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Distinct A2 Regulate the Release of for Eicosanoid Production and Superoxide Anion Generation in Neutrophils

This information is current as Patricia K. Tithof, Marc Peters-Golden and Patricia E. Ganey of October 2, 2021. J Immunol 1998; 160:953-960; ; http://www.jimmunol.org/content/160/2/953

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Distinct Phospholipases A2 Regulate the Release of Arachidonic Acid for Eicosanoid Production and Superoxide Anion Generation in Neutrophils1

Patricia K. Tithof,* Marc Peters-Golden,‡ and Patricia E. Ganey2*†

Arachidonic acid (AA) released from membrane by A2 (PLA2) is important as a substrate for ؊ eicosanoid formation and as a second messenger for superoxide anion (O2 ) generation in neutrophils. Different isoforms of PLA2 in neutrophils might mobilize AA for different functions. To test this possibility, we sought to characterize the PLA2s that are activated by the neutrophil stimuli, Aroclor 1242, a mixture of polychlorinated biphenyls, and A23187, a calcium ionophore. ؊ 3 Both Aroclor 1242 and A23187 caused release of [ H]AA; however, O2 production was seen only in response to Aroclor 1242. Eicosanoids accounted for >85% of the radioactivity recovered in the supernatant of A23187-stimulated cells but <20% of the Downloaded from radioactivity recovered from cells exposed to Aroclor 1242. Omission or chelation of calcium abolished A23187-induced AA ؊ release, but did not alter AA release in Aroclor 1242-stimulated neutrophils. AA release and O2 production in response to

Aroclor 1242 were inhibited by bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2. BEL, however, did not alter

A23187-induced release of AA. Cell-free assays demonstrated both calcium-dependent and calcium-independent PLA2 activity. Calcium-independent activity was inhibited >80% by BEL, whereas calcium-dependent activity was inhibited <5%. Further- more, calcium-independent, but not calcium-dependent, PLA activity was significantly enhanced by Aroclor 1242. These data

2 http://www.jimmunol.org/ suggest that Aroclor 1242 and A23187 activate distinct isoforms of PLA2 that are linked to different functions: Aroclor 1242 ؊ activates a calcium-independent PLA2 that releases AA for the generation of O2 , and A23187 activates a calcium-dependent

PLA2 that mobilizes AA for eicosanoid production. The Journal of Immunology, 1998, 160: 953–960.

3 rachidonic acid (AA) serves as a precursor for the gen- (PLA2)-dependent hydrolysis of sn-2-acyl bonds. Several dif-

eration of a family of bioactive mediators known as ferent isoforms of PLA2 have been described that represent distinct A eicosanoids that includes , , gene products. These include two well-characterized, small-m.w., and . The roles of these products in normal physiologic calcium-dependent that are not selective for AA, the14- function (1) as well as in pathophysiologic states (2) have been by guest on October 2, 2021 kDa secretory PLA2 and pancreatic PLA2 (13, 14); an 85-kDa well documented. More recently, AA itself, as well as other un- cytosolic PLA2 (cPLA2), which is calcium dependent and arachi- saturated fatty acids, has been shown to serve important functions donoyl selective (15–17); and two calcium-independent enzymes, as second messengers. AA regulates such processes as activation one of which is selective for AA (18–20) and one that is not of kinase C (3) and mitogen-activated protein kinases (4), (21–23). Many cell types including neutrophils contain multiple synthesis of heat shock (5), mobilization of intracellular isoforms of PLA2 (24–26); however, the functional significance of calcium and activation of calcium channels (6, 7), and modulation the presence of different isoforms within the cell is not well un- of the activity of potassium channels (8, 9). In addition, AA and derstood. It has been suggested that different PLA s within the cell other unsaturated fatty acids may play an essential role in activa- 2 carry out distinct biologic functions (26). tion of the NADPH oxidase, which is responsible for the Ϫ In neutrophils, PLA -derived AA is important as a substrate for generation of superoxide anion (O ) by neutrophils (10–12). 2 2 eicosanoid production. AA has also been implicated as a second An important pathway for the release of AA or other unsaturated messenger for generation of OϪ, which is essential for neutrophil- fatty acids from pools involves phospholipase A 2 2 mediated killing of microbial (10–12); however, this function remains controversial (27). In support of a role for AA in Ϫ Ϫ † Departments of *Pharmacology and Toxicology, and Medicine and Institute for generation of O2 , virtually all agents that stimulate O2 production Environmental Toxicology, Michigan State University, East Lansing, MI 48824; in neutrophils also cause the release of AA (11, 12, 28). In addi- and ‡Division of Pulmonary and Critical Care Medicine, Department of Medi- cine, University of Michigan, Ann Arbor, MI 48109 tion, experiments with intact cells, as well as with reconstituted Received for publication November 26, 1997. Accepted for publication October NADPH oxidase systems, suggest that AA, rather than its metab- 1, 1997. olites, regulates a hydrogen ion channel that is linked to NADPH The costs of publication of this article were defrayed in part by the payment of oxidase activity (29, 30). It has been suggested that arachidonate page charges. This article must therefore be hereby marked advertisement in plays an important role in assembly of oxidase components by accordance with 18 U.S.C. Section 1734 solely to indicate this fact. exposing SH3 domains of p47phox, thus allowing interaction with 1 This work was supported by National Institutes of Health Grant ESO4911. cytochrome b558 and p67phox (31). 2 Address correspondence and reprint requests to Dr. Patricia E. Ganey, Depart- ment of Pharmacology and Toxicology, B440 Life Sciences, Michigan State Uni- PLA2-mediated AA release appears to play an essential role in versity, East Lansing, MI 48824. Ϫ production of O2 by neutrophils upon exposure to the commercial 3 Ϫ Abbreviations used in this paper: AA, arachidonic acid; O2 , superoxide anion; mixture of polychlorinated biphenyls (PCBs), Aroclor 1242 (28). PLA2, ; cPLA2, cytosolic PLA2; BEL, bromoenol lactone; LDH, lactate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; PCB, polychlori- PCBs are environmental toxicants that cause diverse biologic ef- nated biphenyl; TCB, tetrachlorobiphenyl; SOD, superoxide dismutase. fects, including modulation of inflammatory-mediated responses

