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The role of antioxidant defense mechanisms in the pathogenesis of granulomatous human lung diseases remains open to investigation. In this study we investigated the immunoreactivity of two important superoxide radical scavenging intracellular antioxidant enzymes, manganese superoxide dismutase (MnSOD) and copperzinc superoxide dismutase (CuZnSOD), in pulmonary sarcoidosis and extrinsic allergic alveolitis. In histologically normal lung MnSOD was variable but mostly positive in the cells of bronchial epithelium, alveolar epithelium especially in type II pneumocytes, and alveolar macrophages. Copperzinc SOD showed positive immunoreactivity most markedly in the bronchial epithelium. The biopsies of 22 patients with pulmonary sarcoidosis and 10 with extrinsic allergic alveolitis indicated that MnSOD was highly stained in the granulomas of both diseases, with 60 to 100% of the granulomas showing intensive immunoreactivity. Western blots conducted on the cell samples of bronchoalveolar lavage (BAL) fluid revealed significantly higher amounts of MnSOD in sarcoidosis and extrinsic allergic alveolitis than in the controls. Immunohistochemistry on the cells obtained from BAL fluid showed positive immunoreactivity of MnSOD in the macrophages but not in the lymphocytes. In contrast, copperzinc SOD was not induced in either of these diseases. We conclude that MnSOD is highly expressed in the granulomas of pulmonary sarcoidosis and extrinsic allergic alveolitis, and variable but mostly positive in alveolar macrophages, possibly owing to cytokine mediated induction during the granuloma formation.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The pathogenesis of granulomatous diseases is associated with complex cell-mediated immune reactions. Most important cell types which are involved in granuloma formation are CD4+ T lymphocytes, alveolar macrophages, blood monocytes, and epithelioid cells matured from mononuclear cells (1). The recruitment and activation of the inflammatory cells and lymphocytes as well as the role of various cytokines in the pathogenesis of granulomatous diseases have been relatively well investigated (2), whereas few studies are available on the possible role of free radicals or cellular antioxidant defense mechanisms during granuloma formation (5), and only one of these studies has been conducted on human cells (6).
Activation of inflammatory cells results in generation of
free radicals by NADPH oxidase and nitric oxide synthase
activation (7, 8). Both reactive oxygen metabolites and cytokines are known to induce especially manganese superoxide
dismutase (MnSOD) in various cells. This mitochondrial enzyme is induced by changes in the cellular redox state, inflammatory cytokines such as tumor necrosis factor-alpha (TNF-
,
interleukin-1 (IL-1), interleukin-6 (IL-6) (9), and by high
oxygen tension in the lungs of hyperoxia-exposed animals (17-
19). Copperzinc superoxide dismutase (CuZnSOD), which is
localized in the cytosolic fraction, is usually more abundant than MnSOD, but is not induced by cytokines or hyperoxia to
the same extent as MnSOD (15, 16, 20).
The messenger RNA (mRNA) and/or protein levels of MnSOD are usually low in normal lung and highly expressed in alveolar type II pneumocytes, alveolar macrophages, interstitial fibroblasts, and visceral pleura of hyperoxia-exposed rats (19, 21). CuZnSOD has been shown to be expressed in alveolar epithelial cells, fibroblasts, and capillary endothelial cells in normal rats (19). Both of these enzymes are expressed at low levels in human bronchial epithelium (24) and in human alveolar type II pneumocytes and macrophages (25). According to recent studies, CuZnSOD was decreased in the epithelium of asthmatic patients (26, 27). We are not aware of any studies where MnSOD and CuZnSOD have been assessed in nonmalignant pulmonary parenchymal diseases. It can be hypothesized that MnSOD is also induced in human lung in conditions where inflammatory cells are activated with involvement of cytokine expression of these cells.
We investigated the expression of MnSOD and CuZnSOD
in pulmonary sarcoidosis and extrinsic allergic alveolitis, both
of which represent granulomatous pulmonary diseases with
typical histology and well-preserved lung architecture. In addition, these clinical entities represent situations where inflammatory cells are activated with simultaneous effects of TNF-
and IL-1. The expressions of MnSOD and CuZnSOD were assessed semiquantitatively by immunohistochemistry. In addition, immunohistochemistry and Western blotting were conducted on the cell samples of bronchoalveolar lavage (BAL)
fluid to compare the expression of these enzymes in healthy
lung, sarcoidosis, and extrinsic allergic alveolitis, and to compare the induction of these enzymes in pulmonary lymphocytes and alveolar macrophages.
