INTRODUCTION

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Pulmonary fibrosis is an entity comprising heterogeneous diseases, of which idiopathic pulmonary fibrosis (IPF) is a representative with a very poor prognosis (1, 2). Current therapeutic agents show few or no effects against IPF. Although the pathophysiology of pulmonary fibrosis has not yet been well clarified, it is generally assumed to result from overhealing and remodeling following acute lung injury (3).

Previous studies have shown that many kinds of chemical factors, including cytokines and oxidant/antioxidant imbalance, are involved in the acute lung injury of IPF (4). Active oxygen produced and released from neutrophils and alveolar macrophages (AM) may be involved in this process via tissue-damaging actions and the induction of cytokines and adhesion molecules (7).

In the normal airway mucosa, antioxidant defense systems, such as superoxide dismutase (SOD), glutathione peroxidase, and catalase exist to protect the mucosa from various oxidant stimuli. It has been reported that glutathione levels in bronchoalveolar lavage fluid (BALF) are low in patients with IPF, possibly as a consequence of the epithelial cell damage in the disease (8). Contrastingly, increased serum levels of SOD have been reported to correlate with disease severity in IPF, possibly in conjunction with degranulation of activated neutrophils (9). These findings suggest that an antioxidant such as SOD might be a new therapeutic candidate for IPF. SOD was extracted as an antiinflammatory protein in bovine blood, and was later shown to specifically delete active oxygen in the form of superoxide anion (O₂). Because active oxygen was shown to be involved in various inflammatory conditions, clinical applications of SOD have been attempted, but so far without success (10). The main reason for this is that because the lifespan of many active oxygen species is extremely short. Therefore, the blood concentration of SOD should be maintained, and its affinity for the cell membrane, where active oxygen is mainly detected, should be increased in order to obtain its clinical effects. In rat models of adjuvant-induced arthritis and bleomycin (BLM)-induced lung fibrosis, Mn SOD, with a prolonged half-life in blood, was shown to be more efficient as an antiinflammatory agent than was Cu-Zn SOD, which has a short half-life (11). We therefore conducted an evaluation of the effect on BLM-induced pulmonary fibrosis of modified SOD with a much longer half-life.

Lecithinized SOD (phosphatidylcholine [PC]-SOD) is a chemically modified SOD that was recently synthesized by covalent bonding of the lecithin derivative phosphatidylcholine to recombinant human Cu-Zn SOD to increase the cellular affinity of SOD (12). PC-SOD may accumulate at the site of a lesion after intravenous administration, through the high cell-membrane affinity of lecithin, and efficiently exhibit effects of SOD (13).

BLM-induced pulmonary fibrosis is a popular animal model of human pulmonary fibrosis, and we have reported the effectiveness of neutrophil elastase inhibitor on BLM-induced pulmonary fibrosis in mice (14). Generally, BLM-induced pulmonary fibrosis in mice is almost complete within 4 wk after administration of BLM. During the acute stage of the disease (first 2 wk), persistent acute lung injury follows initial injury by BLM. Then, as a result of overhealing of lung injury, remodeling of normal alveolar structure occurs during the chronic stage of the disease (second 2 wk) (15, 16). In the development of pulmonary fibrosis, proinflammatory cytokines and growth factor are considered to play important roles in cell-cell and cell-matrix interactions. We also found that interleukin (IL)-1 and platelet-derived growth factor (PDGF)-A are closely related to the pathogenesis of BLM-induced pulmonary fibrosis (17).

In the present study, done to examine whether PC-SOD may be a new treatment agent for pulmonary fibrosis, we administered PC-SOD to a mouse model of pulmonary fibrosis prepared by intraperitoneal administration of BLM, and examined the inhibitory effects of this agent alone or in combination with methylprednisolone (mPSL), which is currently used as a standard treatment agent for pulmonary fibrosis. We also measured expression levels of cytokines during the development of pulmonary fibrosis.

	METHODS

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Experiment 1

Experimental protocol. A total of 75 specific-pathogen-free, 8-wk-old male Institute for Cancer Research (ICR) mice, weighing 30 to 38 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.

The protocol for Experiment 1 is shown in Figure 1. BLM (Bleo; Nippon Kayaku, Tokyo, Japan) was dissolved in 200 µl saline solution and administered intraperitoneally at a dose of 10 mg/kg/d for 10 sequential days to mice in four groups (Groups B, C, D, and E). PC-SOD, obtained from the Seikagaku Corporation (Tokyo, Japan) and the Asahi Glass Corporation (Tokyo, Japan), dissolved in 200 µl 10% xylitol solution, was administered intravenously for 10 d at a dose of 1 mg/kg/d to Group C mice and at a dose of 10 mg/kg/d to Group D mice. These doses were based on a previous report (18). mPSL, dissolved in 200 µl 10% xylitol solution, was administered intravenously for 10 d at a dose of 15 mg/kg/d to Group E mice. Saline solution was administered intraperitoneally to mice in Group A. Xylitol solution was administered intravenously to mice in Groups A and B. Thus, Group A mice were treated with saline and xylitol, Group B mice were treated with BLM and xylitol, Groups C and D mice were treated with BLM and PC-SOD, and Group E mice were treated with BLM and mPSL. After measurement of their body weight, five mice in each group were killed by cervical dislocation under ether anesthesia, and were subjected to bronchoalveolar lavage (BAL) at 1 and 29 d after the last administration of the drugs used in the study. Five further mice in each group were killed at 29 d, and subjected to measurement of lung hydroxyproline content and histopathologic evaluation.

View larger version (32K): [in this window] [in a new window]   Figure 1.   Experimental design. Arrows indicate times of killing. ip = intraperitoneal injection, iv = intravenous injection, BLM = bleomycin, mPSL = methylprednisolone.

