Hydrogen peroxide inhibits alveolar macrophage 5-lipoxygenase metabolism in association with depletion of ATP.

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, 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 degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated 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 H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.

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 synthesis of 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 degradation of leukotrienes by Hz02 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that HzOZ dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 PM) and zymosan (100 pg/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane AZ and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. l) HzOz depleted ATP in A23187-and zymosan -s t' 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 HzOZ were compatible with a rate-limiting effect of ATP on leukotriene synthesis in HzOz-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin 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 attenuated both 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 an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase path-way, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is 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 intact laboratory animals (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(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 concentrations in 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 that in most tissues, as well as the large number of oxidant pollutants in the atmosphere, the lung faces a uniquely high oxidant burden 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 oxidant Hz02 on 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)(20)(21). We have previously shown (12) that exogenously added Hz02 triggers AA metabolism in the cultured rat alveolar macrophage. Noncytotoxic concentrations of H2O2 stimulated release of cellular AA and The abbreviations used 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.
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 of H202 on alveolar macrophage 5-lipoxygenase metabolism.
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-lipoxygenase products 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 the alveolar 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.

EXPERIMENTAL PROCEDURES
Macrophage Isolation and Culture-Respiratory disease-free 126-150 g female Wistar rats were obtained from Charles River (Portage, MI) and housed under specific pathogen-free conditions. Following anesthesia with intraperitoneal sodium pentobarbital, lungs were surgically excised and lavaged as previously described (32). Lavage fluid, as well as Hanks' balanced salt solution (HBSS: GIBCO) and medium 199 with modified Earle's salts (M199; GIBCO) all contained 100 units/ml penicillin, 100 pg/ml streptomycin, and 0.25 mg/ml amphotericin B (Antibiotic-Antimycotic Solution, Sigma). Two X lo6 cells (-95% alveolar macrophages) suspended in 1.5 ml of M199 were plated in 35 X 10-mm plastic culture dishes (Falcon Plastics, Oxnard, CA) and cultured at 37 "C in a humidified atmosphere of 5% COZ in air. After 1 h, nonadherent cells were removed by washing twice with HBSS. The resultant adherent cell population has been found to contain 95% macrophages by morphologic criteria and esterase staining (32) with viability always exceeding 90% as assessed by trypan blue exclusion. Macrophage monolayers were then cultured overnight (16 h) in M199 containing 10% heat-inactivated newborn calf serum (GIBCO) prior to experimental incubations. Following overnight culture, these monolayers have been found to contain approximately 8.5 pg of DNA (32) and 100 pg of protein (Bradford method (33), with bovine serum albumin as standard).
Prelabeling of Macrophage Cultures-In selected experiments, cellular lipids were prelabeled by including 0.2 pCi of [1-14C]AA (specific activity 54-57 mCi/mmol; Du Pont-New England Nuclear) in the medium during overnight culture. To remove unincorporated label, cells were washed with HBSS, incubated for an additional hour with M199 containing 10% newborn calf serum, and washed again prior to experimental incubations. The uptake of radiolabel by macrophage cultures, determined as described previously (32), was 34.8 f 2.2% (mean f S.E., n = 4).
Incubations with H z 0 2 and Agonists-Following overnight incubation, duplicate cultures of unlabeled or prelabeled alveolar macrophages were washed twice with HBSS and incubated for 30 min in 1 ml of M199 alone or M199 containing various concentrations of H202 (Malinkrodt). Alternatively, alveolar macrophage cultures were washed and incubated for various times with either calcium ionophore A23187 (Behring Diagnostics) at 10 p~ (in 0.5% dimethyl sulfoxide) or preboiled zymosan A (Sigma) at 100 pg/ml in the presence or absence of various concentrations of Hz02. In certain experiments, 50 nM antimycin A (Sigma) or a combination of 5 mM inosine (Sigma) plus 10 mM supplemental inorganic phosphate (as sodium phosphate, pH 7.4) were added to culture media for a specified pretreatment period and during stimulation with A23187.
