The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region.

Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.

Apoptosis and epithelial injury in the lungs.

Epithelial injury is a critical event in the development of acute lung injury, but the mechanisms that cause death of the alveolar epithelium are not completely understood. Epithelial death occurs by necrosis and apoptosis; more information is needed about the balance between these two types of cell death in the lungs. Direct epithelial necrosis probably occurs in response to bacterial exotoxins and over-distension of alveolar units by mechanical ventilation. Apoptosis is a regulated form of cell death that is mediated by membrane death receptors and direct mitochondrial injury. Apoptosis pathways are activated in the lungs of patients with acute lung injury, in part by activation of the membrane Fas death receptor by soluble Fas ligand (sFasL), which accumulates in biologically active form at the onset of lung injury. Accumulating evidence in humans and experimental models links sFasL and Fas pathway with lung epithelial injury and fibrosis. New strategies to inhibit Fas-mediated epithelial apoptosis need to be developed in order to develop new ways to preserve epithelial function in patients who develop acute lung injury.

Anti-vascular endothelial growth factor gene therapy attenuates lung injury and fibrosis in mice.

Vascular endothelial growth factor (VEGF) is an angiogenesis factor with proinflammatory roles. Flt-1 is one of the specific receptors for VEGF, and soluble flt-1 (sflt-1) binds to VEGF and competitively inhibits it from binding to the receptors. We examined the role of VEGF in the pathophysiology of bleomycin-induced pneumopathy in mice, using a new therapeutic strategy that comprises transfection of the sflt-1 gene into skeletal muscles as a biofactory for anti-VEGF therapy. The serum levels of sflt-1 were significantly increased at 3-14 days after the gene transfer. Transfection of the sflt-1 gene at 3 days before or 7 days after the intratracheal instillation of bleomycin decreased the number of inflammatory cells, the protein concentration in the bronchoalveolar lavage fluid and with von Willebrand factor expression at 14 days. Transfection of the sflt-1 gene also attenuated pulmonary fibrosis and apoptosis at 14 days. Since the inflammatory cell infiltration begins at 3 days and is followed by interstitial fibrosis, it is likely that VEGF has important roles as a proinflammatory, a permeability-inducing, and an angiogenesis factor not only in the early inflammatory phase but also in the late fibrotic phase. Furthermore, this method may be beneficial for treating lung injury and fibrosis from the viewpoint of clinical application, since it does not require the use of a viral vector or neutralizing Ab.

[A case of rapidly deteriorated pulmonary aspergillosis with various clinical manifestations].

A 62-year-old man with chronic hepatitis was admitted to the hospital because of severe asthma, fever, and a chest radiograph abnormality. He had previously been treated unsuccessfully with several antibiotics in another hospital. The chest radiographs and CT films showed multiple infiltrations along the bronchi and in the peripheral regions of both lungs. Aspergillus fumigatus was detected in a sputum culture and the non-specific serum IgE was elevated. Allergic broncho-pulmonary aspergillosis (ABPA) was suspected and then steroids were administered. Although the asthma symptoms improved after the steroid therapy, lung infiltration deteriorated rapidly, affecting both lungs, and cavitations appeared. We concluded that invasive aspergillosis had developed from ABPA. Itraconazole and amphotericin B were administered, resulting in gradual improvements in the bilateral infiltration seen in the chest radiographs. The patient underwent open lung biopsy to rule out systemic vasculitis. The histological diagnosis was organizing pneumonia without vasculitis and without aspergillus or other organisms. The pathological findings resulted from the intensive anti-fungal therapy. There is a possibility that the temporal steroid therapy may have affected the conversion of ABPA to invasive aspergillosis.

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary fibrosis in mice.

Monocyte chemoattractant protein-1 (MCP-1) is a proinflammatory chemokine and may play an important role in the development of pulmonary fibrosis. We examined a new therapeutic strategy that comprises the transfection of the mutant MCP-1 gene into skeletal muscles as a biofactory for anti-MCP-1 therapy against bleomycin-induced pulmonary fibrosis in mice. Overexpression of the mutant MCP-1 gene at 10-14 days after intratracheal instillation of bleomycin resulted in decreased DNA damage, apoptosis, and pulmonary fibrosis at 14 days. However, overexpression of the mutant MCP-1 at 0-4 days after bleomycin instillation did not result in decreased pathological grade, DNA damage, or apoptosis at 7 and 14 days. Because, in this model, inflammatory cell infiltration begins at 3 days and is followed by interstitial fibrosis, it is likely that MCP-1 has an important role to play in the development of fibrogenesis but not in the development of early lung inflammation. This method does not require the use of viral vector or neutralizing antibody, and, as such, it is possible to avoid problems regarding the pathogenicity of the viral vector or immunocomplex. This new strategy may be a beneficial method of treating pulmonary fibrosis from the viewpoint of clinical application.

Induction of CDK inhibitor p21 gene as a new therapeutic strategy against pulmonary fibrosis.

Alveolar epithelial cells are known to be present at the primary site of lung damage in pulmonary fibrosis. Apoptosis has been implicated as being involved in epithelial cell damage and pulmonary fibrosis. Because the cyclin-dependent kinase inhibitor p21 induces G1 arrest and DNA repair and because it also prevents apoptosis in some cells, we hypothesized that p21 gene transfer may attenuate bleomycin-induced pulmonary fibrosis in mice, the pathogenesis of which likely involves epithelial cell apoptosis. Human p21 protein was expressed in mouse alveolar epithelial cells at 1-7 days in vitro and was detected predominantly in lung epithelial cells at 1-7 days in vivo after adenoviral transfer of the human p21 gene. Inflammatory cell infiltration and fibrosis had already begun at 7 days in this model. Adenoviral transfer of the human p21 gene at 7 days after intratracheal instillation of bleomycin led to a decrease in the number of apoptotic cells, lung inflammation, and fibrosis at 14 days. Therefore, the forced expression of p21 exerted both anti-apoptotic and anti-fibrotic effects, which would facilitate the ultimate goal of treatment for pulmonary fibrosis.

