INTRODUCTION

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Idiopathic pulmonary fibrosis (IPF) is commonly lethal, and current therapies for this disease have little effect on patient prognosis (1, 2). Despite many recent studies, its etiology remains uncertain (1, 3). Bleomycin is an antineoplastic agent that causes alveolar damage and pulmonary fibrosis as side effects (4). The pathophysiological findings in bleomycin- induced pulmonary fibrosis in animals resemble those in human IPF, so such fibrosis has been widely used as an animal model for this disease (5).

Generally, experimental bleomycin-induced pulmonary fibrosis completes within 28 d of the last stimulus (6). The first 14 d correspond to the acute stage, in which inflammation dominates, while the second 14 d correspond to the fibrotic stage (5, 6). Neutrophils appear to play a role in the pathogenesis of both IPF and bleomycin-induced pulmonary fibrosis (3, 7). Neutrophil elastase (NE) is an elastolytic enzyme with a broad substrate range that will digest most proteins and is known to be involved in some kinds of human lung injury (8). The level of NE in bronchoalveolar lavage (BAL) fluids is known to be elevated in IPF; therefore, elevated NE has been speculated to contribute to its pathogenesis (9). Although previous studies have demonstrated the inhibitory effects of nonspecific NE inhibitors such as 1-proteinase inhibitor (7) and truncated secretory leukoprotease inhibitor (10) on bleomycin-induced pulmonary fibrosis, the possibility that these effects were induced by mechanisms other than the inhibition of NE remains.

The compound known as ONO-5046 · Na (sodium N-[2-[4- (2,2-dimethylpropionyloxy)phenylsulfonylamino] benzoyl]aminoacetate tetrahydrate) was developed as a low-molecular weight (528.51 D) inhibitor of human NE (11). It is known to be ineffective against trypsin, thrombin, plasmin, kallikrein, chymotrypsin, and cathepsin G (11). Although ONO-5046 · Na inhibits porcine pancreatic elastase, the activity is very weak. Thus, its inhibitory effect against protease is considered to be specific for NE (11). In addition, ONO-5046 · Na has also been shown to inhibit NE effectively in other experimental animals (11), suggesting its usefulness in studying the pathogenic roles of NE in animal models. Although several studies have reported the inhibitory effects of ONO-5046 · Na on experimental acute lung injury (12, 13), there are no reports of its effects on pulmonary fibrosis.

To clarify the potential role of NE in the development of pulmonary fibrosis as well as acute lung injury, we investigated the efficacy of ONO-5046 · Na in treating bleomycin-induced pulmonary fibrosis. In addition, we studied cytokine mRNA expression during this process to evaluate the relationship between NE and other inflammatory cells. We previously showed the importance of cytokines such as interleukin (IL)-1, platelet- derived growth factor (PDGF-A), and insulin-like growth factor (IGF-I) during the development of bleomycin-induced pulmonary fibrosis (14). The level of IL-8 in BAL fluid is known to be elevated in human pulmonary fibrosis, and IL-8 has the capacity to recruit and activate neutrophils (15). However, no molecules with high homology to human IL-8 have been identified in mice. Macrophage inflammatory protein-2 (MIP-2) is the murine functional homolog of human IL-8 and is a potent chemotactic agent for neutrophils (16). To elucidate the possible mechanism by which NE inhibitors suppress pulmonary fibrosis, we measured levels of these cytokines, which are considered to be involved in its pathogenesis.

	METHODS

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Animal Preparation

A total of 98 specific pathogen-free 8-wk-old male Institute for Cancer Research mice, weighing 30 to 37 g, were purchased from Charles River Japan (Yokohama, Japan). All mice were maintained under standard conditions with free access to water and rodent laboratory food.

