eric KRUMP’, Joseph A. MANCINI’, Shaun R. McCOLL’, Philip K. WEECH’ and Pierre BORGEAT‘ ’ Centre de recherche en Rhumatologie et Irnmunologie, Centre de recherche du CHUL, Qutbec, Canada ’ Merck Frosst Center for Therapeutic Research, Pointe-Claire, Qukbec, Canada (Received 18 October 1995/19 February 1996) - EJB 95 169514

The distribution of cytosolic phospholipase A, (cPLA,), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA, and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A231 87, both proteins accumulated primarily in nucleicontaining fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA, were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclearmembrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A231 87 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA,, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state. Keywords: leukotriene; cytosolic phospholipase A,; 5-lipoxygenase; neutrophil ; translocation.

selective for PtdCho and PtdEtn species that contain an arachidonoyl residue in position sn-2 [8-141 suggested that this enzyme is of critical importance to LT synthesis. cPLA, was originally purified in monocytic and macrophage cell lines [ 14 171 and in platelets [12]. Immunologically related cPLA, was also detected in human neutrophils and monocytes [lo, 111. A distinctive feature of cPLA, is that it readily associates with membranes in the presence of calcium [S, 11, 141. Recent studies have suggested that while calcium seems to be required for vesicle binding [18, 191, cPLA, activity depends upon its phosphorylation on serine residues by the p42 mitogen-activated protein kinase 120, 211. The other key enzyme involved in LT synthesis is arachidonate 5-lipoxygenase, which catalyzes the first two reactions Correspizdeiic-e to P. Borgeat, Centre de recherche en Rhumatologie of the conversion of d,Ach into leukotriene A, (LTA,) [22]. et Immunologic, 270.5 boul. Laurier, room 9800, Ste-Foy, QC, Canada Whereas 5-lipoxygenase appears to be predominantly cytosolic GIV 4G2 in resting neutrophils, it has been shown to translocate to a celluFax: + 1 41 8 654 2765. Abhreviutiorrs. LT, leukotriene; PAF, platelet-activating factor; lar-membrane fraction after cell stimulation with calcium ionoA,Ach, arachidonic acid; cPLA,, cytosolic phospholipase A l ; 5-LAP, S- phores or with chemoattractants [23, 241. Activation of 5-lipoxylipoxygenease-activating protein ; Met-Leu-Phe, N-formylmethionylleu- genase in LT-producing cells also requires the participation of cylphenylalanine; Me2S0, dimethylsulfoxide; HBSS, Hanks’ balanced an 18-kDa membrane protein, termed 5-lipoxygenase-activating salt solution; NP-40, ethylphenolpoly(ethyleneglyco1ether)n; ECL, protein (5-LAP) 125, 261. It was proposed that 5-LAP could play enhanced chemiluminescence; 1 S-HETE, (ISS)-15-hydroxy-S,8,11,I 3- a role in the transfer of endogenous A,Ach to the 5-lipoxy(Z,Z,Z,E)-icosatetraenojc acid ; 15-HpETE, (1 SS)-15-hydroperoxygenase, based on its ability to bind &Ach [27] and to promote 5,8,11,13-(Z,Z,Z,E)-icosatetraenoic acid. its utilization by 5-lipoxygenase 1281. E ~ Z Y ~ P .Phospholipase ~. A2 (EC 3.1.1.4); lipoxygenase (EC 1.13.11.12). Despite the considerable amount of information accumulated Note. M. Pouliot and P. P. McDonald conlributed equally to the on the various components involved in LT synthesis, their subpresent study. cellular localization has only been partially elucidated. Because The leukotrienes (LT) are a family of arachidonic acid (d,Ach) derivatives synthesized by activated leukocytes, which are considered as potent mediators of inflammation and allergy [ I , 21. Leukotriene synthesis is thought to be ultimately dependent upon A,Ach availability [3 -51. Early studies established that in human neutrophils whose phospholipid pools had been labeled with [7H]d,Ach, cell activation led to a loss of radiolabel predominantly from the PtdCho species, l-O-alkyl-2-arachidonoyl-sn-glycerophosphocholine [ 61. Moreover, the specific radioactivity associated with leukotriene B, (LTB,) under the same conditions correlated with that of this particular PtdCho species [7]. The identification of a high-molecular-mass phospholipase A? (termed cytosolic PLA, or cPLA,) that is highly

