Effects of In Vitro Amiodarone Exposure on Alveolar Macrophage Inflammatory Mediator Production R.
J. ZITNIK,* J. A. D. COOPER, JR, t J. A. RANKIN,* J. SUSSMAN*
ABSTRACT: Administration of amiodarone, although often lifesaving, is associated with pulmonary side effects. Patients with amiodarone pulmonary toxicity can present with either a chronic disorder that suggests pulmonary fibrosis or a more acute process. Mechanisms of acute pulmonary injury resulting from amiodarone are unclear. Previous studies have demonstrated that the drug is preferentially concentrated in alveolar macrophages. In the present study, the authors examined whether in vitro exposure to amiodarone resulted in alteration of rat alveolar macrophage superoxide, leukotriene B 4 , or fibronectin release. In addition, the authors assessed whether macrophages were ultrastructurally altered by in vitro amiodarone exposure. Twenty four hour exposure to therapeutic tissue concentrations of amiodarone resulted in enhancement of phorbol myristate acetate-stimulated macrophage superoxide release. In addition, 48 hours exposure to amiodarone caused a dose-dependent inhibition of spontaneous fibronectin release by macrophages. Macrophages exposed to 48 hours of 10 p.g/ml amiodarone were ultrastructurally abnormal, containing lamellar inclusions and demonstrating a large degree of vacuolization. The authors concluded that alveolar macrophages are very sensitive to therapeutic tissue concentrations of amiodarone. Alteration of macrophage mediator release by amiodarone may be one mechanism for lung damage induced by the drug. KEY INDEXING TERMS: Alveolar macrophage; Amiodarone; From the *Pulnwnary Sections and the :j:Department of Pathology, Yale University School of Medicine, New HCUJen, Connecticut, the Birmingham V AM.C., Birmingham, Alabama, and tThe University of Alabama at Birmingham. Supported in part by the Birmingham V.AM.C. Research Service (JADC). Presented in part at the American Thoracic Society Annual Meeting, Las Vegas, Nevada, May 1988. Correspondence: J. Allen D. Cooper Jr., MD, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, University Station, Birmingham, AL 35294.
352
Fibronectin; Leukotrienes; Superoxide. [Am J Med Sci 1992; 304(6):352-356.]
A
miodarone is an antiarrhythmic drug that causes pulmonary disease in 6-15% of exposed patients. 1,2 Patients with amiodarone pulmonary toxicity can develop acute, rapidly progressive lung injury or a more insidious syndrome that suggests pulmonary fibrosis. 1,2 Risk factors for developing amiodarone pulmonary toxicity are unclear, although maintenance dose appears to be a major factor.l An increased incidence of acute lung injury upon exposure to iodinecontaining radiographic contrast or surgery requiring general anesthesia in patients receiving amiodarone recently has been noted.3,4 Amiodarone is lipophilic and distributes in lipid-rich organs, including the lung.5 Within the lung, amiodarone has a particular propensity to distribute in alveolar macrophages. In one study,6 alveolar macrophages (AM) from rats treated with amiodarone had significantly greater concentrations of amiodarone compared to type IT pneumocytes from the same animals. Within the cell, amiodarone is a potent inhibitor of phospho. lipase A activity.7 Inhibition of this enzyme results m accumulation of phospholipids of all classes. Some of these phospholipids are potential substrates for phospholipase C, which cleaves between the phosphate molecule and the glycerol backbone of phospholipids to form phosphoinositol and diacylglycero1.8 Each of these lipids plays a role in activation of protein kinase C, an enzyme that activates an oxidase important in the production of superoxide, a reactive oxygen metabolite.9 A recent studyl° also demonstrated that 40 JLmoljl amiodarone causes a rise in cellular cytosolic calcium concentrations, another important modulator of protein kinase C activity. Although mechanisms by which amiodarone can cause acute pulmonary toxicity are obscure, reactive oxygen species produced by resident pulmonary macrophages or in£luxing polymorphonuclear neutrophils may be important in other causes of acute pulmonary December 1992 Volume 304 Number 6
