A23187 stimulates translocation of 54ipoxygenase to membrane in human alveolar macrophages
from cytosol
ROBERT J. PUERINGER, CHILING C. BAHNS, MARTHA M. MONICK, AND GARY W. HUNNINGHAKE Pulmonary Disease Division, Department of Internal Medicine, Department of Veterans Affairs Medical Center, and The University of Iowa College of Medicine, Iowa City, Iowa 52242 Pueringer, Robert J., Chiling C. Bahns, Martha M. Monick, and Gary W. Hunninghake. A23187 stimulates translocation of 5lipoxygenase from cytosol to membrane in human alveolar macrophages. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L454-L458, 1992.-Human alveolar macrophages stimulated with the calcium ionophore A23187 selectively release large amounts of leukotriene Bq (LTB,) and (+)&hydroxy-(6E, 82-112, 142)-eicosatetraenoic acid. To determine whether LTB, release by human alveolar macrophages following A23187 stimulation required the de novo production of 5-lipoxygenase, alveolar macrophages were stimulated under conditions that would preclude a role for new enzyme production. We found that A23187-stimulated alveolar macrophages release LTB* within 10 min following stimulation, that LTB, release is not inhibited by protein synthesis inhibitors, and that release of LTB, does not correlate with the de novo synthesis of the first committed enzyme, 5-lipoxygenase. In contrast, LTB, release correlated with the translocation of li-lipoxygenase from the cytosol to the membrane fraction of the cells following A23187 stimulation and was inhibited by MK-886. These findings show that A23187 stimulation of alveolar macrophages results in translocation of a preexistent Ei-lipoxygenase from the cytosol to the membrane fraction of the cell and that this translocation of 5-lipoxygenase is associated with release of LTB, from the cells. arachidonic
acid; leukotriene
B,; lung, eicosanoids;
MK-886
ALVEOLAR MACROPHAGES (HAMS), stimulated with the calcium ionophore AZ3187, selectively release large amounts of (+)-5hydroxy-(GE, 82-112, 142)-eicosatetraenoic acid (5HETE) and leukotriene Bq (LTB& which are potent inflammatory mediators (1, 3, 4, 6-10, 12, 16). Prior studies demonstrated that HAMS, stimulated with lipopolysaccharide (LPS), selectively release prostaglandins and thromboxanes (1). Further, the release of these products is regulated, in part, by the de novo synthesis of prostaglandin H synthase (PGH synthase), the first committed enzyme in the metabolism of an arachidonic acid to prostaglandins and thromboxanes (18). The mechanisms for the selective metabolism of arachidonic acid to products of 5lipoxygenase in A23187-stimulated HAMS are unknown. Leukotriene A, (LTA,) and 5HETE are formed from arachidonic acid by the action of the enzyme 5-lipoxygenase, which is a calcium- and ATP-requiring enzyme (19, 23-25). LTB, is then formed from LTA4 by the enzyme LTA4 hydrolase (25). In polymorphonuclear cells, 5lipoxygenase translocates to the cell membrane after A23187 stimulation (22). Membrane translocation involves binding of 5-lipoxygenase to a membraneassociated protein, 5-lipoxygenase-activating protein (FLAP), and coincides with 5-lipoxygenase activation (5, 17). If membrane translocation is inhibited with MK886, a specific inhibitor of the translocation of 5-lipoxyHUMAN
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genase, polymorphonuclear cells do not express 5-lipoxygenase activity (2 1). There is no information regarding the regulation of 5lipoxygenase in HAMS. Therefore, we evaluated if de novo synthesis of 5-lipoxygenase and/or translocation of 5-lipoxygenase from cytosol to membrane is associated with activation of enzyme activity in these cells. We found that de novo synthesis of 5-lipoxygenase was not required for expression of enzyme activity in these cells. We also found that 5-lipoxygenase does translocate to the membrane fraction of the cells after stimulation with A23187 and that translocation of the enzyme coincides with activation of enzyme activity. Further, MK-886, an inhibitor of 5-lipoxygenase translocation, significantly decreased LTB, release by these cells after stimulation with A23187. Thus it is likely that translocation of a preexistent 5lipoxygenase to the membrane is associated with 5-lipoxygenase activation in human alveolar macrophages. METHODS NormaZ subjects. HAMS were obtained from normal volunteers with a lifetime nonsmoking history. They had no medical illnesses and were not taking medications, including nonsteroida1 anti-inflammatory agents. These studies were approved by the Committee for Investigations Involving Human Subjects at the University of Iowa, and there were no complications. Isolation of alveolar macrophages. HAMS were obtained by bronchoalveolar lavage (BAL) as previously described (27). The subjects were premeditated with meperidine and atropine sulfate. The upper airways were anesthetized with 5% lidocaine, and the fiberoptic bronchoscope was inserted, transorally, into the tracheobronchial tree and wedged into a subsegmental bronchus of the lingula or the right middle lobe. The BAL consisted of five separate 25-ml aliquots of sterile saline infused and retrieved by low-pressure suction. The BAL was repeated in three different subsegments; the first 25-ml lavage from each subsegment was discarded. The lavage fluid was filtered through two layers of gauze and centrifuged at 250 g for 5 min. The cell pellet was washed twice in Rosewell Park Memorial Institute tissue culture medium (RPM1 1640) containing 5% endotoxin-free fetal calf serum (FCS, Hyclone Laboratories, Logan, UT), 0.3 mg/ml glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. Cell counts and differentials were determined with a hemacytometer and Wright-Giemsa-stained cytocentrifuge preparations, respectively. Cell culture. The HAMS were cultured at a density of 1 x lo6 cells/ml of RPM1 1640 supplemented with 0.3 mg/ml glutamine, 100 U/ml penicillin, 5% FCS, and 100 pg/ml streptomycin in 12-well flat plate culture dishes (Costar, Cambridge, MA) in an atmosphere of 95% humidified air-5% CO, at 37°C. After an initial 4 h of culture, the cells were cultured in the presence or absence of the calcium ionophore A23187 (1 ,uM, Sigma
