Abstract
Idiopathic pulmonary fibrosis (IPF) is a devastating disease with poor prognosis. Leukotrienes play an important role in IPF, and leukotriene (LT)B4 is one of the key eicosanoids in IPF. In this study, we investigated whether ONO-4057, a LTB4 receptor (BLTR) antagonist is capable of preventing bleomycin-induced pulmonary fibrosis.
On day 1, C57BL/6 male mice were given a single intratracheal injection of bleomycin (2.5 mg·kg−1), and ONO-4057 (1.0 mg·kg−1) or vehicle alone, administered by intraperitoneal injection on days 1–5 each week for 3 weeks after the bleomycin injection.
ONO-4057 reduced the total cell count in bronchoalveolar lavage fluid (BALF) on days 7, 14 and 21 and the Ashcroft score and the lung hydroxyproline content on days 14 and 21. The LTB4, interleukin (IL)-6, IL-13, transforming growth factor (TGF)-β levels in BALF and the TGF-β expression in lung tissue, assessed by immunohistochemistry were decreased on day 7, whereas interferon (IFN)-γ level in BALF was increased on day 14.
The results of this study indicated that the BLTR antagonist inhibited the development of bleomycin-induced pulmonary fibrosis in mice by decreasing inflammation and altering TGF-β, IL-6, IL-13 and IFN-γ.
Idiopathic pulmonary fibrosis (IPF) is a deleterious disease with very poor prognosis despite all known methods of treatment 1. The pathological features of IPF are fibroblast proliferation, increased amounts of extracellular matrix and varying degrees of persistent inflammation of the alveolar septa 2. Recent reports have suggested that leukotrienes (LTs), which are arachidonic acid metabolites, are important regulators of pulmonary fibrosis 3, 4. The synthesis of LTB4 and cysteinyl LT (cysLT) is catalysed by 5-lipoxygenase. LTB4 plays an important role in the host defence system against infection and invasion by foreign bodies 5. LTB4 is thought to be a cause of various inflammatory disorders, and it is produced by alveolar cells in patients with idiopathic pulmonary fibrosis (IPF) 6. In fact, LTB4 level is elevated in bronchoalveolar lavage fluid (BALF) and lung tissues in patients with IPF 7, 8. There are two LTB4 receptor (BLTR) subtypes, BLT1 and BLT2; both subtypes are G-protein-coupled receptors and are present on the cell surface 9. BLT1 is primarily expressed in leukocytes whereas BLT2 is expressed more ubiquitously. There have been several reports concerning the role of BLTR in airway disease. According to one report, BLT1 mediates LTB4-induced T helper (Th) type 1 and Th2 cell chemotaxis and firm adhesion to endothelial cells exposed to flow, and mediates CD4+ and CD8+ T cell recruitment into the airway in an asthma model 10. BLT1-mediated T cell trafficking is critical to the development of rejection and obliterative bronchiolitis after lung transplantation by mediating airway fibroproliferation 11. However, the role of BLTR in pulmonary fibrosis has remained unclear. We recently reported that montelukast, one of the cysLT1 receptor antagonists that have been widely used in the treatment of bronchial asthma, inhibits the development of bleomycin-induced pulmonary fibrosis 12.
In the present study, we attempted to determine whether ONO-4057, a BLTR antagonist, has any preventive effect on bleomycin-induced pulmonary fibrosis in mice. ONO-4057 (5-(2-(2-carboxyethyl)­3-(6-(4-methoxyphenyl)-5E-hexenyl) oxyphenoxy) valeric acid) (ONO Pharmaceutical, Osaka, Japan) is a nonselective BLTR antagonist that predominantly antagonises BLT1 and has been reported to inhibit human neutrophil aggregation, chemotaxis and degranulation induced by LTB4 9, 13. We also focused on the effects of ONO-4057 on prostaglandin (PG)E2, active transforming growth factor (TGF)-β1, interleukin (IL)-4, IL-6, IL-13 and interferon (IFN)-γ levels in BALF and on TGF-β expression in lung tissue in pulmonary fibrosis, because these mediators have been found to be strongly associated with the pathogenesis of pulmonary fibrosis 14–16.