Study Design and Experimental Protocol

As shown in Figure 1, bleomycin (Bleo, Nippon Kayaku, Tokyo, Japan) was dissolved in 200 µl saline solution and administered intraperitoneally (ip) at a dose of 7.5 mg/kg body weight for 10 sequential d (6) to 10 wk old group A and group B mice. The ONO-5046 · Na, dissolved in 200 µl saline solution, was administered subcutaneously (sc) for 10 d at a dose of 100 mg/kg body weight to group B mice. Saline solution was administered to groups A and S mice as a control. Thus, group A mice (n = 30) were treated with BLM (ip) and saline (sc), group B mice (n = 30) with BLM (ip) and ONO-5046 · Na (sc), and group S mice (n = 30) with only saline (ip and sc). After their weight was measured, 10 mice in each group were sacrificed by cervical dislocation under ether anesthesia 1, 15, and 29 d after the last administration of drugs. Five mice from each of these groups were subjected to BAL. The remaining five were used for measurement of lung hydroxyproline content and histopathological evaluation.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Study design and experimental protocol. Arrows indicate times of sacrifice. BLM, bleomycin; ONO, ONO-5046 · Na; ip, intraperitoneal injection; sc, subcutaneous injection.

To determine the efficacy of ONO-5046 · Na administration after exposure to bleomycin, we used two additional groups as follows: ONO-5046 · Na (100 mg/kg body weight) was administered subcutaneously from days 1 to 14 to group C (n = 4) and from days 15 to 28 to group D (n = 4) after 10 d of bleomycin administration. Group C therefore corresponds to a trial of ONO-5046 · Na in the acute stage and Group D to a trial in the fibrotic stage. At 29 d, these mice were sacrificed, evaluated histopathologically, and their lung hydroxyproline contents measured.

Measurement of Wet Lung Weight

The heart and lungs were removed en bloc from the five mice in each group that were not subjected to BAL at 1, 15, and 29 d, and the wet lung weight was measured as an indicator of lung inflammation (17) after careful excision of extraneous tissues. The wet lung weight to body weight ratio was then calculated.

Histopathological Evaluation

After the measurement of wet lung weight, each right lung was fixed with 10% formaldehyde neutral buffer solution over at least 48 h, then embedded in paraffin. Sequential 3 µm sections were stained with hematoxylin-eosin and Azan-Mallory stains. Severity of fibrosis was semiquantitatively assessed, according to the method of Ashcroft and co-workers (18). Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by examining 30 randomly chosen regions per sample at a magnification of ×100. Criteria for grading lung fibrosis were as follows: Grade 0, normal lung; Grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; Grade 3, moderate thickening of walls without obvious damage to lung architecture; Grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; Grade 7, severe distortion of structure and large fibrous areas; Grade 8, total fibrous obliteration of the fields. If there was any difficulty in deciding between two odd-numbered categories, the field would be given the intervening even-numbered grade. The score of lung fibrosis was expressed as a mean grade of fibrosis for each sample.

Measurement of Lung Hydroxyproline Content

To estimate the total amount of collagen deposited as an indicator of pulmonary fibrosis (19), the hydroxyproline content of the left lung was measured in each group at 29 d as described by Nakazawa and colleagues (20). Briefly, after their wet weights were measured, the homogenized left lungs were hydrolyzed with 0.5 ml 12 N hydrochloric acid in capped tubes for 20 h at 100° C. After neutralization with sodium hydroxide, a 0.1 ml aliquot of supernatant was mixed in 1.5 ml 0.3 N lithium hydroxide solution. The hydroxyproline contents of the hydrolysates were analyzed using high-performance liquid chromatography and expressed as µmol/g.

Bronchoalveolar Lavage

Bronchoalveolar lavage was carried out in five mice from each group. After excision of the trachea, a plastic cannula was inserted into the trachea and 1 ml saline solution was gently injected via a syringe, then withdrawn. This procedure was repeated five times. A 500 µl aliquot of BAL fluid was reserved for total cell count and cell differentiation, and the remainder was centrifuged immediately at 1,100 rpm for 10 min. The cell pellet was preserved at 80° C for extraction of mRNA. The total cell number was counted with a standard hemocytometer. Cell differentiation was examined by counting at least 200 cells on a smear prepared using cytospin and Wright-Giemsa staining.