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METHODS
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Patients and Handling of the Specimens
Histopathologically typical cases of sarcoidosis and extrinsic allergic alveolitis were retrieved from the files of the Department of Pathology, Oulu University Hospital, by reevaluating diagnostic lung or lymph node biopsies taken either as open, thoracoscopic, transbronchial, or lymph node biopsies between 1982 and 1996. Thirty-two patients (15 women and 17 men) representing sarcoidosis (n = 22) and extrinsic allergic alveolitis, such as farmer's lung (n = 10), were included in the study (Tables 1 and 2). Diagnoses of all patients were based on light microscopic evaluations using the histologic criteria presented by Dail and Hammar (28). Two of the patients received oral corticosteroid treatment, and four of the patients received inhaled corticosteroid treatment before the lung biopsy. All the patients had sarcoidosis or extrinsic allergic alveolitis with parenchymal involvement, as the main reason for the diagnostic lung biopsy. The lymph node biopsies were taken during mediastinoscopy due to hilar lymph node enlargement in four patients and parenchymal involvement in two patients.
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Biopsies were usually obtained from the right middle lobe or the lingula of the left lung. The biopsy material was fixed in 10% formalin under vacuum in order to expand the tissue and to remove air bubbles or perfused by injecting the fixative, using a small syringe into bronchioles as described by Dail and Hammar (28). The lymph node biopsies were also fixed in 10% formalin. The specimens were then dehydrated and embedded in paraffin. Uninvolved peripherial lung tissue, used as a control, was obtained from five patients operated on for malignant lung tumors.
In additional experiments, samples of BAL fluid were stained in
order to evaluate the localization of MnSOD and CuZnSOD in different cells. BAL fluid samples of five patients with histologically confirmed sarcoidosis and five patients with extrinsic allergic alveolitis
were included in the study (Table 3). A control group consisted of five
patients, who had been investigated for minor respiratory symptoms
and fever for unknown etiology (Table 3). The cell number and distribution as well as chemical inflammatory parameters of the BAL fluid
of the control subjects were normal. BAL fluid was centrifuged and
the cells were harvested, fixed with 10% formalin, embedded in 2%
agar, and after solidification, further embedded in paraffin. For the
Western blotting the BAL fluid was centrifuged and the cells were
collected, frozen and stored at 
80° C.
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Immunohistochemistry
One representative paraffin block was selected for immunohistochemical stainings. Four-micrometer-thick sections were cut and processed further within a few days. First, they were deparaffinized in xylene, and rehydrated in graded alcohol. Then, endogenous peroxidase was consumed by incubating sections in 0.1% hydrogen peroxide in absolute methanol for 10 min. For the immunostainings, the avidin- biotin-peroxidase complex method was used as described (29). The polyclonal antibodies for MnSOD and CuZnSOD were a gift from Prof. James D. Crapo (National Jewish Medical and Research Center, Denver, CO) (24, 30). The sections were incubated with the primary antibodies (anti-MnSOD with a dilution 1:1,000 and anti-CuZnSOD with a dilution of 1:100) at room temperature for 2 h, followed by a biotinylated swine anti-rabbit secondary antibody (at a dilution of 1:200 for 30 min) and the avidin-biotin-peroxidase complex (both from Dakopatts, Glostrup, Denmark). The color was developed with diaminobenzidine. The sections were counterstained with a light hematoxylin stain. The negative controls consisted of substituting phosphate-buffered saline (PBS) at pH 7.2 and normal rabbit serum for the primary antibody.
Western Blotting
The cell pellets were melted, mixed with the electrophoresis sample buffer, and boiled for 5 min at 95° C. Protein concentration of the samples was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA), and 50 µg of cell protein per lane was applied to a 12% sodium dodecyl sulfate polyacrylamide gel. The gel was electrophoresed for 1.5 h (90 V), and the protein transferred (45 min, 100 V) onto Hybond ECL nitrocellulose membrane (Amersham, Buckinghamshire, UK) in a Mini Protean II Cell (Bio-Rad). The blotted membrane was incubated with the antibody to MnSOD (diluted 1:10,000), or CuZnSOD (diluted 1:10,000) followed by a donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham) (dilution 1:30,000). Manganese SOD and CuZnSOD were detected using the enhanced chemoluminescence system (ECL; Amersham), and the luminol excitation was imaged on X-ray film. Manganese SOD and CuZnSOD expressions of the patients and normal control subjects were compared quantitatively by scanning densitometry using 300A Computing Densitometer and Image Quant Software v3.0 Fast Scan (Molecular Dynamics, Sunnyvale, CA).