MATERIALS AND METHODS
Animals and bleomycin-induced pulmonary fibrosis model
The animal protocol was approved by the Animal Care and Use Committee of Tokyo Women’s Medical University (Tokyo, Japan). We performed the study in 6-week-old C57BL/6 male mice. On day 1, the mice were given a single intratracheal injection of 50 μL of saline containing bleomycin (2.5 mg·kg−1) (Nippon Kayaku, Tokyo, Japan) or 50 μL of saline alone. ONO-4057 (1.0 mg·kg−1) was supplied by Ono Pharmaceutical Co., and the animals were intraperitoneally injected with ONO-4057 or with vehicle (0.14% sodium bicarbonate) alone on days 1–5 of each week for 3 weeks starting 2 h after the bleomycin injection, or on days 15–19 after bleomycin injection. Thus, there were four groups of mice in this study: a saline-injected group treated with the vehicle (vehicle-treated group, n = 10), a bleomycin-injected group treated with the vehicle (bleomycin group, n = 10), a bleomycin-injected group treated with ONO-4057 starting 2 h after the intratracheal injection of bleomycin (ONO-4057 group, n = 10), and a bleomycin-injected group treated with ONO-4057 starting 15 days after the intratracheal injection of bleomycin (ONO-4057 delayed initiation group, n = 10). We evaluated the severity of the lung inflammation by performing a BALF analysis on days 7, 14 and 21, and we evaluated the effect of ONO-4057 on pulmonary fibrosis by histological evaluation by means of the Ashcroft score 17 and based on the hydroxyproline content of the right lung on days 14 and 21 18.
BALF analysis
After intraperitoneally anaesthetising mice with pentobarbital (50 mg·kg−1), a tracheotomy was performed and a custom-made cannula was inserted. The lungs were lavaged with 1.0 mL of PBS and then with 0.8 mL of PBS. The BALF was centrifuged at 360×g for 10 min, and the supernatant was stored at -80°C for the subsequent measurements. A total cell count was performed manually with a haemocytometer. Slides of BALF cells were prepared with cytospin, stained with May-Grünwald–Giemza stain, and a differential count of 1,000 cells per sample was made. Eicosanoid and cytokine levels in BALF were measured on days 7 and 14 by using ELISA. The LTB4 level in BALF was measured using ELISA (Amersham Biosciences, Piscataway, NJ, USA), and the detection limit of the assay was 6 pg·mL−1. The PGE2 level in BALF was measured with a mouse PGE2 ELISA kit (GE Healthcare, Amersham, UK) and the detection limit was 50 pg·mL−1. The IL-4, IL-6, IL-13, IFN-γ, and active TGF-β1 levels in BALF were measured with a mouse IL-4 ELISA kit, a mouse IL-6 ELISA kit, a mouse IL-13 ELISA kit, a mouse IFN-γ ELISA kit, and a mouse TGF-β1 ELISA kit (R&D system, Minneapolis, MN, USA), and the detection limits were 2.0 pg·mL−1, 1.6 pg·mL−1, 1.5 pg·mL−1, 2.0 pg·mL−1 and 4.2 pg·mL−1, respectively.
Histological analysis
The left lung was fixed by inflation with 4% paraformaldehyde and embedded in paraffin (n = 10). Sections were cut 5 μm thick and stained with haematoxylin and eosin. The Ashcroft score was used for semiquantitative analysis of fibrotic change on day 14 and on day 21 as reported previously 17.
Immunostaining for TGF-β
For immunostaining of TGF-β, after deparaffinising sections in xylene and dehydrating them in ethanol, they were washed three times with PBS, reacted with peroxidase-blocking solution (DakoCytomation, A/S, Glostrup, Denmark) for 10 min at room temperature to block endogenous peroxidase activity, and then washed three times with PBS. Next, they were reacted with Protein Block Serum-Free (DakoCytomation) for 10 min at room temperature. Some sections were incubated at 4°C overnight with anti-mouse TGF-β antibody (diluted 1:50; Santa Cruz Biotechnology, Inc. San Diego, CA, USA) and control sections were incubated at 4°C overnight with a rabbit immunoglobulin fraction as a negative control (diluted 1:1000; DakoCytomation). The following day, all sections were washed three times with PBS. Antibody that had bound to TGF-β was detected by incubation for 30 min at room temperature with dextran polymer reagent conjugated with peroxidase and secondary antibody (DAKO EnVision+; DakoCytomation). The sections were then washed three times with PBS, and colour development was achieved by exposure to 3,3′-diaminobenzidine (DAKO DAB+ Liquid System; DakoCytomation) for 2 min. The tissues were counterstained with Mayer’s hematoxylin.
