Hydrogen Peroxide Inhibits Alveolar Macrophage 5-Lipoxygenase ...

Vol. 263,No. 29,Issue of October 15,pp. 14776-14783.1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Hydrogen Peroxide Inhibits Alveolar Macrophage 5-Lipoxygenase Metabolism in Association with Depletion of ATP* (Received for publication, January 19, 1988)

Peter H. S . SpornS and MarcPeters-Golden8 From the Division of Pulmonary andCritical Care Medicine, Department of Internal Medicine, University of Michigan and Veterans Administration Medical Centers, Ann Arbor, Michigan 48109-0360

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (HzOZ)stimulates arachidonic acid (AA) release and thromboxane Az synthesis in the rat alveolar macrophage. We have now investigated the effects of HzOZ on alveolar macrophage 5-lipoxygenase metabolism. Hz02 failed to stimulate detectable synthesisof leukotriene Bat leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays(RIAs). This was not explained by oxidative degradationof leukotrienes by Hz02 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that HzOZdose-dependently inhibited synthesis of leukotriene B4,leuby the agonists kotriene C4, and5-HETEinduced A23187 (10 PM) and zymosan (100 pg/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxaneAZ and 12-hydroxy-5,8,10-heptadecatrienoicacid. Four lines of evidence suggestedthat H2O2inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. l) HzOz depleted ATP in A23187- and zymosan-st’1mulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by HzOZwere compatible with a rate-limiting effect of ATP on leukotriene synthesis in HzOz-exposed cultures. 3) Treatment of alveolar macrophages with the electron transportinhibitorantimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane Az production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuatedboth ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of HzOz. Our results show that Hz02 has the capacity to act both as anagonist for macrophageAA metabolism, and as a selective inhibitorof the 5-lipoxygenase path-

* This work was supported by funds from the American Lung Association, the Research Service of the Veterans Administration, the Rackham Faculty Grant of the University of Michigan, and the Michigan Memorial Phoenix Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. $ Edward Livingston Trudeau Scholar of the American Lung Association. To whom correspondence should be addressed: Division of Pulmonary and Critical Care Medicine, 3916 Taubman Center, University of Michigan Medical Center, Ann Arbor, MI 48109-0360. §Recipient of Clinical Investigator Award HL-01638 from the National Heart, Lung, and Blood Institute.

way, probably as a result of its ability to deplete ATP. Depletion of cellular energy storesby oxidants generated during inflammation in vivo may be a means by which the inflammatory responseis self-limited.

Reactive species derived from molecular oxygen have been implicated in the pathogenesis of diverse types of inflammatory reactions and tissue injury (1).Likewise, metabolites of arachidonic acid (AA)’ are thought to function as mediators of many aspects of the inflammatory response (2). A number of studies have now demonstrated that reactive oxygen metabolites can themselves trigger the release and metabolism of AA in experimental systems ranging from cell-free reaction mixtures to intactlaboratory animals (3-16). The majority of these investigations (3-14) have documented the ability of reactive oxygen intermediates to activate release of AA from phospholipid and its conversion via cyclooxygenase to prostaglandins and related compounds. Metabolism of AA via lipoxygenase pathways in response to reactive oxygen species has been less well-studied, although Burghuber and associates (15) provided evidence for 5-lipoxygenase product formation in isolated perfused lungs injured with hydrogen peroxide (H202),and Taniguchi and co-workers (16) found increased leukotriene B4 concentrationsin bronchoalveolar lavage fluid of rats maintained in a hyperoxic environment. The lung is an organ in which an interaction between oxidants and AA metabolism may be particularly important. By virtue of the high oxygen tension in the alveolar space relative to thatin most tissues, as well as thelarge number of oxidantpollutants in the atmosphere, the lung faces a uniquely high oxidantburden even under normal circumstances. Furthermore, many studies suggest that reactive oxygen metabolites (reviewed in 17) and eicosanoids (reviewed in 18) are important in initiating or propagating various forms of pulmonary inflammation and injury, both experimentally and in human disease. We have therefore studied the effects of the biologically important oxidantHz02on AA metabolism in the pulmonary alveolar macrophage. The alveolar macrophage is the major resident inflammatory cell in the alveolus of the lung and actively metabolizes AA to both cyclooxygenase and lipoxygenase products (19-21). We have previously shown (12) that exogenously added Hz02 triggers AA metabolism in the cultured rat alveolar macrophage. Noncytotoxic concentrations of H2O2stimulated release of cellular AA and The abbreviationsused are:AA, arachidonic acid; H202, hydrogen peroxide; HETE, hydroxyeicosatetraenoic acid; HBSS, Hanks’ balanced salt solution; M199, medium 199 with modified Earle’s salts; RIA, radioimmunoassay; RP-HPLC, reverse-phase high performance liquid chromatography; PG, prostaglandin; HHT, 12-hydroxy-5,8,10heptadecatrienoic acid.

