Ritsuko Kunitake

Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis.

The Fas ligand is predominantly expressed in activated T lymphocytes and is one of the major effector molecules of cytotoxic T lymphocytes and natural killer cells. Previously, we found excessive apoptosis of epithelial cells and infiltrating lymphocytes expressing Fas ligand mRNA in the lung tissue of bleomycin-induced pulmonary fibrosis in mice. Here we demonstrated that the administration of a soluble form of Fas antigen or anti-Fas ligand antibody prevented the development of this model and that lpr and gld mice were resistant against the induction of pneumopathy. These results suggest that the Fas-Fas ligand pathway plays an essential role in the development of pulmonary fibrosis and that preventing this pathway could have therapeutic value in lung injury and fibrosis.

Protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor

LPS (lipopolysaccharide) is one of the major factors that induce acute lung injury. Recently, it was reported that LPS induced disseminated endothelial apoptosis, preceding nonendothelial tissue damage. Caspases play important roles in apoptosis, including tumor necrosis factor-alpha-induced apoptosis, in several systems. We therefore investigated whether the injection of a caspase inhibitor prevents LPS-induced apoptosis and acute lung injury in mice. LPS (30 mg/kg) was administered intravenously to Institute for Cancer Research mice. Electron microscopic findings demonstrated characteristic features of apoptosis in endothelial cells and alveolar epithelial cells. The caspase-3 activity and the number of terminal dUTP nick-end labeling-positive cells in lung tissues were significantly increased after LPS administration. Benzyloxycarbonil-Val-Ala-Asp fluoromethylketone (Z-VAD.fmk), which is a broad-spectrum caspase inhibitor, was injected before and after the administration of LPS. The injection of Z-VAD.fmk suppressed the caspase-3 activity in lung tissues, and significantly decreased the number of terminal dUTP nick-end labeling-positive cells. Furthermore, the survival rate of mice was prolonged significantly by the injection of Z-VAD.fmk. These results indicate that apoptosis may play an important role in acute lung injury, and thus that inhibition of caspase activity may constitute a new therapeutic approach for treatment of this disease.

Expression of apoptosis-regulatory genes in epithelial cells in pulmonary fibrosis in mice.

Up‐regulation of Fas and Fas ligand and excessive apoptosis of bronchiolar and alveolar epithelial cells were identified in bleomycin‐induced pulmonary fibrosis in mice. This study hypothesized that apoptosis‐regulatory genes other than Fas–Fas ligand, such as p53, p21 (Waf1/Cip1), bcl‐2, bcl‐x, and bax, may also participate in epithelial cell apoptosis in this model. The expression of these genes was assessed by reverse transcription polymerase chain reaction (RT‐PCR), RT in situ PCR, or immunohistochemistry. The expression of p53 and p21 mRNA was concurrently up‐regulated in the alveolar epithelial cells at 1 h to 7 days after intratracheal instillation of bleomycin. The expression of bcl‐2 mRNA was weakly up‐regulated at 1 h to 14 days, while the expression level of bcl‐2 protein was not changed. The expression of bcl‐x(L) and bax mRNA was strongly up‐regulated at 1 h to 7 days. The expression of bcl‐x protein was up‐regulated in lymphocytes and macrophages, whereas bax protein was up‐regulated in both epithelial and inflammatory cells. It is concluded that epithelial cell apoptosis in this model may also be induced by the up‐regulation of p53 and bax and by the imbalance between apoptosis‐inducible and ‐inhibitory genes, in addition to the up‐regulation of the Fas–Fas ligand pathway. Copyright

Upregulation of Fas-signalling molecules in lung epithelial cells from patients with idiopathic pulmonary fibrosis

The caspase cascade is an executioner of apoptosis, mediated by Fas. Fas-associating protein with death domain (FADD) interacts with Fas and initiates apoptosis through activating caspase-8. It has previously been demonstrated that the Fas-Fas ligand pathway may be involved in the pathophysiology of idiopathic pulmonary fibrosis (IPF). The aim of this study was to investigate Fas-signalling molecules in epithelial cells in IPF. The immunohistochemistry for FADD and caspase-1 and -3 and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick endlabelling (TUNEL) methods were performed in lung tissues from 10 patients with IPF obtained by thoracoscopic biopsy and in seven normal lung parenchyma specimens. The induction of caspases expression and activation by Fas-ligation on lung epithelial cell line A549 was also investigated. The immunoreactivity grade for FADD and caspase-1 and -3, and positive signals for TUNEL were significantly increased in epithelial cells of IPF compared with controls. Fas-ligation induced upregulation of caspase-1 and -3 expression in the nucleus and cytoplasm in A549 cells. Procaspase-1, -3, and -8 were activated in apoptotic cells, but not in viable cells. Although direct measurement of the caspase activity in lung epithelial cells of idiopathic pulmonary fibrosis could not be made, these results suggest that the Fas-signalling pathway is upregulated in lung epithelial cells of idiopathic pulmonary fibrosis.

