Complex Effects of In Vitro Hyperoxia on Alveolar Macrophage Arachidonic Acid Metabolism Peter H. S. Sporn, Teresa M. Murphy, and Marc Peters-Golden Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan and Veterans Administration Medical Centers, Ann Arbor, Michigan
Metabolites of arachidonic acid (AA) released into bronchoalveolar lavage fluid of animals exposed to hyperoxia have previously been implicated as mediators of pulmonary oxygen toxicity. The alveolar macrophage (AM) represents an important potential source of these eicosanoids. We have therefore investigated the effects of in vitro hyperoxia (95 % O 2 /5 % CO2) versus normoxia (95 % air/5 % CO 2) on tlle metabolism of AA in the AM of the rat. Exposure to 95 % O2 for up to 72 h did not impair the viability or affect the protein content of cultured AMs. Hyperoxia for 24 to 72 h increased the accumulation of free AA liberated from endogenous stores in cultures of resting AMs. Despite this increase in free AA, no changes in synthesis of thromboxane B2 , prostaglandin (PG) E2 , PGF2a , leukotriene (LT) B4 , or LTC were observed in resting AMs exposed to hyperoxia for up to 72 h. This was not due to degradation of eicosanoids in hyperoxia. However, formation of cyclooxygenase metabolites from exogenously supplied AA was reduced in hyperoxia-incubated AMs, suggesting that hyperoxia inhibited the cyclooxygenase enzyme. In AMs stimulated with calcium ionophore A23187, both AA release and synthesis of cyclooxygenase and lipoxygenase eicosanoids were augmented after incubation in hyperoxia for 24 to 72 h. The increase in A23187-stimulated LTB4 synthesis caused by hyperoxia was inhibited by the antioxidants catalase, superoxide dismutase, and the intracellular cysteine loading agent L-2-oxothiazolidine-4carboxylic acid, suggesting that the augmentation by hyperoxia of A23187-induced AA metabolism was mediated by reactive oxygen metabolites. Thus, hyperoxia has complex effects on AA metabolism in the AM, which include the ability to augment the release of AA and formation ofbioactive eicosanoids. These findings support a possible role for eicosanoid synthesis by the AM in the pathogenesis of oxygen toxicity of the lung.
Continuous exposure to high concentrations of inspired oxygen causes neutrophilic alveolitis and lung injury in both animals and humans (1). A possible role for metabolites of arachidonic acid (AA) in the pathogenesis of pulmonary oxygen toxicity has been suggested by recent reports of increased bronchoalveolar lavage (BAL) fluid prostaglandin (2, 3) and leukotriene (4-6) levels preceding the developKey Words: oxygen toxicity, arachidonate, prostaglandins, leukotrienes (Received in original form April 24, 1989 and in revised form September I, 1989) Address correspondence to: Peter H. S. Sporn, M.D., Division of Pulmonary and Critical Care Medicine, 3916 Taubman Center, University of Michigan Medical Center, Ann Arbor, MI 48109-0360. Abbreviations: arachidonic acid, AA; alveolar macrophage, AM; bronchoalveolar lavage, BAL; Hanks' balanced salt solution, HBSS; high performance liquid chromatography, HPLC; hydroxyeicosatetraenoic acid, HETE; 12-hydroxy-5,8,IO-heptadecatrienoic acid, HHT; lactate dehydrogenase, LDH; leukotriene, LT; Medium 199 with modified Earle's salts, M199; newborn calf serum, NCS; phosphate-buffered saline, PBS; prostaglandin, PG; radioimmunoassay, RIA; superoxide dismutase, SOD; thin layer chromatography, TLC; thromboxane, TX. Am. J. Respir. CeU Mol. BioI. Vol. 2. pp. 81-90, 1990