Semiquantification of Cytokine mRNA by Reverse Transcription Polymerase Chain Reaction

The poly (A)⁺ RNA was directly extracted from the BAL cell pellet using guanidinium thiocyanate solution and an oligo (dT)-cellulose spun-column (Quick Prep Micro mRNA Purification kit; Pharmacia Biotech, Tokyo, Japan). One quarter of the poly (A)⁺ RNA was reverse-transcribed into cDNA in a 20 µl reaction mixture containing 1.2 µM oligo (dT)₁₈ primers (Sigma, St. Louis, MO), 0.5 mM each deoxynucleotide (TaKaRa, Kyoto, Japan), 20 units RNase inhibitor (RNasin; Promega, Madison, WI), 200 units Molony murine leukemia virus RNase H reverse transcriptase (Superscript; GIBCO/BRL, Gaithersburg, MD), 10 mM DTT, 50 mM Tris-HCl buffer (pH 8.3), 75 mM KCl, and 3 mM MgCl₂. The reaction mixture was incubated at 37° C for 60 min, then at 95° C for 5 min to inactivate the reverse transcriptase.

Oligonucleotide primers used in the present study are as follows: -actin (540 bp) 5' primer 5'-GTGGGCCGCTCTAGGCACCAA-3', 3' primer 5'-CTCTTTGATGTCACGCACGATTTC-3'; IL-1 (625 bp) 5' primer 5'-ATGGCCAAAGTTCCTGACTTGTTT-3', 3' primer 5'-CCTTCAGCAACACGGGCTGGTC-3'; IL-1 (382 bp) 5' primer 5'-GCAACTGTTCCTGAACTCA-3', 3' primer 5'-CTCGGAGCCTGTAGTGCAG-3'; MIP-2 (357 bp) 5' primer 5'-GCTGGCCACCAACCACCAGG-3', 3' primer 5'-AGCGAGGCACATCAGGTACG-3'; PDGF-A (225 bp) 5' primer 5'-TGTGCCCATTCGCAGGAAGAG-3', 3' primer 5'-TTGGCCACCTTGACACTGCG-3'; PDGF-B (299 bp) 5' primer 5'-GCAATAACCGCAATGTGCAATGCC-3', 3' primer 5'-CGCCTTGTCATGGGTGTGCTTAAA-3'; and IGF-I (321 bp) 5' primer 5'-TCGTCTTCACACCTCTTCTACCTG-3', 3' primer 5'-CTTCTGAGTCTTGGGCATGTCAGT-3'. The primers for -actin and IL-1 were purchased from Clontech (Palo Alto, CA), while those for IL-1 (21), MIP-2 (16), PDGF-A (GenBank, accession number M29464), PDGF-B (22), and IGF-I (23) were synthesized according to their published sequences and purified by a manufacturer (Toyobo, Osaka, Japan). The optimal number of polymerase chain reaction (PCR) cycles for each primer set was determined in preliminary experiments so that the amplification process was carried out during the exponential phase of amplification. The numbers of PCR cycles were as follows: 30 for -actin, 34 for IL-1, 36 for IL-1, 36 for MIP-2, 35 for PDGF-A, 36 for PDGF-B, and 35 for IGF-I. Coamplification of the cDNA for each cytokine and -actin was then performed in one tube following published methods (14, 24). The -actin primers were added after several cycles with only cytokine primer so that the final number of PCR cycles was optimal for both the cytokine and -actin. Coincident amplifications were carried out with 1 µl cDNA solution in a total of 100 µl reaction mixture containing 0.5 µM of each primer, 200 µM of each deoxynucleotide, 2.5 units Taq DNA polymerase (AmpliTaq; Perkin-Elmer, Foster City, CA), 10 mM Tris-HCl buffer (pH 8.3), 0.001% gelatin, 50 mM KCl, and 1.5 mM MgCl₂.