Hydroxyproline assays
Lung homogenates were prepared and assayed for hydroxyproline content as previously described 18. In brief, lung tissues were hydrolysed with 12 N hydrochloric acid at 110°C for 24 h. After neutralisation with sodium hydroxide, the hydrolysates were diluted with distilled water. The absorbance at 560 nm was measured.
Statistical analysis
Data are reported as means±sem. Statistical analysis was performed with ANOVA followed by Sheffe’s F test as a post hoc analysis test. A p-value of <0.05 was considered statistically significant.
RESULTS
Cell analysis of BALF
The total cell count in BALF was markedly higher in the bleomycin group than in the vehicle-treated group, and the count on days 7, 14 and 21 showed that the increase was significantly inhibited by ONO-4057 (n = 10, p<0.01, fig. 1a⇓). The increase in neutrophil count on day 7 was attenuated significantly in the ONO-4057 group (n = 10, p<0.01, fig. 1b⇓). The increase in macrophage count on day 14 was attenuated in the ONO-4057 group (n = 10, p<0.01, fig. 1c⇓). The increase in lymphocyte count was attenuated in the ONO-4057 group on day 14 and day 21 and in the ONO-4057 delayed initiation group on day 21 (n = 10, p<0.05, p<0.01 and p<0.01, respectively, fig. 1d⇓).
Effects of ONO-4057 on cell counts of bronchoalveolar fluid on day 7, 14 and 21 after the bleomycin injection. a) Total cell count. The increase in total cell count in bronchoalveolar lavage fluid on day 7, 14 and 21 was attenuated in the ONO-4057 group (░), and was also attenuated on day 21 in the ONO-4057 delayed initiation group (▒). b) Differential neutrophil cell count. The increase in neutrophil count in bronchoalveolar lavage fluid on day 7 was attenuated in the ONO-4057 group. c) Differential macrophage cell count. The increase in macrophage count in bronchoalveolar lavage fluid on day 14 was attenuated in the ONO-4057 group. d) Differential lymphocyte cell count. The increase in lymphocyte count in bronchoalveolar lavage fluid on day 14 and day 21 was attenuated in the ONO-4057 group, and was also attenuated on day 21 in the ONO-4057 delayed initiation group. Data are presented as mean±sem (n = 10 in each group). □: vehicle-treated group; ▪: bleomycin group. *: p<0.05 versus vehicle-treated group; **: p<0.01 versus vehicle-treated group; #: p<0.05 versus bleomycin group; ##: p<0.01 versus bleomycin group.
Histological analysis
Histological evaluation of haematoxylin and eosin-stained sections in the bleomycin group revealed extensive accumulation of numerous inflammatory cells, thickening of alveolar walls, and fibrotic lesions on day 21 after the bleomycin injection (fig. 2a⇓). By contrast, less severe inflammatory and fibrotic changes in the subpleural areas of the lung were observed in the ONO-4057 group (fig. 2b⇓). Histological analysis by the Ashcroft score showed a lower degree of pulmonary fibrosis in the ONO-4057 group than in the bleomycin group on days 14 and 21 after the bleomycin injection (n = 10, p<0.01 and p<0.01, respectively, fig. 3a⇓). But the ONO-4057 delayed initiation group failed to attenuate the lung fibrosis by the Ashcroft score on day 21 (figs 2c⇓ and 3a⇓).
Effects of ONO-4057 on histopathological changes of the lung on day 21 after the bleomycin injection. Representative lung sections stained with haematoxylin and eosin. a) A lung section from the bleomycin group showing widespread accumulation of numerous inflammatory cells, thickening of the alveolar walls, and fibrotic lesions on day 21 after the bleomycin injection. b) By contrast, less severe inflammatory and fibrotic changes in the subpleural areas of the lung were observed in the ONO-4057 group. c) The histological changes in the ONO-4057 delayed initiation group were the middle grade of the bleomycin group and the ONO-4057 group. Scale bars = 200 μm.