Detection of adenovirus E1A DNA in pulmonary fibrosis using nested polymerase chain reaction

The history of patients with idiopathic pulmonary fibrosis (IPF) shows that the disease may be preceded by a viral-like illness. Although viruses have not been demonstrated, it is possible that viruses were not detected in culture because they do not replicate during latency. We investigated the presence of adenovirus in IPF and interstitial pneumonia associated with collagen vascular disease (CVD-IP), using the nested polymerase chain reaction (PCR) and in situ hybridization (ISH) for the E1A region of the adenovirus genome. Studies were performed on lung tissues obtained by transbronchial lung biopsy from 19 patients with IPF, 10 patients with CVD-IP and, for comparison, from 20 patients with sarcoidosis. The E1A DNA was present in 3 out of 19 (16%) cases of IPF, in 5 of 10 (50%) cases of CVD-IP, and in 2 of 20 (10%) cases of sarcoidosis. The incidence of E1A DNA in CVD-IP was significantly higher than that in sarcoidosis (p<0.05). In patients with IPF and CVD-IP, E1A DNA was more prevalent in patients treated with corticosteroids (6 out of 9 cases; 67%) than in those without it (2 out of 20 cases; 10%) (p<0.01). ISH studies showed that 1 out of 8 cases of IPF and CVD-IP, in which E1A DNA was detected by PCR, was positive for E1A DNA. We conclude that adenovirus E1A is unlikely to be aetiologically involved in the pathogenesis of idiopathic pulmonary fibrosis or interstitial pneumonia associated with collagen vascular disease. However, a latent adenovirus infection may be reactivated or may newly infect the host following corticosteroid administration.

Apoptosis in the course of granulomatous inflammation in pulmonary sarcoidosis.

Sarcoidosis is a chronic inflammatory disease of unknown aetiology characterized by the formation of non-necrotizing granulomas. The course of disease is usually self-limiting with the spontaneous resolution of granuloma. In the immune system, Fas antigen (Fas) and Fas ligand (FasL) are involved in the down regulation of immune reactions by inducing apoptosis. Therefore, it was hypothesized that the Fas/FasL pathway and apoptosis may be associated with the course of granulomatous inflammation in sarcoidosis. Terminal deoxynucleotidyl transferase-mediated biotin nick end-labelling (TUNEL) was performed to assess deoxyribonucleic acid strand breakages as a characteristic of apoptosis. Immunohistochemistry was also performed to detect Fas and FasL protein, and reverse transcriptase polymerase chain reaction (RT-PCR) and RT in situ PCR to detect FasL messenger ribonucleic acid (mRNA). Positive signals for TUNEL were detected in epithelioid histiocytes and lymphocytes within granulomas and in bronchoalveolar lavage (BAL) lymphocytes from patients with sarcoidosis. Positive signals for Fas were also detected in these cells. FasL mRNA was expressed in BAL lymphocytes from 15 of 20 patients with sarcoidosis, but from only one of 10 patients with normal lung parenchyma. FasL protein was expressed in lymphocytes surrounding and within the granuloma. There was a significant correlation between the result of TUNEL and clinical course in patients with sarcoidosis. Apoptosis in epithelioid histiocytes and inflammatory cells seems to participate in the course of granulomatous inflammation. Further studies are needed to determine the role of Fas, FasL and other regulatory factors in apoptosis in the granulomatous inflammation in pulmonary sarcoidosis.

KL-6, Surfactant Protein A and D in Bronchoalveolar Lavage Fluid from Patients with Pulmonary Sarcoidosis

Background: KL-6, and surfactant protein A (SP-A) and surfactant protein D (SP-D) derived from alveolar type II cells and/or bronchiolar epithelial cells have been reported to be useful markers for interstitial lung diseases. Objective: The aim of this study was to measure the levels of these molecules in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis to investigate their relationship with other markers of inflammatory activity. Methods: We measured KL-6, SP-A and SP-D levels in BALF from patients with pulmonary sarcoidosis using an ELISA. Results: KL-6 and SP-D, but not SP-A levels were significantly increased in pulmonary sarcoidosis compared with controls. KL-6, SP-A and SP-D levels were significantly correlated with each other. KL-6 and SP-D levels were relatively and significantly correlated with the percentage of lymphocytes in BALF. KL-6, SP-D, but not SP-A levels were significantly correlated with the concentration of albumin in BALF. There was no significant correlation between KL-6, SP-A, or SP-D levels and chest X-ray findings, angiotensin-converting enzyme levels, or CD4/CD8 ratio in BALF. Conclusions: We conclude that KL-6 and SP-D levels in BALF were increased in pulmonary sarcoidosis. Since these markers are specifically derived from epithelial cells, it is considered that KL-6 and SP-D levels are reflecting damage or release of these markers from epithelial cells due to the inflammatory response.

