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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pulmonary fibrosis begins with alveolitis, which progresses to destruction of lung tissue and excess collagen deposition. This process could be the result of DNA damage and a form of apoptosis. Therefore, we hypothesized that Fas ligand (FasL), which induces apoptosis in cells expressing Fas antigen (Fas), is associated with pulmonary fibrosis. We examined frozen lung tissues from seven patients with idiopathic pulmonary fibrosis (IPF), and bronchoalveolar lavage fluid (BALF) cells from 19 patients with IPF and from 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP). We used five frozen lungs with normal lung parenchyma and BALF cells from 10 patients with solitary pulmonary nodule as controls. Reverse transcription-polymerase chain reaction (RT-PCR) showed that FasL messenger RNA (mRNA) was expressed in BALF cells from all patients with IPF and from 15 of 16 patients with CVD-IP. FasL mRNA was not detected in BALF cells except in one of 10 controls. RT in situ PCR detected FasL mRNA in inflammatory cells in BALF from patients with IPF. Immunohistochemistry detected FasL protein in infiltrating lymphocytes and granulocytes in all of seven frozen lung tissues of IPF, but in none of five control lung tissues. Additionally, the expression of Fas appeared to be upregulated in bronchiolar and alveolar epithelial cells in IPF compared with normal lung parenchyma by immunohistochemistry. We conclude that Fas and FasL were upregulated in fibrosing lung diseases and may associate with DNA damage or apoptosis of bronchiolar and alveolar epithelial cells in this disorder.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Fas antigen (Fas) is a cell surface protein that mediates apoptosis and is expressed in various cells and tissues, including the thymus, liver, ovary, heart, and lung (1). It has structural homology with several cell surface receptors, including tumor necrosis factor (TNF) receptor and nerve growth factor receptor (1). Fas ligand (FasL), a cell surface molecule belonging to the TNF family, binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells (2). FasL was expressed in predominantly activated T lymphocytes and in tissues including small intestines, kidney, testis, and lung (2). In the immune system, Fas and FasL are involved in downregulation of immune reactions (3). Malfunction of the Fas-FasL pathway causes lymphoproliferative disorders (2, 6) and accelerates autoimmune diseases, whereas its exacerbation may cause tissue destruction (7, 8).
Although apoptosis has been implicated before as a homeostatic mechanism, it may have a role in human diseases in two different ways. First, diseases may be caused by a malfunction of apoptosis mechanism. Repair after an acute lung injury requires the elimination of proliferating mesenchymal and inflammatory cells from the alveolar airspace or alveolar walls (9). Failure to clear unwanted cells by apoptosis will prolong the inflammation because of the release of their toxic contents. Second, excessive apoptosis may cause diseases. An intraperitoneal injection of agonistic anti-Fas antibody into adult mice caused hepatic failure and death, suggesting that acute fulminant hepatitis in humans may be caused by excessive apoptosis mediated by the Fas-FasL system (7).
Although the etiology of idiopathic pulmonary fibrosis (IPF) is still uncertain, it is felt that the initial lesion prior to the formation of fibrosis is probably alveolitis, which is characterized by the loss of type I epithelial cells, and type II pneumocyte hyperplasia (10, 11). We demonstrated previously that there was DNA damage or apoptosis in the bronchial and alveolar epithelial cells in IPF (12) using the in situ DNA nick end-labeling method, a commonly accepted method for the detection of the apoptotic process (13). It has been demonstrated that Fas is expressed in bronchiolar and alveolar epithelial cells in normal lung tissues (14). In this study, we hypothesized that excessive expression of FasL participates in the loss of lung parenchyma and in pulmonary fibrosis, and examined the expression of FasL messenger RNA (mRNA), FasL protein, and Fas in lung tissues from patients with IPF.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patients
Patients' characteristics, including sex, age, smoking, and means of obtaining the specimens, are summarized in Table 1. The study of IPF was performed on 19 patients. Bronchoalveolar lavage fluid (BALF) cells were obtained from all of 19 patients, and lung specimens were obtained from seven patients by open lung biopsy and from 12 patients by transbronchial lung biopsy. The study of BALF cells was also performed on 16 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP). There were nine cases of rheumatoid arthritis, two cases of polymyositis, and one case each of dermatomyositis with Sjögren's syndrome, rheumatoid arthritis with progressive systemic sclerosis, polymyositis with progressive systemic sclerosis, Sjögren's syndrome, and systemic lupus erythematosus. The diagnoses of IPF and CVD-IP were established by a combination of medical history, physical examination, laboratory tests, chest roentgenograms, pulmonary function tests, and the results of pulmonary biopsies, according to previously described criteria (10, 11, 15). Patients with CVD-IP were all in the chronic stage and exhibited the features of fibrosing alveolitis in transbronchial lung biopsy specimens. The results from those with IPF and CVD-IP were compared with those in 10 control patients with normal lung parenchyma obtained by lobectomy for solitary pulmonary nodule.