Effects of ONO-4057 on the Ashcroft score and the lung hydroxyproline content of the right lung on day 14 and day 21 after the bleomycin injection. a) The Ashcroft score after the bleomycin injection showed a lower degree of pulmonary fibrosis in the ONO-4057 group (░) than in the bleomycin group (▪) on day 14 and day 21. b) The hydroxyproline content of the right lung was lower in the ONO-4057 group than in the bleomycin group on day 14 and day 21. Data are presented as mean±sem (n = 10 in each group). □: vehicle-treated group; ▒: ONO-4057 delayed initiation group. *: p<0.05 versus vehicle-treated group; **: p<0.01 versus vehicle-treated group; #: p<0.05 versus bleomycin group; ##: p<0.01 versus bleomycin group.
Hydroxyproline
The hydroxyproline content of the right lung on day 14 and on day 21 was lower in the ONO-4057 group than in the bleomycin group (day 14; bleomycin group versus ONO-4057 group; 522.5±31.9 μg·right lung−1 versus 383.4±32.5 μg·right lung−1, n = 10, p<0.05, day 21; bleomycin group versus ONO-4057 group; 601.3±31.8 μg·right lung−1 versus 430.5±40.9 μg·right lung−1, n = 10, p<0.01) (fig. 3b⇑). However, the ONO-4057 delayed initiation group on day 21 did not result in a significant reduction in the hydroxyproline (584.1±34.8 μg·right lung−1, n = 10, fig. 3b⇑).
Eicosanoid levels in BALF
On day 7, the LTB4 level in the BALF was significantly higher in the bleomycin group than the vehicle-treated group (vehicle-treated group versus bleomycin group; 35.9±10.2 pg·mL−1 versus 192.1±56.4 pg·mL−1, n = 10, p<0.05) (fig. 4a⇓), and was lower in the ONO-4057 group than in the bleomycin group (bleomycin group versus ONO-4057 group; 192.1±56.4 pg·mL−1 versus 59.7±26.3 pg·mL−1, n = 10, p<0.05) (fig. 4a⇓). Although the LTB4 level tended to be higher in the bleomycin group than that in the vehicle-treated group on day 14 (n = 10, p = 0.053, fig. 4a⇓), there were no significant differences between the bleomycin group and the ONO-4057 group on day 14 (bleomycin group versus ONO-4057 group; 160.2±61.1 pg·mL−1 versus 97.1±38.0 pg·mL−1, n = 10, p = 0.21) (fig. 4a⇓). PGE2 levels were increased after bleomycin injection but there were no significant differences in PGE2 level between the bleomycin group and the ONO-4057 group on day 7 and on day 14 (day 7; bleomycin group versus ONO-4057 group; 3506.8±873.9 pg·mL−1 versus 3713.0±824.0 pg·mL−1, n = 10, p = 0.999, day 14; bleomycin group versus ONO-4057 group; 4431.8±729.7 pg·mL−1 versus 3780.9±912.0 pg·mL−1, n = 10, p = 0.985) (fig. 4b⇓).
Effects of ONO-4057 on the leukotriene (LT)B4 and prostaglandin (PG)E2 levels in bronchoalveolar fluid on day 7 and day 14 after the bleomycin injection. a) The LTB4 levels were significantly higher in the bleomycin group (▪) than in the vehicle-treated group (□) on day 7 (p<0.05, n = 10), but not on day 14 (p = 0.053, n = 10). LTB4 level was lower in the ONO-4057 group (░) than in the bleomycin group on day 7 (p<0.05, n = 10), but not on day 14 (p = 0.21, n = 10). b) There were no significant differences in PGE2 level between the ONO-4057 group and the bleomycin group on day 7 and day 14. Data are presented as mean±sem (n = 10). *: p<0.05 versus vehicle-treated group; #: p<0.05 versus bleomycin group.