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 954 PHOSPHOLIPASE A2 AND NEUTROPHIL FUNCTION such as neutrophil-mediated liver injury (32). Aroclor 1242 con- incubation period, neutrophils were washed two times with Mg2ϩ- and ϩ sists of a complex mixture of coplanar and ortho-substituted, non- Ca2 -free HBSS. The cell count was adjusted so that the final concentra- ϫ 6 3 coplanar PCB congeners. Release of AA and production of OϪ can tion of neutrophils was 2 10 /ml. The incorporation of H-AA was 2 approximately 60% and that of [3H] was approximately 70% be attributed to the ortho-substituted congeners within the mixture, of the total radioactivity added to the cells. since congeners such as 2,2Ј,4,4Ј-tetrachlorobiphenyl (TCB) stim- Ϫ ulate both the release of AA and the generation of O2 , whereas coplanar congeners such as 3,3Ј,4,4Ј-TCB cause neither Determination of release from prelabeled effect (28). neutrophils The magnitude of release of AA in response to Aroclor 1242 is Prelabeled neutrophils were suspended in HBSS containing 0.1% BSA. In similar to that observed from neutrophils stimulated with the cal- these experiments BSA was used to trap released fatty acids, thus inhibiting cium ionophore, A23187. However, the fate of released AA in subsequent and reacylation: as a result, the radioactivity in the supernatant reflects cumulative deacylation of [3H]fatty acid from phos- response to these two stimuli may be different. AA released from pholipid pools. Release of [3H]fatty acid was determined in the presence

A23187-stimulated neutrophils is metabolized extensively to eico- and absence of the inhibitor of the calcium-independent PLA2, BEL. Neu- sanoids (33); however, A23187 does not stimulate the production trophils were incubated for 15 min with BEL before addition of stimuli. To Ϫ determine the role of extracellular Ca2ϩ in stimulated release of AA, in- of O2 (34). In contrast, AA released in response to Aroclor 1242 2ϩ Ϫ cubations were conducted in the presence or absence of extracellular Ca . appears to be linked to O generation (28). It is unknown whether ϩ 2 To determine the role of intracellular Ca2 in release of AA, prelabeled AA mobilized by Aroclor 1242 results in eicosanoid production. neutrophils were loaded for 1 h with the cell-permeant calcium chelator One possible explanation for the different fates of AA released in BAPTA-AM. Release of [3H]fatty acids from prelabeled cells was mea-

response to these two stimuli may be that two discrete pools of AA sured in neutrophils stimulated for 20 min at 37°C with Aroclor 1242, Downloaded from exist, one destined to serve as a precursor for metabolism to eico- A23187, or the appropriate vehicle as described previously (28). Ϫ sanoids and the other to serve as a second messenger for O2 generation. Determination of PLA2 activity The purpose of the present study was to characterize the PLA2s PLA2 activity was measured using whole cell sonicates of fresh neutrophils activated upon stimulation of rat neutrophils with A23187 and (7 ϫ 107 cells/ml) according to the method of Smith and Waite (35). 2ϩ