Zitnlk et al
injury.ll In addition, fibronectin has been implicated as an important modulator of inflammatory cells and fibroblast function.12 Elevated alveolar levels of fibronectin have been noted in patients with acute lung injury, and a pathogenic role for this molecule has been proposed.13 However, fibronectin also is important in the repair process that must occur after acute tissue injury.14 Thus, lower concentrations of fibronectin might prevent normal healing mechanisms that must occur after acute lung injury. Finally, leukotriene B4, an arachidonic acid metabolite, is a powerful chemoattractant for polymorphonuclear leukocytes and can activate phagocytes to release oxidants.IS Because pulmonary macrophages are potential sources of these three mediators that may participate in acute lung injury, we examined whether in vitro exposure to amiodarone results in altered AM production of these molecules. Methods Isolation of Alveolar Macrophages. Male Fischer rats
were anesthetized with LD100 pentobarbital (200-250 mg/kg). Bronchoalveolar lavage (BAL) was performed as previously described.16 The trachea was cannulated, 100 ml of room temperature Hanks' balanced salt solution (HBSS) without calcium or magnesium were instilled in 5 ml aliquots, and eluates were collected by gravity drainage. Bp~ cells were separated from BAL fluid by centrifugation (400 X g for 10 minutes), washed twice in HBSS without calcium or magnesium, and resuspended in HBBS with calcium. and magnesium at 2 X 106 cells/ml. Cell CUlture and Amiodarone Exposure. One hundred microliters of the cell suspension was placed in wells of a sterile 96 well microtiter plate (Nunc Inc., Naperville, IL) and cells were allowed to adhere for 1 hour at 37° C in 5% CO2, Adherent cell monolayers were washed once with pre-warmed HBSS without calcium. or magnesium. Then, 200 1'1 McCoy's media with 5% fetal calf serum and penicillin/streptomycin were added. Finally, appropriate concentrations of amiodarone (gift of Wyeth Pharmaceuticals, Philadelphia, PA) or drug diluent (0.85% dimethyl sulfoxide, 0.15% tween 20) were added and the cultures were incubated for 24 hours at 37° C in 5% CO2, In some cases, 200 JLl of fresh media containing amiodarone or diluent were added and the cells were cultured for an additional 24 hours. At the end of the incubation, supernatants were removed and saved for fibronectin determination or quantitation of cellular detachment. After washing once with pre-warmed HBSS without calcium or magnesium, fresh appropriate media, devoid of amiodarone or diluent, were added for determination of superoxide or leukotriene release. Assessment of Superoxide Anion Release. Superoxide anion (02-) production at rest or in the presence of phorbol myristate acetate was assessed using a previously described techniqueI6 that employs the reducTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
tion of ferricytochrome C in the presence of superoxide. After exposure to amiodarone or diluent, fresh HBSS with calcium. and magnesium containing cytochrome C (80 JLmol/l) and phorbol myristate acetate (2 JLmol/ I) were added. Superoxide dismutase (12.5 ~/ml final concentration) was added to duplicate wells to allow calculation of superoxide dismutase-inhibitable cytochrome C reduction. Cultures were incubated for an additional 45 minutes and the degree of cytochrome C reduction was quantitated by determining absorbance at 550 nm on a microplate spectrophotometer. Results are expressed as nanomoles/liter X 106 cells/45 min. Determination of Fibronectin Production. Spontaneous fibronectin release by adherent macrophage cultures exposed to amiodarone or diluent alone was assessed using an enzyme linked immunoassay. A commercially available antibody to rat fibronectin (Calbiochem Corp., San Diego, CA) was coupled to horseradish peroxidase (HRP) by the periodate method.17 Microtiter plates were coated overnight at 4° C with 600 ng/ml fibronectin. UnknoWIi samples or known concentrations of rat fibronectin also were incubated overnight at 4°C with an optimal