0 1992 the American
Physiological
Society
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ALVEOLAR
MACROPHAGE
Chemical, St. Louis, MO), MK-886 (0.5 PM, Merck Frosst, Quebec, Canada), and/or L 224 (1 ,uM, Merck Frosst). At various intervals after stimulation with A23187, the supernatants were aspirated and centrifuged at 250 g to remove nonadherent cells. The resultant supernatant was stored at -70°C and used to determine LTB, release (see LTB, release.). The pelleted nonadherent cells were added to the adherent cells and scraped into 1 ml of 50 mM potassium phosphate, 0.1 M NaCl, and 5 mM EDTA. The cells were then pelleted at 250 g and used to determine the total cellular amount and subcellular distribution of 5-lipoxygenase (see Preparation of subceLLular fractions. ). LTB, release. LTB, release was measured with a commercially available radioimmunoassay kit (Advanced Magnetics, Cambridge, MA). Cross reactivity with other leukotrienes, thromboxanes, and prostaglandins is ~1%. Preparation of subcellular fractions. The cell pellet from above was either solubilized with sample buffer to determine total cellular 5-lipoxygenase (see Immunoblot detection of 5Zipoxygenase) or separated into cytosolic and membrane fractions as previously described (22). In brief, to separate the total cell pellet into separate fractions, the pellets were resuspended in 150 ~1 of 20 mM potassium phosphate, 4 mM EDTA, and 0.2 mg/ml cy2macroglobin. The suspension was then sonicated and centrifuged at 100,000 g for 60 min at 4°C. The resultant supernatant was used to determine the relative amounts of cytoplasmic 5-lipoxygenase (see ImmunobZot detection of 5lipoxygenase). The pellet was used to determine relative amounts of membrane-associated 5-lipoxygenase. Immunoblot detection of 54poxygenase. Total cellular, cytoplasmic, and membrane-associated immunoreactive 5-lipoxygenase were determined by immunoblot analysis as previously described (22). In brief, the total cell pellet or respective subcellular fractions were solubilized in 100 ~1 of sample buffer [lo% glycerol, 2% sodium dodecyl sulfate (SDS), 5% ,&mercaptoethanol, 0.25% bromophenol blue, and 0.625 M tris(hydroxymethyl)aminomethane hydrochloride (Tris . HCl), pH 6.81. The proteins were separated using 10% SDS polyacrylamide gel electrophoresis as previously described (14). The separated proteins were transferred to nitrocellulose at 50 V and 4°C overnight using the Western technique (2). The transferred 5lipoxygenase was immunolabeled with a 1:lOO dilution of rabbit antisera specific for 5-lipoxygenase, kindly provided by C. A. Rouzer (22), for 2 h at 25°C followed by 1251-labeled protein A (100 &i/ml, New England Nuclear, Du Pont) for 1 h. The immunolabel was detected by autoradiography using Kodak XAR2 radiographic film at -70°C. Relative amounts of 5lipoxygenase were estimated by optical densitometry (Eiconix). Statistics. Results are expressed as means t SE. Significant differences were identified using Student’s paired t test. Each data point was reproduced in cells from at least three separate volunteers. Reagents. MK-886, L 224, and the polyclonal antibody specific for 5-lipoxygenase were kindly provided by the Merck Frosst Centre for Therapeutic Research (21, 22).
L455
5-LIPXOYGENASE
in the presence of protein synthesis inhibitors. We observed that HAMS stimulated with A23187 released LTB4 as early as 5 min after stimulation, with a peak occurring at 10 min after stimulation (Fig. 1). In addition, the release of LTB, after A23187 stimulation was not inhibited by either cycloheximide or actinomycin D at concentrations that previously showed inhibited protein synthesis in alveolar macrophages (Fig. 2) (13). These results suggest that HAMS do not require new protein synthesis to release LTB, after A23187 stimulation. We also evaluated the relative amounts of alveolar macrophage 5-lipoxygenase by Western analysis, in the presence and absence of A23187. There was no measurable difference in total cellular 5-lipoxygenase after A23187 stimulation when compared with unstimulated cells (Fig. 3). Because the Western analysis reflects the sum of total cellular enzyme synthesis and degradation, the amounts of [35S]methionine incorporation into alveolar macrophage proteins were measured after A23187 stimulation. There was no detectable 35S incorporation into any protein at a molecular mass of 60-90 kDa after A23187 stimulation (data not shown). These observations further suggest that A23187 stimulation of alveolar macrophages to release products of 5-lipoxygenase does
ctl Time
1 (minutes)
5 after
30
10 exposure
to
A23187
Fig. 1. Kinetics of leukotriene B4 (LTB,) release in A23187-stimulated alveolar macrophages. Alveolar macrophages were stimulated with A23187 (1 PM) for various periods of time. Supernatant LTB, was measured by radioimmunoassay. n = 3; * P < 0.05 compared with control (unstimulated alveolar macrophages).