Aroclor 1242. The results of this study indicate that A23187 and Neutrophils were isolated as described above, washed once in Ca -free http://www.jimmunol.org/ PBS containing 5 mM EDTA and 1 mM PMSF, resuspended in cold ho- Aroclor 1242 activate different PLA2 isoforms. In addition, the data suggest that, in neutrophils, activation of distinct PLA iso- mogenizing buffer (deionized water, 5 mM EDTA, and 1 mM PMSF), 2 placed on ice, and sonicated two times (80% duty) for 30 s. Light micros- forms results in AA hydrolysis that subserves distinct biologic copy was used to verify that cells were broken. The substrates, 14C-AA-PC functions. and 14C-LA-PC, were dried under nitrogen and resuspended by sonication (90% duty for 5 min) in assay buffer containing 120 mM NaCl and 40 mM Materials and Methods Tris-HCl (pH 9) to a final concentration of 3 ␮M radiolabeled substrate/test Materials tube. To determine the role of calcium in activation of PLA2, assays were performed in the presence of either 5 mM CaCl2 or 5 mM EGTA. To Aroclor 1242 was obtained from ChemService (West Chester, PA). determine the effect of PCBs on PLA2 activity, whole cell sonicates were Cytochrome c, superoxide dismutase (SOD), , xanthine oxidase, incubated in the presence or absence of Ca2ϩ with 10 ␮g/ml of Aroclor by guest on October 2, 2021 and A23187 were obtained from Sigma Chemical Company (St. Louis, 1242 or vehicle for 2 min before addition of substrate. In addition, soni- MO). [3H][5,6,8,9,11,12,14,15]AA (3H-AA; 180–240 Ci/mmol) was pur- cates were preincubated in the presence or absence of Ca2ϩ with 10 ␮M chased from DuPont NEN (Boston, MA). [3H][9,10,12,13]linoleic acid BEL or vehicle for 2 min to determine the effect of this inhibitor on cal- 3 ( H-LA; 60–120 Ci/mmol), 1-palmitoyl-2-arachidonoyl [arachidonoyl-1- cium-dependent and calcium-independent PLA2 activities. Optimal condi- 14 14 ϭ C] ( C-AA-PC; sp. act. 55 mCi/mmol), and tions for PLA2 were determined in preliminary experiments by measuring 1-palmitoyl-2-linoleoyl [linoeloyl-1-14C]phosphatidylcholine (14C-LA-PC; activity in the presence of various concentrations of substrate and protein. sp. act. ϭ 55 mCi/mmol) were obtained from American Radiolabeled These optimal conditions, i.e., 3 ␮M substrate and 60 ␮g protein/250 ␮l Chemicals (St. Louis, MO). 1,2-bis-(o-aminophenoxy)-ethane-N,N,NЈ,NЈ- assay buffer, were used thereafter. Reactions were initiated by addition of tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) was from either 3 ␮M 14C-AA-PC or 14C-LA-PC to whole cell sonicates that were Calbiochem (San Diego, CA). E-6-(bromomethylene)tetrahydro-3-(1- then incubated for 30 min at 37°C in a shaking water bath. Reactions were naphthalenyl)2H-pyran-2-one (BEL) was purchased from Biomol (Ply- terminated by addition of 1.2 ml of chloroform-methanol, 2:1 (v/v). The mouth Meeting, PA). Sep-pak cartridges were obtained from Waters (Mil- chloroform layer was extracted, dried under nitrogen, resuspended in 60 ␮l ford, MA). For all experiments, Aroclor 1242 was dissolved in methanol. of chloroform, and spotted on silica gel thin layer chromatography plates. Neutrophils received 1 ␮l of methanol. A23187 and BEL were dissolved in The plates were chromatographed in a neutral lipid solvent system con- DMSO and diluted so that the final concentration of DMSO was Ͻ 1%. taining hexane, diethyl ether, and glacial acetic acid (70:30:2, by volume).

The were visualized with I2 vapor, and the zones corresponding to Neutrophil isolation fatty acid and phospholipid were cut out and radioactivity determined by Glycogen-elicited neutrophils were obtained from the peritoneal cavities of scintillation counting. Results were expressed as percent of total radioac- male Sprague-Dawley rats (Charles River Laboratories, Portage, MI). Rats tivity present as free fatty acid. were anesthetized with diethyl ether, and 30 ml of 1% glycogen were injected i.p. The rats were anesthetized again 4 h later and killed by de- capitation. The peritoneal cavity was washed with 30 ml of heparinized (1 Determination of metabolism of released AA by neutrophils U/ml) 0.1 M PBS and the peritoneal fluid collected, filtered through gauze, For determination of metabolic products of AA, neutrophils prelabeled ϫ and centrifuged at 500 g for 7 min. Contaminating RBCs were lysed with 0.5 ␮Ci/ml [3H]AA were stimulated for 20 min at 37°C with Aroclor ϩ ϩ with 15 ml of 0.15 M NH4Cl, and neutrophils were suspended to a final 1242, A23187, or the appropriate vehicle in Ca2 - and Mg2 -containing ϫ volume of 50 ml with PBS and centrifuged for 7 min at 300 g. Cells were HBSS. For determination of metabolic products of AA, experiments were washed once with PBS and suspended in HBSS of the following compo- performed in the absence of albumin. Eicosanoids were extracted from sition: 4.5 mM KCl, 0.6 mM Na2HPO4, 0.62 mM KH2PO4, 120 mM NaCl, cell-free supernatant fluids with C18 Sep-pak cartridges, separated by re- 23 mM Tris, 1.6 mM CaCl2, 0.68 mM MgCl2, 10 mM glucose, and 14 mM verse-phase HPLC, and identified by coelution with known standards (36). NaHCO3. The percentage of neutrophils in this preparation is routinely Products were quantified via an on-line radioactivity detector. To verify Ͼ 95% (28). that of radiolabeled eicosanoids in prelabeled cells reflected Labeling of neutrophils with 3H-fatty acids the total mass of eicosanoids from endogenous stores of AA, selected ei- cosanoids released into the extracellular medium from unlabeled neutro- Neutrophils (107/ml) were suspended in Mg2ϩ- and Ca2ϩ-free HBSS con- phils were measured by enzyme immunoassay (Cayman Chemical, Ann taining 0.1% BSA and incubated in the presence of 0.5 ␮Ci/ml [3H]AA or Arbor, MI). For each sample, the average of duplicate determinations was [3H]linoleic acid for 90 min, gently shaking at 37°C. At the end of the used. Experimental incubations were the same as those described above. The Journal of Immunology 955

