Oxygen Radical Production by Alveolar Inflammatory Cells in Idiopathic Pulmonary Fibrosis 1- 3
JANOS STRAUSZ, JOACHIM MOLLER-QUERNHEIM, HARALD STEPPlING, and RUDOLF FERlINZ
Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, inflammatory disorder of the lower respiratory tract characterized by the injury of parenchymal cells and connective tissue components of the alveolar wall and a progressive fibrosis of the lung structure (1-6). Evaluation of the inflammatory process in IPF demonstrated that the accumulation of alveolar macrophages (AMs) and neutrophils in the lower respiratory tract is a characteristic feature of IPF (3, 5, 6). Both cell types have a potent armamentarium of mediators, proteases, and other secretory products capable either of modulating the inflammatory process or damaging the lung parenchyma directly (7-11, 13). Reactive oxygen intermediates (ROI) are claimed to be a major cause of tissue damage in IPF (1, 2, 12, 15). Activated AMs, as well as neutrophils, are capable of releasing O2 - , H 202 , and other highly active oxygen metabolites spontaneously(8,1O, 11, 13-15). However,the accurate source and the relative contribution of these cell types to the total ROI burden of IPF are not unequivocally identified (1, 2, 12).
In this context, we evaluated the ROI release of total bronchoalveolar lavage (BAL) cells and isolated AMs in IPF patients by means of luminol-enhanced chemiluminescence (CL). The correlation between the oxidant release of BAL cells and the clinical status of IPF patients before and after therapy were also investigated. Methods Study Population The diagnosis of IPF was established in 14 patients (11 men, 3 women; age 51 ± 4 yr, mean ± SEM) based on clinical symptoms, physical examination, chest X-ray, lung function test, and histological examination (1, 2, 124
SUMMARY Idiopathic pUlmon.ry fibrosis (IPF) Is • chronic Infl.mmatory Intlmltl.llung dl..... ch.racterlzed by the accumulation of .lveol.r macroph.gas (AMs) .nd n.utrophlls In th.lower respiratory tract, parenchym.1celliniury, .nd fibrosis of the .Iveol.r structure. Reactlw oxygen Intlrmediates (ROI) .re cl.lmed to be • m.jor ceu.. of tlssu. d.mag. In IPF; howwer, the source of ROI hss not been unequlvoc.11yId.ntlfled. AM., .s _II ss n.utrophll., .re capabl. of releasing the.. agentl. Th. contributions of th... posslbl. sources are not known. Toeddress this qu.stlon, _ _Iuated the spontlneous .nd stimulated (pMA or zymossn) ROI rei.... of totll bronchoalveoI.r c.lls .nd Isol.ted AMs In 14 patl.nte with IPF by m••ns of lumlnol-enh.nced ch.mllumln... cence. Broncho.lwol.r I~. (BAL) c.lIs from 17 Indlvldu.ls without .ny signs of Inflammation ..rved ss controls. In comparison with the controls, the spontlneous ss _II .s the stimulated ROI rei.... of totll BAL cells In IPF.re m.rkildly Incre88ed (20,783.9 :t 5,079.3 WIlIUS2,509.5 :t 300.6count8l10 sI2 • 10· cells, spontlneously, IPFV.IlIUScontrol; 106,819.3 :t 33,802.8WIlIUS8,919 :t 1,357.9 PMA Induced; 41,597.1 :t 8,442.6WIlIUS6,223.8 :t 1,025.1 zymo..n Induced, p < 0.001). Me.surem.nt of the ROI rei.... of pUrified AMs _led that th... cells produce the bulk part of ROI released by BAL cells (84~). In spite of the fact that, on • per cell bAls, the ROI rele... of neutrophllsls1.7·101d of that of AM., there Is no correlation between the ROI production of totll BAL cells .nd the percentage of neutrophlls In BAL, demonstrating. minor role of th... cells In the generation of the totel ROI burden In IPF.Addltlon.IIy, It _s not possible to suppress the ROI rele... by 10-· M prednisolone; howwer, some c...s of clinically effectlw therapy with prednisolone resulted In • m.rkiId dacre... of the ROI burden. Our results demonstrst. th.t In IPF, AMs .nd neutrophlls produce heightened .mounts of ROI;the moat Importent source, howwer, .re AM.. AM REV RESPIR DIS 19110; 141:124-128
5, 6). Seven patients were smokers, 2 were exsmokers, and 5 werenonsmokers. The patients with IPF under therapy weregiven 20 to 60 mg prednisolone per day orally. Six months after initiating prednisolone therapy, the responsivenesswas assessed by changes in symptoms, chest X-ray, and pulmonary function tests.
Control Group The control group consisted of 17 nonsmoking patients (10 men, 7 women, mean age: 37 ± 15 yr) with hamartoma (n = 7) and cough without any morphological alteration (n = 10). The results of the lung function tests were within the normal range. BAL and Cell Separation BAL was performed by flexible fiberoptic bronchoscopy with a total volume of 200 ml of 0.9070 sterile saline in four 50-ml a1iquots as previously described (16). The cells were separated by centrifugation (500 g, 4° C, 10 min), washed three times in RPMI 1640 (Gibco Europe GmbH, Karlsruhe, FRG). The differential cell counts were performed on Wright's stained cyt.ocentrifugepreparations.