The PCR was carried out using a Program Temp Control System PC-800 (ASTEC, Fukuoka, Japan) under the following conditions: denaturation at 95° C for 1 min, annealing at 63° C for 1.5 min, and extension at 72° C for 2.5 min. Then 8 µl of each PCR product was electrophoresed on a 2% agarose gel and stained with ethidium bromide. The intensity of ethidium bromide luminescence for each PCR product was measured by a charge-coupled device imaging system (Densitograph AE-6900MF; ATTO, Tokyo, Japan). The cytokin/-actin ratio of the intensity of ethidium bromide luminescence for each PCR product was calculated. To evaluate the relative amount of cytokine mRNA in each mouse, the cytokine/-actin ratio of each animal was then divided by that of one control mouse sacrificed on the same day (14). These procedures were repeated and the reproducibility was confirmed.

Statistical Analysis

All data were expressed as means ± standard error of the means. Statistical analysis was performed using StatView-J II software on a Macintosh computer. One-way analysis of variance followed by Fisher's least significant difference test was used to detect differences between groups, and a probability value of less than 0.05 was considered statistically significant.

	RESULTS

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Wet Lung Weight

Wet lung weights and wet lung weight/body weight ratios are listed in Table 1. Both wet lung weight and the wet lung weight/body weight ratio in group A were significantly higher than those in group S at 1, 15, and 29 d and those in group B at 15 and 29 d (p < 0.01).

Histopathological Evaluation

Infiltration of alveolar macrophages and neutrophils into the alveolar space was noted in group A at 1 and 15 d after treatment. The alveolar wall was also thickened by edema and infiltration of fibroblasts, and some inflammatory cells including neutrophils, alveolar macrophages, and lymphocytes were observed. These histological changes were mild in group B (not shown). At 29 d, multifocal fibrosis was observed mainly in the subpleural regions in group A (Figure 2a). In group B, mild infiltration of inflammatory cells into the alveolar and interstitial regions with slightly thickened and edematous alveolar walls were observed but there were no apparent fibrotic regions (Figure 2b). In group C at 29 d, the alveolar wall was thickened and edematous with some inflammatory cells, but no fibrotic changes were observed, similar to the condition of group B (Figure 2c). In group D at 29 d, fibrotic changes as severe as those in group A occurred (Figure 2d). Throughout the whole experimental period, there were no abnormal histological alterations in group S (Figure 2e).

View larger version (155K): [in this window] [in a new window]   Figure 2.   Histologic sections of right lung fields 29 d after the last bleomycin administration. (a) Group A: subpleural pulmonary fibrosis was observed. (b) Group B: the alveolar wall was thickened with some inflammatory cells, but it had no fibrotic regions. (c ) Group C: similar appearance to that of B. (d ) Group D: patchy fibrotic changes as severe as those in group A occurred. (e ) Group S: the normal alveolar structure was maintained. (Hematoxylin-eosin stain; original magnification ×50)

The grades of fibrosis in groups A, B, C, D, and S are presented in Table 2. All bleomycin-treated groups (A, B, C, and D) demonstrated significantly higher scores than group S. Among them, the scores in group A were significantly higher than those in group D (p < 0.05) and groups B and C (p < 0.01). The scores in groups B and C were comparable and significantly decreased compared with those in group A. The scores in group D were significantly higher than those in group C (p < 0.01).

Lung Hydroxyproline Analysis

A comparison of the hydroxyproline contents in groups A, B, C, D, and S are presented in Table 2. The hydroxyproline contents in groups B and C exhibited no significant differences compared with group S, while they were significantly decreased compared with group A. The value in group C was somewhat low compared with group B, but there was no significant difference between them. No significant difference was observed between groups A and D, and the content in group D was significantly higher than that in groups C (p < 0.01) and S (p < 0.05).

Total Cell Number and Cell Differentiation of Bronchoalveolar Lavage Fluid

The recovery rates of BAL fluids in all groups were over 85%. At 1, 15, and 29 d, total cell numbers and the proportion of neutrophils in the BAL fluids significantly increased in group A compared with group S (p < 0.01) (Table 3). Although the percentages of neutrophils in group B were also higher than those in group S, with a peak on day 1, they were significantly lower than those in group A throughout the course of the study, as were the total cell numbers (p < 0.05).