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 cultures were dissolved in 500 pl of acetonitrile/water/ trifluoroacetic acid (33:67:0.1, v/v/v) and subjected to RP-HPLC using a Waters HPLC system equipped with a Waters 5-pm Bondapak CIS column (30 X 0.4 cm) eluted with acetonitrile/water/trifluoroacetic acid at 1 ml/min, as previously described (35). Using this system, cyclooxygenase metabolites are eluted during an initial isocratic 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 1OO:O:O.l (v/v/v). The eluate was continuously monitored for UV absorbance (210 nm for cyclooxygenase products and free AA, 280 nm for leukotrienes, and 235 nm for mono-HETEs). Authentic thromboxane Bz, prostaglandin (PG)DP PGEz, PGFz,, and 6-keto-PGFl, were generous gifts of Dr. J. Pike (Upjohn Co., Kalamazoo, MI), and lipoxygenase standards leukotrienes B4, C,, and D,, 5-, 12-, and 15-HETE of Dr. J. Rokach (Merck Frosst, Inc., Quebec, Canada). Authentic 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was obtained from Cayman Chemical Co. (Ann Arbor, MI), and arachidonic acid from Nu-Chek Prep, Inc. (Elysian, MN). Eluate fractions of 1 ml were collected and radioactivity quantitated in 6 ml of ACS scintillant (Amersham) using a Beckman LS1801 scintillation counter (Beckman Instruments, Inc., Fullerton, CA) with a counting efficiency for 14C of approximately 90%. Radiolabeled eicosanoids were identified by their co-elution with authentic standards (35).
Adenosine Triphosphate Assay-Cellular ATP was determined by the luciferase-luciferin assay (36), as described (37). After experimental incubation, culture media were removed and monolayers scraped with a rubber policeman into 1 ml of iced 10 mM potassium phosphate, 4 mM MgS04 buffer, pH 7.4; samples were transferred to glass tubes on ice and plates washed with an additional 1.5 ml of buffer. Cell suspensions were placed in a 90-95 "C water bath for 4 min, and then on ice until assay, within 4 h. At the time of assay, 1 ml of sample was added to 2 ml of 50 mM NazHAs04, 20 mM MgSO, buffer, pH 7.4, in a glass scintillation vial. Fifty pl of luciferase-luciferin (Sigma), reconstituted in sterile glycine buffer (195 mM glycine, 20 mM MgS04, 2 mM EDTA, 1.3 mM NazHAs04, 2.6 mM NazHP04, 4.4 mg/ml bovine serum albumin, pH 7.1), were added to the assay mixture and light emission immediately quantitated in a Beckman LS1801 counter using the single photon monitor mode. ATP (Sigma) was diluted in the 10 mM potassium phosphate, 4 mM MgSO, buffer, and the concentration of standards was confirmed spectrophotometrically at 260 nm based on a molar extinction coefficient of 15,400. Standard curves of log cpm versus log [ATP] were linear over the range lo-' to lo+' M ATP.
H z 0 2 Assay-Hz02 was measured by the homovanillic acid-horseradish peroxidase assay of Ruch et al. (38). Following adherence and overnight culture, alveolar macrophage monolayers were incubated with added HZOZ (0.15 mM) in Dulbecco's phosphate-buffered saline containing 0.1% glucose (1 ml/plate), from which duplicate 50-pl aliquots were taken for assay at time points up to 1 h. The tr for HZOZ added to alveolar macrophage cultures was determined from the linear regression equation for a plot of log [H2OZ] versus time.