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cPLA,, 5-LAP and 5-lipoxygenase have complementary actions, it is probable that they lie in close proximity in activated cells. In activated human neutrophils, 5-lipoxygenase and 5-LAP have been co-localized to the nuclear envelope and endoplasmic reticulum [29]. There is only indirect evidence for association of cPLA, with nuclear structures in activated neutrophils ; nuclear cPLA, has been detected in activated rat macrophages [30], and most of the d,Ach released upon activation of murine fibroblasts is derived from the nucleus [31]. In the present study, we used different cell-disruption techniques, immunoblot procedures, photoaffinity labeling and enzymatic assays to examine the subcellular distribution of the key proteins involved in LT synthesis.

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3.5 mM MgCl,, 1 mM ATP, 1.25 mM EGTA (relaxation buffer) containing an anti-protease cocktail (1 mM phenylmethanesulphony1 fluoride, 10 pg/ml leupeptin, 10 pg/ml aprotinin) at 2X lo7 celldml. Platelet preparations were obtained by sedimentation of erythrocytes and centrifugation of the resulting platelet-rich plasma (2000Xg, 15 min). Pellets were suspended in HBSS containing 10 mM Hepes, pH 7.40, and platelet numbers and purity were evaluated by means of a Coulter cell counter model T-890 (Coulter Electronics). Suspensions were diluted to 1X10' platelets/ml, warmed ( 5 min, 37°C) and incubated with 100 nM Met-Leu-Phe (1 min) or 2 U/ml thrombin (5 min). Cell disruption. Sonication. Neutrophils (2 X lo7 cells/ml in ice-cold relaxation buffer) were sonicated on ice (4x20 s, at a power setting of 2 and 60% duty cycle) in a Johns Scientific Inc. Ultrasonic Processor (Biotech Scientifique). Sonicates were MATERIALS AND METHODS centrifuged (12000Xg, 10 min, 4°C) and the resulting supernaChemicals. Rabbit polyclonal anti-(5-lipoxygenase) serum tants were centrifuged (180000Xg, 2.5 min, 4°C) in an Airfuge (SLO 32) and anti-(5-LAP) serum (HS) were supplied by Drs ultracentrifuge (Beckman Instruments). The 18000OXg supernaJillian F. Evans and Philip Vickers (Merck Frosst, Pointe-Claire, tants (referred to as cytosol), the corresponding pellets (referred QC, Canada) ; the specificity of these antibodies has been to as cellular membranes) and the 12000Xg pellets were quickly extensively characterized [23, 32, 331. The anti-cPLA, serum processed for assessment of 5-lipoxygenase activity and/or for (MF-142) was raised to a synthetic peptide that corresponded to SDS/PAGE analysis. Nitrogen cavitation. The procedure was conducted essenresidues S731-A749 of cPLA,, and homogenous cPLA, was purified essentially as described [34, 351. Horseradish-peroxi- tially as described [37] and all steps were performed at 4°C. dase-linked antibodies and the enhanced-chemiluminescence Briefly 2X 10' neutrophils in 10 ml ice-cold relaxation buffer (ECL) detection kit were from Amersham Canada. Immobilon-P were pressurized (2.4 Wa, 20 min, with constant stirring) in a poly(viny1idene fluoride) blotting membrane was from Milli- nitrogen bomb ( P a r Instrument Co.). Cavitates were spun at pore, Ficoll-Paque and protein-A- Sepharose were from Phar- 500Xg for 10 min to pellet unbroken cells and intact nuclei. The macia-LKB, and thrombin (thrombostat, DIN 023108) was from 500x9 pellet was suspended in 2 ml relaxation buffer, laid onto Parke-Davis Canada. Calcium ionophore (A23 187), ATP, EDTA, 2 x 4 ml of a Percoll step gradient (2.050 and 1.120 g/ml) and EGTA, Percoll, Hepes, Pipes, ethylphenolpoly(ethyleneg1ycol- centrifuged (1500Xg, 10 min). The nuclei-rich fraction at the ether)n (NP-40), Tween 20, and antibody PMN 13F6 were interface of the Percoll layers was diluted in relaxation buffer purchased from Sigma Chemical