RESULTS
The average cellular yield for the bronchoalveolar lavages was 46.6 t 4.5 X lo6 cells. Alveolar macrophages comprised 88 t 2%, whereas lymphocytes (10 t 2.6%) and polymorphonuclear cells (Cl%) consists of the remainder of the cells recovered. To determine whether release of LTB, required de novo synthesis of 5lipoxygenase, we stimulated HAMS with A23187 (1 PM) under conditions that should preclude new protein synthesis; i.e., at early time points and
Control
A231 87
A231 87 + Cycloheximide
A23 187 + Actinomycin
Fig. 2. Inhibitors of protein synthesis do not reduce A23187-stimulated LTB, release in alveolar macrophages. Alveolar macrophages were stimulated for 30 min with A23187 (1 PM) in presence and absence of actinomycin D (1 PM) or cycloheximide (1 pg/ml). Supernatant LTB4 was measured by radioimmunoassay. n = 5; * P < 0.05 compared with control (unstimulated alveolar macrophages).
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L456
ALVEOLAR
MACROPHAGE
5LIPXOYGENASE
A
Cytosol
Membrane
92Kd-
68Kd68Kd-
A23187
-
A231 87 Fig. 3. Immunoreactive 5lipoxygenase in alveolar macrophages. Total cellular 5-lipoxygenase was estimated by Western analysis of detergentlysed alveolar macrophages cultured in presence and absence of A23187 (1 PM). Representative study from 3 separate experiments; time, 10 min.
not require de novo synthesis of the enzyme. To evaluate if cytosol-to-membrane translocation correlated with LTB4 release, the relative amounts of associated membrane Slipoxygenase were compared in alveolar macrophages in the presence and absence of A23187. The amounts of membrane-associated Slipoxygenase increased in alveolar macrophages stimulated with A23187 when compared with controls (Fig. 4). This increase in membrane-associated Slipoxygenase correlated with LTBl release and suggested that A23187 activated preexistent 5-lipoxygenase by translocating the enzyme to the membrane fraction. To evaluate this further, LTB, release following A23187 stimulation was determined in the presence of MK-886, an inhibitor of 5-lipoxygenase translocation. MK-886 (0.5 /*M) inhibited the A23187-stimulated release of LTBI in human alveolar macrophages (Fig. 5). L 224, a direct inhibitor of 5lipoxygenase, also markedly decreased release of LTBI by the alveolar macrophages.
a
-
COfltKl
A23187
(1uM)
Conditions
Fig. 4. Membrane-associated 5lipoxygenase is increased in alveolar macrophages stimulated with A23187. A: membrane and cytosolic 5lipoxygenase. Membrane- and cytosolic-associated 5lipoxygenase was estimated by Western analysis of respective subcellular fractions in both presence and absence of A23187 (1 PM). Representative study from 3 separate experiments; time, 10 min. B: membrane-associated 5lipoxygenase. Relative amounts of membrane associated 5-lipoxygenase were estimated by optical densitometry of the above autoradiographs. n = 3; * P < 0.05.
DISCUSSION
These studies evaluated the regulation of 5-lipoxygenase activity in human alveolar macrophages. We found that, after A23187 stimulation, human alveolar macrophages release LTB, maximally within 10 min and that the release of LTB4 is not inhibited by protein synthesis inhibitors. In addition, A23187 stimulation did not increase the mass of cellular 5-lipoxygenase. These observations showed that A23187 does not regulate 5-lipoxygenase activity by altering total amounts of the enzyme within the cell. 5-Lipoxygenase activity did correlate, however, with translocation of the enzyme to the membrane fraction of the cells. In addition, inhibition of translocation of 5-lipoxygenase from cytosol to membrane with MK-886 blocked the ability of the cells to
COllld
A23187
A23187 M&S
A23187
&.I
Fig. 5. Inhibition of 5-lipoxygenase membrane translocation inhibits release of LTB, from A23187-stimulated alveolar macrophages. LTBl was measured by radioimmunoassay from supernatants of A23187stimulated alveolar macrophages in presence of MK-886 (0.5 PM), an inhibitor of 5-lipoxygenase translocation, or L 224 (1 PM), a direct inhibitor of 5-lipoxygenase. n = 5; time, 10 min; *P < 0.05 compared with control (unstimulated alveolar macrophages).