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Analysis of BALF Cells
BALF cells were obtained from 19 patients with IPF, 16 patients with CVD-IP, and 10 patients with normal lung parenchyma. BAL was performed using a total of 150 ml of sterile physiologic saline solution. The recovered fluid was filtered through a single layer of gauze to remove mucus. Cells in the lavage fluid were counted using a hemocytometer. Differential counts were performed on a total of 100 cells stained with Wright and Giemsa stain.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis for FasL mRNA in BALF Cells
RNA from BALF cells was prepared by the method of Wilkinson (16). Briefly, cytoplasmic membrane of BALF cells was treated with lysis buffer (22.5 mM Tris-HCl, 0.12 M NaCl, 4.5 mM KCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.01% dextran sulfate) and centrifuged, and supernatant was subjected to phenol/chloroform extraction. For PCR analysis of RNA, complementary DNA (cDNA) was prepared by RT of 1 µg of each RNA sample. The cDNAs were then diluted to 50 µl, and the same DNA mixtures were used in all PCRs. PCRs were performed in a 50-µl reaction volume containing 5 µl of each cDNA, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 or 2.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM dNTPs, and 1.25 U of Taq polymerase (Takara Biochemicals, Otsu, Japan). The primers and probes used are as follows.
(1) 
-actin:
sense 5'-TCCTGTGGCATCCATGAAACT-3',
antisense 5'-CTTCGTGAACGCCACGTGCTA-3'.
(2) FasL: sense 5'-ATCCCTCTGGAATGGGAAGA-3', antisense 5'-CCATATCTGTCCAGTAGTGC-3', probe 5'-GAGGATCTGGTGCTAATGGA-3'.
The condition for amplification of -actin and FasL was
as follows: 93°C for 3 min for one cycle, 93°C for 1 min,
55°C for 1 min, 72°C for 2 min for 35 cycles, and 72°C for
7 min for one cycle. Cycle curve studies confirmed that, for
the amounts of cDNA being amplified, the reactions had
not reached the plateau of the amplification curve for any
primer pair. Negative controls performed with no RT yielded
no detectable fragments with either primer pair. PCR
products for FasL cDNA were transferred to filters and
hybridized with an oligonucleotide probe labeled with
digoxigenin-ddUTP by using the DIG Oligonucleotide
3'-End Labeling Kit (Boehringer Mannheim, Mannheim,
Germany). Hybridizations were performed according to
the manufacturer's recommendation, and digoxigenin-labeled probe that hybridized with PCR products was detected
with the DIG Nucleic Acid Detection Kit (Boehringer
Mannheim).