Co. Monoclonal antibody CC- and centrifuged (1500Xg, 10 min) to rid the nuclei of remaining 3 was a generous gift from Dr Michel Vincent (OntogCnkse et Percoll. The 500Xg supernatants were laid onto 2 x 1 4 ml of a GCnCtique molCculaire, Centre de Recherche du CHUL and Uni- Percoll step gradient (1.050 g/ml and 1.120 g/ml) and centriversit6 Laval, QC, Canada). Hank's balanced salt solution fuged (48000Xg, 15 min) in a Beckman L8-70 M ultracentri(HBSS) was from GIBCO, ['HH]d,Ach was from NEN Products, fuge. This procedure separates azurophil granules, specific and all organic solvents were HPLC grade from Anachemia. The granules, a plasma-membrane-enriched fraction and cytosol reversible 5-lipoxygenase inhibitor, BI-L-0239, was provided by [37]. Each fraction was aspirated with a syringe and centrifuged Dr Peter Farina of Boehringer-Ingelheim (Ridgefield CT, USA). (18OOOOXg, 120min) in a Beckman TL 100 ultracentrifuge. Soybean lipoxygenase was used to synthesize (1 5S)-15-hydro- Particulate fractions were suspended in a minimal volume of peroxy-5,8,11,I 3-(Z,Z,Z,E)-icosatetraenoic (1 5-HpETE) from HBSS containing 10 mM Hepes, pH 7.40, and promptly d,Ach, as previously described for (13S)-13-hydroperoxy-octa- analyzed by SDS/PAGE. Cytosolic fractions were dialyzed decadienoic acid [36]. d,Ach was purified on a silicic acid overnight, lyophilyzed and suspended in a minimal volume of column prior to use for enzymatic synthesis. 15-HpETE was HBSS containing 10 mM Hepes, pH 7.40, prior to SDS/PAGE reduced to (1 5S)-15-hydroxy-5,8,11,13-(Z.Z,Z,E)-icosatetra- analysis. NP-40 lysis. Neutrophils (2X lo7 cells) were pelleted, briefly enoic acid (15-HETE), by incubation with 4mg/ml SnCI, in ethanol/H,O ( 5 : 1) for 2 min at room temperature. 15-HpETE vortexed, and suspended in 600 pl ice-cold 10 mM Tris/HCI, and 15-HETE were purified by normal-phase HPLC and stored pH 7.40, 10 mM NaCI, 3 mM MgCl,, 1 mM EDTA containing 0.1 % NP-40 and the antiprotease cocktail. The cells were vorat 3 mM in distilled ethanol at -80°C under it N, atmosphere. Cell isolation and incubations. Peripheral blood was texed for 1.5 s, kept on ice for 5 min and centrifuged (SOOXg, collected from healthy donors, neutrophils were isolated as de- 10 min, 4°C). The resulting supernatants (non-nuclear fractions) scribed [5] and suspended in HBSS containing 10 mM Hepes, and the washed pellets (nuclei-containing fractions) were immepH 7.40, 1.6 mM CaC1, and 1 mM MgCI,, at 5x10' cells/ml. diately processed for assessment of 5-lipoxygenase activity and Neutrophil suspensions consistently contained fewer than 1 % for SDSPAGE analysis. Nuclear integrity was routinely verified monocytes and their viability was greater than 98%, as deter- by light microscopy. In some experiments, nuclear fractions mined by esterase staining and trypan-blue exclusion. Platelet were suspended in a minimal volume of relaxation buffer, sonicontamination never exceeded 3 %, as determined by Wright cated and centrifuged (180000Xg, 20 min, 4°C) in a Beckman staining. Neutrophil suspensions were warmed (5 min at 37°C) Airfuge. The resulting supernatants and pellets are referred to as and incubated with 1.0 pM A23187 or its diluent, dimethyl- nucleoplasmic and nuclear-membrane-containing fractions, resulfoxide (Me,SO), for 5 min at 37°C. Alternatively, neutrophils spectively. Photoaffinity labeling and immunoprecipitation of 5were treated with 100 nM N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) (5 min) or 2 U/ml thrombin (1 min). Reac- LAP. The procedures followed were as described 1271. SDSmAGE and immunoblots. Samples were analyzed by tions were stopped on ice, and cells were pelleted and suspended in ice-cold 10 mM Pipes, pH 7.30, 0.1 M KCl, 3 mM NaCI, SDWPAGE and immunoblotting as described previously [32]

