Nature Medicine
8, 480 - 484 (2002)
doi:10.1038/nm0502-480
A pivotal role of cytosolic phospholipase A2 in bleomycin-induced pulmonary fibrosisTakahide Nagase1, Naonori Uozumi2, 3, Satoshi Ishii2, 3, Yoshihiro Kita2, 3, Hiroshi Yamamoto1, Eijiro Ohga1, Yasuyoshi Ouchi1
& Takao Shimizu2, 31 Department of Geriatric Medicine; Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan 2 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan 3 CREST of Japan Science and Technology Corporation, Tokyo, Japan
Correspondence should be addressed to Takahide Nagase takahide-tky@umin.ac.jpPulmonary fibrosis is an interstitial disorder of the lung parenchyma whose mechanism is poorly understood. Potential mechanisms include the infiltration of inflammatory cells to the lungs and the generation of pro-inflammatory mediators. In particular, idiopathic pulmonary fibrosis is a progressive and fatal form of the disorder characterized by alveolar inflammation, fibroblast proliferation and collagen deposition. Here, we investigated the role of cytosolic phospholipase A2 (cPLA2) in pulmonary fibrosis using cPLA2-null mutant mice, as cPLA2 is a key enzyme in the generation of pro-inflammatory eicosanoids. Disruption of the gene encoding cPLA2 (Pla2g4a) attenuated IPF and inflammation induced by bleomycin administration. Bleomycin-induced overproduction of thromboxanes and leukotrienes in lung was significantly reduced in cPLA2-null mice. Our data suggest that cPLA2 has an important role in the pathogenesis of pulmonary fibrosis. The inhibition of cPLA2-initiated pathways might provide a novel therapeutic approach to pulmonary fibrosis, for which no pharmaceutical agents are currently available.Pulmonary fibrosis comprises a group of interstitial disorders of the lung parenchyma1,
2,
3,
4,
5. Idiopathic pulmonary fibrosis (IPF) is the most common form of the disorder and is characterized by progressive development of alveolar inflammation, fibroblast proliferation and collagen deposition. IPF is clinically manifested as increased lung elasticity, restrictive ventilation impairment and respiratory failure1. Although IPF is a progressive and fatal disease, no useful drugs are currently available for its treatment. Current medical intervention for IPF is either lung transplantation or supportive treatment, such as oxygen therapy. Although the molecular mechanisms underlying development of IPF remains to be elucidated, potential mechanisms might be the infiltration of inflammatory cells to the lungs, and the generation of pro-inflammatory and proliferative mediators, leading to fibroblast proliferation and accumulation of extracellular collagen1,
2.
Platelet-activating factor (PAF) and metabolites of arachidonic acid might be involved in pathogenesis of pulmonary fibrosis, although little attention has been paid to these mediators. PAF is a pro-inflammatory phospholipid mediator that has various biological effects including cell proliferation and the production of cytokines and eicosanoids via activation of the G protein−coupled PAF receptor (PAFR)6,
7,
8,
9,
10,
11,
12,
13,
14,
15,
16,
17. Thromboxanes (Txs) and leukotrienes (LTs) are potent mediators generated from arachidonic acid by cyclooxygenase and 5-lipoxygenase, respectively. TxA2 may induce lung inflammation, whereas LTB4 is a potent neutrophil chemoattractant. Cysteinyl LTs (LTC4, LTD4 and LTE4) are among the most important mediators in inflammatory lung diseases. A crucial enzyme for generation of these pro-inflammatory mediators, including eicosanoids and PAF, is phospholipase A2. In particular, cytosolic phospholipase A2 (cPLA2) is a key player, as it is activated by submicromolar concentration of Ca++ and by phosphorylation by mitogen-activated protein kinases18,
19,
20,
21,
22.
Our aim was to elucidate the role of cPLA2 in the experimental model of pulmonary fibrosis induced by bleomycin. To perform this study, we used mutant mice lacking the gene encoding cPLA2 (ref. 23; also known as Pla2g4a) and their wild-type littermates as controls.