Data Analysis-For all experiments in which eicosanoid levels were measured by RIA or macrophage ATP content quantitated, duplicate culture plates were utilized for each experimental condition and the resulting values averaged to yield a single data point. All data for which n 2 3 are expressed as mean values f S.E. The effects of HzO, and antimycin A on eicosanoid and ATP levels are expressed as the percent of the corresponding level found in cultures exposed to agonist alone. The significance of differences between group means was assessed by paired Student's t test, or by one-way analysis of variance and the Newman-Keuls multiple range test, as appropriate (39). A p value < 0.05 was considered significant. prelabeled alveolar macrophages were incubated in medium containing 0.15 mM HzOP for 30 min, and pooled media from triplicate cultures were extracted and subjected to RP-HPLC. The eluate was collected in 1-ml fractions and radioactivity quantitated. The total radioactivity (minus background), given in cpm, contained within each peak is thromboxane B,, 362; HHT, 258; AA, 251.

Effect of H 2 0 2 Alone on Alveolar Macrophage 5-Lipoxygenme
Metabolism-Previous results (12)' indicated that HzOz optimally stimulated alveolar macrophage synthesis of thromboxane Az (measured as the stable metabolite thromboxane Bz) at a concentration of 0.15 mM. Reverse-phase HPLC analysis of media from [14C]AA-prelabeled alveolar macrophage cultures exposed for 30 min to 0.15 mM H202 confirmed the production of ['4C]thromboxane BQ, and further showed release of ["CIHHT (a nonenzymatic 17-carbon derivative of the endoperoxide prostaglandin intermediate) and [14C]AA (Fig. 1). However, no measurable 14C-labeled leukotrienes or mono-HETEs were detected following this H202 exposure.
The absence of detectable leukotrienes in HzOz-exposed alveolar macrophage cultures which released AA and the cyclooxygenase products thromboxane BP and HHT was confirmed in unlabeled cultures utilizing RIAs sensitive to <0.03 ng of leukotriene B4 and <0.10 ng of leukotriene C4/ml of culture medium. No immunoreactive leukotriene B d or leukotriene C4 was detected in cultures exposed to medium alone or to H202 at concentrations from 0.001 to 1 mM ( n = 6 for each leukotriene, data not shown). Because in some experimental systems cyclooxygenase inhibition enhances 5-lipoxygenase product formation (40, 41), alveolar macrophage monolayers were exposed to 0.1 mM Hz02 in the presence of * P. H. S. Sporn and M. Peters-Golden, unpublished results.
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 Hz02 in the presence of indomethacin.
Oxygen metabolites, including H202, have the capacity to degrade leukotrienes in various systems (42)(43)(44). Therefore, to determine whether the absence of detectable leukotrienes in Hz02-exposed alveolar macrophage cultures actually reflected a lack of leukotriene synthesis as opposed to degradation of synthesized leukotrienes, we measured the recovery of exogenously added leukotrienes from media of resting alveolar macrophage cultures incubated in the presence or absence of HzOz. In these experiments, exposure of alveolar macrophage cultures to 1 mM 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 H202 versus 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 Hz02 uersus 0.79 f 0.15 ng/plate in the absence of HzOz, n = 4, p = not significant) recovered from control cultures not exposed to HzOz. On the other hand, the addition of 10 mM Hz02 to cultures 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 C4 ( n = 2) recovered from non-HzOz-exposed controls. Thus, although 10 mM H202 does cause loss of leukotriene immunoreactivity, leukotriene B4 and C4 are not degraded by HZ02 exposures up to 1 mM, the range of relevant concentrations at which H202 functions as an agonist for AA metabolism. Therefore, the failure to detect immunoreactive leukotriene B4 and C4 in these experiments reflects a lack of leukotriene synthesis by alveolar macrophages challenged with H202.