Light Microscopic Evaluation
For immunohistochemical stainings the whole tissue section was evaluated by light microscopy and the results were assessed semiquantitatively by grading the staining intensity as follows: weak (1), moderate (2), and intense (3) immunoreactivity. Whenever possible we also evaluated the percentage of the whole cell population showing any immunoreactivity with an accuracy of 10%.
Statistical Analysis
Two separate groups in Western blotting were compared using a two-tailed Student's t test, and p < 0.05 was considered significant.
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MnSOD and CuZnSOD Immunohistochemistry in Normal Lung
Manganese SOD was variable but mostly weakly positive in alveolar macrophages. Positive immunoreactivity was also found in the cuboidal cells of alveolar epithelium, evidently alveolar type II pneumocytes, the staining intensity being moderate in one and weak in two biopsies. Two of the biopsies showed no immunohistochemical staining for MnSOD in the pneumocytes (Table 4). The cells of BAL fluid of the healthy control subjects showed weak MnSOD immunoreactivity with the exception of one case. Clinically, this particular case, which revealed positive MnSOD immunoreactivity, was later diagnosed as sarcoidosis, but was never confirmed histologically. The staining pattern of MnSOD was intracytoplasmic and granular.
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CuZnSOD stained positively in the bronchial epithelium of histologically normal lung. The CuZnSOD staining was moderate in one and weak in four biopsies, 50 to 100% of the bronchial epithelial cells showing immunoreactivity (Table 4). CuZnSOD stained the ciliated bronchial epithelial cells more intensively than the peripheral bronchial epithelium. In contrast, usually no or occasionally very weak immunoreactivity for CuZnSOD was found in alveolar macrophages (data not shown). The immunostaining for CuZnSOD was intracytoplasmic and homogenous.
MnSOD and CuZnSOD in Sarcoidosis
The lung biopsies of 16 patients with pulmonary sarcoidosis showed that MnSOD was highly stained in the granulomas. The staining was intense in nine and moderate in seven biopsies, 60 to 100% of the granulomas showing immunoreactivity for MnSOD, especially in Langhans type giant cells and to a lesser degree in epithelioid cells (Table 5) (Figures 1A, 1B, and 1C). When compared with the controls, manganese SOD was positive in type II pneumocytes (Figure 1D). In one case (Patient 5) numerous proliferating type II pneumocytes were present and all observed cells were positively stained for MnSOD. However, the number of type II pneumocytes varied considerably from one patient to another. In two cases (Patients 8 and 12) the smooth muscle in the media of arterioles showed immunoreactivity for MnSOD. Immunohistochemistry conducted on the BAL cell samples revealed positive MnSOD immunoreactivity in the macrophages (Table 6) but not in the lymphocytes (Figures 1E and 1F). The intensity varied from weak to intense in 30 to 90% of the macrophages. In agreement, Western blotting of MnSOD indicated significantly higher reactivity in the BAL cell samples of sarcoidosis patients than in the control subjects (Figure 2).
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In contrast to MnSOD, the granulomas showed no immunoreactivity for CuZnSOD. The bronchial epithelium was stained from 30 to 100%, and the intensity was moderate in five biopsies and weak in nine biopsies (Table 5). In contrast to the finding with MnSOD, CuZnSOD was also low or negative in the BAL cell samples of these patients (data not shown).