Pulmonary fibrosis induced by bleomycin administration
Fig. 1 demonstrates the lung elasticity in bleomycin and saline-treated groups (n = 4-6). In the bleomycin-treated wild-type group, the value of lung elastance (EL) was significantly greater than those in the other groups, whereas there was a no difference in EL values among bleomycin-treated cPLA2-null group and saline-treated groups. In terms of alterations in lung parenchyma, physiological data suggest that bleomycin-induced responses in cPLA2-null mice were significantly reduced compared with their wild-type controls.
 | | |
We assessed collagen content of the lungs by measuring hydroxyproline-content values. Values in bleomycin-treated wild-type group were significantly greater than those in bleomycin-treated cPLA2-null group (83.6 4.4 and 56.03.0  g per left lung; n = 6, respectively; P  0.05), indicating that bleomycin-induced collagen synthesis in cPLA2-null mice was significantly attenuated compared with bleomycin-treated wild-type group. There was no difference in hydroxyproline content among bleomycin-treated cPLA2-null group and saline-treated groups.
Histopathological assessment of pulmonary fibrosis
Fig. 2 shows lung histology 14 days after bleomycin or saline administration. Bleomycin treatment elicited alveolar thickening and subpleural fibrous foci in wild-type mice. In cPLA2-null mice, histological changes including alveolar thickening and fibrosis were minimal. We performed further histopathological evaluation of pulmonary fibrosis using an established scoring method3. There was a significant difference in the scores of fibrotic lesions between bleomycin-treated wild-type and cPLA2-null mice (Fig. 3).
Bleomycin-induced inflammation
In the wild-type group, bleomycin administration increased protein content and the numbers of macrophages, polymorphonuclear leukocytes (PMNs) and lymphocytes in bronchoalveolar lavage fluid (BALF), indicating bleomycin-induced protein leakage and leukocyte infiltration (Table 1 and Fig. 4). The protein leakage was significantly attenuated in cPLA2-null mice, which had lesser macrophage, PMN and lymphocyte sequestration compared with the control mice (Fig. 4).
| | Table 1. Physiological and biochemical data after saline or bleomycin treatment. | | |  |
 Full Table |
| |
Thromboxane and leukotriene content
To assess the biosynthesis of cPLA2 products, we performed BALF assays of TxA2 (measured as TxB2), LTB4 and LTC4/D4/E4. Table 1 summarizes the results of BALF TxB2, LTB4 and LTC4/D4/E4 assays in each experimental group. Each eicosanoid level in bleomycin-treated wild-type mice was significantly greater than that in any other group, whereas any eicosanoid measured in similarly treated cPLA2-null mice was reduced to the same level as the saline-treated groups. Bleomycin administration increased TxB2, LTB4 and LTC4/D4/E4 levels in BALF, whereas the levels of these eicosanoids were significantly reduced in cPLA2-null mice.
Relative contribution of downstream cPLA2 mediator, PAF
To assess the relative contribution of downstream mediators of cPLA2, we examined the role of PAF in bleomycin-induced pulmonary fibrosis using mutant mice lacking the gene encoding the PAF receptor (Pafr-/- mice)17 and their wild-type littermates as controls. The disruption of Pafr reduced the score of fibrotic lesions, although there was a marked difference between bleomycin-treated Pafr-/- mice and saline-treated groups (Table 2). However, there was no significant difference in either EL or hydroxyproline content between bleomycin-treated wild-type and Pafr-/- mice.
Discussion
Our results suggest that cPLA2 has a pivotal role in the pathogenesis of pulmonary fibrosis. Disruption of the gene encoding cPLA2 significantly attenuated pulmonary fibrosis and inflammation induced by bleomycin. These observations indicate that cPLA2 may be involved in pulmonary fibrosis by mediating overproduction of inflammatory mediators including Txs and LTs.
The fibrotic lung disorders such as IPF are characterized by chronic lung parenchymal injury and inflammation, that is, alveolitis1,
2. The inflammation of pulmonary fibrosis is associated with infiltration of leukocytes including macrophages, lymphocytes and neutrophils. It is thought that these inflammatory cells might generate and release chemical mediators, leading to proliferation of mesenchymal cells and deposition of extracellular matrix protein. This pathologic process is associated with clinical findings including reduced vital capacity and impaired gas exchange1. In investigating the pathogenesis of pulmonary fibrosis, the efficacy of the pharmacological approach is limited as the progression of fibrotic disorders is a chronic and continuous process. We therefore used a mutant mouse model to examine whether cPLA2 would mediate development of IPF.