Apoptosis and Fas/Fas ligand mRNA expression in acute immune complex alveolitis in mice

Deoxyribonucleic acid (DNA) strand breaks as a characteristic of apoptosis, and Fas antigen (Fas)/Fas ligand (FasL) expression may participate in acute immune complex alveolitis in mice. Male Institute for Cancer Research (ICR) mice were injected intravenously with immunoglobulin G (IgG) antibodies against ovalbumin and inhaled an aerosolized oval albumin (OA) solution. They were killed at 4, 6, 12, 24, 48 h and 7 days after aerosolization. We assessed DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL). The expression of Fas and FasL messenger ribonucleic acid (mRNA) in lung tissues was assessed by reverse transcriptase (RT) polymerase chain reaction, and by in situ hybridization (ISH) to localize Fas mRNA, and RT in situ polymerase chain reaction to localize FasL mRNA. The fragmentation of DNA extracted from lung tissue was found 6-24 h after OA inhalation. TUNEL detected positive signals in bronchial and alveolar epithelial, endothelial and inflammatory cells in the lung tissue. These positive signals had disappeared 7 days after OA inhalation. TUNEL also detected positive signals in apoptotic neutrophils in bronchoalveolar lavage fluid at 6-12 h. Fas mRNA was expressed in the alveolar epithelial and inflammatory cells, while the expression of FasL mRNA appeared to be upregulated in infiltrating inflammatory cells at 6-24 h. These results suggest that apoptosis may be associated with the resolution of inflammation and with tissue repair and also suggest the involvement of the Fas antigen/Fas ligand pathway in acute immune complex alveolitis in mice.

Expression of p53, p21 (Waf1/Cip1/Sdi1) and Fas antigen in collagen vascular and granulomatous lung diseases

Fas is expressed in various cells and transduces the cell death signal. p21 is a mediator of p53-dependent G1 arrest associated with deoxyribonucleic acid (DNA) damage. The upregulation of p53 and p21 associated with DNA damage in idiopathic pulmonary fibrosis has been described previously. In this study, p53, p21, and Fas expression and DNA damage were examined in interstitial pneumonia associated with collagen vascular diseases (CVD-IP). DNA damage was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end-labelling (TUNEL) and p53, p21 and Fas proteins were detected by immunohistochemistry in 13 cases of CVD-IP, 13 of sarcoidosis, seven of hypersensitivity pneumonitis (HP) and eight control patients with normal lung parenchyma. TUNEL-positive signals were found in bronchiolar or alveolar epithelial cells in 11 of 13 (85%) specimens of CVD-IP, but not in sarcoidosis, HP or controls, except for a case of chronic HP with pulmonary fibrosis. p53, p21 and Fas were detected in bronchiolar or alveolar epithelial cells in nine (69%), 10 (77%) and 12 (92%) of 13 specimens of CVD-IP, respectively, but not in sarcoidosis, HP or controls, except for a case of chronic HP. These results suggest that the upregulation of p53, p21 and Fas in bronchiolar and alveolar epithelial cells associated with deoxyribonucleic acid damage may participate in the process of pulmonary fibrosis in interstitial pneumonia associated with collagen vascular diseases and chronic hypersensitivity pneumonitis.

Detection of group C adenovirus DNA in small-cell lung cancer with the nested polymerase chain reaction.

Group C adenovirus is latent in human tissues and can malignantly transform cells. The purpose of this study was to investigate the association between this virus and lung cancer. We investigated latent adenoviral infection using the nested polymerase chain reaction and in situ hybridization in transbronchial biopsy specimens from patients with small-cell lung cancer and non-small-cell lung cancer. The polymerase chain reaction was performed on DNA extracts with two sets of primers directed at a 261-base-pair target sequence of the E1A region of the adenoviral genome. In situ hybridization was performed on histological sections using DNA representing the entire adenovirus type 5 genome. E1A target DNA was present in 11 (31%) of 35 cases of small-cell lung cancer but in none of the 40 cases of non-small-cell lung cancer (P<0.01). Of the 11 cases found positive by PCR, 8 were positive for adenovirus DNA by in situ hybridization. Adenovirus was prominent in tumor cells in 5 of the 8 cases, and in normal epithelial cells in the 3 remaining cases. Adenovirus DNA was not detected by in situ hybridization in specimens in which E1A DNA was not detected by the polymerase chain reaction. Small-cell lung cancer has mutations or deletions in the p53 and retinoblastoma genes more frequently than are found in non-small-cell lung cancer. Therefore, we speculate that adenovirus infection might participate in the pathogenesis of SCLC by producing mutation in these genes, rather than by inhibiting the function of these proteins.