Ϫ Generation and detection of O2 Ϫ O2 production was measured in neutrophils stimulated for 20 min at 37°C with Aroclor 1242, A23187, or the appropriate vehicle. Previous studies have demonstrated that A23187 does not cause significant production of Ϫ O2 in rat neutrophils (34). To determine whether the failure of A23187 to Ϫ generate O2 was related to the extensive metabolism of AA by cyclo- Ϫ oxygenase and 5- pathways in A23187-stimulated cells, O2 production was determined in neutrophils pretreated with the cyclooxy- genase inhibitor, (100 ␮M; 37) and the 5-lipoxygenase inhibitor, (10 ␮M; 38). Neutrophils were preincubated for 30 min with as- pirin and for 15 min with zileuton before stimulation with A23187. To determine the role of calcium-independent PLA2 in Aroclor 1242-induced Ϫ neutrophil activation, O2 production was measured in the presence and absence of the calcium-independent PLA2 inhibitor, BEL. Neutrophils were preincubated with BEL or vehicle for 15 min before stimulation with Ϫ Aroclor 1242. Cumulative O2 production was measured by the SOD-sen- sitive reduction of cytochrome c as described previously (28).

Certain PLA2 inhibitors have been shown to scavenge radicals Ϫ (28). Therefore, to determine whether BEL inhibited O2 production by a Ϫ mechanism related to this effect, O2 generated during oxidation of xan- thine by xanthine oxidase was measured in the presence and absence of BEL as described previously (28). In addition, absorbance of reduced cy- Downloaded from tochrome c was measured in the presence and absence of BEL to ensure Ϫ that apparent inhibition of neutrophil-generated O2 was not a result of quenching of absorbance by BEL. There were no differences in absorbance values in the presence and absence of BEL, thus indicating that BEL does not interfere with the cytochrome c assay.

Determination of cytotoxicity

Ϫ http://www.jimmunol.org/ To ensure that inhibition of O2 by BEL did not result from injury to the cells, cytotoxicity was determined in neutrophils exposed to BEL. Neutro- phils were incubated with BEL as described above, and activity of the cytosolic enzyme, lactate dehydrogenase (LDH), was determined in the cell-free supernatant fluids as described previously (39, 40).

Statistical analysis Results are expressed as mean Ϯ SEM. [3H]AA or [3H]linoleic acid results are expressed as percent of total cellular radioactivity released into the

medium. Data were analyzed by ANOVA, and group means were com- by guest on October 2, 2021 pared using Student-Newman-Keuls’ test. Appropriate transformations were performed on all data that did not follow a normal distribution. If transformation failed to normalize the data, then nonparametric statistics were used (Mann-Whitney test). For all studies, the criterion for statistical significance was p Յ 0.05. Ϫ FIGURE 1. Concentration-dependent generation of O2 and release Results of [3H]arachidonate in rat peritoneal neutrophils. Cumulative genera- Ϫ ⅷ 3  Arachidonic acid release and metabolism tion of O2 ( ) and release of [ H]arachidonate ( ) were measured in 3 neutrophils stimulated for 20 min with the indicated concentrations of A minimal amount of [ H]AA was released into the medium from Ϫ 3 A23187 (A) or Aroclor 1242 (B). O2 production was measured as the unstimulated neutrophils (Fig. 1, A and B). The release of [ H]AA SOD-sensitive reduction of cytochrome c, as described in Materials increased in a concentration-dependent manner in neutrophils ex- and Methods. Release of [3H]arachidonate into the extracellular me- posed to the calcium ionophore A23187 (Fig. 1A) or Aroclor 1242 dium was measured in the presence of 0.1% albumin. Results are ex- (Fig. 1B). The magnitude of release of [3H]AA was not signifi- pressed as the percent of total incorporated cellular radioactivity. n ϭ cantly different between neutrophils stimulated with maximal con- 5 to 7. a, Statistically different from respective value obtained in the centrations of A23187 (20 ␮M; 22.8 Ϯ 0.6%) or Aroclor 1242 (10 absence of stimulus. ␮g/ml; 22.5 Ϯ 4.7%). Radioactivity elution profiles of supernatants taken from neu- phils exposed to Aroclor 1242 was 10-fold higher than that seen trophils stimulated with A23187 or Aroclor 1242 are shown in with unstimulated neutrophils (5168 dpm; Fig. 2C). However, in Figure 2. Unstimulated neutrophils released small amounts of ra- contrast to A23187, 82% of the radioactivity released in response dioactivity (435 dpm in the representative experiment shown in to Aroclor 1242 remained as free AA while only 18% was metab-