In bleomycin-treated wild-type mice, we observed increases in EL, pulmonary fibrosis, protein leakage and infiltration of inflammatory cells including macrophages, lymphocytes and neutrophils. Consistently, increases in Txs and LTs were found in the BAL fluid of bleomycin-treated wild-type mice. These observations were markedly attenuated by cPLA2 gene disruption, suggesting that cPLA2 could mediate the process of lung fibrotic disorder via the production of pro-inflammatory mediators. Moreover, cPLA2 products are related to infiltration of various leukocytes, which may lead to the fibrotic disorder. Indeed, increases in leukocytes, especially PMNs, are found in BAL fluid of patients with IPF (ref. 1). It has been reported that lung homogenates from IPF patients contain much higher levels of LTB4 and LTC4 compared with non-fibrotic controls2. LTB4 is an important mediator of neutrophil-mediated inflammation including lung injury24,
25,
26. It is possible that not only PMN, but also macrophages and lymphocytes, may be necessary to induce the bleomycin-induced lung inflammation. The cPLA2-initiated pathways may mediate both infiltration and activation of these leukocytes triggered by bleomycin, resulting in pulmonary fibrosis. Our results suggest that cPLA2 has a dominant role in leukocyte sequestration, and potential interaction between mediators such as PAF and eicosanoids may further upregulate the development of fibrotic process.
To investigate the relative contribution of downstream pathways of cPLA2 mediators, we investigated the role of PAF in bleomycin-induced fibrosis using mutant mice established in our laboratory17. We observed that the disruption of the PAF receptor modestly attenuated the histopathological changes induced by bleomycin, although we found no significant differences in physiological data or collagen content between bleomycin-treated mutant mice and controls. These findings suggest that PAF may be potentially involved in the development of bleomycin-induced pulmonary fibrosis. However, the relative contribution of PAF is only partial and limited when compared with the crucial role of cPLA2. We therefore suggest that other downstream mediators of cPLA2, that is, eicosanoids produced by cyclooxygenase and/or lipoxygenase, may also contribute to the development of bleomycin-induced pulmonary fibrosis.
Here we measured TxB2, LTB4 and cysteinyl LTs (LTC4, LTD4 and LTE4) in BALF to confirm the generation of cPLA2 products. We observed significant increases in each eicosanoid in the model of bleomycin-induced pulmonary fibrosis, but the LTC4/D4/E4 levels were much higher than the other eicosanoids. As there is abundant cysteinyl LT receptors in the lung27, the coupling of these cysteinyl LTs and receptors may induce inflammatory process including increased permeability and leukocyte infiltration in the lung28. Our findings suggest that the cysteinyl LTs might have an important role in the development of pulmonary fibrosis.
Recent studies have shown that cPLA2 may be involved in the pathogenesis of various inflammatory diseases including bronchial asthma and adult respiratory distress syndrome (ARDS)23,
29. Although there are extreme differences in clinical features between ARDS and IPF, both diseases are fatal disorders with no useful drug therapies. Whereas no effective cPLA2 inhibitors are available, cPLA2 might be a potential target to intervene development of pulmonary fibrosis as well as acute lung injury.
Bleomycin is an antibiotic agent with antitumor activity and commonly used to treat various types of tumors30. It has been reported that bleomycin is an essential component of combination chemotherapy in patients with germ-cell tumors31. The most important and severe side effects of bleomycin are pulmonary injuries including bleomycin-induced pulmonary fibrosis30. In terms of both clinical and pathological features, bleomycin-induced pulmonary fibrosis resembles IPF. Histologically, bleomycin-induced pulmonary fibrosis is associated with infiltration of inflammatory cells into alveoli, focal collagen depositions and fibrotic lesions30. Recent studies have shown that bleomycin stimulates cytokine secretion by alveolar macrophage or fibroblast proliferation in human cells32,
33. However, the exact mechanism underlying the development of bleomycin-induced pulmonary fibrosis also remains to be elucidated.