Effects of HzOz on Agonist-Stimulated 5-Lipoxygenase Metabolism-To further evaluate the lack of detectable 5-lipoxygenase product formation in alveolar macrophages exposed to Hz02 alone, immunoreactive leukotriene synthesis was assessed and compared to immunoreactive thromboxane A2 (measured as thromboxane B2) production in alveolar macrophage cultures stimulated with either calcium ionophore A23187 or the particulate zymosan, in the presence and absence of varying concentrations of H202. Cultures stimulated with A23187 (10 PM) for 30 min in the absence of Hz02 synthesized 33.50 f 7.30 ng of leukotriene B4 ( n = 61, 5.29 f 1.48 ng of leukotriene C4 ( n = 5), and 1.64 k 0.33 ng of thromboxane BP (n = 7) per plate as determined by RIAs. Cultures stimulated with zymosan (100 pglrnl) for 30 min produced 4.42 f 0.86 ng of leukotriene Ba (n = 4), 0.39 f 0.09 ng of leukotriene C4 ( n = 4), and 0.81 f 0.11 ng of thromboxane BP ( n = 4) per plate. When stimulation with either agonist was carried out in the presence of simultaneously added H202, there was dose-dependent inhibition of A23187-and zymosaninduced synthesis of immunoreactive leukotriene B4 and C4 over the concentration range 0.01 to 1 mM Hz02 (Fig. 2, A  and B ) . The HzOz concentration causing 50% inhibition (1%) of A23187-stimulated leukotriene production was 0.13 mM for leukotriene B4 and 0.11 mM for leukotriene c q . Corresponding H2O2 ICs0 values in the case of zymosan stimulation were 0.14 mM for leukotriene B4 and 0.13 mM for leukotriene c4. In contrast to its inhibitory effect on agonist-stimulated leukotriene synthesis, H202 caused dose-related augmentation of A23187-and zymosan-induced synthesis of the cyclooxygenase metabolite thromboxane BP over the range from 0.001 to 1 mM Hz02 (Fig. 3). Peak augmentation of thromboxane BZ production in response to both A23187 (5.6 f 1.0-fold increase, n = 6, p < 0.001) and zymosan (2.6 f 0.8-fold increase, To corroborate inhibition by H202 of agonist-induced leu-kotriene B4 and C4 synthesis as determined by RIAs, and to assess the effect of H202 on alveolar macrophage HETE synthesis, radiolabeled metabolites produced by prelabeled alveolar macrophage cultures stimulated with A23187 were separated by RP-HPLC. As the radioactivity elution profile in Fig. 4A shows, A23187 stimulated the synthesis of cyclooxygenase metabolites thromboxane Bz and HHT, 5-lipoxygenase metabolites leukotriene B4, C4, and 5-HETE, and the 12lipoxygenase metabolite 12-HETE. 5-Lipoxygenase products accounted for the vast majority of the recovered radiolabeled eicosanoids. On the other hand, when A23187 stimulation was carried out in the presence of 0.15 mM H202, quantities of leukotriene B4, C4 and 5-HETE recovered were reduced in each case by approximately 80% (Fig. 423). Of note, Hz02 at 0.15 mM decreased 12-HETE to a similar degree. However, in contrast to its inhibition of lipoxygenase products, 0.15 mM H202 caused substantial augmentation of cyclooxygenase Pooled media from triplicate cultures were extracted and subjected to RP-HPLC as described in the legend for Fig. 1. A AA (Fig. 4B). Thus, analysis by RP-HPLC of radiolabeled metabolites produced by A23187-stimulated cells confirmed H202-induced inhibition of leukotriene B4 and C4 synthesis and augmentation of thromboxane B2 production as demonstrated by RIAs; in addition, similar inhibition by H202 of both 5-and 12-HETE synthesis and augmentation by Hz02 of release of HHT and free AA were documented.