Mitochondria-mediated apoptosis of lung epithelial cells in idiopathic interstitial pneumonias.

We previously demonstrated that the up-regulation of p53, Fas, and DNA damage are present in lung epithelial cells from patients with idiopathic interstitial pneumonias (IIP). Fas ligation induces apoptosis of lung epithelial cells predominantly through the direct activation of the caspase cascade via caspase-8 activation, whereas the up-regulation of p53 and other cellular stresses can induce mitochondria-mediated apoptosis. In this study, we investigated the incidence of mitochondria-mediated apoptosis of epithelial cells in IIP. We performed TUNEL staining to detect apoptotic cells and western blot analysis and immunohistochemistry to assess the expression and activation of caspases and the cytochrome c release from mitochondria in lung tissues from eight patients with usual interstitial pneumonia, five patients with nonspecific interstitial pneumonia, and eight patients with normal lung parenchyma. The expressions of pro- and cleaved caspase-8, 9, 3, and cytochrome c release from the mitochondria were all significantly increased in the lung tissues of IIP compared with normal lung parenchyma. The positive signals for caspases in epithelial cells were increased in IIP compared with normal lung parenchyma by immunohistochemistry. The results of TUNEL and electron microscopy suggested that apoptotic cells were predominantly epithelial cells. TUNEL-positive cells in % of epithelial cells were significantly increased in IIP compared with normal lung parenchyma, and significantly correlated with cytochrome c release from the mitochondria and with the expression of cleaved caspase-3 in epithelial cells. We conclude that mitochondria-mediated apoptosis may be involved in the pathophysiology of IIP.

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.

[The roles of apoptosis in lung injury].

Apoptosis serves important roles in organ development, cell differentiation, and the maintenance of homeostasis. Lung injury studies have underlined the role of fibroblast and endothelial cell apoptosis during lung repair from acute lung injuries, and demonstrated apoptosis of alveolar epithelial cells in association with diffuse alveolar damage. Pulmonary fibrosis is characterized by the loss of lung epithelial cells and the proliferation of fibroblasts. It is possible that Fas, Fas ligand, p 53, p 21, and other apoptosis-regulating proteins may play important roles in the pathophysiology of lung injury and fibrosis.

TGF-beta 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells.

Transforming growth factor-beta 1 (TGF-beta 1) 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-beta1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and down-regulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-beta 1 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-beta 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-beta 1 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-beta 1 and HP BALF created proapoptotic effects comparable with the IPF BALF. In vivo, the administration of TGF-beta 1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-beta 1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.

Increased circulating levels of soluble Fas ligand are correlated with disease activity in patients with fibrosing lung diseases.

OBJECTIVE: The Fas-Fas ligand (FasL) pathway is one of the important apoptosis-signalling molecule systems. We previously determined that this pathway may be involved in the pathogenesis of fibrosing lung diseases. In the present study, we evaluated the clinical significance of the levels of soluble forms of Fas (sFas) and FasL (sFasL) in serum from patients with fibrosing lung diseases. METHODOLOGY: We measured sFas, sFasL, KL-6 (a measure of alveolar type II cell damage), surfactant protein D (SP-D), and surfactant protein A (SP-A) levels in serum from 35 patients with idiopathic pulmonary fibrosis (IPF), 17 patients with interstitial pneumonia associated with collagen vascular diseases (CVD-IP), and 13 normal healthy controls using enzyme-linked immunosorbent assays (ELISA). RESULTS: The serum levels of sFasL were significantly increased in patients with active IPF and CVD-IP, compared with those with inactive disease and controls. There was no significant difference in sFasL levels between patients with inactive disease and controls. Serum sFasL levels were significantly correlated with lactate dehydrogenase and KL-6 levels in IPF. The decrease in sFasL levels following corticosteroid therapy was not correlated with the clinical course of IPF. There was no significant difference in serum sFas levels between IPF or CVD-IP patients and controls. CONCLUSIONS: Although further studies need to be performed on a large number of patients with histologically proven IPF or CVD-IP, it would seem that serum sFasL levels may reflect the activity of IPF and CVD-IP.

[A case of sarcoidosis and idiopathic thrombocytopenic purpura accompanied with invasive pulmonary aspergillosis].

A 54-year-old woman with a 21-year history of sarcoidosis was admitted to our hospital with dyspnea on exertion, weight loss, and the appearance of consolidation in chest radiographs. The serum level of soluble IL-2 receptor was high, and CT findings demonstrated mediastinal, hilar and abdominal lymphadenopathy. The histological findings of subpleural consolidation in a transbronchial lung biopsy of the left lung showed giant cells; and those of a CT-assisted biopsy of a retroperitoneal lymph node revealed non-caseous epithelioid cell granulomas. After the biopsy, severe thrombocytopenia (6,000/microliter) developed. With prednisolone treatment, the platelet count rose to normal and the subpleural consolidation on chest radiography was improved. Five weeks later, the had a productive cough with fever, rapidly progressive cavitary lesions and consolidation on chest radiography. Aspergillus fumigatus was detected in the sputum by PCR, and Aspergillus antigen was detected in the serum. She died of progressive respiratory failure, in spite of therapy with amphotericin B and itraconazole. We report a rare case of sarcoidosis and idiopathic thrombocytopenic purpura accompanied with invasive pulmonary aspergillosis.