dilution of HRP-labeled anti-fibronectin antibody. After the coated plate with phosphate buffered saline (PBS) containing tween, bovine serum albumin (BSA; .002%), and phenylmethylsulfonyl fluoride (PMSF) (0.1 mmol/ I) was washed, the solutions containing specific antibody in the presence of known or unknown samples were added and the plates were incubated for 1 hour at 37° C. Wells were washed twice with PBS-tweenPMSF; then peroxidase substrate was added and the amount of bound peroxidase was quantitated by measuring the absorbance at 490 nm. Absorbance was inversely related to the amount of fibronectin in the soluble phase. The concentration of fibronectin in the unknown samples was estimated by comparison to a curve generated using known concentrations of fibronectin. Quantitation of Immunoreactive Leukotriene B••
Leukotriene B4 (LTB4) release by macrophages exposed to varying concentrations of amiodarone was quantitated using a monospecific antibody to L TB4 (provided by Dr. E. Hayes, Merck Frosst, Rahway, NJ), as previously described.1s Protein in samples was precipitated by adding 1.6 ml methanol to 400 JLl supernatant. The mixture was incubated at 4 ° C for 30 minutes, deproteinated by centrifugation at 2000 X g for 10 minutes, dried by evaporation (Savant Instruments, Farmingdale, NY), and resuspended in 250 JLl of PBS. The radioimmunoassay was performed by adding 200 1'1 of immune rabbit serum, diluted to give 40-60% binding of 3H-L TB4, added to a mixture of 200 JLl PBS containing BSA, azide, approximately 7000 counts per minute 3H-LTB4 (New England Nuclear, Boston, MA), and 100 JLl of sample or synthetic LTB4 standard. The mixture was incubated for 2 hours at 4°C before 500 JLl of goat anti-rabbit IgG (Bio-Rad, Melville, NY) was added, and the incubation continued for 19-20 hours
353
In Vitro Amiodarone and Alveolar Macrophages
.5 E
Table 1. Alveolar Macrophage Leukotriene B. Production after 24 Hour Amiodarone Exposure
4
10
-*** •
'1:1'
o G,
3
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J. ZITNIK,* J. A. D. COOPER, JR, t J. A. RANKIN,* J. SUSSMAN*
ABSTRACT: Administration of amiodarone, although often lifesaving, is associated with pulmonary side effects. Patients with amiodarone pulmonary toxicity can present with either a chronic disorder that suggests pulmonary fibrosis or a more acute process. Mechanisms of acute pulmonary injury resulting from amiodarone are unclear. Previous studies have demonstrated that the drug is preferentially concentrated in alveolar macrophages. In the present study, the authors examined whether in vitro exposure to amiodarone resulted in alteration of rat alveolar macrophage superoxide, leukotriene B 4 , or fibronectin release. In addition, the authors assessed whether macrophages were ultrastructurally altered by in vitro amiodarone exposure. Twenty four hour exposure to therapeutic tissue concentrations of amiodarone resulted in enhancement of phorbol myristate acetate-stimulated macrophage superoxide release. In addition, 48 hours exposure to amiodarone caused a dose-dependent inhibition of spontaneous fibronectin release by macrophages. Macrophages exposed to 48 hours of 10 p.g/ml amiodarone were ultrastructurally abnormal, containing lamellar inclusions and demonstrating a large degree of vacuolization. The authors concluded that alveolar macrophages are very sensitive to therapeutic tissue concentrations of amiodarone. Alteration of macrophage mediator release by amiodarone may be one mechanism for lung damage induced by the drug. KEY INDEXING TERMS: Alveolar macrophage; Amiodarone; From the *Pulnwnary Sections and the :j:Department of Pathology, Yale University School of Medicine, New HCUJen, Connecticut, the Birmingham V AM.C., Birmingham, Alabama, and tThe University of Alabama at Birmingham. Supported in part by the Birmingham V.AM.C. Research Service (JADC). Presented in part at the American Thoracic Society Annual Meeting, Las Vegas, Nevada, May 1988. Correspondence: J. Allen D. Cooper Jr., MD, Division of Pulmonary and Critical Care Medicine, University of Alabama at Birmingham, University Station, Birmingham, AL 35294.