Role of ATP in H202-induced Inhibition of 5-Lipoxygenme
Metabolism-To evaluate the hypothesis that HzOz-induced depletion of cellular ATP might be responsible for inhibition of alveolar macrophage 5-lipoxygenase metabolism by H202, we used the luciferase-luciferin assay to measure the ATP content of alveolar macrophages following stimulation for 30 min with either A23187 or zymosan in the presence or absence of varying concentrations of Hz02. The effect of H202 on cellular ATP levels was assessed in agonist-stimulated cultures in order to duplicate the conditions under which inhibition of 5-lipoxygenase metabolism was demonstrated. Of interest, A23187 had an ATP-depleting effect of its own (control, 352 -C 34 pmol/plate; A23187, 136 f 14 pmol/plate; n = 6, p < 0.001), whereas zymosan was not associated with such an effect (control, 333 k 40 pmol/plate; zymosan. 319 f 44 pmol/plate; n = 6, p = not significant). Importantly, the addition of HZ02 to alveolar macrophage cultures during agonist stimulation resulted in dose-dependent depletion of cellular ATP (Fig. 5). Despite the difference in ATP content of cultures stimulated with A23187 or zymosan, the Hz02 dose-response curves for ATP depletion with the two agonists were nearly identical when ATP levels were expressed as a percentage of the ATP content in cultures incubated with each agonist alone. Moreover, the dose-response relationships for depletion of ATP by Hz02 closely resembled those for inhibition of agonist-induced synthesis of leukotriene B4 and Cq (Fig. 2). The IC6o values of Hz02 for ATP depletion in A23187-and zymosan-stimulated alveolar macrophages, 0.11 and 0.18 mM, respectively, closely matched the ICs0 values for inhibition by H202 of A23187-and zymosan-induced leukotriene B, and C4 synthesis (range 0.11 to 0.14 mM, see above).
After observing such close concordance of the dose-response relationships for inhibition of leukotriene synthesis and depletion of ATP by H2O2, we investigated the time course of the development of these two Hn02-induced phenomena. As seen in Fig. 6A, following stimulation with A23187, leuko- triene B4 levels in culture media rose rapidly for the first 10 min and then reached a plateau. In cultures stimulated with A23187 in the presence of 0.15 mM HzOz, leukotriene B4 synthesis increased more slowly during the 10 min following challenge, and then leveled off. Fig. 6B shows that relative to alveolar macrophages stimulated with A23187 alone, ATP levels in cultures challenged with both A23187 and 0.15 mM H202 were reduced as early as 1 min and fell progressively during the first 10 min following stimulation. Of note, by 10 min, the time at which the steep rise in leukotriene B4 synthesis culminated, the reductions in leukotriene B4 production and cellular ATP caused by Hz02 were of similar magnitude (to 45 and 52%, respectively, of values in cultures stimulated with A23187 alone). Thus, the time courses of ATP depletion and leukotriene B4 accumulation in H202exposed cultures are compatible with a rate-limiting effect of the lower ATP levels on leukotriene B4 synthesis in alveolar macrophages challenged with A23187 in the presence of H202.
To further evaluate the importance of ATP depletion in the inhibition of agonist-stimulated 5-lipoxygenase metabolism, we examined eicosanoid production in alveolar macrophage cultures depleted of ATP by means other than HZOZ exposure. In these experiments, alveolar macrophage cultures were treated with or without the electron transport inhibitor antimycin A (50 nM) for 5 min before and 30 min during stimulation with A23187, after which cellular ATP content and media immunoreactive eicosanoid levels were determined.