Fig. 2) that coeluted exclusively with AA (Fig. 2A). Total radio- olized to eicosanoids, predominantly B2, 12-hy- activity released into the medium by A23187-treated neutrophils droxyheptadecatrienoic acid, and 12-HETE (Fig. 2C). A similar was approximately 30-fold higher than control (14847 dpm). Only lack of metabolism was observed with a PCB congener, 2,2Ј,4,4Ј- Ϫ 14% of the released radioactivity remained as free AA, while 86% TCB, which stimulates O2 production by neutrophils (data not was metabolized to eicosanoids. The products that shown). were detected included E2, , throm- To ensure that the results obtained with radiolabeled neutrophils boxane B2, 12-hydroxyheptadecatrienoic acid, and the stable me- were reflective of those seen in unlabeled cells, the total mass of tabolite of , 6-keto prostaglandin F1␣. Also detected selected eicosanoids was measured in supernatants from neutro- were products of 5-lipoxygenase, B4 and 5-hydroxy- phils stimulated with A23187 or Aroclor 1242 by enzyme immu- eicosatetraenoic acid (HETE), and of 12-lipoxygenase, 12-HETE noassay (Table I). was chosen as a cyclooxy- (Fig. 2B). Total radioactivity released into the medium by neutro- genase product and as a 5-lipoxygenase product. 956 PHOSPHOLIPASE A2 AND NEUTROPHIL FUNCTION

Table I. Production of thromboxane B2 (TXB2) and leukotriene B4 (LTB4) in neutrophils stimulated with A23187 and Aroclor 1242

Treatment LTB4 (ng/ml) TXB2 (ng/ml) Control 0.015 Ϯ 0.004 5.2 Ϯ 0.6 A23187 81 Ϯ 20a,b 146 Ϯ 32a,b Aroclor 1242 0.43 Ϯ 0.14 14.8 Ϯ 0.8a

Neutrophils (2 ϫ 106/ml) were stimulated for 20 min with 20 ␮M A23187, 10 ␮g/ml Aroclor 1242, or vehicle, and the concentration of eicosanoids in the extracellular medium was determined by enzyme immunoassay as described in Materials and Methods (n ϭ 3).a Significantly different from respective value obtained in the absence of stimulus.b Statistically different from respective value obtained in the presence of Aroclor 1242.

Table II. Lack of effect of aspirin and zileuton on superoxide anion production in neutrophils stimulated with A23187

Superoxide anion production Downloaded from (nmol/106 cells/20 min)

Treatment Control A23187

Vehicle 2.3 Ϯ 1.7 1.6 Ϯ 0.5 Aspirin 0.9 Ϯ 0.2 2.3 Ϯ 1.2 Zileuton 0 Ϯ 0 1.2 Ϯ 0.8 ϩ Ϯ Ϯ

Aspirin zileuton 1.6 1.0 0.6 0.4 http://www.jimmunol.org/ Superoxide anion was measured in neutrophils (2 ϫ 106/ml) that were pre- treated with 100 ␮M aspirin and 10 ␮M zileuton alone or in combination, and ϭ FIGURE 2. Metabolism of [3H]arachidonate released by neutrophils stimulated with A23187 or vehicle as described in Materials and Methods (n 3). No significant differences between groups were observed. stimulated with A23187 or Aroclor 1242. Neutrophils were prelabeled with [3H]arachidonate and exposed for 20 min to vehicle (A), 20 ␮M A23187 (B), or 10 ␮g/ml Aroclor 1242 (C). Radiolabeled products in the cell-free supernatant were extracted, separated by reverse-phase the activation of different isoforms of PLA2, experiments were HPLC, and identified by coelution with known standards. Radioactiv- performed to determine the characteristics of the PLA activated ity was measured as dpm by an on-line radioactivity detector. These 2 by these stimuli. The preference for AA was investigated by com- by guest on October 2, 2021 results are representative of two separate experiments. paring release of [3H]AA and [3H]linoleic acid (3H-LA) from pre- labeled neutrophils stimulated with A23187 or Aroclor 1242 (Fig. 3). Similar to results in Figure 1, significant release of [3H]AA was Exposure to A23187 caused release of large amounts of throm- observed in neutrophils stimulated with either A23187 (Fig. 3A)or boxane B2 and leukotriene B4 from neutrophils; however, signif- Aroclor 1242 (Fig. 3B). Neither compound, however, caused sig- icantly less of both products was released from neutrophils stim- nificant deacylation of [3H]LA (Fig. 3, A and B). ulated with Aroclor 1242. Thus, results using unlabeled To study the role of Ca2ϩ in A23187- and Aroclor 1242-medi- neutrophils were consistent with those obtained with cells labeled ated release of [3H]AA, experiments were performed in the pres- 3 with [ H]AA. ence and absence of extracellular Ca2ϩ and in cells preloaded with BAPTA-AM or vehicle. Significant release of [3H]AA was seen in Generation of OϪ 2 neutrophils treated with A23187 or Aroclor 1242 in the presence Ϫ 2ϩ No O2 was generated from unstimulated neutrophils (Fig. 1, A and of 1.6 mM CaCl2. Omission of Ca from the extracellular me- Ϫ B). Aroclor 1242 stimulated the production of O2 with a concen- dium or treatment with BAPTA-AM caused significant reduction tration-response relation that was similar to the [3H]AA response in [3H]AA release from A23187-treated neutrophils. In contrast, induced by this compound (Fig. 1B). A23187, however, did not Aroclor 1242-induced deacylation of [3H]AA was not significantly Ϫ stimulate neutrophils to produce O2 , despite significant release of affected by the removal of either extracellular or intracellular cal- 3 Ϫ [ H]AA. The failure of A23187 to stimulate production of O2 in cium (Fig. 4). rat neutrophils might be due to extensive metabolism of AA via the Recently, a PLA2 has been characterized in cardiac myocytes cyclooxygenase and 5-lipoxygenase pathways, resulting in inade- that is calcium independent and arachidonoyl selective (18–20). quate levels of free AA to activate the NADPH oxidase. To test This enzyme is inhibited by BEL with 1000-fold greater potency Ϫ this hypothesis, O2 production was determined in A23187-stim- than are calcium-dependent isoforms of PLA2 (20). The effect of ulated neutrophils in the presence and absence of aspirin and zileu- BEL on [3H]AA release in neutrophils stimulated with A23187 or Ϫ 3 ton. No O2 was produced by neutrophils stimulated with A23187, Aroclor 1242 is shown in Figure 5. [ H]AA release stimulated by and pretreatment with aspirin and/or zileuton did not alter this Aroclor 1242 was inhibited in a concentration-dependent manner response (Table II). by BEL; significant inhibition was achieved with a concentration of BEL as low as 0.1 ␮M. In contrast, the release of [3H]AA Characterization of arachidonate release by A23187 and evoked by 20 ␮M A23187 was not significantly altered by BEL at Aroclor 1242 in intact cells concentrations up to 10 ␮M (Fig. 5). To examine whether the differences in the metabolism of AA in In light of the reported specificity of BEL for calcium-indepen- neutrophils stimulated with A23187 or Aroclor 1242 resulted from dent PLA2 activity (20), the inhibition of Aroclor 1242-induced The Journal of Immunology 957