Cytokine levels in BALF
There were no significant differences between the IL-4 level in BALF in each group (bleomycin group versus ONO-4057 group, day 7: 14.6±1.2 pg·mL−1 versus 11.5±1.3 pg·mL−1, day 14: 11.3±0.9 pg·mL−1 versus 13.6±0.8 pg·mL−1 n = 10) (fig. 5a⇓). On day 7, the IL-6 level in BALF was lower in the ONO-4057 group than in the bleomycin group (bleomycin group versus ONO-4057 group, 1003.9±151.3 pg·mL−1 versus 508.2±135.0 pg·mL−1, n = 10, p<0.01) (fig. 5b⇓). On day 7, the IL-13 level in BALF was lower in the ONO-4057 group than in the bleomycin group (bleomycin group versus ONO-4057 group, 24.0±2.3 pg·mL−1 versus 17.4±1.1 pg·mL−1, n = 10, p<0.05) (fig. 5c⇓), but there were no significant differences in the IL-13 level between the bleomycin group and the ONO-4057 group on day 14 (bleomycin group versus ONO-4057 group, 20.2±1.2 pg·mL−1 versus 22.6±1.2 pg·mL−1, n = 10) (fig. 5c⇓). On day 14, the IFN-γ level in BALF was higher in the ONO-4057 group than in the bleomycin group (bleomycin group versus ONO-4057 group, 11.8±5.2 pg·mL−1 versus 53.8±17.3 pg·mL−1, n = 10, p<0.05) (fig. 5d⇓), but the IFN-γ level in BALF on day 7 tended to be higher, but not significantly different (bleomycin group versus ONO-4057 group, 27.6±11.5 pg·mL−1 versus 51.2±16.1 pg·mL−1, n = 10, p = 0.14) (fig. 5d⇓).
Effects of ONO-4057 on interleukin (IL)-4, IL-6, IL-13 and interferon (IFN)-γ levels in bronchoalveolar fluid on day 7 and day 14 after the bleomycin injection. a) There were no significant differences in the IL-4 level between each group on day 7 and day 14. b) The IL-6 level on day 7 was significantly reduced by ONO-4057. c) The IL-13 level on day 7 was significantly reduced by ONO-4057. d) The IFN-γ level on day 14 was significantly increased by ONO-4057. Data are reported as mean±sem (n = 10 in each group). □: vehicle-treated group; ▪: bleomycin group; ░: ONO-4057 group. *: p<0.05 versus vehicle-treated group; #: p<0.05 versus bleomycin group; ##: p<0.01 versus bleomycin group.
Assessment of TGF-β
TGF-β was evaluated by immunohistochemical staining of lung sections on day 7 after the bleomycin injection. Stronger TGF-β expression was detected in the neutrophils and pulmonary epithelial cells of the lung sections in the bleomycin group than in the ONO-4057 group (fig. 6a⇓ and b), and active TGF-β1 level in BALF was increased on day 7 and on day 14 in the bleomycin group, and ONO-4057 inhibited active TGF-β1 level on day 7 (bleomycin group versus ONO-4057 group: 204.5±24.3 pg·mL−1 versus 98.1±11.4 pg·mL−1, p<0.01, n = 10) (fig. 6c⇓). However, there were no significant differences in active TGF-β1 level between the bleomycin group and the ONO-4057 group on day 14 (n = 10, fig. 6c⇓).
Effects of ONO-4057 on immunohistochemically staining for transforming growth factor (TGF)-β and on the active TGF-β1 level of bronchoalveolar fluid. Lung sections immunohistochemically stained for TGF-β on day 7 after the bleomycin injection. a) A lung section from the bleomycin group shows stronger TGF-β expression in the neutrophils and pulmonary epithelial cells. b) TGF-β expression was weaker in the ONO-4057 group. Scale bars = 100 μm. c) The active TGF-β1 level on day 7 was significantly lower in the ONO-4057 group (░) than in the bleomycin group (▪). Data are presented as mean±sem (n = 10 in each group). □: vehicle-treated group. *: p<0.05 versus vehicle-treated group; **: p<0.01 versus vehicle-treated group; ##: p<0.01 versus bleomycin group.
DISCUSSION
The results of our study show that ONO-4057 attenuated pulmonary inflammation and fibrosis, reduced IL-6, IL-13, active TGF-β1 level, increased IFN-γ level in BALF and attenuated TGF-β expression in the lung tissue of bleomycin-treated mice.