ment of inflammation and injury in lungs of animals exposed to hyperoxia. In particular, the potent chemoattractant leukotriene (LT) B4 has beenjmplicated by the finding that inhibition of either phospholipase (4) or 5-lipoxygenase (5) decreased pulmonary neutrophil influx and mortality in hyperoxia-exposed rats, in association with reduced BAL fluid concentrations of LTB4 • Additional studies have shown that in vitro hyperoxia increases eicosanoid synthesis in renal medullary tissue (7) and in cultured pulmonary artery endothelial cells (8). The alveolar macrophage (AM) is the predominant resident inflammatory cell in the alveolar space, and has the capacity to synthesize large amounts of both cyclooxygenaseand 5-lipoxygenase-derived eicosanoids (9-11). Moreover, recent evidence suggests that LTB4 is the major neutrophil chemotaxin produced by the human AM upon stimulation in culture (12). In addition, we have previously demonstrated that hydrogen peroxide (H20 2), the intracellular generation of which is increased in hyperoxia (13), triggers AA release and cyclooxygenase metabolism in the cultured rat AM (14). Although these observations suggest the AM as a likely source of eicosanoids in pulmonary oxygen toxicity, the di-
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rect effects of hyperoxia on AA metabolism in the AM have not previously been examined. Therefore, in the current study we investigated the effects of in vitro exposure to hyperoxia (95% O2) on AA metabolism in the cultured rat AM. Our results indicate that hyperoxia exerts complex effects on AA metabolism in both resting and agonist-stimulated AMs, including the ability to augment AA release and eicosanoid synthesis. These findings support a potential role for the AM as a source of eicosanoids that may participate in the genesis of oxygen toxicity of the lung.
Materials and Methods Macrophage Isolation and Culture Respiratory disease-free female Wistar rats weighing 126 to 150 g were obtained from Charles River (Portage, MI) and housed under specific pathogen-free conditions. After anesthesia with intraperitoneally administered sodium pentobarbital (200 mg/kg), lungs were surgically excised and lavaged as previously described (14). Lavage fluid, as well as Hanks' balanced salt solution (HBSS; GIBCO, Grand Island, NY) and Medium 199 with modified Earle's salts (MI99; GIBCO), all contained 100 U/rnl penicillin, 100 p.g/rnl streptomycin, and 0.25 mg/ml amphotericin B (AntibioticAntimycotic Solution; Sigma Chemical Co., S1. Louis, MO). Two million cells suspended in 1.5 rnl of Ml99 were plated in 35 X 10 mm plastic culture dishes (Falcon Plastics, Oxnard, CA) and cultured at J7 0 C in a humidified atmosphere of 5 % CO2 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 % AMs by morphologic criteria and esterase staining, with viability always exceeding 90% as assessed by trypan blue exclusion. Macrophage monolayers were then cultured overnight (16 to 18 h) in M199 containing 10% heat-inactivated newborn calf serum (NCS) (GIBCO) prior to experimental incubations. Prelabeling of Macrophage Cultures with ['''C]AA In certain experiments, cellular lipids were prelabeled by including 0.2 p.Ci of [1- 14C]AA (sp act, 54 to 57 mCi/mmol; New England Nuclear, Boston, MA) in the medium during overnight culture. To remove unincorporated label, cells were washed with HBSS, incubated for an additional hour with M199 containing 10% NCS, and washed again prior to experimental incubations. The uptake of [i4C]AA by macrophage cultures after overnight labeling was 30.0 ± 1.2 % (mean ± SE, n = 7) of the added radiolabel. Of the radiolabel taken up by AMs, > 90% was incorporated into phospholipids and only "-'0.5% remained as free [i4C]AA. In all cases, AMs were prelabeled prior to experimental incubations in normoxia or hyperoxia in order to avoid potential effects of hyperoxia on [i4C]AA incorporation, which might confound comparison of labeled products formed in normoxic and hyperoxic cultures. Normoxic and Hyperoxic Incubations After overnight incubation, prelabeled or unlabeled AM monolayers were washed twice with HBSS, fresh Ml99 containing 10% NCS (2 mllplate) was added, and parallel cultures were exposed to normoxia or hyperoxia, each in dupli-