release LTBl after A23187 stimulation. We were unable, however, to detect any significant differences in the amounts of cytosolic protein after A23187 stimulation. This is likely explained by the finding that only small amounts of cytosolic enzyme actually translocate to
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ALVEOLAR
MACROPHAGE
membrane fractions of the cells. Thus the relative increase in the membrane fraction of &lipoxygenase, after stimulation is much greater than the relative decrease in cytosolic enzyme. These studies suggest that LTB, release after A23187 stimulation is, in part, due to the activation of a preexistent enzyme, a process that correlates with membrane translocation. In previous studies, we showed that stimulation of human alveolar macrophages with A23187 resulted in the release of only 5lipoxygenase products of arachidonate acid, whereas stimulation with LPS resulted in release of only products of the cyclooxygenase pathway of arachidonic acid (1). We also demonstrated that LPS-stimulated release of prostaglandins and thromboxanes in human alveolar macrophages was dependent upon the de novo synthesis of PGH synthase, the first committed enzyme in that metabolic pathway of arachidonic acid (18). Using LPS as a probe, we observed that PGH synthase product release was delayed for up to 6 h after stimulation, was inhibited by protein synthesis inhibitors, and correlated with the new synthesis of PGH synthase (18). In contrast, human alveolar macrophages stimulated with A23187 release LTB4 early, LTB4 release is not affected by protein synthesis inhibitors, and LTB4 release is not dependent on the de novo synthesis of 5lipoxygenase. Although the latter findings are not surprising inasmuch as the promoter-regulatory region of the 5lipoxygenase gene is similar to previously described housekeeping genes (11)) previous studies have correlated 5-lipoxygenase product release with enzyme mass (13, 20). These observations show that these two pathways of arachidonic acid metabolism (cyclooxygenase vs. 5-lipoxygenase) in human alveolar macrophages are regulated in very different ways. In conclusion, our studies demonstrate that human alveolar macrophages release LTB, after A23187 stimulation by activating preexistent 5-lipoxygenase. The activation of 5-lipoxygenase correlates with the translocation of the enzyme from the cytosol to the membrane fraction of the cells. These studies show that alveolar macrophages metabolize arachidonic acid to products of either PGH synthase or 5-lipoxygenase due in part to distinct mechanisms of enzyme activation. Special thanks to Jeanne Hunter and Deborah Jarrard for their secretarial assistance. This study was supported by the following grants: IA-89-F-8, a fellowship grant from the American Heart Association, Iowa Affiliate; institutional NRSA Fellowship Training Grant HL-07638 and Specialized Center of Research Grant HL-37121 from the National Heart, Lung, and Blood Institute; a Merit Review Grant from the Department of Veterans Affairs; and Grant RR59 from the Clinical Research Center, National Institutes of Health. Address for reprint requests: R. J. Pueringer, Pulmonary Disease Division, C33, University of Iowa College of Medicine, Iowa City, IA 52242. Received
14 June
1991; accepted
in final
form
8 October
1991.
REFERENCES 1. Brown, Human Physiol. 2. Burnette, nroteins
G. P. , M. M. Monick, and G. W. Hunninghake. alveolar macrophage arachidonic acid metabolism. Am. J. 254 (Cell Physiol. 23): C809-C815, 1988. W. N. Western blotting: electrophoretic transfer of from sodium dodecvl sulfate-nolvacrvlamide eels to un-
L457
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3. 4.
5.
6.
modified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195-203, 1981. Chang, J., M. C. Liu, and D. S. Newcombe. Identification of two monohydroxyeicosatetraenoic acids synthesized by human pulmonary macrophages. Am. Rev. Respir. Dis. 126: 457-459, 1982. Damon, M., C. Chavis, P. Godard, F. B. Michel, and A. Crastes de Paulet. Purification and mass spectrometry identification of leukotriene Dq synthesized by human alveolar macrophages. Biochem. Biophys. Res. Commun. 111: 518-524,1983. Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature Lond. 343: 282-284, 1990. Fels, A. 0. S., N. A. Pawlowski, E. B. Cramer, T. K. C. King, Z. A. Cohn, and W. A. Scott. Human alveolar macrophages produce leukotriene Bq. Proc. N&Z. Acad. Sci. USA 79:
7866-7870,1982. 7. Goetzl, E. J., and W. C. Pickett.
The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J. ImmunoZ. 125: 1789-91, 1980. 8. Henderson, W. R., Jr. Eicosanoids and lung inflammation. Am. Rev. Respir. Dis. 135: 1176-1185, 1987. 9. Holtzman, M. J. State of the art: arachidonic acid metabolism. Am. Rev. Respir. Dis. 143: 188-203, 1991. 10. Hoover, R. L., M. J. Karnovsky, K. F. Austen, E. J. Corey, and R. A. Lewis. Leukotriene Bq action on endothelium mediates augmented neutrophil/endothelial adhesion. Proc. NatZ. Acad. Sci. USA 81: 2191-2193, 1984. 11. Hoshiko, S., 0. Radmark, and B. Samuelsson. Characterization of the human 5-lipoxygenase gene promoter. Proc. N&Z. Acad. Sci. USA 87: 9073-9077, 1990. 12. Hsueh, W., and F. F. Sun. Leukotriene B, biosynthesis by alveolar macrophages. Biochem. Biophys. Res. Commun. 106: 1085-
1091,1982. 13. Kargman, S., and C. A. Rouzer. Studies on the regulation, biosynthesis, and activation of 5-lipoxygenase in differentiated HL60 cells. J. BioZ. Chem. 264: 13313-13320, 1989. 14. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680-685,
1970. 15. Lewis, R. A., K. F. Austen, and R. J. Soberman. Leukotrienes and other products of the 5-lipoxygenase pathway; biochemistry and relation to pathobiology in human diseases. N. EngZ. J. Med.