LTs are important regulators of pulmonary fibrosis 3, 4. LTB4 and cysLT were increased in BALF and lung homogenates of patients with IPF and the levels of these mediators were found to correlate with the extent of fibrosis in histological sections 7, 8. In the human IPF lung tissue, the content of LTB4 is greater than that of cysLT. By contrast, Peters-Golden et al. 19 reported that the cysLT level greatly exceeded the LTB4 level in the BALF in a bleomycin-induced mouse model. Furthermore, Failla et al. 20 reported the efficacy of pharmacological inhibition of LT activity in the development of bleomycin-induced lung injury with mice treated with zileuton, a 5-lipoxygenase inhibitor and MK-571, a cysLT1 receptor antagonist. As the potency of zileuton for the inhibition of bleomycin-induced injury was similar to that of MK-571, they speculated that LTB4 does not have a predominant role in mediating inflammation and fibrosis in their bleomycin-treated mice 20. However, our results showed that the LTB4 level was significantly higher on day 7 and tended to be higher on day 14 in the bleomycin-treated mice than in the vehicle-treated mice, and the level was comparable to the cysLT level as shown in our previous report 12. Furthermore, ONO-4057, a BLT inhibitor, decreased the LTB4 level in BALF on day 7 and ameliorated bleomycin-induced pulmonary inflammation and fibrosis. These results suggest that LTB4, as well as cysLT, plays an important role in bleomycin-induced pulmonary fibrosis in our mouse model. The reason for the discrepancy between others and ours is uncertain but the difference in mouse strain, bleomycin dose, and the pharmacological mechanism of action (a synthetic enzyme inhibitor or a receptor antagonist) may have been influences.
Alternatively, the PGE2 level between the bleomycin group and the ONO-4057 group was not different, suggesting that the effects of ONO-4057 are unrelated to prostaglandin production. Thus, ONO-4057 did not affect the cycloxygenase pathway. This result was similar to a previous report that nordihydroquiaretic acid, 5-lipoxygenase inhibitor did not affect macrophage-derived PGE2 production in bleomycin-induced pulmonary fibrosis 21.
ONO-4057 is a specific LTB4 receptor antagonist that inhibits the human neutrophil aggregation, chemotaxis and degranulation induced by LTB4 13. In our study, the neutrophil number and the LTB4 level in BALF on day 7 was decreased by ONO-4057, indicating that neutrophil chemotaxis, aggregation and degranulation were suppressed. Alternatively, LTB4 plays an important role in antimicrobial lung defence through its production by alveolar macrophages and neutrophils, and the use of a BLT1 antagonist may compromise host immune response to pulmonary infection 3, although ONO-4057 (1 mg·kg−1) did not decrease the neutrophil number and the LTB4 level below the levels in the vehicle-treated group on day 7. Further examination may be necessary for clinical application of a BLT1 antagonist.
In the present study, LTB4 and active TGF-β1 levels in BALF were increased on day 7 and on day 14 in the bleomycin group, but were inhibited by ONO-4057 only on day 7. As neutrophils were accumulated on day 7, the source of TGF-β on day 7 may have been derived from neutrophils 22. In fact, neutrophils and epithelial cells on day 7 immunostained positive for TGF-β in our study, suggesting that these cells are the main source of TGF-β production. By contrast, the source of LTB4 and TGF-β on day 14 may have been derived from macrophages that were predominant cells on day 14. Although ONO-4057 failed to inhibit LTB4 and active TGF-β1 levels on day 14 in BALF, ONO-4057 inhibited macrophage number on day 14. In view of the fact that BLTRs are expressed in the spleen and on leukocytes 23, ONO-4057 probably inhibits neutrophil- and macrophage-mediated inflammation by suppressing BLTRs on neutrophils and macrophages and, as a result, inhibits TGF-β production especially by neutrophils. TGF-β is well known to be a critical growth factor in the fibrotic stage of pulmonary fibrosis and to play an important role in its pathogenesis, including the pathogenesis of bleomycin-induced fibrosis. As neutralisation of TGF-β with antibodies mitigates bleomycin-induced pulmonary fibrosis, and exogenous over-expression of Smad7, an inhibitor of the TGF-β signalling pathway, also mitigates bleomycin-induced pulmonary fibrosis, inhibition of the effects of TGF-β may provide an effective means of treating pulmonary fibrosis 24.