FIGURE 4. Calcium dependence of [3H]arachidonate release in neutrophils stimulated with A23187 or Aroclor 1242. The release of [3H]arachidonate from prelabeled neutrophils was measured in cells ϩ 2ϩ Ϫ 2ϩ suspended in HBSS with ( Ca ) or without ( Ca ) 1.6 mM CaCl2. Downloaded from Before stimulation, cells were loaded for 60 min with BAPTA-AM or vehicle as indicated in the figure. The concentrations of A23187 and Aroclor 1242 used for stimulation were 20 ␮M and 10 ␮g/ml, respec- tively. Radioactivity released into the medium was measured as de- scribed in Materials and Methods. n ϭ 5. a, Statistically different from respective value obtained in the absence of stimulus. b, Statistically

different from respective value obtained in the presence of calcium. http://www.jimmunol.org/

3 3 FIGURE 3. Preferential release of [ H]arachidonic acid over [ H]li- by guest on October 2, 2021 noleic acid by neutrophils stimulated with A23187 or Aroclor 1242. Neutrophils were prelabeled with [3H]arachidonic acid or [3H]linoleic acid and stimulated with A23187 (A) or Aroclor 1242 (B) at the indi- cated concentrations for 20 min in HBSS containing 0.1% albumin. Radioactivity released into the medium was quantified by scintillation counting, and results expressed as percent of total incorporated cellu- lar radioactivity as described in Materials and Methods. n ϭ 3to4.a, Statistically different from respective value obtained in the absence of stimulus.

[3H]AA release by BEL (Fig. 5) is consistent with the hypothesis that AA release in response to Aroclor 1242 is due to activation of 3 a calcium-independent PLA . To test whether this enzyme is im- FIGURE 5. Effect of BEL on release of [ H]arachidonate in neutro- 2 phils stimulated with A23187 or Aroclor 1242. Prelabeled neutrophils portant in the mechanism of Aroclor 1242-induced stimulation of Ϫ were suspended in HBSS containing 0.1% albumin, incubated for 15 neutrophils, the production of O2 evoked by Aroclor 1242 was min in the presence or absence of BEL at the indicated concentrations, measured in the presence and absence of BEL. Aroclor 1242-in- and stimulated for 20 min with 20 ␮M A23187 (ⅷ), 10 ␮g/ml Aroclor Ϫ 3 duced O2 generation decreased in a concentration-dependent man- 1242 (), or the appropriate vehicle. Release of [ H]arachidonate into Ϫ ner in neutrophils exposed to BEL (Fig. 6). BEL inhibited O2 the extracellular medium was quantified as described in Materials and production (Fig. 6) with a concentration-response relation that was Methods and results are expressed as percent of the response obtained similar to BEL-induced inhibition of [3H]AA release (Fig. 5). The in the absence of inhibitor. [3H]arachidonate release was 2.2 Ϯ 0.3% inhibition of [3H]AA release and OϪ production by BEL was not in neutrophils incubated with methanol (vehicle for Aroclor 1242) and 2 Ϯ a result of cytotoxicity since LDH values were not different in cells 2.1 0.2% in neutrophils incubated with DMSO (vehicle for preincubated with BEL at maximal concentrations and stimulated A23187). These values were not significantly different in the presence and absence of BEL. The release of [3H]arachidonate by neutrophils with Aroclor 1242 (LDH ϭ 6.7 Ϯ 0.8%) when compared with Ϯ stimulated with A23187 and Aroclor 1242 in the absence of BEL was neutrophils incubated with Aroclor 1242 alone (6.7 1.6%) or 21.9 Ϯ 1.8% and 31.4 Ϯ 2.1%, respectively. n ϭ 3; a, Significantly Ϯ vehicle alone (4.9 0.4%). different from respective value obtained in the absence of BEL.