cate, for all experimental conditions. Normoxic incubations were carried out in a standard incubator atmosphere consisting of humidified 95 % air/5 % CO 2 at J7 0 C. Hyperoxic incubations were carried out simultaneously in a gastight Modular Incubator Chamber (Forma Scientific, Marietta, OH) placed inside the same incubator used for normoxic incubations. The Modular Incubator Chamber contained a humidity source, and was flushed continuously through an intake line with 95% O2 /5% CO2 at a flow rate of approximately 1.2 L/min during experimental incubations. An exhaust line from the Modular Incubator Chamber was connected to an S-3A Oxygen Analyzer (Applied Electrochemistry, Sunnyvale, CA), which was used to confirm that the O2 concentration in the chamber was maintained at 95 % throughout each experiment. The oxygen tension of the culture media (determined with a 1312 Blood Gas Manager; Instrumentation Laboratory, Lexington, MA) was 154 ± 2· mm Hg (n = 6) under normoxic incubation conditions and 640 ± 50 mm Hg (n = 8) under hyperoxic conditions. AA Metabolism in Resting AM Cultures Metabolism of endogenous unlabeled AA and [14C]AA by resting AMs was assessed in unlabeled and [i4C]AA-prelabeled cultures, respectively, by analysis of accumulated free AA or eicosanoids in media after culture in normoxia or hyperoxia for 24, 48, or 72 h. Metabolism of exogenous AA by resting AMs was assessed after culture of unlabeled cells in normoxia or hyperoxia for 72 h, at which point monolayers were washed and fresh serum-free Ml99 (1 mll plate) containing either 2 p.g/ml unlabeled AA (Nu-Chek Prep, Elysian, MN; in 0.1 % ethanol) or 2 p.g/ml (= 0.34 p.Ci/ml) [14C]AA (0.05 % ethanol) was added. After incubation with exogenous AA for 30 min, again in normoxia or hyperoxia, media were removed and analyzed for unlabeled or 14C-labeled AA metabolites.
AA Metabolism Stimulated by A23187 After culture of unlabeled or [i4C]AA-prelabeled AMs in normoxia or hyperoxia for 24, 48, or 72 h, monolayers were washed, and fresh serum-free Ml99 (1 ml/plate), either alone or containing calcil,lm ionophore A23187 (Calbiochern-Behring, La Jolll;l(CA) at 10 p.M (in 0.5 % dimethyl sulfoxide), was added to stimulate metabolism of endogenous AA. After incubation for 30 min with or without A23187, again in normoxia or hyperoxia, media were removed for analysis of free AA or eicosanoids. In the case of prelabeled cultures, monolayers were then scraped into 1 ml of methanol that was added to 9 ml of ACS scintillant (Amersham, Arlington Heights, IL) and counted in an LS18011iquid scintillation counter (Beckman Instruments, Fullerton, CA) for determination of remaining cell-associated radioactivity. In selected experiments, AMs were incubated in normoxia or hyperoxia for 72 h in the presence and absence ofthe following antioxidants, alone and in combination, prior to stimulation with A23187: catalase (Sigma), 1,760 U/ml (100 p,g/ ml); superoxide dismutase (SOD; Sigma), 310 U/ml (100 p.g/ml); and L-2-oxothiazolidine-4-carboxylic acid (Aldrich Chemical, Milwaukee, WI), an intracellular cysteine delivery agent that augments AM glutathione levels (15), 5 mM.