MnSOD and CuZnSOD in Extrinsic Allergic Alveolitis
The granulomas of extrinsic allergic alveolitis showed marked MnSOD immunoreactivity. However, the staining was patchy and less intense than that observed in sarcoidosis (Table 7, Figure 3A). Also, immunohistochemistry of the BAL fluid cell samples showed similar findings as in sarcoidosis: macrophages but not lymphocytes were positive for MnSOD (Table 6). Neither immunohistochemistry nor Western blotting showed any upregulation of CuZnSOD expression in the granulomas of extrinsic allergic alveolitis (Figure 3B), whereas bronchial epithelium was positive for CuZnSOD immunoreactivity (Figures 3B, 3C, and 3D). By Western blotting the reactivity for MnSOD was significantly higher in the BAL fluid samples of patients with allergic alveolitis than in the BAL fluid samples of the control subjects (Figure 4).
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INTRODUCTION
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DISCUSSION
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We found for the first time that MnSOD is highly expressed in the granulomas of sarcoidosis and extrinsic allergic alveolitis in human lung. This finding was constant and repeatable not only in the granulomas, but also in the lymph nodes of six patients with sarcoidosis. In contrast to MnSOD, CuZnSOD showed similar positive immunoreactivity in the bronchial epithelium of healthy control subjects and in the patients of sarcoidosis and extrinsic allergic alveolitis, but no expression in the granulomas of either of these diseases. CuZnSOD was also low or negative in the BAL cell samples of these patients. Interestingly, additional unpublished experiments in our laboratory have shown enhanced MnSOD, but not CuZnSOD, immunoreactivity also in the granulomas of pulmonary tuberculosis.
The localization and regulation of MnSOD and CuZnSOD in the lungs of experimental animals have been relatively well elucidated (19, 21, 22, 31), and they may have similarities in human and animal lung. In rat lung, Chang and coworkers (19) and Coursin and coworkers (22) have demonstrated immunohistochemical positivity for MnSOD in ciliated bronchial epithelial cells, Clara cells, alveolar macrophages, and type II pneumocytes. By immunogold technique MnSOD appears to be highly localized to the mitochondria. In human lung Coursin and coworkers (22) were not able to demonstrate any immunohistochemical positivity by immunoperoxidase for CuZnSOD in any of the previously mentioned cells while by immunogold technique CuZnSOD was found throughout mesenchymal cells, arterioles, and epithelial cells of the alveoli of rat lung (22). In later studies, however, the same group detected CuZnSOD immunoreactivity in bronchial epithelium of normal human lung (25). Furthermore, other studies conducted on human fetal and adult lung have exhibited weak positivity for CuZnSOD in bronchial epithelial cells and Clara cells (24, 32, 33). The present findings on healthy human lung are in agreement with previous studies on human lung showing most intense MnSOD reactivity in alveolar macrophages and type II pneumocytes and positive CuZnSOD reactivity in the bronchial epithelium.
The pathogenesis of hypersensitivity granulomatous diseases is still partially unclear, but it can be hypothesized that
free radicals may have a central contributing role in these diseases. Granuloma formation involves several cytokines and
lymphokines, such as TNF-, 1L-1, and IL-6, all of which can
cause MnSOD induction (11). The present study showed
high MnSOD immunoreactivity in alveolar macrophages and
type II pneumocytes both in the patients with sarcoidosis and
extrinsic allergic alveolitis. Both of these cells were also more
resistant to exogenous oxidants than alveolar type I cells or
endothelial cells (34). Western blotting revealed significantly
higher level of MnSOD in the BAL cell samples in sarcoidosis
and extrinsic allergic alveolitis than in the controls, and immunohistochemistry indicated that MnSOD was expressed in alveolar macrophages, but not in the lymphocytes. Because the percentage of macrophages in the BAL samples of healthy
control subjects is higher than in granulomatous diseases associated with relative lymphocytosis, the results of the Western
blotting underestimate the real expression of MnSOD in the
macrophages of these diseases. Since these granulomatous diseases usually follow a benign course and well-preserved lung
architecture, the enhanced expression of MnSOD may play an
important role in protection of the lung during macrophage
activation and granuloma formation. It is known that MnSOD
is localized in alveolar macrophages, and against this background, it is natural that MnSOD is also expressed in granulomas, which originate from monocyte lineages, i.e., macrophages.
CuZnSOD has been found especially in ciliated epithelial cells and Clara cells (22, 25). CuZnSOD is not usually induced by cytokines (15, 16) and these previous findings are also in agreement with our study, which shows relatively weak immunoreactivity of CuZnSOD both in the cells of BAL samples and alveolar macrophages and positive staining especially in the ciliated epithelium. Thus the localization and induction of these two superoxide radical scavenging enzymes appears to differ in various cells in human lung.