Effect of Aroclor 1242 and BEL on PLA2 activity in neutrophil lysates against either 14C-AA-PC or 14C-LA-PC, both in the presence and

To confirm results obtained with intact neutrophils, cell-free PLA2 absence of calcium; however, PLA2 activity was significantly 14 assays were performed. PLA2 activity was present when assayed greater when assayed against C-AA-PC and in the presence of 958 PHOSPHOLIPASE A2 AND NEUTROPHIL FUNCTION

Table III. Effect of PCBs and BEL on calcium-dependent and calcium-independent PLA2 activity in neutrophil lysates

Phospholipase A2 Activity (pmol/mg/30 min)

Treatment 14C-AA-PC 14C-LA-PC

5 mM CaCl2 ϩ vehicle 687.7 Ϯ 4.3 67.7 Ϯ 1.2c ϩ Aroclor 1242 435.7 Ϯ 2.3a 36.0 Ϯ 0.6a,c ϩ BEL 655.3 Ϯ 2.7a 56.3 Ϯ 0.7a,c 5 mM EGTA ϩvehicle 152.7 Ϯ 3.2b 20.0 Ϯ 1.5b,c ϩ Aroclor 1242 241.7 Ϯ 2.3a 22.3 Ϯ 0.9c ϩ BEL 30.0 Ϯ 1.0a 1.7 Ϯ 0.7a,c

PLA2 activity in neutrophil lysates was measured in the presence of 5 mM ␮ CaCl2 or EGTA. Lysates were pretreated with Aroclor 1242 (10 g/ml), BEL (10 ␮M), or vehicle for 2 min at 37°C before addition of 3 ␮M 1-palmitoyl-2-arachi- donyl-[arachidonyl-1-14C]-phosphatidylcholine (14C-AA-PC) or 1-palmitoyl-2- linoleoyl-[linoleoyl-1-14C]-phosphatidylcholine (14C-LA-PC) for 30 min. Reac- Ϫ tions were stopped by addition of 1.2 ml chloroform:methanol (2:1), and lipids FIGURE 6. Inhibition of Aroclor 1242-induced O2 production by were separated by TLC as described in Materials and Methods. PLA2 activity is Downloaded from BEL. Neutrophils were incubated for 15 min in the presence or ab- expressed as pmol/mg protein/30 min. The results represent mean Ϯ SEM from sence of BEL at the concentrations indicated and stimulated for 20 min triplicate determinations in a single experiment. These results are representative ␮ Ϫ a with 10 g/ml Aroclor 1242 or vehicle. Cumulative O2 production of four experiments. Significantly different from results obtained in the presence of vehicle. bSignificantly different from respective values obtained in the pres- was measured as described in Materials and Methods, and results ex- c ence of CaCl2. Significantly different from results obtained in the presence of pressed as percent of response obtained in the absence of inhibitor. No 14 Ϫ C-AA-PC. O2 was produced by unstimulated neutrophils either in the presence or absence of BEL. Neutrophils stimulated with Aroclor 1242 pro- Ϯ Ϫ 6 ϭ http://www.jimmunol.org/ duced 19.8 2.0 nmol O2 /10 cells/20 min. n 4 to 5. a, Signifi- pled to subsequent metabolic pathways responsible for production cantly different from response obtained in the absence of BEL. Ϫ of eicosanoids, but does not serve to stimulate O2 production. In contrast, AA release in response to Aroclor 1242 results in gen- eration of OϪ, but not production of eicosanoids. Ca2ϩ (Table III). Pretreatment of lysates with Aroclor 1242 sig- 2 The hypothesis that Aroclor 1242 activates a calcium-indepen- nificantly enhanced calcium-independent PLA activity, whereas 2 dent PLA is corroborated by results in a cell-free system. BEL calcium-dependent PLA activity was significantly attenuated in 2 2 inhibited activity of this enzyme in cell lysates and also inhibited the presence of Aroclor 1242. BEL (10 ␮M) inhibited calcium- Ϫ Aroclor 1242-induced O production in whole cells, further sup- independent activity by Ͼ80%; however, calcium-dependent ac- 2 porting the interpretation that activation of calcium-independent by guest on October 2, 2021 tivity was inhibited by Ͻ5% in the presence of BEL. Similar re- Ϫ PLA and O generation by Aroclor 1242 are linked. The possi- sults were obtained when 14C-LA-PC was used as substrate, 2 2 bility cannot be excluded, however, that BEL inhibited AA release although less PLA2 activity was observed under these conditions. Ϫ and O2 production by a mechanism that was unrelated to inhibi- tion of calcium-independent PLA2. BEL has been reported to in- Discussion hibit ␣-chymotrypsin activity (41) as well as to inhibit cytosolic, In the present study both the PCB mixture, Aroclor 1242, and the magnesium-dependent phosphatidic acid phosphohydrolase activ- calcium ionophore A23187 stimulated the release of AA from iso- ity (42). These effects of BEL are unlikely to be involved in the lated neutrophils. However, the characteristics of AA release in inhibition of AA release by BEL in our study, given the observa- response to these two agents were markedly different. Moreover, tion that it significantly inhibited and Aroclor 1242 significantly

the consequences of mobilization of AA in response to the two enhanced calcium-independent PLA2 activity under cell-free con- stimuli were different, suggesting that different isoforms of PLA2 ditions that were optimized specifically for this enzyme (Table III). were activated by A23187 and Aroclor 1242, and that activation of Moreover, there is no evidence that phosphatidic acid phosphohy-

these different PLA2s serves distinct functions within the neutro- drolase plays any role in NADPH oxidase activation. These results phil. Several lines of evidence support this hypothesis. First, provide strong evidence that Aroclor 1242 stimulates AA release