Sporn, Murphy, and Peters-Golden: Hyperoxia and Macrophage Arachidonate Metabolism
Extraction and Quantitation of Free Arachidonic Acid One milliliter of medium from (14C]AA-prelabeled cultures was acidified to pH 3 with 1 N HCl and extracted with 6 ml chloroform:methanol (2:1, vol/vol) followed by an additional 2 ml chloroform. The combined chloroform phases were evaporated under nitrogen, and the dried lipid extracts stored under nitrogen at -70 0 C. Free [14C]AA was then separated by thin layer chromatography (TLC) on Silica Gel 60 plates (E. Merck, Darmstadt, Federal Republic of Germany) using hexane:diethyl ether:acetic acid (70:30:2, vol/vol/ vol), and quantitated by liquid scintillation spectrometry, as described (14). Extraction and Quantitation of Eicosanoids Eicosanoids were extracted from medium of unlabeled, as well as [14C]AA-labeled, cultures using Sep-pak Cs cartridges (Waters Associates, Milford, MA), as described (16). Recoveries for this extraction procedure, determined by radioimmunoassay (RIA; see below), were as follows: thromboxane (TX) B2 (the stable breakdown product of TXA 2), 93.8 ± 0.7% (n = 8); prostaglandin (PG) E2 , 104.0 ± 5.5% (n = 8); PGF 20 , 94.6 ± 2.0% (n = 8); LTB 4, 87.3 ± 0.8% (n = 4); and LTC., 70.8 ± 1.7% (n = 4). TXB 2 , PGE2, PGF 2a , LTB 4, and LTC. in media from unlabeled cultures were quantitated by RIA. Dried lipid extracts were dissolved in 1 ml of phosphate-buffered saline (PBS) containing 0.1% gelatin (pH 7.4), and l00-Jd aliquots assayed in duplicate for each sample. The antibody sources, cross-reactivities, and sensitivities for the TXB 2 , PGEh LTB 4, and LTC. assays have been described previously (15). The PGF 20 antibody (Upjohn Co., Kalamazoo, MI) has negligible cross-reactivity with related prostaglandins, and the assay in which it is utilized is sensitive to 0.9 pg/100 JLl sample. The specificity of each RIA has been confirmed by reverse-phase high performance liquid chromatography (HPLC) analysis in our laboratory. Quantities of immunoreactive eicosanoids reported were corrected for recoveries during extraction. For separation of radiolabeled metabolites produced by [14C]AA-prelabeled AMs or by unlabeled AMs incubated with exogenous [14C]AA, lipid extracts of pooled media from replicate cultures were dissolved in 500 JLl of acetonitrile/water/trifluoroacetic acid (33:67:0.1, vol/vol/vol) and subjected to reverse-phase HPLC using a Waters HPLC system equipped with a 5-JLrn Bondapak Cs column (30 X 0.4 cm) eluted with acetonitrile/water/trifluoroacetic acid at 1 ml/min, as previously described (17). Using this system,. cyclooxygenase metabolites are eluted during an initial isocratic phase (33:67:0.1, vol/vol/vol), followed by lipoxygenase metabolites and free AA, which elute during a stepwise gradient increase of acetonitrile to 100:0:0.1 (vol/vol/ vol). Radiolabeled AA and its metabolites were identified by their coelution with authentic standards. The eluate was continuously monitored for UV absorbance at 210 nm for cyclooxygenase products and free AA, 280 nm for LTs, and 235 nm for mono-hydroxyeicosatetraenoic acids (monoHETEs). Authentic TXB2, PGD 2 , PGE h PGF20 , and 6-ketoPGF lo were generous gifts of Dr. 1. Pike (Upjohn Co.), and lipoxygenase standards LTB4, LTC 4, LTD4, 5-HETE, 12HETE, and 15-HETE of Dr. 1. Rokach (Merck Frosst, Inc.,
83