Treatment with antimycin A reduced the ATP content of alveolar macrophages to 34.7 f 4.4% of that of cultures stimulated with A23187 alone (antimycin A plus A23187, 47

Effect of inosine plus phosphate on ATP depletion and inhibition of leukotriene synthesis induced by Hz02
Alveolar macrophages were incubated for 30 min in medium containing A23187 (10 p M ) alone, or A23187 plus HzOz (0.15 mM), in the absence or presence of inosine (5 mM) plus supplemental inorganic phosphate (10 mM). Cellular ATP content was then determined by the luciferase-luciferin assay, and leukotrienes in culture media were quantitated by RIA. ATP is expressed as pmol/plate, and leukotriene B4 and leukotriene C4 as ng/plate. For cultures incubated with A23187 plus HZOz, ATP and leukotriene levels are also given as percentages of the plate; n = 3, p < 0.001). Although this reduction in A23187stimulated thromboxane B2 synthesis by antimycin A is statistically significant, the percent decrease of thromboxane Bz in the presence of the electron transport inhibitor (21.0 & 10.1%) was significantly less than the decreases in leukotriene Bq and leukotriene C4 (72.8 f 5.3 and 63.4 f 5.6%, respectively; n = 3, p < 0.005 for thromboxane Bz versus both leukotrienes), which correlated closely with the degree to which the inhibitor depleted ATP (65.3 f 4.4%; n = 3, p < 0.005 versus thromboxane Bz, p = not significant versus both leukotrienes). In order to examine whether depletion of ATP and inhibition of 5-lipoxygenase metabolism were causally related, we sought to augment ATP levels in H202-exposed alveolar macrophage cultures and determine the effect of such a manipulation on 5-lipoxygenase metabolite production. To accomplish this, alveolar macrophage cultures were incubated with the ATP precursors inosine (5 mM) and supplemental inorganic phosphate (10 mM) in M19g3 for 30 min prior to and 30 min during stimulation with A23187. Although inosine plus phosphate treatment had no effect on the ATP content of alveolar macrophages stimulated with A23187 in the absence of H202, these additives significantly attenuated ATP depletion in cells stimulated with A23187 plus HzOz (Table I). The possibility that inosine plus phosphate increased ATP levels in HzOz-exposed cultures by scavenging H202 was evaluated by determining the tlh of H202 following its addition at 0.15 mM to alveolar macrophage cultures containing or not containing inosine plus supplemental phosphate. The t1,? of HZO2 in alveolar macrophage cultures was 8.8 min without and 8.7 min with inosine plus phosphate, indicating that attenuation of Hz02-induced ATP depletion by these additives did not The concentration of phosphate in unsupplemented M199 is 0.9 mM. result from scavenging of H202. As shown in Table I, inhibition by H202 of A23187-stimulated immunoreactive leukotriene B4 and C4 synthesis was attenuated in cultures treated with inosine plus phosphate as compared to those not incubated with the ATP precursors. This attenuation was significant whether the amount of each leukotriene formed in H202exposed cultures was expressed as actual ng/plate, or as a percentage of the level in corresponding non-HzOz-exposed cultures with or without inosine plus phosphate. However, because inosine plus phosphate themselves reduced A23187stimulated leukotriene synthesis in the absence of H202, the magnitude of their effect on inhibition of leukotriene synthesis caused by H202 is most appropriately determined from comparison of the percentage data. This analysis shows that in the presence of HZ02, leukotriene B4 and C4 synthesis was increased 1.9 f 0.3-fold and 2.7 f 0.2-fold, respectively ( p < 0.001 for both leukotrienes), by inosine plus phosphate. Thus, in association with their protective effect on cellular ATP, inosine plus phosphate attenuated the inhibition of A23187induced leukotriene synthesis resulting from exposure to H20z.

DISCUSSION
In a previous study (12), we showed that H2Oz triggered release of cellular AA and synthesis of the cyclooxygenase product thromboxane Az in the rat alveolar macrophage. HzOZ also augmented thromboxane AZ synthesis stimulated by the agonists calcium ionophore A23187 and zymosan. However, in the present study, using a combination of HPLC and sensitive RIA techniques, we have shown that HzOz-exposed alveolar macrophages do not produce measurable amounts of leukotriene B4, leukotriene C4, or 5-HETE. Inhibition of the cyclooxygenase pathway by indomethacin did not shunt AA to leukotriene synthesis in alveolar macrophages exposed to H202. The failure to recover leukotriene B4 and C. , in H202exposed cultures was shown to represent lack of synthesis, and not degradation, of leukotrienes by H202. This finding is consistent with the observation of Neil1 and colleagues (44) that oxidative degradation of leukotrienes in mononuclear cell cultures is dependent on both HZ02 and myeloperoxidase, an enzyme which the macrophage loses during differentiation from its monocytic precursor (45,46). Thus, HzOz failed to stimulate 5-lipoxygenase metabolism, despite the fact that the alveolar macrophage is a cell with the capacity for synthesis of large quantities of 5-lipoxygenase-derived eicosanoids (20, 21).