A23187 stimulated the release of AA by a mechanism that was by activating a calcium-independent PLA2. dependent on calcium and resistant to inhibition by BEL, a PLA2 A23187, which released similar amounts of AA as Aroclor Ϫ inhibitor that shows 1000-fold greater potency against calcium- 1242, failed to cause the production of O2 . The failure of A23187 Ϫ independent PLA2 (20). In contrast, Aroclor 1242 stimulated re- to elicit O2 production was not related to insufficient levels of AA lease of AA by a mechanism that was independent of calcium and due to extensive metabolism via the cyclooxygenase and lipoxy- inhibited by BEL. Second, the fate of AA released in response to genase pathways, since pretreatment with inhibitors of these path- Ϫ A23187 and Aroclor 1242 was different. A minimal amount of AA ways did not unmask generation of O2 by A23187-treated cells. released by A23187 stimulation remained as free AA, while Ͼ80% These data are consistent with the hypothesis that in neutrophils, Ͼ was metabolized to eicosanoids. In contrast, 80% of the AA activation of calcium-dependent PLA2 does not lead to generation Ϫ released in response to Aroclor 1242 remained as free arachidonate of O2 . Inhibition of calcium-dependent PLA2 by Aroclor 1242 in with little metabolism to eicosanoids. Third, AA released by Aro- cell-free experiments (Table III) further supports this hypothesis. If Ϫ clor 1242, but not A23187, was associated with the production of calcium-dependent PLA2 was important in the production of O2 , Ϫ Ϫ Ϫ O2 . Both Aroclor 1242-induced AA release and O2 production Aroclor 1242, an agent that stimulates O2 , should not inhibit its occurred with similar concentration-response relations and both activity. were inhibited by BEL. Taken together, these data suggest that, in Cell-free assays confirmed the presence of both calcium-depen-

neutrophils, A23187 causes the release of AA that is tightly cou- dent and -independent PLA2 activities suggested by experiments in The Journal of Immunology 959

intact cells. Our results are most consistent with the possibility that The results of this study suggest that different PLA2 release AA the enzyme activated by Aroclor 1242 in rat neutrophils is the that subserves distinct functions within the cell. Why might AA calcium-independent PLA2 first identified in cardiac myocytes by mobilized by different PLA2 have different biologic fates? One Wolf and Gross (18); Hazen, Stuppy, and Gross (19); and Hazen possibility is that subcellular compartmentalization of PLA2 de- et al. (20). The myocardial enzyme, like the neutrophil enzyme, termines whether released AA is coupled to eicosanoid synthesis Ϫ does not require calcium for activity and is inhibited by BEL (Fig. or O2 production. A number of eicosanoid-synthesizing enzymes 4 and Table III; Refs. 19 and 20). In addition, the myocardial are located at the (48–51), and the activation- enzyme displays a preference for hydrolysis of phospholipids that associated translocation of cPLA2 to the nuclear envelope (48, 52, contain arachidonate at the sn-2 position (19). [3H]LA was not 53) with concomitant nuclear membrane phospholipid hydrolysis released from neutrophils in response to activation with Aroclor (53) would facilitate efficient coupling of released AA to eico- 1242, whereas [3H]AA was (Fig. 3). Moreover, calcium-indepen- sanoid synthesis. On the other hand, the NADPH oxidase is lo- cated at the plasma membrane (54). It is tempting to speculate that dent PLA2 activity under cell-free conditions was 10-fold greater 14 when C-AA-PC was used as substrate compared with the re- the calcium-independent PLA2 translocates to this site upon cel- sponse obtained in the presence of 14C-LA-PC (Table III). These lular activation, so that plasma membrane-derived AA hydrolyzed data are consistent with the hypothesis that Aroclor 1242 activates by this enzyme is in closer proximity to the NADPH oxidase than to eicosanoid-forming enzymes. Thus, nuclear membrane AA and a calcium-independent, arachidonoyl-selective PLA2 in neutro- phils (18–22). This study is the first to identify and define a role for plasma membrane AA would represent discrete pools of AA des- tined to serve distinct functions. This speculation will require di- a calcium-independent, arachidonoyl-selective PLA2 in rat neutro-

rect examination. Downloaded from phils. Smith and Waite described a calcium-independent PLA2 in human neutrophils that was optimally active at pH 9; however, the function of this enzyme was not elucidated (35). The calcium- Acknowledgments independent PLA2 described here may be the same as or similar to Special thanks to Rob McNish for expert technical assistance. the enzyme described in human neutrophils. The effect of Aroclor 1242 to activate the calcium-independent

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