Semiquantification of Cytokine mRNA

Cytokine mRNA levels in BAL cells were analyzed and compared among groups A, B, and S. In all samples examined, the reverse transcription polymerase chain reaction (RT-PCR) products amplified from -actin and each cytokine mRNA could be detected by ethidium bromide staining. The relative amounts of IL-1 mRNA were increased by approximately 4.7 and 2.1 times in group A at 1 and 15 d, respectively, compared with those in group S mice (p < 0.01), but had normalized at 29 d (Figure 3). The level of IL-1 mRNA in group B on day 1 was significantly lower (p < 0.01) than that in group A. The MIP-2 mRNA level was increased about 2.0 times in group A on day 1 compared with that in group S, but the values were similar in all three groups at 15 and 29 d. The MIP-2 level in group B at 1 d was somewhat decreased compared with that in group A. At 1, 15, and 29 d, the levels of PDGF-A and IGF-I mRNA in group A were significantly higher than those in group S (p < 0.01). The levels of PDGF-A and IGF-I mRNA in group B at 1 and 15 d were significantly lower than those in group A, whereas there were no significant differences at 29 d. No significant differences in the levels of IL-1 or PDGF-B mRNA were observed among the three groups.

View larger version (38K): [in this window] [in a new window]   Figure 3.   Relative amounts of cytokine mRNA in BAL cells from bleomycin-treated mice (group A), bleomycin and ONO-5046 · Na-treated mice (group B), and saline solution-treated mice (group S). Bars represent the mean; extensions show + SEM. *p < 0.05; **p < 0.01.

	DISCUSSION

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In the present study, we demonstrated that ONO-5046 · Na attenuated the severity of BLM-induced acute lung injury, as shown by the wet lung weight to body weight ratios and BAL findings, and also that it reduced the severity of subsequent pulmonary fibrosis, as shown by the lung hydroxyproline contents and the results of the histopathological evaluation. The fact that the proportion of neutrophils in group A was significantly increased compared with that in group S from day 1 implies that neutrophils contribute to lung injury. Neutrophil elastase is known to cause endothelial cell injury (25), and a previous study showed that it increased epithelial permeability (26). One possible reason for the decrease in the proportion of neutrophils in group B could therefore be suppression of epithelial permeability due to ONO-5046 · Na-induced inhibition of NE.

We initially tried to measure the NE activity of concentrated BAL fluid using a previously reported method (27), but contrary to our expectations, activity was undetectable in all animals. This might have been due to the current lack of proper methods for evaluating mouse NE activity, as no specific substrate for mouse NE has so far been identified. We considered that the dosage of ONO-5046 · Na used in this experiment would have been capable of inhibiting NE activity in vivo. This is supported by the results of an in vitro experiment (11) that showed that a very low concentration of ONO-5046 · Na could inhibit the hydrolysis of substrate by mouse NE.

The period from 1 to 14 d after the last stimulus has been reported to represent the acute stage, and the period from 15 to 28 d the fibrotic stage of experimental bleomycin-induced pulmonary fibrosis (5, 6). The suppressive effects of ONO-5046 · Na in groups B and C, in which the drug was administered before or during the acute stage, indicate that NE might play an important role in acute lung inflammation. However, ONO-5046 · Na did not inhibit pulmonary fibrosis in group D mice, suggesting that NE does not have much influence on the fibrotic stage (the period from 15 to 29 d in our model). Shen and colleagues (28) indicated that the severity of fibrosis induced by bleomycin was closely related to the severity of acute lung injury in hamsters. It might therefore be possible to attenuate fibrosis by inhibiting acute lung injury. Thus, we speculate that ONO-5046 · Na had its major effect in the acute stage, but only a minimal effect in the fibrotic stage. Furthermore, excessive NE activity might be involved in the development of acute lung injury and fibrosis in groups A and D, and ONO-5046 · Na may have ameliorated acute lung injury and subsequent fibrosis by inhibiting NE activity in groups B and C.