Pointe Claire-Dorval, Quebec, Canada). Authentic 12-hydroxy-5,8,IO-heptadecatrienoic acid (HHT) was obtained from Cayman Chemical Co. (Ann Arbor, MI), and the AA standard from Nu-Chek Prep. Eluate fractions of 1 ml were collected, and radioactivity quantitated in 6 ml of ACS scintillant. Lactate Dehydrogenase Assay After specified periods of incubation in normoxia or hyperoxia, supernatant culture media were removed, and AM monolayers scraped into fresh Ml99 (2 ml/plate) and sonicated. Lactate dehydrogenase (LDH) activity in culture supernatants and in sonicated cell suspensions was determined spectrophotometrically by monitoring the NADH-dependent conversion of pyruvate to lactate at 340 nm (18). Because NCS was found to contain high levels of LDH activity, normoxic and hyperoxic incubations in these experiments were carried out in serum-free medium. Exposure of cell-free medium containing LDH to hyperoxia for up to 72 h was shown not to affect the activity of the enzyme. Data are expressed as percent LDH release, calculated as (LDH..p
Metabolites of arachidonic acid (AA) released into bronchoalveolar lavage fluid of animals exposed to hyperoxia have previously been implicated as mediators of pulmonary oxygen toxicity. The alveolar macrophage (AM) represents an important potential source of these eicosanoids. We have therefore investigated the effects of in vitro hyperoxia (95 % O 2 /5 % CO2) versus normoxia (95 % air/5 % CO 2) on tlle metabolism of AA in the AM of the rat. Exposure to 95 % O2 for up to 72 h did not impair the viability or affect the protein content of cultured AMs. Hyperoxia for 24 to 72 h increased the accumulation of free AA liberated from endogenous stores in cultures of resting AMs. Despite this increase in free AA, no changes in synthesis of thromboxane B2 , prostaglandin (PG) E2 , PGF2a , leukotriene (LT) B4 , or LTC were observed in resting AMs exposed to hyperoxia for up to 72 h. This was not due to degradation of eicosanoids in hyperoxia. However, formation of cyclooxygenase metabolites from exogenously supplied AA was reduced in hyperoxia-incubated AMs, suggesting that hyperoxia inhibited the cyclooxygenase enzyme. In AMs stimulated with calcium ionophore A23187, both AA release and synthesis of cyclooxygenase and lipoxygenase eicosanoids were augmented after incubation in hyperoxia for 24 to 72 h. The increase in A23187-stimulated LTB4 synthesis caused by hyperoxia was inhibited by the antioxidants catalase, superoxide dismutase, and the intracellular cysteine loading agent L-2-oxothiazolidine-4carboxylic acid, suggesting that the augmentation by hyperoxia of A23187-induced AA metabolism was mediated by reactive oxygen metabolites. Thus, hyperoxia has complex effects on AA metabolism in the AM, which include the ability to augment the release of AA and formation ofbioactive eicosanoids. These findings support a possible role for eicosanoid synthesis by the AM in the pathogenesis of oxygen toxicity of the lung.
Continuous exposure to high concentrations of inspired oxygen causes neutrophilic alveolitis and lung injury in both animals and humans (1). A possible role for metabolites of arachidonic acid (AA) in the pathogenesis of pulmonary oxygen toxicity has been suggested by recent reports of increased bronchoalveolar lavage (BAL) fluid prostaglandin (2, 3) and leukotriene (4-6) levels preceding the developKey Words: oxygen toxicity, arachidonate, prostaglandins, leukotrienes (Received in original form April 24, 1989 and in revised form September I, 1989) Address correspondence to: Peter H. S. Sporn, M.D., Division of Pulmonary and Critical Care Medicine, 3916 Taubman Center, University of Michigan Medical Center, Ann Arbor, MI 48109-0360. Abbreviations: arachidonic acid, AA; alveolar macrophage, AM; bronchoalveolar lavage, BAL; Hanks' balanced salt solution, HBSS; high performance liquid chromatography, HPLC; hydroxyeicosatetraenoic acid, HETE; 12-hydroxy-5,8,IO-heptadecatrienoic acid, HHT; lactate dehydrogenase, LDH; leukotriene, LT; Medium 199 with modified Earle's salts, M199; newborn calf serum, NCS; phosphate-buffered saline, PBS; prostaglandin, PG; radioimmunoassay, RIA; superoxide dismutase, SOD; thin layer chromatography, TLC; thromboxane, TX. Am. J. Respir. CeU Mol. BioI. Vol. 2. pp. 81-90, 1990