RT In Situ PCR Analysis for FasL mRNA in BALF Cells
The BALF cells from all patients from IPF, CVD-IP, and control groups were analyzed by RT in situ PCR for FasL mRNA. RT in situ PCR was performed as previously described (17) according to the method of Nuovo (18). Briefly, poly-L-lysin-coated slides containing cells were fixed in 10% buffered formalin for 8 h. The specimen was immersed in 20 µg/ml proteinase K, then treated overnight with an RNase-free DNase I (GIBCO BRL, Gaithersburg, MD) at 37°C. The specimens were incubated on the glass slide at 42°C for 30 min with 10 µl of a solution which contained the downstream primer (1 µM) and Molony-murine leukemia virus-reverse transcriptase (GIBCO BRL). The solution for the amplification of the FasL cDNA contained 4.5 mM MgCl2, 200 µM each of dNTPs, 10 µM digoxigenin-11-dUTP, 1 µM of each primer, 100 µg/ml of bovine serum albumin, and 5 U of Taq polymerase (Takara)/25 µl amplifying solution. The condition for amplification was 93°C for 1 min, and 55°C for 2 min, for 20 cycles. The digoxigenin-11-dUTP-labeled PCR product was detected after incubation with an alkaline phosphatase antidigoxigenin conjugate for 30 min and development in NBT/BCIP color reaction solution for 30 min in a dark room. The positive control for RT in situ PCR was to eliminate DNase digestion. An intense nuclear signal was generated from target-specific amplification, DNA repair, and mispriming (18). This control demonstrated that the PCR reaction and the subsequent detection steps worked. The negative control was RT in situ PCR in which the tissue was treated with DNase and the RT step was eliminated. The absence of a signal demonstrated that amplification of genomic DNA was not occurring.
Immunohistochemistry for FasL, CD4, CD8, and CD15
Seven frozen lung tissues of IPF obtained by open lung biopsy, and five frozen normal lung tissues obtained by lobectomy were used. Four-micrometer cryostat sections of lung tissues were used for immunohistochemistry (IHC) with rat anti-FasL monoclonal antibody (Alexis, San Diego, CA), mouse antihuman CD15 monoclonal antibody (Cosmo Bio., Tokyo, Japan), mouse antihuman CD4 monoclonal antibody (Dako, Ltd., Kyoto, Japan), and mouse antihuman CD8 monoclonal antibody (Novocastra, Newcastle, UK). The FasL-specific monoclonal antibody was immunolocalized with biotin-conjugated rabbit antirat IgM (Zymed, San Francisco, CA). The anti-CD15, -CD4, and -CD8 were immunolocalized with biotin-conjugated rabbit antimouse immunoglubin G (IgG; Nichirei, Tokyo, Japan). These stains were visualized with a Nichirei SAB-PO kit (Nichirei). The dual labeling for FasL with CD4, CD8, or CD15 was visualized with a Nichirei SAB-PO kit or avidin-alkaline phosphatase-substrate system (Vector Laboratories, Burlingame, CA). For control incubations, specific antibodies were replaced by nonimmune rat IgM or nonimmune mouse IgG.
IHC for Fas
IHC for Fas was performed on formalin-fixed, paraffin-embedded lung tissues, which consisted of specimens obtained by open lung biopsy from seven patients with IPF and specimens obtained by lobectomy from 10 controls. We used hydrated autoclaving as a pretreatment to immunostaining for Fas as previously described by Shin and colleagues (19). Following deparaffinization, the tissue sections were autoclaved at 120°C for 20 min in a stainless-steel pot filled with distilled water to completely immerse the sections (hydrated autoclaving). IHC was performed using a modified streptavidin-biotinylated peroxidase technique using a Histofine SAB-PO kit from Nichirei Corp. The sections were incubated with mouse antihuman Fas monoclonal antibody (UB2) (MBL, Nagoya, Japan) at 4°C overnight. The sections were rinsed with phosphate-buffered saline and incubated with biotinylated antimouse IgG for 30 min. For control incubations, anti-Fas antibody was replaced by nonimmune mouse IgG.
Statistics
To determine statistical significance for differential counts of BALF cells, a Student's t test for nonpaired data was performed. For RT-PCR analysis, the positivity of FasL mRNA in the IPF, CVD-IP, and control groups was analyzed by chi-square test. A P value of less than 0.05 was considered statistically significant.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Differential Percentage Counts of BALF Cells
The increased percentage counts of inflammatory cells in the BALF samples of the patients with IPF and CVD-IP were contrasted with those in samples from control subjects (Table 2). The majority of cells in the control samples were macrophages. By contrast, the fibrosing lung diseases were clearly distinguished from controls by their significant increases in lymphocytes, neutrophils, and eosinophils. These results were compatible with previous studies (20).