352
Fibronectin; Leukotrienes; Superoxide. [Am J Med Sci 1992; 304(6):352-356.]
A
miodarone is an antiarrhythmic drug that causes pulmonary disease in 6-15% of exposed patients. 1,2 Patients with amiodarone pulmonary toxicity can develop acute, rapidly progressive lung injury or a more insidious syndrome that suggests pulmonary fibrosis. 1,2 Risk factors for developing amiodarone pulmonary toxicity are unclear, although maintenance dose appears to be a major factor.l An increased incidence of acute lung injury upon exposure to iodinecontaining radiographic contrast or surgery requiring general anesthesia in patients receiving amiodarone recently has been noted.3,4 Amiodarone is lipophilic and distributes in lipid-rich organs, including the lung.5 Within the lung, amiodarone has a particular propensity to distribute in alveolar macrophages. In one study,6 alveolar macrophages (AM) from rats treated with amiodarone had significantly greater concentrations of amiodarone compared to type IT pneumocytes from the same animals. Within the cell, amiodarone is a potent inhibitor of phospho. lipase A activity.7 Inhibition of this enzyme results m accumulation of phospholipids of all classes. Some of these phospholipids are potential substrates for phospholipase C, which cleaves between the phosphate molecule and the glycerol backbone of phospholipids to form phosphoinositol and diacylglycero1.8 Each of these lipids plays a role in activation of protein kinase C, an enzyme that activates an oxidase important in the production of superoxide, a reactive oxygen metabolite.9 A recent studyl° also demonstrated that 40 JLmoljl amiodarone causes a rise in cellular cytosolic calcium concentrations, another important modulator of protein kinase C activity. Although mechanisms by which amiodarone can cause acute pulmonary toxicity are obscure, reactive oxygen species produced by resident pulmonary macrophages or in£luxing polymorphonuclear neutrophils may be important in other causes of acute pulmonary December 1992 Volume 304 Number 6
Zitnlk et al
injury.ll In addition, fibronectin has been implicated as an important modulator of inflammatory cells and fibroblast function.12 Elevated alveolar levels of fibronectin have been noted in patients with acute lung injury, and a pathogenic role for this molecule has been proposed.13 However, fibronectin also is important in the repair process that must occur after acute tissue injury.14 Thus, lower concentrations of fibronectin might prevent normal healing mechanisms that must occur after acute lung injury. Finally, leukotriene B4, an arachidonic acid metabolite, is a powerful chemoattractant for polymorphonuclear leukocytes and can activate phagocytes to release oxidants.IS Because pulmonary macrophages are potential sources of these three mediators that may participate in acute lung injury, we examined whether in vitro exposure to amiodarone results in altered AM production of these molecules. Methods Isolation of Alveolar Macrophages. Male Fischer rats
were anesthetized with LD100 pentobarbital (200-250 mg/kg). Bronchoalveolar lavage (BAL) was performed as previously described.16 The trachea was cannulated, 100 ml of room temperature Hanks' balanced salt solution (HBSS) without calcium or magnesium were instilled in 5 ml aliquots, and eluates were collected by gravity drainage. Bp~ cells were separated from BAL fluid by centrifugation (400 X g for 10 minutes), washed twice in HBSS without calcium or magnesium, and resuspended in HBBS with calcium. and magnesium at 2 X 106 cells/ml. Cell CUlture and Amiodarone Exposure. One hundred microliters of the cell suspension was placed in wells of a sterile 96 well microtiter plate (Nunc Inc., Naperville, IL) and cells were allowed to adhere for 1 hour at 37° C in 5% CO2, Adherent cell monolayers were washed once with pre-warmed HBSS without calcium. or magnesium. Then, 200 1'1 McCoy's media with 5% fetal calf serum and penicillin/streptomycin were added. Finally, appropriate concentrations of amiodarone (gift of Wyeth Pharmaceuticals, Philadelphia, PA) or