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Inhibition of Macrophage 5-Lipoxygenase by Hz02

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Radioimmunoassays-Leukotriene B4, leukotriene C4, and thromboxane BP in media from unlabeled alveolar macrophage cultures were quantitated by radioimmunoassays (RIAs) performed by the Ligand Core Laboratory of the University of Michigan Diabetes Research and Training Center. Dried lipid extracts were dissolved in 1 ml of phosphate-buffered saline containing 0.1% gelatin, pH 7.4, and 100-pl aliquots assayed in duplicate for each sample. The antibody sources and cross-reactivities and the assay sensitivities for these RIAs have been described previously (35). The specificities of the RIAs have been confirmed by analyses utilizing reverse-phase high performance liquid chromatography (RP-HPLC) (12,351. In all cases, quantities of immunoreactive eicosanoids reported were corrected for recovery. Eicosanoid Separation by Reverse-phase High Performance Liquid Chromatography-For separation of [“CIAA metabolites produced by prelabeled alveolar macrophages, lipid extracts of pooled media from triplicate cultureswere dissolved in 500 pl of acetonitrile/water/ trifluoroacetic acid (33:67:0.1, v/v/v) and subjected to RP-HPLC using a WatersHPLC system equipped with a Waters5-pm Bondapak CIScolumn (30 X 0.4 cm) eluted with acetonitrile/water/trifluoroacetic acid a t 1ml/min, as previously described (35). Using this system, cyclooxygenasemetabolites are eluted during an initialisocratic phase (33:67:0.1, v/v/v), followed by lipoxygenase metabolites and free AA, which elute during a stepwise gradient increase of acetonitrile to EXPERIMENTALPROCEDURES 1OO:O:O.l (v/v/v). The eluate was continuously monitored for UV Macrophage Isolation and Culture-Respiratory disease-free 126- absorbance (210 nm for cyclooxygenase products and free AA,280 150 g female Wistar rats were obtained from Charles River (Portage, nm for leukotrienes, and 235nm for mono-HETEs).Authentic MI) and housed under specific pathogen-free conditions. Following thromboxane Bz, prostaglandin (PG)DPPGEz, PGFz,, and 6-ketoanesthesia with intraperitoneal sodium pentobarbital, lungs were PGFl, were generous gifts of Dr. J. Pike (Upjohn Co., Kalamazoo, surgically excised and lavaged as previously described (32). Lavage MI), and lipoxygenase standards leukotrienes B4, C,, and D,, 5-, 12-, fluid, as well as Hanks’ balanced salt solution (HBSS: GIBCO) and and 15-HETEof Dr. J. Rokach (Merck Frosst, Inc., Quebec, Canada). acid (HHT) was obmedium 199 with modified Earle’s salts (M199; GIBCO) all contained Authentic 12-hydroxy-5,8,10-heptadecatrienoic 100 units/ml penicillin, 100 pg/ml streptomycin, and 0.25 mg/ml tained from Cayman Chemical Co. (Ann Arbor, MI), andarachidonic amphotericin B (Antibiotic-Antimycotic Solution, Sigma). Two X lo6 acid from Nu-Chek Prep, Inc. (Elysian, MN). Eluate fractions of 1 cells (-95% alveolar macrophages) suspended in 1.5 ml of M199 were mlwere collected and radioactivity quantitatedin6 mlofACS plated in 35 X 10-mm plastic culture dishes (Falcon Plastics, Oxnard, scintillant (Amersham) using a Beckman LS1801 scintillation counCA) and cultured at 37 “C in a humidified atmosphere of 5% COZ in ter (Beckman Instruments, Inc., Fullerton, CA) with a counting air. After 1h, nonadherent cells were removed bywashing twice with efficiency for 14Cof approximately 90%. Radiolabeled eicosanoids HBSS. The resultant adherent cell population has been found to were identified by their co-elution with authentic standards (35). Adenosine Triphosphate Assay-Cellular ATP was determined by contain 95% macrophages by morphologic criteria and esterase staining (32) with viability always exceeding 90% as assessed by trypan the luciferase-luciferin assay (36), as described (37). After experimenblue exclusion. Macrophage monolayers were then cultured overnight tal incubation, culture media were removed and monolayers scraped (16 h) in M199 containing 10% heat-inactivatednewborn calf serum with a rubber policeman into 1ml of iced 10 mM potassium phosphate, (GIBCO) prior to experimental incubations. Following overnight 4 mM MgS04 buffer, pH 7.4; samples were transferred to glass tubes culture, these monolayers