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RT-PCR and RT In Situ PCR Analysis for FasL mRNA in BALF Cells
RT-PCR analysis detected the specific bands of FasL mRNA with a molecular weight of 209 base pairs in BALF cells obtained from all of 19 (100%) patients with IPF and 14 of 16 (87.5%) cases with CVD-IP, but only one of 10 (10%) control patients (Figure 1). The positivity of FasL mRNA in IPF and CVD-IP groups was significantly higher than that in the control group (P < 0.05). We repeated RT-PCR for FasL mRNA three times, and these results were consistent. The results of RT in situ PCR for FasL mRNA demonstrated positive signals in BALF cells from 14 of 19 (73.7%) patients with IPF and from 10 of 16 (62.5%) patients with CVD-IP, but not in control subjects (Figure 2).
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IHC for FasL, CD4, CD8, CD15, and Paired IHC for FasL with CD4, CD8, or CD15
Figure 3 shows the results of IHC for FasL protein and paired IHC for FasL protein with CD4, CD8, or CD15. FasL protein was detected in a substantial number of infiltrating inflammatory cells in frozen lung tissues obtained from all of seven cases with IPF, but in none of five normal lung parenchyma. To investigate whether FasL is expressed in CD4+ or CD8+ T lymphocytes, and in granulocytes in vivo, we performed paired IHC for FasL with CD4, CD8, or CD15. The numbers of T lymphocytes positively stained with anti-CD4 or anti-CD8 antibody were almost equal, and a substantial number of lymphocytes positively stained with anti-FasL antibody were also stained with anti-CD4 or anti-CD8 antibody. FasL protein was also detected in granulocytes positively stained with anti-CD15 antibody.
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IHC for Fas
Figure 4 shows the representative results of IHC for Fas. The prominent signals appeared to be detected in bronchiolar and alveolar epithelial cells as well as macrophages in all of seven cases of IPF, but in none of the specimens from 10 controls except weak signals in some macrophages and epithelial cells.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have demonstrated here that FasL protein was expressed in lymphocytes and granulocytes infiltrating into lung tissues obtained from patients with IPF, and that FasL mRNA was also detected in inflammatory cells in BALF obtained from patients with IPF and CVD-IP. In contrast, FasL mRNA or protein was not detected or was detected at significantly lower levels in normal lung parenchyma and in BALF cells. Fas appeared to be expressed in bronchiolar and alveolar epithelial cells as well as macrophages in IPF, whereas no or very weak signals were detected in normal lung parenchyma. We therefore speculate that FasL expressed by infiltrating lymphocytes and granulocytes present at close to bronchiolar and alveolar epithelial cells may be associated with DNA damage or apoptosis of epithelial cells in fibrosing lung diseases.
FasL is expressed mainly in activated T lymphocytes, and cytotoxic T cells can deploy FasL as a death effector molecule in their strategies to induce killing of target cells (23, 24). Fas-dependent lysis appears to be a secondary pathway with CD8+ cytotoxic T lymphocytes (23) but a primary mechanism with many CD4+ lytic effectors (26, 27). We have also demonstrated that FasL was expressed in both CD4+ and CD8+ T lymphocytes in IPF. Recently, Conrad-Liles and associates demonstrated that FasL was expressed significantly in neutrophils in vitro (28). We found that FasL protein appeared to be expressed in granulocytes as well as lymphocytes in lung tissues from patients with IPF. FasL expressed by granulocytes may also be one important mediator of tissue damage in fibrosing lung diseases.
Previously we showed Fas mRNA expression and apoptosis in alveolar epithelial cells, and excessive expression of FasL mRNA in infiltrating inflammatory cells in bleomycin-induced pulmonary fibrosis, which is a mouse model of IPF (17). We also demonstrated that the inhalation of agonistic anti-Fas antibody induced pulmonary fibrosis in mice (29). Therefore, apoptosis and excessive expression of FasL appear to be consistent in animal models of pulmonary fibrosis and human diseases.