10:645-655,199O. 16. Martin, T. R., L. C. A&man, R. K. Albert, and W. R. Henderson. Leukotriene B, production by the human alveolar macrophage: a potential mechanism for amplifying inflammation in the lung. Am. Rev. Respir. Dis. 129: 106-111, 19844. 17. Miller, D. K., J. W. Gillard, P. J. Vickers, S. Sadowski, C. Leveille, J. A. Mancini, P. Charleson, R. A. F. Dixon, A. W. Ford-Hutchinson, R. Fortin, J. Y. Gauthier, J. Rodkey, R. Rosen, C. Rouzer, I. S. Sigal, C. D. Strader, and J. F. Evans. Identification and isolation of a membrane protein necessary for leukotriene production. Nature Lond. 343: 278-281, 1990. 18. Pueringer, R. J., and G. W. Hunninghake. Lipopolysaccharide stimulates de novo synthesis of PGH synthase in human alveolvar macrophages. Am. J. Physiol. 262 (Lung CeZZ. MOL. Physiol. 6): L78L85,1992. 19. Puustinen, T., M. M. Scheffer, and B. Samuelsson. Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles. Biochim. Biophys. Acta 960: 261-267, 1988. 20. Reid, G. K., S. Kargman, P. J. Vickers, J. A. Mancini, C. Leveille, D. Ethier, D. K. Miller, J. W. Gillard, R. A. F. Dixon, and J. F. Evans. Correlation between expression of 5lipoxygenase-activating protein, 5-lipoxygenase, and cellular leukotriene synthesis. J. BioZ. Chem. 265: 19818-19823, 1990. 21. Rouzer, C. A., A. W. Ford-Hutchinson, H. E. Morton, and J. W. Gillard. MK-886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in inophore-challenged leukocytes. J. BioZ. Chem.
265:1436-1442,199O. 22. Rouzer, C. A. , and S. Kargman. ase to the membrane
in human
Translocation of 5-lipoxygenleukocytes challenged with iono-
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phore A23187. J. BioZ. Chem. 263: 10980-10988,1988. 23. Rouzer, C. A., T. Matsumoto, and B. Samuelsson. Single protein from human leukocytes possesses 5lipoxygenase and leukotriene A4 synthase activities. Proc. Natl. Acad. Sci. USA 83: 857861, 1986. 24. Rouzer, C. A., and B. Samuelsson. On the nature of the 5 lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors. Proc. Natl. Acad. Sci. USA 82: 6040-6044, 1985. 25. Samuelsson, B., and C. D. Funk. Enzymes involved in the
5LIPXOYGENASE biosynthesis of leukotriene Bq. J. Biol. Chem. 264: 19469-19472, 1989. 26. Yoss, E. B., E. W. Spannhake, J. T. Flynn, J. E. Fish, and S. P. Peters. Arachidonic acid metabolism in normal human alveolar macrophages: stimulus specificity for mediator release and phospholipid metabolism, and pharmacologic modulation in vitro and in vivo. Am. J. Respir. Cell MOL. Biol. 2: 69-80, 1990. 27. Zavala, D. C., and G. W. Hunninghake. Lung lavage. In: Recent Advances in Respiratory Medicine. Edinburgh: Churchill Livingstone, 1983, p. 21-33.
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 20, 2019.
from cytosol
ROBERT J. PUERINGER, CHILING C. BAHNS, MARTHA M. MONICK, AND GARY W. HUNNINGHAKE Pulmonary Disease Division, Department of Internal Medicine, Department of Veterans Affairs Medical Center, and The University of Iowa College of Medicine, Iowa City, Iowa 52242 Pueringer, Robert J., Chiling C. Bahns, Martha M. Monick, and Gary W. Hunninghake. A23187 stimulates translocation of 5lipoxygenase from cytosol to membrane in human alveolar macrophages. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L454-L458, 1992.-Human alveolar macrophages stimulated with the calcium ionophore A23187 selectively release large amounts of leukotriene Bq (LTB,) and (+)&hydroxy-(6E, 82-112, 142)-eicosatetraenoic acid. To determine whether LTB, release by human alveolar macrophages following A23187 stimulation required the de novo production of 5-lipoxygenase, alveolar macrophages were stimulated under conditions that would preclude a role for new enzyme production. We found that A23187-stimulated alveolar macrophages release LTB* within 10 min following stimulation, that LTB, release is not inhibited by protein synthesis inhibitors, and that release of LTB, does not correlate with the de novo synthesis of the first committed enzyme, 5-lipoxygenase. In contrast, LTB, release correlated with the translocation of li-lipoxygenase from the cytosol to the membrane fraction of the cells following A23187 stimulation and was inhibited by MK-886. These findings show that A23187 stimulation of alveolar macrophages results in translocation of a preexistent Ei-lipoxygenase from the cytosol to the membrane fraction of the cell and that this translocation of 5-lipoxygenase is associated with release of LTB, from the cells. arachidonic
acid; leukotriene
B,; lung, eicosanoids;
MK-886
ALVEOLAR MACROPHAGES (HAMS), stimulated with the calcium ionophore AZ3187, selectively release large amounts of (+)-5hydroxy-(GE, 82-112, 142)-eicosatetraenoic acid (5HETE) and leukotriene Bq (LTB& which are potent inflammatory mediators (1, 3, 4, 6-10, 12, 16). Prior studies demonstrated that HAMS, stimulated with lipopolysaccharide (LPS), selectively release prostaglandins and thromboxanes (1). Further, the release of these products is regulated, in part, by the de novo synthesis of prostaglandin H synthase (PGH synthase), the first committed enzyme in the metabolism of an arachidonic acid to prostaglandins and thromboxanes (18). The mechanisms for the selective metabolism of arachidonic acid to products of 5lipoxygenase in A23187-stimulated HAMS are unknown. Leukotriene A, (LTA,) and 5HETE are formed from arachidonic acid by the action of the enzyme 5-lipoxygenase, which is a calcium- and ATP-requiring enzyme (19, 23-25). LTB, is then formed from LTA4 by the enzyme LTA4 hydrolase (25). In polymorphonuclear cells, 5lipoxygenase translocates to the cell membrane after A23187 stimulation (22). Membrane translocation involves binding of 5-lipoxygenase to a membraneassociated protein, 5-lipoxygenase-activating protein (FLAP), and coincides with 5-lipoxygenase activation (5, 17). If membrane translocation is inhibited with MK886, a specific inhibitor of the translocation of 5-lipoxyHUMAN