Pure AMs were prepared by Ficoll-Hypaque density gradient centrifugation (17)and subsequent plastic adherence. The percentage of AMs was determined by esterase staining. The usual yield was > 99.5070 pure AM preparations. Preparations with less than 98070 AM were discarded. Peripheral blood neutrophils wereprepared by gradient centrifugation and subsequent dextran-sedimentation (17). The immunocytological characterization of alve-
(Received in original form January 30, 1989 and in revised form June 8, 1989) 1 From the IIIrd Department of Internal Medicine, Division of Pneumology, Johannes Gutenberg University, Mainz, Federal Republic of Germany. • Supported by Naturwissenschaftlich - Medizinisches Forschungszentrum, Maim, and by the Humboldt Foundation (J. S.). 3 Address correspondence and reprint requests to Dr. J. Strausz, IIIrd Department of Internal Medicine, Division of Pneumology, Johannes Gutenberg University, Langenbeckstr. 1. 6500 Maim, Federal Republic of Germany.
125
OXYGEN FlADICAL PRODUCTION
olar lymphocytes was performed on adherence slides (18), using monoclonal antibodies against CD3, CD4, CD8 differentiation antigens (Ortho Pharmaceutical Corp., Sommerville, NJ) and peroxidase-antiperoxidase staining.
CL Assay The CL assay described by Trush (19) was slightly modified. Briefly, the reaction mixture consisted of 100 ul freshly prepared luminol (Sigma Chemical Co., St. Louis, MO) solution (stock solution 10 mM luminol in dimethylsulfoxide was diluted 1:50 in aqua dest); 100 ul RPMI 1640,or 100 ul stimulant: either 2.4 ~M phorbol myristate acetate(PMA) (Sigma), or 1 mg/ml zymosan (Z) (Sigma); 100 ul RPMI 1640 or other additives, and 100 ul cell suspension (containing 2 x l()5 cells). The reaction mixture without cells was prewarmed to 37° C, and the cell suspension was pipetted into the reaction vials. The addition of the cells denoted the beginning of the reaction period. The CL was measured over the next 50 min by a Packard Luminometer (packard Instruments, Frankfurt, FRO). The peak wasusually observed within the first 15min. The data are presented as peak counts per 10 sl200,OOO cells. Superoxide dismutase (SOD) 100 ug/ml (Sigma) or 10-
JANOS STRAUSZ, JOACHIM MOLLER-QUERNHEIM, HARALD STEPPlING, and RUDOLF FERlINZ
Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, inflammatory disorder of the lower respiratory tract characterized by the injury of parenchymal cells and connective tissue components of the alveolar wall and a progressive fibrosis of the lung structure (1-6). Evaluation of the inflammatory process in IPF demonstrated that the accumulation of alveolar macrophages (AMs) and neutrophils in the lower respiratory tract is a characteristic feature of IPF (3, 5, 6). Both cell types have a potent armamentarium of mediators, proteases, and other secretory products capable either of modulating the inflammatory process or damaging the lung parenchyma directly (7-11, 13). Reactive oxygen intermediates (ROI) are claimed to be a major cause of tissue damage in IPF (1, 2, 12, 15). Activated AMs, as well as neutrophils, are capable of releasing O2 - , H 202 , and other highly active oxygen metabolites spontaneously(8,1O, 11, 13-15). However,the accurate source and the relative contribution of these cell types to the total ROI burden of IPF are not unequivocally identified (1, 2, 12).