have been found to contain approximately on ice and plates washed with an additional 1.5 ml of buffer. Cell 8.5 pgof DNA (32) and 100 pgof protein (Bradford method (33), suspensions were placed in a 90-95 “C water bath for 4 min, and then with bovine serum albumin as standard). on ice until assay, within 4 h. At the time of assay, 1 ml of sample Prelabeling of Macrophage Cultures-In selected experiments, cel- was added to 2 ml of 50 mM NazHAs04,20 mM MgSO, buffer, pH lular lipids were prelabeled by including 0.2 pCi of [1-14C]AA(specific 7.4, in aglass scintillation vial. Fifty pl of luciferase-luciferin (Sigma), activity 54-57 mCi/mmol; Du Pont-New England Nuclear) in the reconstituted in sterileglycine buffer (195 mM glycine, 20 mM MgS04, medium during overnight culture. To remove unincorporated label, 2 mM EDTA, 1.3 mM NazHAs04,2.6 mM NazHP04,4.4 mg/ml bovine cells were washed with HBSS, incubated for an additional hour with serum albumin, pH 7.1), were added to the assay mixture and light M199 containing 10% newborn calf serum, and washed again prior emission immediately quantitatedina Beckman LS1801 counter to experimental incubations. The uptake of radiolabel by macrophage using the single photon monitor mode. ATP (Sigma) was diluted in cultures, determined as described previously (32), was 34.8 f 2.2% the 10 mM potassium phosphate,4 mM MgSO, buffer, andthe (mean f S.E., n = 4). concentration of standards was confirmed spectrophotometrically a t Incubations with H z 0 2 and Agonists-Following overnight incuba- 260 nm based on a molar extinction coefficient of 15,400. Standard tion, duplicate cultures of unlabeled or prelabeled alveolar macro- curves of log cpm versus log [ATP] were linear over the range lo-’ to phages were washed twice with HBSS and incubated for 30 min in 1 lo+’M ATP. ml of M199 alone or M199containing various concentrations of H202 H z 0 2 Assay-Hz02 was measured by the homovanillic acid-horse(Malinkrodt). Alternatively, alveolar macrophage cultures were radish peroxidase assay of Ruch et al. (38). Following adherence and washed and incubated for various times with either calcium ionophore overnight culture, alveolar macrophage monolayers were incubated A23187 (Behring Diagnostics) at 10 p~ (in 0.5% dimethyl sulfoxide) with added HZOZ(0.15 mM) in Dulbecco’s phosphate-buffered saline or preboiled zymosan A (Sigma) at 100 pg/ml in the presence or containing 0.1%glucose (1 ml/plate), fromwhich duplicate 50-pl absence of various concentrations of Hz02. In certain experiments, aliquots were taken for assay at time points up to 1 h. The tr for 50 nM antimycin A (Sigma) or combination a of 5 mM inosine (Sigma) HZOZadded to alveolar macrophage cultures was determined from the plus 10 mM supplemental inorganic phosphate (as sodium phosphate, linear regression equation for a plot of log [H2OZ]versus time. pH 7.4)were added to culture media for a specified pretreatment Data Analysis-For all experiments inwhich eicosanoid levels were period and during stimulation with A23187. measured by RIAor macrophage ATP content quantitated, duplicate Eicosanoid Extractions-Eicosanoids were extracted from culture culture plates were utilized for each experimental condition and the medium using Sep-pak C18 cartridges (Waters Associates, Milford, resulting values averaged to yield a single data point. All data for MA) according to themethod of Westcott etal. (34). Methanol/water which n 2 3 are expressed as mean values f S.E. The effects of HzO, (80:20, v/v) eluates containingeicosanoids were dried under nitrogen and antimycin A on eicosanoid and ATP levels are expressed as the and stored at -70 “C. Recoveries for this extraction procedure, as- percent of the corresponding level found in cultures exposed to agonist sessed using tritiated standards (Du Pont-New England Nuclear) alone. The significance of differences between group means was added to M199, were as follows: leukotriene B4, 67.6 f 2.7% ( n = 3); assessed by paired Student’s t test, or by one-way analysis of variance leukotriene C4, 67.3 f 1.8%( n = 6), andthromboxane Bz(the stable and the Newman-Keuls multiple range test, as appropriate (39). A p breakdown product of thromboxane Az), 64.6 f 2.7% ( n = 3). value < 0.05 was considered significant.