drug diluent (0.85% dimethyl sulfoxide, 0.15% tween 20) were added and the cultures were incubated for 24 hours at 37° C in 5% CO2, In some cases, 200 JLl of fresh media containing amiodarone or diluent were added and the cells were cultured for an additional 24 hours. At the end of the incubation, supernatants were removed and saved for fibronectin determination or quantitation of cellular detachment. After washing once with pre-warmed HBSS without calcium or magnesium, fresh appropriate media, devoid of amiodarone or diluent, were added for determination of superoxide or leukotriene release. Assessment of Superoxide Anion Release. Superoxide anion (02-) production at rest or in the presence of phorbol myristate acetate was assessed using a previously described techniqueI6 that employs the reducTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
tion of ferricytochrome C in the presence of superoxide. After exposure to amiodarone or diluent, fresh HBSS with calcium. and magnesium containing cytochrome C (80 JLmol/l) and phorbol myristate acetate (2 JLmol/ I) were added. Superoxide dismutase (12.5 ~/ml final concentration) was added to duplicate wells to allow calculation of superoxide dismutase-inhibitable cytochrome C reduction. Cultures were incubated for an additional 45 minutes and the degree of cytochrome C reduction was quantitated by determining absorbance at 550 nm on a microplate spectrophotometer. Results are expressed as nanomoles/liter X 106 cells/45 min. Determination of Fibronectin Production. Spontaneous fibronectin release by adherent macrophage cultures exposed to amiodarone or diluent alone was assessed using an enzyme linked immunoassay. A commercially available antibody to rat fibronectin (Calbiochem Corp., San Diego, CA) was coupled to horseradish peroxidase (HRP) by the periodate method.17 Microtiter plates were coated overnight at 4° C with 600 ng/ml fibronectin. UnknoWIi samples or known concentrations of rat fibronectin also were incubated overnight at 4°C with an optimal dilution of HRP-labeled anti-fibronectin antibody. After the coated plate with phosphate buffered saline (PBS) containing tween, bovine serum albumin (BSA; .002%), and phenylmethylsulfonyl fluoride (PMSF) (0.1 mmol/ I) was washed, the solutions containing specific antibody in the presence of known or unknown samples were added and the plates were incubated for 1 hour at 37° C. Wells were washed twice with PBS-tweenPMSF; then peroxidase substrate was added and the amount of bound peroxidase was quantitated by measuring the absorbance at 490 nm. Absorbance was inversely related to the amount of fibronectin in the soluble phase. The concentration of fibronectin in the unknown samples was estimated by comparison to a curve generated using known concentrations of fibronectin. Quantitation of Immunoreactive Leukotriene B••
Leukotriene B4 (LTB4) release by macrophages exposed to varying concentrations of amiodarone was quantitated using a monospecific antibody to L TB4 (provided by Dr. E. Hayes, Merck Frosst, Rahway, NJ), as previously described.1s Protein in samples was precipitated by adding 1.6 ml methanol to 400 JLl supernatant. The mixture was incubated at 4 ° C for 30 minutes, deproteinated by centrifugation at 2000 X g for 10 minutes, dried by evaporation (Savant Instruments, Farmingdale, NY), and resuspended in 250 JLl of PBS. The radioimmunoassay was performed by adding 200 1'1 of immune rabbit serum, diluted to give 40-60% binding of 3H-L TB4, added to a mixture of 200 JLl PBS containing BSA, azide, approximately 7000 counts per minute 3H-LTB4 (New England Nuclear, Boston, MA), and 100 JLl of sample or synthetic LTB4 standard. The mixture was incubated for 2 hours at 4°C before 500 JLl of goat anti-rabbit IgG (Bio-Rad, Melville, NY) was added, and the incubation continued for 19-20 hours
353
In Vitro Amiodarone and Alveolar Macrophages
.5 E
Table 1. Alveolar Macrophage Leukotriene B. Production after 24 Hour Amiodarone Exposure
4
10
-*** •
'1:1'
o G,
3
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