Inhibition of Macrophage 5-Lipoxygenme by H202
The failure of HzOz-exposed alveolar macrophages to produce 5-lipoxygenase metabolites was explored further by examining the effect of Hz02 on 5-lipoxygenase metabolism in alveolar macrophages challenged with agonists which themselves stimulate 5-lipoxygenase product formation. Experiments utilizing RIA analyses demonstrated that H202 dose dependently inhibited leukotriene B4 and C4 synthesis stimulated by both A23187 and zymosan, two agonists which activate AA metabolism by different mechanisms. Inhibition by Hz02 of A23187-stimulated leukotriene B4 and C4 synthesis was confirmed by RP-HPLC of eicosanoids from cultures of ['4C]AA-prelabeled alveolar macrophages. RP-HPLC analysis also showed that A23187-induced 5-HETE production was inhibited by H202; at the H202 concentration used (0.15 mM), 5-HETE and leukotriene synthesis were inhibited to an equivalent degree.
Inhibition of 5-lipoxygenase metabolism by Hz02 was not the result of nonspecific toxicity caused by the oxidant for the following reasons. First, H2O2 inhibited formation of 5lipoxygenase products over a dose range which we have previously shown, using 51Cr-labeled cells, is not cytotoxic for cultured rat alveolar macrophages (12). Second, 5-lipoxygenase inhibition was seen with the particulate zymosan, as well as with A23187, ruling out the possibility that this observation could be accounted for solely by toxicity caused by A23187 or the combination of A23187 plus HZ02. Finally, and most importantly, over the same concentration range at which it inhibited 5-lipoxygenase metabolism, H202 markedly augmented cyclooxygenase metabolism in response to both A23187 and zymosan. Thus the lesion in alveolar macrophage arachidonate metabolism caused by HzOz is a selective one.
Mammalian 5-lipoxygenase is known to be dependent on ATP for activity, both in cell-free assay systems (22-26) and in the intact cell (27). Cyclooxygenase, on the other hand, is not ATP dependent. In addition, H2O2 exposure has been shown to cause depletion of cellular ATP in human platelets (28,29) and lymphocytes (31) and in a murine macrophagelike cell-line (30, 31), shown in the latter case to be due to interference with both glycolytic and mitochondrial pathways for ATP generation (30,31,47,48). In view of these observations, we explored the possibility that depletion of cellular ATP might be responsible for the selective inhibition of 5lipoxygenase metabolism by HzO2. Four lines of experimental evidence suggest that this mechanism is likely to play a role. First, in alveolar macrophages stimulated with two different agonists (A23187 and zymosan), Hz02 depleted cellular ATP with a dose dependency very similar to that for inhibition of leukotriene synthesis. Second, the time courses of both ATP depletion and inhibition of leukotriene B4 production caused by H202 in A23187-stimulated cultures were similar, and therefore compatible with a rate-limiting effect of ATP on leukotriene synthesis in such cultures. Third, exposure of alveolar macrophages to the electron transport inhibitor antimycin A caused proportionally similar depletion of ATP and reductions in leukotriene B4 and C4 synthesis, with relative sparing of production of the cyclooxygenase metabolite thromboxane Az. And fourth, treatment with the ATP precursors inosine and phosphate reduced inhibition of leukotriene B4 and C4 synthesis at the same time that it attenuated ATP depletion by Hz02 in A23187-stimulated alveolar macrophages. Of note, A23187-induced 12-HETE synthesis was inhibited in the presence of H2Oz, as well (Fig. 4). Since mammalian 12-lipoxygenase shares with 5-lipoxygenase a dependence on ATP (49), inhibition of 12-HETE production may also have resulted from HzOz-induced ATP depletion. This interpretation is consistent with our findings in another study (371, in which we showed