synthesis of the cyclooxygenase product thromboxane AS, and also augmented alveolar macrophage thromboxane Az synthesis induced by the agonists calcium ionophore A23187 and zymosan. In the current study, we have evaluated the effects on alveolar macrophage 5-lipoxygenase metabolism. of H202 Our results show that, despite its ability to trigger AA release and metabolism via cyclooxygenase, Hz02 failed to stimulate alveolar macrophage synthesis of the 5-lipoxygenaseproducts leukotriene B4,leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE). Furthermore, H202 inhibited the formation of all of these 5-lipoxygenase metabolites in cells stimulated with A23187 and zymosan, classical agonists of arachidonate 5-lipoxygenase metabolism in the macrophage. Because 5lipoxygenase depends on adenosine triphosphate (ATP) for activity (22-27), and since HzOz has been shown to deplete ATP in cells other than thealveolar macrophage (2&31), we hypothesized that depletion of cellular ATP might play a role in 5-lipoxygenase inhibition by H202. Indeed, our findings suggest that such a mechanism is operative.

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Inhibition of Macrophage 5-Lipoxygenase

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indomethacin (1 PM). Despite 94.0% inhibition of HzOp-induced thromboxane BP synthesis, no detectable immunoreactive leukotriene B4 or leukotriene C4 ( n = 2 for each leukotriene) could be recovered from media of cultures exposed to Hz02in the presence of indomethacin. Oxygen metabolites, including H202,have the capacity to degrade leukotrienes in various systems (42-44). Therefore, to determine whether theabsence of detectable leukotrienes in Hz02-exposed alveolarmacrophage cultures actually reflected a lack of leukotriene synthesis as opposed to degradation of synthesized leukotrienes, we measured the recovery of exogenously added leukotrienesfrom media of resting alveolar macrophage cultures incubated in the presence or A A I 762 absence of HzOz. In these experiments, exposure of alveolar 200 macrophage cultures to 1mM H z 0 2 for 30 min resulted in the recovery of 93.3 f 7.7% of the immunoreactive leukotriene B4 (2.44 f 0.93 ng/plate in the presence of H202versus 2.46 f 0.74 ng/plate in the absence of HzOz, n = 4, p = not significant) and 94.4 f 6.5% of the immunoreactive leukotriene C4 (0.76 f 0.18 ng/plate in the presence of Hz02uersus 0.79 f 0.15 ng/plate in the absence of HzOz, n = 4, p = not signifi0 20 60 40 80 100 120 cant) recovered from control cultures not exposed to HzOz. On the other hand, the addition of 10 mM Hz02 to cultures ELUTION TIME (MIN) for 30 min resulted in the recovery by RIA of only 53.7% of added leukotriene B4 ( n = 2) and 4.8% of added leukotriene FIG. 1. RP-HPLC analysis of eicosanoids produced by [“C] AA-prelabeled alveolar macrophages exposed to H202. RP- C4 ( n = 2) recovered from non-HzOz-exposed controls. Thus, HPLC using acetonitrile/water/trifluoroacetic acid was carried out although 10 mM H202 does cause loss of leukotriene immuas detailed under “Experimental Procedures.” A, tracing of UV abnoreactivity, leukotriene B4 and C4 are not degraded by HZ02 sorbance (monitored at 210 nm, 0-35 rnin; 280 nm, 35-55 min; 235 