Hepatocyte transformed with human hepatitis C virus would express virus antigens and activate cytotoxic T lymphocytes to express FasL, which would then bind to Fas on hepatocytes, inducing them to undergo apoptosis (8). This process normally occurs to remove virus-infected cells, but excessive apoptosis may lead to fulminant hepatitis. In polycystic human kidneys, it also has been demonstrated that apoptotic nuclei can be detected in noncystic tubular epithelial cells, in cells within glomeruli, and in cells lining renal cysts, but not in kidneys from normal humans (30). In addition, apoptotic myocardial cell death occurs in arrythmogenic right ventricular dysplasia (31) and in end-stage cardiomyopathy (32). These studies in other organs also suggest that excessive apoptosis and/or FasL overexpression may be associated with the damage and loss of normal tissue, and may lead to secondary interstitial fibrosis.
Khalil and coworkers have demonstrated increased
production of transforming growth factor (TGF)- in the
epithelial cells of the terminal airways and alveoli in IPF
(33). TGF- inhibits cell growth and is associated with alterations in the phosphorylation and subcellular localization
of p53 protein in vitro (34). It also has been shown that
TGF- upregulates p21 (35). Anti-Fas antibody crosslinking induces Fas-mediated apoptosis through upregulation of p53 and p21 in p53-transfected human colon carcinoma
cells (36), and the induction of Fas expression by exogenous wild-type p53 has been demonstrated (37). We previously noted that the expression of p53 and p21 (WAF1/
Cip1/Sdi1) was upregulated in bronchiolar and alveolar
epithelial cells (12) as well as Fas in IPF. The localization
of TGF-, p53, p21, and Fas are consistent, that is, predominantly in the epithelial cells of the terminal airways
and alveoli. We speculate that these cyclin-dependent kinase inhibitory proteins and the Fas-FasL pathway, which
may be linked to each other, regulate the cell proliferation
and death of bronchial and alveolar epithelial cells.
Although many factors are known to promote growth,
differentiation, or survival, only a few cytokines, including
FasL and TNF-
, have been found to induce apoptosis. It
is known that TNF- mediates bleomycin-induced pulmonary fibrosis (38), and that the expression of TNF- transgene in murine lung causes lymphocytic and fibrosing alveolitis (39). TNF- causes inflammation by damaging the
tissue and by inducing the expression of adhesion molecules and cytokines in epithelial and endothelial cells as well as in inflammatory cells. Agonistic anti-Fas antibody
administration can lead to more production of interleukin-8 than does TNF- administration by colonic epithelial cells in vitro (40), which represents the possibility of
cross-linking of functions of FasL other than apoptosis. As
well as TNF-, FasL may play a role as a proinflammatory cytokine in the pathophysiology of pulmonary fibrosis.
We have reported in vivo evidence indicating that expression of FasL was upregulated in lymphocytes and granulocytes in lung tissues or BALF from patients with IPF and CVD-IP. Fas also appeared to be upregulated in bronchiolar and alveolar epithelial cells in IPF. FasL may participate in tissue remodeling to remove unrepairable resident cells and inflammatory cells; however, overexpression of FasL may participate in tissue destruction and fibrosis by inducing apoptosis or by modulating inflammatory mediators. These findings may shed new light on the pathogenesis of pulmonary fibrosis. If the Fas-FasL system is involved in the destruction of bronchiolar and alveolar epithelial cells, this knowledge may be useful in finding a way to prevent the progression of pulmonary fibrosis.
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Footnotes |
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Address correspondence to: Kazuyoshi Kuwano, M.D., Research Institute for Diseases of the Chest, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka, 812 Japan. E-mail: kkuwano{at}kokyu.med.kyushu-u.ac.jp
(Received in original form February 26, 1997 and in revised form May 15, 1998).
Abbreviations: bronchoalveolar lavage fluid, BALF; interstitial pneumonia associated with collagen vascular disease, CVD-IP; Fas antigen, Fas; Fas ligand, FasL; immunoglobulin G, IgG; immunohistochemistry, IHC; idiopathic pulmonary fibrosis, IPF; reverse transcription-polymerase chain reaction, RT-PCR; transforming growth factor, TGF; tumor necrosis factor, TNF.Acknowledgments: This work was supported by a Grant-in-Aid for Scientific Research (09670620) from the Ministry of Education, Science and Culture of Japan.
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References |
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Introduction
Materials and Methods
Results
Discussion
References
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