Tokuji Matsuba

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.

MAP kinase activation and apoptosis in lung tissues from patients with idiopathic pulmonary fibrosis.

Three major MAP kinases (MAPKs), including extracellular signal‐regulated kinase (ERK), c‐jun N‐terminal kinase (JNK), and p38 kinase (p38 MAPK), are involved in the regulation of lung inflammation and injury. This study investigated whether MAPKs are activated and associated with lung injury in lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The expression of the active ERK, JNK, and p38 MAPK was examined using western blot analysis and immunohistochemistry and apoptosis was also examined by the TUNEL method, in lung tissues from ten patients with IPF obtained by thoracoscopic biopsy and in eight normal lung parenchyma specimens obtained by lobectomy for lung cancer. Activated MAPKs are significantly increased in lung homogenates from patients with IPF compared with controls. Activated ERK in epithelial and endothelial cells, but not in fibroblasts or smooth muscle cells, was decreased, accompanied by the progression of fibrosis. Activated JNK in epithelial and endothelial cells, but not in fibroblasts, was increased, accompanied by the progression of fibrosis. Activated p38 MAPK in epithelial, endothelial, smooth muscle cells, and fibroblasts was increased at the intermediate stage of fibrosis, in which the TUNEL‐positive cells were predominantly detected. This is the first study to suggest that MAPKs may be associated with the regulation of inflammation and lung injury in IPF. Copyright

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.

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.

CT diagnosis of tracheobronchopathia osteochondroplastica.

Accessible online at: www.karger.com/journals/res A 72-year-old man with a persistent dry cough was admitted for further evaluation. He had a 50 pack-year history of cigarette use with no occupational exposure to dust. The chest radiograph revealed overinflation of both lungs. A chest computerized tomography (CT) scan showed a thickened and irregular wall of the distal trachea (fig. 1a) extending to the level of the carina (fig. 1b) with multiple calcified nodules and plaques of bony density projecting into the lumen. These lesions, prominent in the anterior wall, gave rise to a suspicion of TO (tracheobronchopathia osteochondroplastica). Bronchoscopy disclosed multiple hard, yellow-white papilla-like formations on the anterolateral wall of the trachea and main bronchi, with an intact membranous portion (fig. 2). The mucosa overlaying the lesions appeared normal with no abnormality in the more distal bronchi. A microscopical examination of the biopsy specimens showed irregular deposits of bone with fatty marrow tissue below the basement membrane of the squamous metaplastic epithelium, consistent with the diagnosis of TO. A chest CT scan, which had been performed during an episode of bronchopneumonia 1 year previously, also showed nearly identical findings. These findings, however, were overlooked. Thus, detection and diagnosis of TO require careful interpretation of CT findings with extensive knowledge of the disorder. Sparing of the posterior membrane – nicely demonstrated on CT and bronchoscopy – is typical of TO.

Immunohistochemical Localization of B7 Costimulating Molecules and Major Histocompatibility Complex Class II Antigen in Pulmonary Sarcoidosis

Background: Alveolar macrophages (AM) of sarcoidosis have an enhanced capacity to mediate antigen-induced T lymphocyte proliferation. To induce an effective immune response, antigen-presenting cells have to not only present antigenic peptide with MHC molecules to T lymphocytes, but also express B7 costimulating molecules. Objective: The purpose of this study was to investigate the expression of B7 and MHC molecules in lung tissues from patients with sarcoidosis. Methods: We performed immunohistochemistry for B7-1, B7-2 and MHC class II antigens using transbronchial lung biopsy specimens obtained from patients with sarcoidosis and normal lung parenchyma obtained by lobectomy for solitary pulmonary nodule as controls. Results: B7-1, B7-2 and MHC class II antigen were expressed in epithelioid cells in granulomas in 14 (93.3%), 2 (13.3%) and 9 (60.0%) of 15 patients with sarcoidosis, respectively. These were also expressed in AM in 14 (93.3%), 5 (33.3%) and 12 (80.0%) of 15 patients with sarcoidosis, respectively. The positivitiy of B7-1 was significantly higher than that of B7-2 in both epithelioid cells and AM in sarcoidosis (p < 0.01). Positive signals for B7-1, B7-2 and MHC class II antigen were also found in AM in 9 (90%), 8 (80%) and 8 (80%) of 15 of controls, respectively. However, the intensity of positive signals for B7-1, but not B7-2 or MHC class II antigen in AM was significantly increased in sarcoidosis compared to controls (p < 0.05). Conclusions: These results suggested that epithelioid cells in granulomas and AM from patients with sarcoidosis had the capability to act as accessory cells and that the accessory function of these cells was shifted to B7-1 rather than B7-2 in sarcoidosis.

Expression of B7–1, B7–2, and Interleukin–12 in Anti–Fas Antibody–Induced Pulmonary Fibrosis in Mice

Background: We have previously reported that the inhalation of anti–Fas antibody induced pulmonary fibrosis in mice. To induce an effective immune response, antigen–presenting cells have to not only present antigenic peptide with MHC molecules to T lymphocytes, but also express B7 costimulating molecules. The purpose of this study is to investigate whether B7 family costimulating molecules and interleukin–12 (IL–12), which primarily promote cellular immunity, are associated with anti–Fas antibody–induced pulmonary fibrosis. Methods: We examined the expression of B7–1, B7–2, and IL–12 using the revese transcription–polymerase chain reaction (RT–PCR), RT–in situ PCR, and immunohistochemistry. Results: We observed the upregulation of B7–1, B7–2, and IL–12 p40 mRNA after anti–Fas antibody inhalation. B7–2 and IL–12 p40 mRNA appeared to be expressed in mononuclear cells, while B7–1 mRNA and protein were expressed in bronchiolar epithelial cells as well as macrophages. Conclusion: These findings indicate that the T–cell–mediated immune response in this model involved the upregulation of B7–1, B7–2, and IL–12, and that the aberrant expression of B7–1 in bronchiolar epithelial cells may induce autoreactive T cell proliferation against themselves.

Immunogenetics of Allergy: Towards Prevention and Care

International Archives of Allergyand Immunology Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com F 1999 S.Karger AG, Basel 1018–2438/99/1192–0143