exposures up to 1 mM, the range of relevant concentrations nrn, 55-95 min; and 210 nm, 95min through completion of run) at which H202functions as an agonist for AA metabolism. illustrating retention times of authentic standards: a, solvent front; b, 6-keto-PGF1,; c, thromboxane (Tn) BP;d, PGFz,; e, PGEP;f , PGD2; Therefore, the failure to detect immunoreactive leukotriene g, leukotriene (LT)C4; h, leukotriene D4; i, leukotriene B4; j , HHT; B4 and C4 in these experiments reflects a lack of leukotriene k , 15-HETE; I , 12-HETE; m, 5-HETE;and n, AA. B , [14C]AA- synthesis by alveolar macrophages challenged with H202. prelabeled alveolar macrophages were incubated in medium containEffects of HzOz on Agonist-Stimulated 5-Lipoxygenase Meing 0.15 mM HzOP for 30 min, and pooled media from triplicate tabolism-To further evaluate the lack of detectable 5-lipoxcultures were extracted and subjected to RP-HPLC. The eluatewas ygenase product formation in alveolar macrophages exposed collected in 1-ml fractions and radioactivity quantitated. The total to Hz02 alone,immunoreactiveleukotriene synthesis was radioactivity (minus background), given in cpm, contained within each peak is thromboxane B,, 362; HHT, 258; AA, 251. assessed and compared to immunoreactive thromboxane A2 (measured as thromboxane B2) production in alveolar macrophage cultures stimulated with either calciumionophore RESULTS A23187 or the particulate zymosan, in the presence and abEffect of H 2 0 2Alone on Alveolar Macrophage 5-Lipoxygenme sence of varying concentrations of H202. Cultures stimulated Metabolism-Previous results (12)’ indicated that HzOz opti- with A23187 (10 PM) for 30 min in the absence of Hz02 mally stimulated alveolar macrophagesynthesis of thrombox- synthesized 33.50 f 7.30 ng of leukotriene B4 ( n = 61, 5.29 f ane Az (measured as the stable metabolite thromboxane Bz) 1.48 ng of leukotrieneC4 ( n = 5), and 1.64 k 0.33 ng of at a concentration of 0.15 mM. Reverse-phase HPLC analysis thromboxane BP (n = 7) per plate as determined by RIAs. of media from [14C]AA-prelabeled alveolar macrophage cul- Cultures stimulated with zymosan (100 pglrnl)for 30 min tures exposed for 30 min to 0.15 mM H202 confirmed the produced 4.42 f 0.86 ng of leukotriene Ba (n = 4), 0.39 f 0.09 production of [‘4C]thromboxane BQ, and further showed reng of leukotriene C4 ( n = 4), and0.81 f 0.11 ng of thromboxlease of [“CIHHT (a nonenzymatic 17-carbon derivative of ane BP( n = 4) per plate. When stimulationwith either agonist the endoperoxide prostaglandinintermediate)and [14C]AA was carried out in the presence of simultaneously added H202, (Fig. 1).However, no measurable 14C-labeledleukotrienes or there was dose-dependent inhibition of A23187- and zymosanmono-HETEs were detected following this H202exposure. The absence of detectableleukotrienes in HzOz-exposed induced synthesis of immunoreactive leukotriene B4 and C4 over the concentration range 0.01 to 1 mM Hz02 (Fig. 2, A alveolarmacrophage cultures which released AA andthe causing 50%inhibition (1%) cyclooxygenase products thromboxaneBPand HHTwas con- and B ) . The HzOz concentration of A23187-stimulated leukotriene productionwas 0.13 mM for firmed in unlabeled cultures utilizing RIAs sensitive to