Notably, although disruption of the gene encoding cPLA2 attenuated bleomycin-induced lung injury, it did not completely abolish the pathology. Indeed, we found a significant difference in histopathologic scores of fibrosis between bleomycin-treated cPLA2-null mice and saline-treated controls. This observation indicates that factors other than cPLA2 should also be involved in the process of pulmonary fibrosis. It has been postulated that oxygen radicals, adhesion molecules, plasminogen-activator inhibitor and cytokines are also involved in this mechanism1,
2,
3,
4,
5. Because no pharmacological agents are available to treat pulmonary fibrosis and increase survival rates, these factors are also potential targets to develop agents. Our data suggest that the intervention of cPLA2 may also be a promising tool to improve management of pulmonary fibrosis.
In summary, the disruption of cPLA2 significantly attenuated lung inflammation and fibrosis induced by bleomycin treatment. cPLA2 pathways might be involved in the pathogenesis of pulmonary fibrosis caused by bleomycin, and inhibition of these pathways may provide a novel and potential therapeutic approach to pulmonary fibrosis.
Methods
Mice.
cPLA2-null mice were established by gene targeting23. Mice heterozygous for cPLA2 mutant allele with the genetic background of the C57BL/6J  129/Ola hybrid were mated. Pafr-/- mice were also established as reported17,
34,
35. Offspring were genotyped at 4 wk of age. For genotyping, genomic DNAs were isolated from biopsied tail and subjected to PCR amplification23. The animals were maintained on a light/dark cycle with light from 7:00 to 20:00 at 25 °C. Mice were fed with a standard laboratory diet and water ad libitum. Mutant homozygous mice and their littermate homozygous controls were used in this study. All animal experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments.
Experimental pulmonary fibrosis induced by bleomycin administration.
Mice were anesthetized with ketamine and tracheostomized. Then, 1.5 ml/kg solution containing bleomycin (5 mg/kg, Nippon Kayaku, Tokyo, Japan) was intratracheally administered. Control animals were treated with saline instead of bleomycin in the same manner. These procedures were performed in a sterile environment. To assess the development of pulmonary fibrosis physiologically, EL (a reciprocal of lung compliance) was measured 7 or 14 d after bleomycin or saline administration as described36,
37,
38,
39,
40. Briefly, we measured the tracheal pressure (Ptr), flow and volume (V). EL and lung resistance (RL, data not shown) were calculated by adjusting the equation of motion: Ptr = EL (V) + RL (dV/dt) + K, where K was a constant. Values in EL reflect lung parenchymal alterations and stiffening of the lungs.
Assessment of collagen synthesis.
To assess collagen synthesis, a hydroxyproline assay was performed. Mice were anesthetized and the lungs were removed 14 d after bleomycin or saline treatment. The left lung was then excised and hydroxyproline content was measured as reported3.
Histopathological assessment of pulmonary fibrosis.
The right lung of each animal was excised 14 d after bleomycin or saline treatment. Following fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. The slices were stained with H&E or with Masson's trichrome stain. Histopathological evaluation of pulmonary fibrosis was performed using a scoring method as described3. Briefly, the scores of fibrotic lesions were defined as follows: 0, absence of lesion; 1, occasional small localized subpleural foci; 2, thickening of interalveolar septa and subpleural foci; 3, thickened continuous subpleural fibrosis and interalveolar septa.
Bronchoalveolar lavage fluid.
7 d after bleomycin or saline treatment, bronchoalveolar lavage (BAL) was performed (1 ml PBS 5). In each animal, 90% (4.5 ml) of the total injected volume was consistently recovered. After BAL fluid was centrifuged at 450g for 10 min, the total and differential cell counts of the BAL fluid were determined from the cell fraction. The supernatant was used for protein determination by Lowry's method using BSA as a standard.
Thromboxane and leukotriene assay.
TxA2 (measured as TxB2), LTB4 and LTC4/D4/E4 in the BAL fluid were determined by using enzymeimmunoassay (EIA) kits (Amersham Pharmacia Biotech, Piscataway, New Jersey). The detection limits of the EIA assays for TxB2, LTB4 and LTC4/D4/E4 were 3.6, 6 and 10 pg/ml, respectively.
Data analysis.
Comparisons of data among each experimental group were carried out with analysis of variance (Scheffé test). Data are expressed as meanss.e. P values less than 0.05 were considered significant.