IFN-γ level in BALF was increased by ONO-4057 on day 14. IFN-γ has several effects that may play an important role at the interface between inflammation and fibrosis in bleomycin-induced pulmonary fibrosis. It has been found to inhibit collagen synthesis when cultures of fibroblasts have been exposed to them 25, and it eliminates the upregulation of collagen synthesis induced by TGF-β 26. Thus, this suggests that the increase in IFN-γ, as well as the decrease in TGF-β, by ONO-4057 may induce the inhibition of collagen synthesis.
Several recent papers have suggested that LTB4 functions not only as a local inflammatory mediator but also as an important chemoattractant for T cells as well as neutrophils 10. In an asthma model, BLT1 mediates LTB4-induced Th1 and Th2 cell chemotaxis and firm adhesion to endothelial cells exposed to flow, and mediates CD4+ and CD8+ T cell recruitment into the airway. In the present study, the number of lymphocytes was inhibited by ONO-4057 on day 14 and on day 21. Although the role of lymphocytes in the pathogenesis of the pulmonary fibrotic response has not been fully clarified, several studies have emphasised the importance and diverse roles of these cells in the fibrotic process 10. There are two major subsets of T-helper lymphocytes. One subset, Th1 cells, produces IL-2 and IFN-γ, and mediates cellular immune responses. The other subset, Th2 cells, produces IL-4, IL-5, IL-6 and IL-13, etc., and augments humoral immune responses. Th1/Th2 imbalance is proposed to be one of the hypotheses related to developing pulmonary fibrosis. ONO-4057 reduced IL-13 level but not IL-4 on day 7, and increased IFN-γ on day 14 (fig. 5⇑). These results suggest that ONO-4057 may improve Th1/Th2 balance. However, compared with these cytokines, the IL-6 level in BALF was extremely elevated on day 7 in our bleomycin-induced mouse model, and ONO-4057 clearly reduced IL-6. Several studies have demonstrated that IL-6 has both pro-inflammatory and anti-inflammatory properties 27. IL-6 also exhibits a negative feedback on the process of fibrosis. In fact, the IL-6 level of BALF is greatly increased in patients with IPF and in the bleomycin-induced model of pulmonary fibrosis 27. High IL-6 levels are correlated with alveolar hypercellularity and neutrophil counts in IPF 28. Recently, IL-6-deficient mice demonstrated attenuation in bleomycin-induced lung injury and fibrosis 29. Therefore, one of the mechanisms for the inhibition of inflammation and fibrosis by ONO-4057 may be associated with the decrease in IL-6.
In the present study, ONO-4057 delayed initiation group decreased the lymphocyte inflammation on day 21, but failed to decrease the hydroxyproline and the Ashcroft score on day 21. These results suggest that the prevention of developing fibrosis by ONO-4057 may be associated with inhibiting the subsequent fibrosis after inflammation rather than fibrotic process per se. However, as a period of ONO-4057 administration in ONO-4057 delayed initiation group was short (on days 15–19), the possibility cannot be denied that longer administration of ONO-4057 may attenuate the development of fibrosis thereafter; further examination is needed.
In conclusion, ONO-4057 inhibited inflammation by the decrease in the inflammatory cells including neutrophils, macrophages and lymphocytes. More importantly, the decrease in Ashcroft score and lung hydroxyproline content, the decrease in active TGF-β1 level in BALF and the reduced TGF-β expression detected immunohistochemically in the lung sections indicated that ONO-4057 mitigated the development of fibrosis. Current therapy for pulmonary fibrosis is ineffective and the disease has a poor outcome and is associated with severe morbidity 30. The BLTR antagonist shows promise of becoming a new means of treating IPF.
Support statement
This research was supported, in part, by The Katano Grant given by the Alumni Association of Kansai Medical University.
Statement of interest
None declared.
Acknowledgments
The authors would like to thank M. Shino and Y. Sugimura (both First Dept of Medicine, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan) for their technical assistance.
- Received September 19, 2008.
- Accepted May 5, 2009.
- © ERS Journals Ltd
References
