Kazuyoshi Kuwano

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.

TGF-β1 as an Enhancer of Fas-Mediated Apoptosis of Lung Epithelial Cells

Transforming growth factor-β1 (TGF-β1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. In this study, we show that TGF-β1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-β1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-β Ab. Otherwise, BALF from patients with hypersensitivity pneumonitis (HP) could not induce apoptosis on bronchiolar epithelial cells, despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-β1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, coincubation with the low concentration of TGF-β1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-β1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-β1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.

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.

Immunohistochemically detected p53 and P-glycoprotein predict the response to chemotherapy in lung cancer

While resistance to chemotherapy is a major problem in lung cancer treatment, there is no useful predictor of treatment response. We thus designed this study to determine the utility of p53 and P-glycoprotein expression in predicting the response to chemotherapy in patients with primary lung cancer, retrospectively. We evaluated transbronchial biopsy (TBB) specimens from 60 patients with lung cancer, who were previously untreated. Formalin-fixed, paraffin-embedded TBB specimens were immunostained using anti-p53 antibody (DO-1) and anti-P-glycoprotein antibody (JSB-1). The positivity of p53 was 63%, and that of P-glycoprotein was 17%. No correlation was observed between p53 and P-glycoprotein immunostaining. Positivity of p53 correlated significantly (P = 0.004) with a lack of response to chemotherapy in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). In contrast, positivity of P-glycoprotein was correlated with chemotherapy resistance in SCLC (P = 0.003), but not in NSCLC. Multiple logistic regression analysis revealed that positive immunostaining for p53 was a significant risk factor for chemotherapy resistance in NSCLC. These results suggest that immunostaining of p53 and P-glycoprotein for TBB specimens may help to predict response to chemotherapy in NSCLC and SCLC, although the results should be confirmed in a larger, more homogeneous series.

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.

Analysis of Fas and Fas ligand expression and function in lung cancer cell lines

The aim of this study was to investigate the expression of Fas and Fas ligand (FasL) and to determine the significance of these molecules in lung cancer cell lines. Immunoblotting, RT-PCR and flow cytometric analyses were carried out to measure the expression of Fas and FasL and to examine their interactions and effects on cell growth and apoptosis. Fas and FasL were co-expressed in most of the cell lines but to varying degrees. Apoptosis induced by the agonistic anti-Fas antibody was significantly correlated with Fas expression (P=0.0075), whereas cisplatin-induced apoptosis was not. Upregulation of Fas and FasL expression by the administration of cisplatin was found in 7 of 11 (64%) and 9 of 11 (82%) cell lines, respectively. However, cisplatin-induced apoptosis was not suppressed by antagonistic anti-FasL antibody. Thus, our data indicated that Fas and FasL were co-expressed in lung cancer cell lines, and that Fas ligation induced by agonistic anti-Fas antibody is functional and induced apoptosis that was dependent on the levels of Fas expression. In contrast, Fas-FasL interactions appeared to be non-functional. Furthermore, our results suggest that cisplatin-induced apoptosis in lung cancer cells was independent of the Fas-FasL interaction.

Expression of FasL and Fas Protein and Their Soluble Form in Patients with Hypersensitivity Pneumonitis

Background: Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis and loosely formed granulomas in lung biopsy specimens. HP improves or disappears altogether after cessation of antigen exposure. The Fas-Fas ligand (FasL) system is one of the representative systems of apoptosis-signaling receptor molecules, and is involved in various inflammatory diseases. We hypothesized that the Fas-FasL system may be associated with this disorder. Methods: We examined the expression of FasL and Fas proteins in lung tissues from patients with HP using immunohistochemistry. We also measured the soluble form of FasL (sFasL) and sFas levels in serum and bronchoalveolar lavage fluid (BALF) from patients with HP using enzyme-linked immunosorbent assay (ELISA). Furthermore, we also measured the cytotoxic activity of BALF sFasL in vitro. Results: FasL was detected in infiltrating mononuclear cells, and Fas was detected in infiltrating mononuclear cells, alveolar macrophages, and epithelioid cells in HP, whereas FasL was not detected and Fas was detected in few alveolar macrophages in controls. The levels of sFasL and sFas in BALF, but not in serum, were significantly increased in HP compared with controls. BALF of HP that included high levels of sFasL had no cytotoxic activity for bronchiolar epithelial cells in vitro. Conclusions: In HP, there is an upregulation of FasL and Fas in lung tissues. Since there is no incidence of apoptosis and no cytotoxic activity for lung epithelial cells in BALF from patients with HP, the increased levels of BALF sFasL and sFas may reflect the activation and sequestration of inflammatory cells rather than apoptosis.