Received 14 February 2002; Accepted 18 March 2002
REFERENCES
- American Thoracic Society. Idiopathic pulmonary fibrosis: Diagnosis and treatment; international consensus statement. Am. J. Respir. Crit. Care Med. 161, 646–664 (2000). | PubMed | ISI |
- Wilborn, J. et al. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 97, 1827–1836 (1996). | PubMed | ISI | ChemPort |
- Lan Tran, P. et al. Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene. J. Clin. Invest. 99, 608–617 (1997). | PubMed |
- Eitzman, D.T. et al. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J. Clin. Invest. 97, 232–237 (1996). | PubMed | ISI | ChemPort |
- Hattori, N. et al. Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. J. Clin. Invest. 106, 1341–1350 (2000). | PubMed | ISI | ChemPort |
- Prescott, S.M., Zimmerman, G.A. & McIntyre, T.M. Platelet-activating factor. J. Biol. Chem. 265, 17381–17384 (1990). | PubMed | ISI | ChemPort |
- Chao, W. & Olson, M.S. Platelet-activating factor: Receptors and signal transduction. Biochem. J. 292, 617–629 (1993). | PubMed | ISI | ChemPort |
- Izumi, T. & Shimizu, T. Platelet-activating factor receptor: Gene expression and signal transduction. Biochim. Biophys. Acta. 1259, 317–333 (1995). | Article | PubMed | ISI | ChemPort |
- Honda, Z. et al. Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. Nature 349, 342–346 (1991). | Article | PubMed | ISI | ChemPort |
- Nakamura, M. et al. Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J. Biol. Chem. 266, 20400–20405 (1991). | PubMed | ISI | ChemPort |
- Ye, R.D., Prossnitz, E.R., Zou, A.H. & Cochrane, C.G. Characterization of a human cDNA that encodes a functional receptor for platelet activating factor. Biochem. Biophys. Res. Commun. 180, 105–111 (1991). | PubMed | ISI | ChemPort |
- Kunz, D., Gerard, N.P. & Gerard, C. The human leukocyte platelet-activating factor receptor. cDNA cloning, cell surface expression & construction of a novel epitope-bearing analog. J. Biol. Chem. 267, 9101–9106 (1992). | PubMed | ISI | ChemPort |
- Sugimoto, T. et al. Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart. Biochem. Biophys. Res. Commun. 189, 617–624 (1992). | PubMed | ISI | ChemPort |
- Bito, H., Honda, Z. -i, Nakamura, M. & Shimizu, T. Cloning, expression and tissue distribution of rat platelet-activating-factor-receptor cDNA. Eur. J. Biochem. 221, 211–218 (1994). | PubMed | ISI | ChemPort |
- Ishii, S. et al. A murine platelet-activating factor receptor gene: Cloning, chromosomal localization and up-regulation of expression by lipopolysaccharide in peritoneal resident macrophages. Biochem. J. 314, 671–678 (1996). | PubMed | ISI | ChemPort |
- Ishii, S. et al. Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor. EMBO J. 16, 133–142 (1997). | Article | PubMed | ISI | ChemPort |
- Ishii, S. et al. Impaired anaphylactic responses but intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J. Exp. Med. 187, 1779–1788 (1998). | Article | PubMed | ISI | ChemPort |
- Leslie, C.C. Properties and regulation of cytosolic phospholipase A2. J. Biol. Chem. 272, 16709–16712 (1997). | Article | PubMed | ISI | ChemPort |
- Clark, J.D. et al. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-de-pendent translocation domain with homology to PKC and GAP. Cell 65, 1043–1051 (1991). | Article | PubMed | ISI | ChemPort |
- Sharp, J.D. et al. Molecular cloning and expression of human Ca2+-sensitive cytosolic phospholipase A2. J. Biol. Chem. 266, 14850–14853 (1991). | PubMed | ISI | ChemPort |
- Lin, L.L. et al. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72, 269–278 (1993). | Article | PubMed | ISI | ChemPort |
- Kramer, R.M. et al. p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. J. Biol. Chem. 271, 27723–27729 (1996). | Article | PubMed | ISI | ChemPort |
- Uozumi, N. et al. Roles of cytosolic phospholipase A2 in allergic response and parturition. Nature 390, 618–622 (1997). | Article | PubMed | ISI | ChemPort |
- Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. & Shimizu, T. A G-protein-coupled receptor for leukotriene B4 that mediates chemotaxis. Nature 387, 620–624 (1997). | Article | PubMed | ISI | ChemPort |
- Chiang, N. et al. Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion. J. Clin. Invest. 104, 309–316 (1999). | PubMed | ISI | ChemPort |
- Levy, B.D., Clish, C.B., Schmidt, B., Gronert, K. & Serhan, C.N. Lipid mediator class switching during acute inflammation: Signals in resolution. Nature Immunol. 2, 612–619 (2001). | Article | PubMed | ISI | ChemPort |
- Lynch, K.R. et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399, 789–793 (1999). | Article | PubMed | ISI | ChemPort |
- Dahlen, S.E. et al. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules; in vitro effects with relevance to the acute inflammatory response. Proc. Natl. Acad. Sci. USA. 78, 3887–3891 (1981). | PubMed | ChemPort |
- Nagase, T. et al. Acute lung injury by sepsis and acid aspiration: A key role for cytosolic phospholipase A2. Nature Immunol. 1, 42–46 (2000). | Article | PubMed | ISI | ChemPort |
- Sleijfer, S. Bleomycin-induced pneumonitis. Chest 120, 617–624 (2001). | Article | PubMed | ISI | ChemPort |
- Loehrer, P.J., Johnson, D., Elson, P., Einhorn, L.H. & Trump, D. Importance of bleomycin in favorable-prognosis disseminated germ cell tumors. An Eastern Cooperative Oncology Group Trial. J. Clin. Oncol. 13, 470–476 (1995). | PubMed | ISI |
- Scheule, R.K., Perkins, R.C., Hamilton, R. & Holian, A. Bleomycin stimulation of cytokine secretion by the human alveolar macrophage. Am. J. Physiol. 262, L386–L391 (1992). | PubMed | ISI | ChemPort |
- Moseley, P.L., Hemken, C. & Hunninghake, G.W. Augmentation of fibroblast proliferation by bleomycin. J. Clin. Invest. 78, 1150–1154 (1986). | PubMed | ISI | ChemPort |
- Ishii, S. & Shimizu, T. Platelet-activating factor (PAF) receptor and genetically engineered PAF receptor mutant mice. Prog. Lipid Res. 39, 41–82 (2000). | Article | PubMed | ISI | ChemPort |
- Shindou, H., Ishii, S., Uozumi, N. & Shimizu, T. Roles of cytosolic phospholipase A2 and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF. Biochem. Biophys. Res. Commun. 271, 812–817 (2000). | Article | PubMed | ISI | ChemPort |
- Nagase, T., Ishii, S., Shindou, H., Ouchi, Y. & Shimizu, T. Airway hyperresponsiveness in transgenic mice overexpressing platelet-activating factor receptor is mediated by an atropine-sensitive pathway. Am. J. Respir. Crit. Care Med. 165, 200–205 (2002). | PubMed | ISI |
- Nagase, T., Aoki, T., Oka, T., Fukuchi, Y. & Ouchi, Y. ET-1-induced bronchoconstriction is mediated via ETB receptor in mice. J. Appl. Physiol. 83, 46–51 (1997). | PubMed | ISI | ChemPort |
- Nagase, T. et al. Airway hyperresponsiveness to methacholine in mutant mice deficient in endothelin-1. Am. J. Respir. Crit. Care Med. 157, 560–564 (1998). | PubMed | ISI | ChemPort |
- Nagase, T. et al. Airway responsiveness in transgenic mice overexpressing platelet-activating factor receptor: Roles of thromboxanes and leukotrienes. Am. J. Respir. Crit. Care Med. 156, 1621–1627 (1997). | PubMed | ISI | ChemPort |
- Nagase, T. et al. Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice. J. Clin. Invest. 104, 1071–1076 (1999). | PubMed | ISI | ChemPort |
Acknowledgments
We thank Y. Tateno, R. Mitsuzono, M. Yoshino, C. Ohkawara, T. Sato, H. Shiozawa, Y. Matsumoto and M. Ito for technical assistance; and F. Takaku and T. Yokomizo for valuable suggestions. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Human Science Foundation.
Competing interests statement:
The authors declare that they have no competing financial interests. |
) or saline (
) in the wild-type (+/+) or cPLA2-null (-/-)mice (n = 4−6). *, P < 0.001 versus saline-treated; #, P < 0.001 versus bleomycin-treated wild-type group.