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with the following modifications. Proteins were transferred overnight at 300 mA onto Immobilon-P poly(viny1idene fluoride) membranes ; transfer efficiency was visualized by Ponceau Red staining. The membranes were soaked for 30 min at 25°C in 25 mM Tris/HCl, pH 7.60, 0.2 M NaC1, 0.15% Tween 20 (NaCl/Tris/Tween 20) containing 5 % (mass/vol.) dried milk and exposed to the anti-(5-lipoxygenase), anti-cPLA, or anti-(5LAP) sera. The membranes were washed twice with NaCl/Tris/ Tween 20 and exposed to a horseradish-peroxidase-linked donkey anti-rabbit Ig. Bound antibodies were revealed with the enhanced-cheniiluminescence (ECL) reagent, according to the manufacturer's instructions. Alternatively, membranes were exposed to the CC-3 or 13F6 mAbs, as recommended [38-401, and treated with a horseradish-peroxidase-linked sheep antimouse IgG prior to ECL detection. Assessment of 5-lipoxygenase activity. Neutrophil suspensions were treated (20 min, 37°C) with 3.0 pM BI-L-0239, a reversible 5-lipoxygenase inhibitor [41], or in its absence when cytosolic fractions were prepared, and stimulated with A231 87 or its diluent ( 5 min, 37°C). The reversibility of the drug towards inhibition of 5-lipoxygenase activity was confirmed in preliminary experiments. After sonication or NP-40 lysis, cell fractions were suspended (material from 4X lo7 cells/ml, or material from 2X 10' cells/ml for cytosolic fractions) in relaxation buffer supplemented with CaCl, and ATP (to 0.7 mM free Ca2+and 3 mM ATP) that had been warmed to 37°C. Aliquots (500 pl) were dispensed into warmed tubes in a rotary water bath, and reactions were immediately started by the addition of 15-HpETE/15-HETE (to 18 pM and 2 pM, respectively). After a 15-min incubation (37 "C, with vigorous swirling), reactions were stopped by addition of 500 p1 ice-cold NaBH, (1 mg/ml in methanol (freshly prepared). The samples were left at room temperature for 20 min, and 6 p1 1.74 M acetic acid (to neutralize the NaBH,) and 1.O ml ice-cold methanol/acetonitrile (50:50, by vol.) containing 12.5 ng/ml prostaglandin B, and 12.5 ng/ml 19-hydroxyprostaglandin B, (as internal HPLC standards) were added. After 4 h -2O"C, the samples were centrifuged (SOOXg, 10 min, 4°C) to remove precipitated material, and stored at -20°C. Samples were analyzed by reversephase HPLC as described previously, by means of an on-line extraction procedure [42]. The elution of lipoxygenase products was monitored by means of fixed-wavelength ultraviolet photometers at 280 nm and 229 nm. Assessment of 5-lipoxygenase activity was performed by monitoring the 5-lipoxygenase-dependent conversion of 15-HpETE into (5S,15S)-5,15-dihydroxy6,8,11,I 3-(E,Z,Z,E)-icosatetraenoicacid, which was detected at 229 nm [5]. Quantitation was performed by comparison of the peak heights of each compound with those of calibrated standards, after correction for recovery of the internal standards. The lower limit of detection was 0.2 ng at 280 nm and 1 ng at 229 nm. Data analysis. Statistics were performed by mean5 of Student's paired t-test (one-tailed).