Obtaining normal human control subjects for research is often difficult. We had to use histologically normal lung biopsies obtained from patients who had undergone surgery because of a malignant lung tumor, or BAL fluids from patients who had undergone bronchoscopy due to minor respiratory symptoms. The patients with lung tumors are often smokers or ex-smokers. Tobacco smoke has been found to decrease the activities of several antioxidant enzymes, one of those being CuZnSOD (35). On the other hand, oxidant burden and inflammation accompanied by cigarette smoke may induce MnSOD activity in the lung. In our study CuZnSOD was low both in the control biopsies and in the granulomatous diseases, and MnSOD reactivity was low in the control biopsies and high in both granulomatous diseases.
In conclusion, we have demonstrated high MnSOD, but not CuZnSOD immunoreactivity in the granulomas of sarcoidosis and extrinsic allergic alveolitis. MnSOD was prominent in alveolar macrophages and type II pneumocytes, whereas CuZnSOD was expressed in bronchial epithelium. MnSOD immunoreactivity was characteristic for the granuloma formation but not for sarcoidosis since the granulomas of sarcoidosis and extrinsic allergic alveolitis showed intensive MnSOD reactivity.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Vuokko Kinnula, M.D., Ph.D., University of Oulu, Department of Internal Medicine, Kajaanintie 50A, 90220 Oulu, Finland.
(Received in original form November 13, 1997 and in revised form March 16, 1998).
Acknowledgments: Supported by grants from the Finnish Anti-Tuberculosis Association Foundation, the Juselius Foundation, and the Emil Aaltonen Foundation.
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References |
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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V L Kinnula, S Lehtonen, R Kaarteenaho-Wiik, E Lakari, P Paakko, S W Kang, S G Rhee, and Y Soini Cell specific expression of peroxiredoxins in human lung and pulmonary sarcoidosis Thorax, February 1, 2002; 57(2): 157 - 164. [Abstract] [Full Text] [PDF]
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B. Nemery, A. Bast, J. Behr, P.J.A. Borm, S.J. Bourke, Ph. Camus, P. De Vuyst, H.M. Jansen, V.L. Kinnula, D. Lison, et al. Interstitial lung disease induced by exogenous agents: factors governing susceptibility Eur. Respir. J., July 1, 2001; 18(32_suppl): 30S - 42s. [Abstract] [Full Text] [PDF]
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T. Koura, Y. Gon, S. Hashimoto, A. Azuma, S. Kudoh, Y. Fukuda, I. Sugawara, J. Yodoi, and T. Horie Expression of thioredoxin in granulomas of sarcoidosis: possible role in the development of T lymphocyte activation Thorax, September 1, 2000; 55(9): 755 - 761. [Abstract] [Full Text]
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 B. L. Weller, H. Witschi, and K. E. Pinkerton Quantitation and Localization of Pulmonary Manganese Superoxide Dismutase and Tumor Necrosis Factor {alpha} following Exposure to Ozone and Nitrogen Dioxide Toxicol. Sci., April 1, 2000; 54(2): 452 - 461. [Abstract] [Full Text] [PDF]
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E. LAKARI, P. PÄÄKKÖ, P. PIETARINEN-RUNTTI, and V. L. KINNULA Manganese Superoxide Dismutase and Catalase Are Coordinately Expressed in the Alveolar Region in Chronic Interstitial Pneumonias and Granulomatous Diseases of the Lung Am. J. Respir. Crit. Care Med., February 1, 2000; 161(2): 615 - 621. [Abstract] [Full Text]
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 P. Pietarinen-Runtti, E. Lakari, K. O. Raivio, and V. L. Kinnula Expression of antioxidant enzymes in human inflammatory cells Am J Physiol Cell Physiol, January 1, 2000; 278(1): C118 - C125. [Abstract] [Full Text] [PDF]
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S. A. A. COMHAIR, M. J. LEWIS, P. R. BHATHENA, J. P. HAMMEL, and S. C. ERZURUM Increased Glutathione and Glutathione Peroxidase in Lungs of Individuals with Chronic Beryllium Disease Am. J. Respir. Crit. Care Med., June 1, 1999; 159(6): 1824 - 1829. [Abstract] [Full Text]
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