L454
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genase, polymorphonuclear cells do not express 5-lipoxygenase activity (2 1). There is no information regarding the regulation of 5lipoxygenase in HAMS. Therefore, we evaluated if de novo synthesis of 5-lipoxygenase and/or translocation of 5-lipoxygenase from cytosol to membrane is associated with activation of enzyme activity in these cells. We found that de novo synthesis of 5-lipoxygenase was not required for expression of enzyme activity in these cells. We also found that 5-lipoxygenase does translocate to the membrane fraction of the cells after stimulation with A23187 and that translocation of the enzyme coincides with activation of enzyme activity. Further, MK-886, an inhibitor of 5-lipoxygenase translocation, significantly decreased LTB, release by these cells after stimulation with A23187. Thus it is likely that translocation of a preexistent 5lipoxygenase to the membrane is associated with 5-lipoxygenase activation in human alveolar macrophages. METHODS NormaZ subjects. HAMS were obtained from normal volunteers with a lifetime nonsmoking history. They had no medical illnesses and were not taking medications, including nonsteroida1 anti-inflammatory agents. These studies were approved by the Committee for Investigations Involving Human Subjects at the University of Iowa, and there were no complications. Isolation of alveolar macrophages. HAMS were obtained by bronchoalveolar lavage (BAL) as previously described (27). The subjects were premeditated with meperidine and atropine sulfate. The upper airways were anesthetized with 5% lidocaine, and the fiberoptic bronchoscope was inserted, transorally, into the tracheobronchial tree and wedged into a subsegmental bronchus of the lingula or the right middle lobe. The BAL consisted of five separate 25-ml aliquots of sterile saline infused and retrieved by low-pressure suction. The BAL was repeated in three different subsegments; the first 25-ml lavage from each subsegment was discarded. The lavage fluid was filtered through two layers of gauze and centrifuged at 250 g for 5 min. The cell pellet was washed twice in Rosewell Park Memorial Institute tissue culture medium (RPM1 1640) containing 5% endotoxin-free fetal calf serum (FCS, Hyclone Laboratories, Logan, UT), 0.3 mg/ml glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. Cell counts and differentials were determined with a hemacytometer and Wright-Giemsa-stained cytocentrifuge preparations, respectively. Cell culture. The HAMS were cultured at a density of 1 x lo6 cells/ml of RPM1 1640 supplemented with 0.3 mg/ml glutamine, 100 U/ml penicillin, 5% FCS, and 100 pg/ml streptomycin in 12-well flat plate culture dishes (Costar, Cambridge, MA) in an atmosphere of 95% humidified air-5% CO, at 37°C. After an initial 4 h of culture, the cells were cultured in the presence or absence of the calcium ionophore A23187 (1 ,uM, Sigma
0 1992 the American
Physiological
Society
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ALVEOLAR
MACROPHAGE
Chemical, St. Louis, MO), MK-886 (0.5 PM, Merck Frosst, Quebec, Canada), and/or L 224 (1 ,uM, Merck Frosst). At various intervals after stimulation with A23187, the supernatants were aspirated and centrifuged at 250 g to remove nonadherent cells. The resultant supernatant was stored at -70°C and used to determine LTB, release (see LTB, release.). The pelleted nonadherent cells were added to the adherent cells and scraped into 1 ml of 50 mM potassium phosphate, 0.1 M NaCl, and 5 mM EDTA. The cells were then pelleted at 250 g and used to determine the total cellular amount and subcellular distribution of 5-lipoxygenase (see Preparation of subceLLular fractions. ). LTB, release. LTB, release was measured with a commercially available radioimmunoassay kit (Advanced Magnetics, Cambridge, MA). Cross reactivity with other leukotrienes, thromboxanes, and prostaglandins is ~1%. Preparation of subcellular fractions. The cell pellet from above was either solubilized with sample buffer to determine total cellular 5-lipoxygenase (see Immunoblot detection of 5Zipoxygenase) or separated into cytosolic and membrane fractions as previously described (22). In brief, to separate the total cell pellet into separate fractions, the pellets were resuspended in 150 ~1 of 20 mM potassium phosphate, 4 mM EDTA, and 0.2 mg/ml cy2macroglobin. The suspension was then sonicated and centrifuged at 100,000 g for 60 min at 4°C. The resultant supernatant was used to determine the relative amounts of cytoplasmic 5-lipoxygenase (see ImmunobZot detection of 5lipoxygenase). The pellet was used to determine relative amounts of membrane-associated 5-lipoxygenase. Immunoblot detection of 54poxygenase. Total cellular, cytoplasmic, and membrane-associated immunoreactive 5-lipoxygenase were determined by immunoblot