ment of inflammation and injury in lungs of animals exposed to hyperoxia. In particular, the potent chemoattractant leukotriene (LT) B4 has beenjmplicated by the finding that inhibition of either phospholipase (4) or 5-lipoxygenase (5) decreased pulmonary neutrophil influx and mortality in hyperoxia-exposed rats, in association with reduced BAL fluid concentrations of LTB4 • Additional studies have shown that in vitro hyperoxia increases eicosanoid synthesis in renal medullary tissue (7) and in cultured pulmonary artery endothelial cells (8). The alveolar macrophage (AM) is the predominant resident inflammatory cell in the alveolar space, and has the capacity to synthesize large amounts of both cyclooxygenaseand 5-lipoxygenase-derived eicosanoids (9-11). Moreover, recent evidence suggests that LTB4 is the major neutrophil chemotaxin produced by the human AM upon stimulation in culture (12). In addition, we have previously demonstrated that hydrogen peroxide (H20 2), the intracellular generation of which is increased in hyperoxia (13), triggers AA release and cyclooxygenase metabolism in the cultured rat AM (14). Although these observations suggest the AM as a likely source of eicosanoids in pulmonary oxygen toxicity, the di-
82
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990
rect effects of hyperoxia on AA metabolism in the AM have not previously been examined. Therefore, in the current study we investigated the effects of in vitro exposure to hyperoxia (95% O2) on AA metabolism in the cultured rat AM. Our results indicate that hyperoxia exerts complex effects on AA metabolism in both resting and agonist-stimulated AMs, including the ability to augment AA release and eicosanoid synthesis. These findings support a potential role for the AM as a source of eicosanoids that may participate in the genesis of oxygen toxicity of the lung.
Materials and Methods Macrophage Isolation and Culture Respiratory disease-free female Wistar rats weighing 126 to 150 g were obtained from Charles River (Portage, MI) and housed under specific pathogen-free conditions. After anesthesia with intraperitoneally administered sodium pentobarbital (200 mg/kg), lungs were surgically excised and lavaged as previously described (14). Lavage fluid, as well as Hanks' balanced salt solution (HBSS; GIBCO, Grand Island, NY) and Medium 199 with modified Earle's salts (MI99; GIBCO), all contained 100 U/rnl penicillin, 100 p.g/rnl streptomycin, and 0.25 mg/ml amphotericin B (AntibioticAntimycotic Solution; Sigma Chemical Co., S1. Louis, MO). Two million cells suspended in 1.5 rnl of Ml99 were plated in 35 X 10 mm plastic culture dishes (Falcon Plastics, Oxnard, CA) and cultured at J7 0 C in a humidified atmosphere of 5 % CO2 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 % AMs by morphologic criteria and esterase staining, with viability always exceeding 90% as assessed by trypan blue exclusion. Macrophage monolayers were then cultured overnight (16 to 18 h) in M199 containing 10% heat-inactivated newborn calf serum (NCS) (GIBCO) prior to experimental incubations. Prelabeling of Macrophage Cultures with ['''C]AA In certain experiments, cellular lipids were prelabeled by including 0.2 p.Ci of [1- 14C]AA (sp act, 54 to 57 mCi/mmol; New England Nuclear, Boston, MA) in the medium during overnight culture. To remove unincorporated label, cells were washed with HBSS, incubated for an additional hour with M199 containing 10% NCS, and washed again prior to experimental incubations. The uptake of [i4C]AA by macrophage cultures after overnight labeling was 30.0 ± 1.2 % (mean ± SE, n = 7) of the added radiolabel. Of the radiolabel taken up