that uncoupling of oxidative phosphorylation in the alveolar macrophage by exogenous unsaturated fatty acids, including arachidonate, caused similar inhibition of both 5and 12-lipoxygenase metabolism, which paralleled depletion of ATP. While low concentrations of fatty acyl hydroperoxides (but not HzOZ) have the capacity to activate 5-lipoxygenase (50)(51)(52)(53), it has been demonstrated that both soybean lipoxygenase (50,51) and the lipoxygenase isolated from rabbit reticulocytes (54) catalyze their own inactivation during the oxidation of fatty acid through the generation of product hydroperoxides. Indirect evidence for a similar mechanism of self-inactivation of the 5-lipoxygenase isolated from RBL-1 cells has also been presented (55). Furthermore, Egan and colleagues (52) have reported dose-dependent inhibition of the RBL-1 5-lipoxygenase by Hz02 (0.1 and 0.2 mM) in a cell-free reaction mixture. The inhibitory effect of H202 in their system was observed over the same concentration range reported here to cause inhibition of leukotriene synthesis and depletion of ATP in alveolar macrophages. These authors suggested that inhibition of the cell-free 5-lipoxygenase by HzOz reflected hydroperoxide-dependent inactivation of the enzyme, as shown for the soybean and reticulocyte 5-lipoxygenases. However, if HzOz depletes ATP in the cell-free assay system, as it does in the intact alveolar macrophage, an alternate explanation for the observed inhibitory effect of H202 on partially purified 5-lipoxygenase, based on depletion of ATP, is possible. Nevertheless, although our data strongly support an important role for ATP depletion in the inhibition of 5-lipoxygenase, they do not rule out an additional direct effect of HzOz on the enzyme itself, even in intact cells. Indeed, the fact that inosine plus phosphate treatment of HzOz-exposed alveolar macrophages restored leukotriene synthesis to a lesser extent than it did ATP levels raises the possibility that there may be both ATP-dependent and ATP-independent mechanisms by which H202 inhibits 5-lipoxygenase metabolism in the alveolar macrophage.
Using intact cells, Wei and co-workers (56) have recently shown that the addition of catalase and superoxide dismutase to culture medium augmented production of 5-lipoxygenase metabolites by mast cells and combined mast cell-macrophage cocultures stimulated with antigen, as well as by zymosanstimulated peritoneal macrophages. Based on the findings reported herein, we would propose that prevention of oxidantinduced ATP depletion through scavenging of Hz02, and possibly other reactive oxygen species, may have contributed to the augmentation by catalase and superoxide dismutase of 5-lipoxygenase metabolism seen in their study.
In summary, we have demonstrated that although H2OZ stimulates AA release and thromboxane A2 synthesis in the rat alveolar macrophage, it fails to stimulate 5-lipoxygenase product formation. Its inability to do so can be explained by its capacity to inhibit 5-lipoxygenase metabolism. This inhibition closely paralleled depletion by H202 of ATP, a cofactor for 5-lipoxygenase. Thus, HZ02 has the capacity to act both as an agonist of macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway by virtue of a specific biochemical interaction. Other examples of agonist-specific heterogeneity in macrophage AA metabolism have been documented as well (57). In this regard, the effects of Hz02 are reminiscent of those of the sulfhydryl reactant N-ethylmaleimide, which also activates metabolism of AA, while inhibiting sulfidopeptide leukotriene synthesis by depleting cellular glutathione (58). Finally, the effect of H2O2 on 5-lipoxygenase metabolism may be of physiologic importance in vivo. In inflamed tissue where there is generation of both reactive oxygen metabolites and eicosanoids, down-regulation of 5-28. Holmsen, H., and Robkin, L.