analysis as previously described (22). In brief, the total cell pellet or respective subcellular fractions were solubilized in 100 ~1 of sample buffer [lo% glycerol, 2% sodium dodecyl sulfate (SDS), 5% ,&mercaptoethanol, 0.25% bromophenol blue, and 0.625 M tris(hydroxymethyl)aminomethane hydrochloride (Tris . HCl), pH 6.81. The proteins were separated using 10% SDS polyacrylamide gel electrophoresis as previously described (14). The separated proteins were transferred to nitrocellulose at 50 V and 4°C overnight using the Western technique (2). The transferred 5lipoxygenase was immunolabeled with a 1:lOO dilution of rabbit antisera specific for 5-lipoxygenase, kindly provided by C. A. Rouzer (22), for 2 h at 25°C followed by 1251-labeled protein A (100 &i/ml, New England Nuclear, Du Pont) for 1 h. The immunolabel was detected by autoradiography using Kodak XAR2 radiographic film at -70°C. Relative amounts of 5lipoxygenase were estimated by optical densitometry (Eiconix). Statistics. Results are expressed as means t SE. Significant differences were identified using Student’s paired t test. Each data point was reproduced in cells from at least three separate volunteers. Reagents. MK-886, L 224, and the polyclonal antibody specific for 5-lipoxygenase were kindly provided by the Merck Frosst Centre for Therapeutic Research (21, 22).
L455
5-LIPXOYGENASE
in the presence of protein synthesis inhibitors. We observed that HAMS stimulated with A23187 released LTB4 as early as 5 min after stimulation, with a peak occurring at 10 min after stimulation (Fig. 1). In addition, the release of LTB, after A23187 stimulation was not inhibited by either cycloheximide or actinomycin D at concentrations that previously showed inhibited protein synthesis in alveolar macrophages (Fig. 2) (13). These results suggest that HAMS do not require new protein synthesis to release LTB, after A23187 stimulation. We also evaluated the relative amounts of alveolar macrophage 5-lipoxygenase by Western analysis, in the presence and absence of A23187. There was no measurable difference in total cellular 5-lipoxygenase after A23187 stimulation when compared with unstimulated cells (Fig. 3). Because the Western analysis reflects the sum of total cellular enzyme synthesis and degradation, the amounts of [35S]methionine incorporation into alveolar macrophage proteins were measured after A23187 stimulation. There was no detectable 35S incorporation into any protein at a molecular mass of 60-90 kDa after A23187 stimulation (data not shown). These observations further suggest that A23187 stimulation of alveolar macrophages to release products of 5-lipoxygenase does
ctl Time
1 (minutes)
5 after
30
10 exposure
to
A23187
Fig. 1. Kinetics of leukotriene B4 (LTB,) release in A23187-stimulated alveolar macrophages. Alveolar macrophages were stimulated with A23187 (1 PM) for various periods of time. Supernatant LTB, was measured by radioimmunoassay. n = 3; * P < 0.05 compared with control (unstimulated alveolar macrophages).
RESULTS
The average cellular yield for the bronchoalveolar lavages was 46.6 t 4.5 X lo6 cells. Alveolar macrophages comprised 88 t 2%, whereas lymphocytes (10 t 2.6%) and polymorphonuclear cells (Cl%) consists of the remainder of the cells recovered. To determine whether release of LTB, required de novo synthesis of 5lipoxygenase, we stimulated HAMS with A23187 (1 PM) under conditions that should preclude new protein synthesis; i.e., at early time points and
Control
A231 87
A231 87 + Cycloheximide
A23 187 + Actinomycin
Fig. 2. Inhibitors of protein synthesis do not reduce A23187-stimulated LTB, release in alveolar macrophages. Alveolar macrophages were stimulated for 30 min with A23187 (1 PM) in presence and absence of actinomycin D (1 PM) or cycloheximide (1 pg/ml). Supernatant LTB4 was measured by radioimmunoassay. n = 5; * P < 0.05 compared with control (unstimulated alveolar macrophages).
Downloaded from www.physiology.org/journal/ajplung by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 20, 2019.
L456
ALVEOLAR
MACROPHAGE
5LIPXOYGENASE
A
Cytosol
Membrane
92Kd-
68Kd68Kd-
A23187
-
A231 87 Fig. 3. Immunoreactive 5lipoxygenase in alveolar macrophages. Total cellular 5-lipoxygenase was estimated by Western analysis of detergentlysed alveolar macrophages cultured in presence and absence of A23187 (1 PM). Representative study from 3 separate experiments; time, 10 min.
not require de novo synthesis of the enzyme. To evaluate if cytosol-to-membrane translocation correlated with LTB4 release, the relative amounts of associated membrane Slipoxygenase were compared in alveolar macrophages in the presence and absence of A23187. The amounts of membrane-associated Slipoxygenase increased in alveolar macrophages stimulated with A23187 when compared with controls (Fig. 4). This increase in membrane-associated Slipoxygenase correlated with LTBl release and suggested that A23187 activated preexistent 5-lipoxygenase by translocating the enzyme to the membrane fraction. To evaluate this further, LTB, release following A23187 stimulation was determined in the presence of MK-886, an inhibitor of 5-lipoxygenase translocation. MK-886 (0.5 /*M) inhibited the A23187-stimulated release of LTBI in human alveolar macrophages (Fig. 5). L 224, a direct inhibitor of 5lipoxygenase, also markedly decreased release of LTBI by the alveolar macrophages.