In this context, we evaluated the ROI release of total bronchoalveolar lavage (BAL) cells and isolated AMs in IPF patients by means of luminol-enhanced chemiluminescence (CL). The correlation between the oxidant release of BAL cells and the clinical status of IPF patients before and after therapy were also investigated. Methods Study Population The diagnosis of IPF was established in 14 patients (11 men, 3 women; age 51 ± 4 yr, mean ± SEM) based on clinical symptoms, physical examination, chest X-ray, lung function test, and histological examination (1, 2, 124
SUMMARY Idiopathic pUlmon.ry fibrosis (IPF) Is • chronic Infl.mmatory Intlmltl.llung dl..... ch.racterlzed by the accumulation of .lveol.r macroph.gas (AMs) .nd n.utrophlls In th.lower respiratory tract, parenchym.1celliniury, .nd fibrosis of the .Iveol.r structure. Reactlw oxygen Intlrmediates (ROI) .re cl.lmed to be • m.jor ceu.. of tlssu. d.mag. In IPF; howwer, the source of ROI hss not been unequlvoc.11yId.ntlfled. AM., .s _II ss n.utrophll., .re capabl. of releasing the.. agentl. Th. contributions of th... posslbl. sources are not known. Toeddress this qu.stlon, _ _Iuated the spontlneous .nd stimulated (pMA or zymossn) ROI rei.... of totll bronchoalveoI.r c.lls .nd Isol.ted AMs In 14 patl.nte with IPF by m••ns of lumlnol-enh.nced ch.mllumln... cence. Broncho.lwol.r I~. (BAL) c.lIs from 17 Indlvldu.ls without .ny signs of Inflammation ..rved ss controls. In comparison with the controls, the spontlneous ss _II .s the stimulated ROI rei.... of totll BAL cells In IPF.re m.rkildly Incre88ed (20,783.9 :t 5,079.3 WIlIUS2,509.5 :t 300.6count8l10 sI2 • 10· cells, spontlneously, IPFV.IlIUScontrol; 106,819.3 :t 33,802.8WIlIUS8,919 :t 1,357.9 PMA Induced; 41,597.1 :t 8,442.6WIlIUS6,223.8 :t 1,025.1 zymo..n Induced, p < 0.001). Me.surem.nt of the ROI rei.... of pUrified AMs _led that th... cells produce the bulk part of ROI released by BAL cells (84~). In spite of the fact that, on • per cell bAls, the ROI rele... of neutrophllsls1.7·101d of that of AM., there Is no correlation between the ROI production of totll BAL cells .nd the percentage of neutrophlls In BAL, demonstrating. minor role of th... cells In the generation of the totel ROI burden In IPF.Addltlon.IIy, It _s not possible to suppress the ROI rele... by 10-· M prednisolone; howwer, some c...s of clinically effectlw therapy with prednisolone resulted In • m.rkiId dacre... of the ROI burden. Our results demonstrst. th.t In IPF, AMs .nd neutrophlls produce heightened .mounts of ROI;the moat Importent source, howwer, .re AM.. AM REV RESPIR DIS 19110; 141:124-128
5, 6). Seven patients were smokers, 2 were exsmokers, and 5 werenonsmokers. The patients with IPF under therapy weregiven 20 to 60 mg prednisolone per day orally. Six months after initiating prednisolone therapy, the responsivenesswas assessed by changes in symptoms, chest X-ray, and pulmonary function tests.
Control Group The control group consisted of 17 nonsmoking patients (10 men, 7 women, mean age: 37 ± 15 yr) with hamartoma (n = 7) and cough without any morphological alteration (n = 10). The results of the lung function tests were within the normal range. BAL and Cell Separation BAL was performed by flexible fiberoptic bronchoscopy with a total volume of 200 ml of 0.9070 sterile saline in four 50-ml a1iquots as previously described (16). The cells were separated by centrifugation (500 g, 4° C, 10 min), washed three times in RPMI 1640 (Gibco Europe GmbH, Karlsruhe, FRG). The differential cell counts were performed on Wright's stained cyt.ocentrifugepreparations.
Pure AMs were prepared by Ficoll-Hypaque density gradient centrifugation (17)and subsequent plastic adherence. The percentage of AMs was determined by esterase staining. The usual yield was > 99.5070 pure AM preparations. Preparations with less than 98070 AM were discarded. Peripheral blood neutrophils wereprepared by gradient centrifugation and subsequent dextran-sedimentation (17). The immunocytological characterization of alve-
(Received in original form January 30, 1989 and in revised form June 8, 1989) 1 From the IIIrd Department of Internal Medicine, Division of Pneumology, Johannes Gutenberg University, Mainz, Federal Republic of Germany. • Supported by Naturwissenschaftlich - Medizinisches Forschungszentrum, Maim, and by the Humboldt Foundation (J. S.). 3 Address correspondence and reprint requests to Dr. J. Strausz, IIIrd Department of Internal Medicine, Division of Pneumology, Johannes Gutenberg University, Langenbeckstr. 1. 6500 Maim, Federal Republic of Germany.
125
OXYGEN FlADICAL PRODUCTION
olar lymphocytes was performed on adherence slides (18), using monoclonal antibodies against CD3, CD4, CD8 differentiation antigens (Ortho Pharmaceutical Corp., Sommerville, NJ) and peroxidase-antiperoxidase staining.
CL Assay The CL assay described by Trush (19) was slightly modified. Briefly, the reaction mixture consisted of 100 ul freshly prepared luminol (Sigma Chemical Co., St. Louis, MO) solution (stock solution 10 mM luminol in dimethylsulfoxide was diluted 1:50 in aqua dest); 100 ul RPMI 1640,or 100 ul stimulant: either 2.4 ~M phorbol myristate acetate(PMA) (Sigma), or 1 mg/ml zymosan (Z) (Sigma); 100 ul RPMI 1640 or other additives, and 100 ul cell suspension (containing 2 x l()5 cells). The reaction mixture without cells was prewarmed to 37° C, and the cell suspension was pipetted into the reaction vials. The addition of the cells denoted the beginning of the reaction period. The CL was measured over the next 50 min by a Packard Luminometer (packard Instruments, Frankfurt, FRO). The peak wasusually observed within the first 15min. The data are presented as peak counts per 10 sl200,OOO cells. Superoxide dismutase (SOD) 100 ug/ml (Sigma) or 10-