RESULTS Subcellular localization of 5-LAP and 5-lipoxygenase in neutrophils. Resting and A231 87-activated neutrophils were sonicated, and the resulting subcellular fractions were analyzed by SDS/PAGE and immunoblotting for 5-LAP and 5-lipoxygenase. Approximately 20% of the cellular 5-LAP content was recovered in cellular membranes (1 800OOXg pellet), while the bulk of the protein was recovered in the 12000Xg pellet, regardless of whether neutrophils had been stimulated or not (Fig. 1A). In contrast, approximately 70 % of immunoreactive 5-lipoxygen-

Fig. 1. Subcellular localization of 5-LAP and 5-lipoxygenase in human neutrophils disrupted by sonication. (A) Neutrophils were treated with 1.0 pM A23187 (+) or its diluent (Me,SO; -) for 5 min at 37°C. Reactions were stopped on ice, and cells were sonicated and submitted to differential centrifugation. The resulting fractions were analyzed by SDSPAGE (fractions from 2 X 10' cells/well) and immunoblotting for S-LAP, as described in Materials and Methods. The data are from one

representative of experiment ten. (B) Samples from the same experiment as in (A) were analyzed by SDS/PAGE and immunoblotting for 5-lipoxygenase. The positions of molecular-mass standards (ma)are indicated on the left. 5-L0, 5-lipoxygenase; Cyt, cytosol.

ase was detected in the cytosol of resting neutrophils, the remainder being associated with the 12OOOXg pellet. In A23187-treated cells, a 40% decrease in the amount of cytosolic 5-lipoxygenase was consistently observed (relative to resting cells), which was accompanied by increased recovery of immunoreactive protein in the 12000Xg pellet. In addition, approximately 10% of total 5-lipoxygenase was recovered in the 180000Xg pellet of stimulated cells (Fig. 1B), as reported previously 1231. Because 5-LAP and 5-lipoxygenase colocalized to fractions that contain primarily nuclei and secretory granules (the 12000Xg pellet) or that comprise membranes of different cellular origins (the 18OOOOXg pellet), we disrupted neutrophils by nitrogen cavitation, a procedure that yields cell fractions that show little cross-contamination [37]. Nearly all 5-LAP was recovered in the nuclei-rich fraction of resting neutrophils, whereas a minor proportion (< 10%) was found in the plasmamembrane-enriched fraction (Fig. 2A). 5-LAP was not detected in the cytosol or in the azurophilic granules, whereas it was sometimes weakly detected in the specific-granule fraction. The subcellular distribution of 5-LAP was unaffected by cell stimulation with A23187 (data not shown). In contrast, the bulk of cellular 5-lipoxygenase was located in the nuclei-rich fraction of resting neutrophils, while the remainder was detected in the cytosolic fraction (Fig. 2 B). 5-Lipoxygenase was undetectable in the plasma-membrane-enriched or granule fractions. After A231 87 stimulation, 5-lipoxygenase became barely detectable in the cytosolic fraction, while the amount of immunoreactive pro-

Pouliot et al. ( E m J . Biochem. 238)

Fig. 2. Subcellular localization of 5-LAP and 5-lipoxygenase in human neutrophils disrupted by nitrogen cavitation. (A) Resting neutrophils were submitted to nitrogen cavitation and centrifugation on discontinuous Percoll density gradients, and the resulting subcellular fractions were analyzed by SDSPAGE (fractions from 10’ cellslwell, except lane 1 which contained fractions from 5 X 10’ cells/well) and immunoblotting for 5-LAP, as described in Materials and Methods. The data are from one representative experiment of four. The positions of molecular-mass standards (kDa) are indicated on the left. (B) Neutrophils were treated with 1.0 pM A23187 or its diluent (Me,SO) for 5 min at 37°C. Reactions were stopped on ice, cells were disrupted in a nitrogen bomb, and cell fractions were prepared as in (A).Samples were analyzed by SDS/PAGE (fractions from lo7 cells/well, except lane I which contained fractions from 2X lo6 cells/well), and immunoblotting for 5-lipoxygenase. The data are from one representative experiment of four. Results shown in (A) and (Bj were obtained by analyses of the same samples. 500 P, nuclei-rich 500Xg pellet; Az, azurophilic granules; Sp, specific granules; PM, plasma-membrane-enriched fraction; Cyt, cytosol; CTRL,

control cells; A23187, ionophore-treated neutrophils; 5-LO, 5-lipoxygenase.

tein significantly increased in the nuclei-rich fraction (Fig. 2 B). A weak signal (