a
-
COfltKl
A23187
(1uM)
Conditions
Fig. 4. Membrane-associated 5lipoxygenase is increased in alveolar macrophages stimulated with A23187. A: membrane and cytosolic 5lipoxygenase. Membrane- and cytosolic-associated 5lipoxygenase was estimated by Western analysis of respective subcellular fractions in both presence and absence of A23187 (1 PM). Representative study from 3 separate experiments; time, 10 min. B: membrane-associated 5lipoxygenase. Relative amounts of membrane associated 5-lipoxygenase were estimated by optical densitometry of the above autoradiographs. n = 3; * P < 0.05.
DISCUSSION
These studies evaluated the regulation of 5-lipoxygenase activity in human alveolar macrophages. We found that, after A23187 stimulation, human alveolar macrophages release LTB, maximally within 10 min and that the release of LTB4 is not inhibited by protein synthesis inhibitors. In addition, A23187 stimulation did not increase the mass of cellular 5-lipoxygenase. These observations showed that A23187 does not regulate 5-lipoxygenase activity by altering total amounts of the enzyme within the cell. 5-Lipoxygenase activity did correlate, however, with translocation of the enzyme to the membrane fraction of the cells. In addition, inhibition of translocation of 5-lipoxygenase from cytosol to membrane with MK-886 blocked the ability of the cells to
COllld
A23187
A23187 M&S
A23187
&.I
Fig. 5. Inhibition of 5-lipoxygenase membrane translocation inhibits release of LTB, from A23187-stimulated alveolar macrophages. LTBl was measured by radioimmunoassay from supernatants of A23187stimulated alveolar macrophages in presence of MK-886 (0.5 PM), an inhibitor of 5-lipoxygenase translocation, or L 224 (1 PM), a direct inhibitor of 5-lipoxygenase. n = 5; time, 10 min; *P < 0.05 compared with control (unstimulated alveolar macrophages).
release LTBl after A23187 stimulation. We were unable, however, to detect any significant differences in the amounts of cytosolic protein after A23187 stimulation. This is likely explained by the finding that only small amounts of cytosolic enzyme actually translocate to
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ALVEOLAR
MACROPHAGE
membrane fractions of the cells. Thus the relative increase in the membrane fraction of &lipoxygenase, after stimulation is much greater than the relative decrease in cytosolic enzyme. These studies suggest that LTB, release after A23187 stimulation is, in part, due to the activation of a preexistent enzyme, a process that correlates with membrane translocation. In previous studies, we showed that stimulation of human alveolar macrophages with A23187 resulted in the release of only 5lipoxygenase products of arachidonate acid, whereas stimulation with LPS resulted in release of only products of the cyclooxygenase pathway of arachidonic acid (1). We also demonstrated that LPS-stimulated release of prostaglandins and thromboxanes in human alveolar macrophages was dependent upon the de novo synthesis of PGH synthase, the first committed enzyme in that metabolic pathway of arachidonic acid (18). Using LPS as a probe, we observed that PGH synthase product release was delayed for up to 6 h after stimulation, was inhibited by protein synthesis inhibitors, and correlated with the new synthesis of PGH synthase (18). In contrast, human alveolar macrophages stimulated with A23187 release LTB4 early, LTB4 release is not affected by protein synthesis inhibitors, and LTB4 release is not dependent on the de novo synthesis of 5lipoxygenase. Although the latter findings are not surprising inasmuch as the promoter-regulatory region of the 5lipoxygenase gene is similar to previously described housekeeping genes (11)) previous studies have correlated 5-lipoxygenase product release with enzyme mass (13, 20). These observations show that these two pathways of arachidonic acid metabolism (cyclooxygenase vs. 5-lipoxygenase) in human alveolar macrophages are regulated in very different ways. In conclusion, our studies demonstrate that human alveolar macrophages release LTB, after A23187 stimulation by activating preexistent 5-lipoxygenase. The activation of 5-lipoxygenase correlates with the translocation of the enzyme from the cytosol to the membrane fraction of the cells. These studies show that alveolar macrophages metabolize arachidonic acid to products of either PGH synthase or 5-lipoxygenase due in part to distinct mechanisms of enzyme activation. Special thanks to Jeanne Hunter and Deborah Jarrard for their secretarial assistance. This study was supported by the following grants: IA-89-F-8, a fellowship grant from the American Heart Association, Iowa Affiliate; institutional NRSA Fellowship Training Grant HL-07638 and Specialized Center of Research Grant HL-37121 from the National Heart, Lung, and Blood Institute; a Merit Review Grant from the Department of Veterans Affairs; and Grant RR59 from the Clinical Research Center, National Institutes of Health. Address for reprint requests: R. J. Pueringer, Pulmonary Disease Division, C33, University of Iowa College of Medicine, Iowa City, IA 52242. Received
14 June
1991; accepted
in final
form
8 October
1991.
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L457
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