by AMs, > 90% was incorporated into phospholipids and only "-'0.5% remained as free [i4C]AA. In all cases, AMs were prelabeled prior to experimental incubations in normoxia or hyperoxia in order to avoid potential effects of hyperoxia on [i4C]AA incorporation, which might confound comparison of labeled products formed in normoxic and hyperoxic cultures. Normoxic and Hyperoxic Incubations After overnight incubation, prelabeled or unlabeled AM monolayers were washed twice with HBSS, fresh Ml99 containing 10% NCS (2 mllplate) was added, and parallel cultures were exposed to normoxia or hyperoxia, each in dupli-
cate, for all experimental conditions. Normoxic incubations were carried out in a standard incubator atmosphere consisting of humidified 95 % air/5 % CO 2 at J7 0 C. Hyperoxic incubations were carried out simultaneously in a gastight Modular Incubator Chamber (Forma Scientific, Marietta, OH) placed inside the same incubator used for normoxic incubations. The Modular Incubator Chamber contained a humidity source, and was flushed continuously through an intake line with 95% O2 /5% CO2 at a flow rate of approximately 1.2 L/min during experimental incubations. An exhaust line from the Modular Incubator Chamber was connected to an S-3A Oxygen Analyzer (Applied Electrochemistry, Sunnyvale, CA), which was used to confirm that the O2 concentration in the chamber was maintained at 95 % throughout each experiment. The oxygen tension of the culture media (determined with a 1312 Blood Gas Manager; Instrumentation Laboratory, Lexington, MA) was 154 ± 2· mm Hg (n = 6) under normoxic incubation conditions and 640 ± 50 mm Hg (n = 8) under hyperoxic conditions. AA Metabolism in Resting AM Cultures Metabolism of endogenous unlabeled AA and [14C]AA by resting AMs was assessed in unlabeled and [i4C]AA-prelabeled cultures, respectively, by analysis of accumulated free AA or eicosanoids in media after culture in normoxia or hyperoxia for 24, 48, or 72 h. Metabolism of exogenous AA by resting AMs was assessed after culture of unlabeled cells in normoxia or hyperoxia for 72 h, at which point monolayers were washed and fresh serum-free Ml99 (1 mll plate) containing either 2 p.g/ml unlabeled AA (Nu-Chek Prep, Elysian, MN; in 0.1 % ethanol) or 2 p.g/ml (= 0.34 p.Ci/ml) [14C]AA (0.05 % ethanol) was added. After incubation with exogenous AA for 30 min, again in normoxia or hyperoxia, media were removed and analyzed for unlabeled or 14C-labeled AA metabolites.
AA Metabolism Stimulated by A23187 After culture of unlabeled or [i4C]AA-prelabeled AMs in normoxia or hyperoxia for 24, 48, or 72 h, monolayers were washed, and fresh serum-free Ml99 (1 ml/plate), either alone or containing calcil,lm ionophore A23187 (Calbiochern-Behring, La Jolll;l(CA) at 10 p.M (in 0.5 % dimethyl sulfoxide), was added to stimulate metabolism of endogenous AA. After incubation for 30 min with or without A23187, again in normoxia or hyperoxia, media were removed for analysis of free AA or eicosanoids. In the case of prelabeled cultures, monolayers were then scraped into 1 ml of methanol that was added to 9 ml of ACS scintillant (Amersham, Arlington Heights, IL) and counted in an LS18011iquid scintillation counter (Beckman Instruments, Fullerton, CA) for determination of remaining cell-associated radioactivity. In selected experiments, AMs were incubated in normoxia or hyperoxia for 72 h in the presence and absence ofthe following antioxidants, alone and in combination, prior to stimulation with A23187: catalase (Sigma), 1,760 U/ml (100 p,g/ ml); superoxide dismutase (SOD; Sigma), 310 U/ml (100 p.g/ml); and L-2-oxothiazolidine-4-carboxylic acid (Aldrich Chemical, Milwaukee, WI), an intracellular cysteine delivery agent that augments AM glutathione levels (15), 5 mM.