To our knowledge, the relationship between NE and cytokine production in alveolar macrophages has not been thoroughly evaluated. Furthermore, there are few reports that discuss the effects of NE or NE inhibitors on cytokine production (29, 30). We have previously demonstrated the significance of the cytokine network in bleomycin-induced pulmonary fibrosis in mice (14). To clarify the relationship between NE and cytokine expression, which are both considered to be involved in the pathogenesis of bleomycin-induced pulmonary fibrosis, we examined the expression of several kinds of cytokine mRNA. In this experiment, we used the RT-PCR technique (14, 24) to compare expression levels of cytokine mRNA. These cytokines were considered to originate mainly from alveolar macrophages, as these cells composed about 90% of the BAL cell population in all groups. To avoid any artificial influence on the expression of cytokines during the sorting of alveolar macrophages, we did not separate alveolar macrophages from other types of cells. The fact that levels of IL-1, MIP-2, PDGF-A, and IGF-I mRNA were lower in group B than in group A suggested that ONO-5046 · Na reduced the expression of these cytokines' mRNA, either directly or indirectly. Macrophage inflammatory protein-2 is a member of the C-X-C subgroup of chemokines, which are 6 kD basic proteins and are important mediators of inflammation with chemotactic activity for neutrophils (16, 31). Interleukin-1 also has the potential to cause the accumulation of inflammatory cells (32). These results suggest that MIP-2 and IL-1 were responsible, at least in part, for the recruitment of neutrophils. Both PDGF and IGF-I play significant roles in the accumulation of extracellular matrix and generation of fibrosis (32). As in our previous study (14), enhanced expression of PDGF-A mRNA but not PDGF-B mRNA was observed during the development of pulmonary fibrosis in the present study. There were no significant changes in IL-1 and PDGF-B mRNA levels in any group, suggesting that these cytokines are not likely to be important in the pathogenesis of lung injury in our model. In addition, the fact that ONO-5046 · Na did not suppress IL-1 or PDGF-B mRNA levels but did suppress those of other cytokine mRNA in bleomycin-treated mice indicates that the inhibitory effect of ONO-5046 · Na may be due to a specific mechanism. These results, together with the fact that macrophages carry surface receptors for NE (25), suggest a possible interaction between NE and macrophage-releasing cytokines. Because levels of IL-1 and MIP-2 mRNA were lower in group B than in group A at 1 d but not at 15 or 29 d, IL-1 and MIP-2 may play important roles in the early inflammatory phase of IPF. On the other hand, PDGF-A and IGF-I, whose mRNA levels were higher in group A than in group B at 1 and 15 d, may play central roles in fibrogenesis. At 29 d, ONO-5046 · Na did not suppress PDGF-A or IGF-I mRNA levels. Although it is difficult to explain this phenomenon, it supports our hypothesis that ONO-5046 · Na has a major effect in the acute stage but little effect in the fibrotic stage. Another possibility is that the lack of suppression at 29 d could be due to a rebound-like phenomenon caused by withdrawing ONO-5046 · Na. Alternatively, ONO-5046 · Na's effects depend on other kinds of cells and their chemical mediators.

Our results suggest that ONO-5046 · Na could be a promising new therapeutic agent for treating IPF, especially active IPF without obvious fibrosis. It could also be potentially useful as prophylaxis against anticancer-drug-induced pneumonitis during chemotherapy. It has been reported that IPF patients with a high proportion of neutrophils among their BAL cells exhibit resistance to corticosteroid therapy (35). ONO-5046 · Na represents a possible new therapeutic agent for these patients. In the present study, we could not rule out the possibility that ONO-5046 · Na has an unknown anti-inflammatory action as well as an NE inhibitory effect, and further study is needed to clarify the precise mechanisms underlying its protective effect in pulmonary fibrosis.

In conclusion, we demonstrated the inhibitory effects of ONO-5046 · Na on bleomycin-induced pulmonary fibrosis in mice. The fact that IL-1, MIP-2, PDGF-A, and IGF-I mRNA levels were suppressed by administration of an NE inhibitor indicates that these cytokines are involved in the pathogenesis of pulmonary fibrosis and that NE is possibly involved in the expression of these cytokines. We also propose that NE might be involved in the early stages of lung inflammation during the development of pulmonary fibrosis.