Sporn, Murphy, and Peters-Golden: Hyperoxia and Macrophage Arachidonate Metabolism
Extraction and Quantitation of Free Arachidonic Acid One milliliter of medium from (14C]AA-prelabeled cultures was acidified to pH 3 with 1 N HCl and extracted with 6 ml chloroform:methanol (2:1, vol/vol) followed by an additional 2 ml chloroform. The combined chloroform phases were evaporated under nitrogen, and the dried lipid extracts stored under nitrogen at -70 0 C. Free [14C]AA was then separated by thin layer chromatography (TLC) on Silica Gel 60 plates (E. Merck, Darmstadt, Federal Republic of Germany) using hexane:diethyl ether:acetic acid (70:30:2, vol/vol/ vol), and quantitated by liquid scintillation spectrometry, as described (14). Extraction and Quantitation of Eicosanoids Eicosanoids were extracted from medium of unlabeled, as well as [14C]AA-labeled, cultures using Sep-pak Cs cartridges (Waters Associates, Milford, MA), as described (16). Recoveries for this extraction procedure, determined by radioimmunoassay (RIA; see below), were as follows: thromboxane (TX) B2 (the stable breakdown product of TXA 2), 93.8 ± 0.7% (n = 8); prostaglandin (PG) E2 , 104.0 ± 5.5% (n = 8); PGF 20 , 94.6 ± 2.0% (n = 8); LTB 4, 87.3 ± 0.8% (n = 4); and LTC., 70.8 ± 1.7% (n = 4). TXB 2 , PGE2, PGF 2a , LTB 4, and LTC. in media from unlabeled cultures were quantitated by RIA. Dried lipid extracts were dissolved in 1 ml of phosphate-buffered saline (PBS) containing 0.1% gelatin (pH 7.4), and l00-Jd aliquots assayed in duplicate for each sample. The antibody sources, cross-reactivities, and sensitivities for the TXB 2 , PGEh LTB 4, and LTC. assays have been described previously (15). The PGF 20 antibody (Upjohn Co., Kalamazoo, MI) has negligible cross-reactivity with related prostaglandins, and the assay in which it is utilized is sensitive to 0.9 pg/100 JLl sample. The specificity of each RIA has been confirmed by reverse-phase high performance liquid chromatography (HPLC) analysis in our laboratory. Quantities of immunoreactive eicosanoids reported were corrected for recoveries during extraction. For separation of radiolabeled metabolites produced by [14C]AA-prelabeled AMs or by unlabeled AMs incubated with exogenous [14C]AA, lipid extracts of pooled media from replicate cultures were dissolved in 500 JLl of acetonitrile/water/trifluoroacetic acid (33:67:0.1, vol/vol/vol) and subjected to reverse-phase HPLC using a Waters HPLC system equipped with a 5-JLrn Bondapak Cs column (30 X 0.4 cm) eluted with acetonitrile/water/trifluoroacetic acid at 1 ml/min, as previously described (17). Using this system,. cyclooxygenase metabolites are eluted during an initial isocratic phase (33:67:0.1, vol/vol/vol), followed by lipoxygenase metabolites and free AA, which elute during a stepwise gradient increase of acetonitrile to 100:0:0.1 (vol/vol/ vol). Radiolabeled AA and its metabolites were identified by their coelution with authentic standards. The eluate was continuously monitored for UV absorbance at 210 nm for cyclooxygenase products and free AA, 280 nm for LTs, and 235 nm for mono-hydroxyeicosatetraenoic acids (monoHETEs). Authentic TXB2, PGD 2 , PGE h PGF20 , and 6-ketoPGF lo were generous gifts of Dr. 1. Pike (Upjohn Co.), and lipoxygenase standards LTB4, LTC 4, LTD4, 5-HETE, 12HETE, and 15-HETE of Dr. 1. Rokach (Merck Frosst, Inc.,
83
Pointe Claire-Dorval, Quebec, Canada). Authentic 12-hydroxy-5,8,IO-heptadecatrienoic acid (HHT) was obtained from Cayman Chemical Co. (Ann Arbor, MI), and the AA standard from Nu-Chek Prep. Eluate fractions of 1 ml were collected, and radioactivity quantitated in 6 ml of ACS scintillant. Lactate Dehydrogenase Assay After specified periods of incubation in normoxia or hyperoxia, supernatant culture media were removed, and AM monolayers scraped into fresh Ml99 (2 ml/plate) and sonicated. Lactate dehydrogenase (LDH) activity in culture supernatants and in sonicated cell suspensions was determined spectrophotometrically by monitoring the NADH-dependent conversion of pyruvate to lactate at 340 nm (18). Because NCS was found to contain high levels of LDH activity, normoxic and hyperoxic incubations in these experiments were carried out in serum-free medium. Exposure of cell-free medium containing LDH to hyperoxia for up to 72 h was shown not to affect the activity of the enzyme. Data are expressed as percent LDH release, calculated as (LDH..p