Lung wet weight. In each group of mice (n = 5), the heart and lungs were removed en bloc on Day 29 after the last administration of BLM. After excision of extraneous tissues, the lung wet weight was measured as an indicator of lung inflammation, and the lung wet weight-to-body weight ratio (as a percent) was calculated.

BAL. BAL was performed as previously reported (14, 17). A 500-µl aliquot of BALF was used for total cell count and cell differentiation, and the remainder of the fluid was centrifuged immediately at 1,100 rpm for 10 min. The cell pellet was stored at 80° C until extraction of messenger RNA (mRNA). The total cell number was counted with a hemocytometer. Cell differentiation was done by counting 200 cells on a cytospin-prepared smear with Wright-Giemsa staining.

Pathologic evaluation. After the measurement of wet lung weight, each right lung was fixed with 10% formaldehyde/neutral buffer solution over a period of 48 h. Sequential 3-µm sections were embedded in paraffin and stained with hematoxylin and eosin and Azan-Mallory stain. Severity of fibrosis was evaluated semiquantitatively according to the method of Ashcroft and colleagues (19). Briefly, the grade of lung fibrosis was scored on a scale of 0 to 8 after examination of 30 randomly chosen regions in each 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 lung fields. In cases of difficulty in deciding between two odd-numbered grades of fibrosis, the field was given the intervening, even-numbered grade. The score for lung fibrosis was expressed as the mean grade of fibrosis in each sample.

Measurement of lung hydroxyproline content. To estimate the total amount of collagen deposited in the lung as an indicator of pulmonary fibrosis, we measured the hydroxyproline content of the left lung in each group of mice at 29 d, using the method described by Woessner (20). After measurement of its wet weight, the left lung was digested in a 0.25% protease (Actinase; Kaken Pharmaceutical, Chiba, Japan) solution at 55° C for more than 12 h, and the absorbance at 557 nm was measured spectrophotometrically.

Semiquantification of cytokine mRNA by reverse transcription- polymerase chain reaction. From the BALF cell pellet, polyadenine (poly[A])-bearing RNA was extracted with guanidinium thiocyanate solution and an oligodeoxythymidine (oligo[dT])-cellulose spun column (Quick Prep Micro mRNA Purification Kit; Pharmacia Biotech, Tokyo, Japan). The poly(A)-bearing RNA was then reverse-transcribed into complementary DNA (cDNA) in 20 µl of reaction mixture containing 1.2 µM oligo(dT)₁₈ primers (Sigma, St. Louis, MO), 0.5 mM of each deoxynucleotide (TaKaRa, Kyoto, Japan), 20 U of ribonuclease (RNase) inhibitor (RNasin; Promega, Madison, MI), 200 units of Moloney murine leukemia virus ribonuclease H reverse transcriptase (Superscript; GIBCO/BRL, Gaithersburg, MD), 10 mM dithiothreitol, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl₂. The reaction mixture was incubated at 37° C for 60 min and then heated at 95° C for 5 min to inactivate the reverse transcriptase. Oligonucleotide primers used in the study were as follows: -actin (540 bp) 5' primer: 5'-GTGGGCCGCTCTAGGCACCAA-3', 3' primer: 5'-CTCTTTGATGTCACGCACGATTTC-3'; IL-1 (382 bp) 5' primer: 5'-GCAACTGTTCCTGAACTCA-3', 3' primer: 5'- CTCGGAGCCTGTAGTGCAG-3'; PDGF-A (225 bp) 5' primer: 5'-TGTGCCCATTCGCAGGAAGAG-3', 3' primer: 5'-TTGGCCACCTTGACACTGCG-3'. The primers for -actin were purchased from Clontech (Palo Alto, CA) and those for IL-1 and PDGF-A (GenBank, accession no. M29464) were synthesized according to their published sequences and purified by the 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 conducted during the exponential phase of amplification. The numbers of PCR cycles were 32 for -actin and 38 each for IL-1 and PDGF-A. Coincident amplifications were conducted with 1 µl cDNA solution in a total of 100 µl reaction mixture containing 0.5 M of each primer, 200 M of each deoxynucleotide, 2.5 units Taq DNA polymerase (AmpliTaq Gold; Perkin-Elmer, Foster City, CA), 10 mM Tris-HCl (pH 8.3), 0.001% gelatin, 50 mM KCl, and 1.5 mM MgCl₂. PCR was done with the PC-800 programmed temperature control system (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. Following this, 8 ml of each PCR product was electrophoresed on a 2% agarose gel and stained with ethidium bromide. Five microliters of Low DNA Mass Ladder (GIBCO/BRL) with a known amount of DNA was also electrophoresed on every gel with the PCR reaction products in order to adjust for intergel differences in staining intensity. The intensity of ethidium bromide luminescence for each PCR product was measured with a charge-coupled device (CCD)-based imaging system (Densitograph AE-6900MF; ATTO, Tokyo, Japan). The cytokine-to--actin ratio of the intensity of ethidium bromide luminescence for each PCR product was calculated. These procedures were repeated and the reproducibility of the results was confirmed.

Experiment 2

To estimate the inhibitory effects of combination therapy with PC-SOD and corticosteroid on pulmonary fibrosis, we established a group of mice treated with 1 mg/kg/d of PC-SOD, which was shown to be effective in Experiment 1, and a group treated with 1 mg/kg/d of PC-SOD and 15 mg/kg/d of mPSL, using a mouse BLM-induced model of pulmonary fibrosis. All mice were killed on Day 29 after BLM administration.

Samples were prepared as described for Experiment 1. As a control group, we established a group designated Group B' (n = 5), which was given BLM intraperitoneally and xylitol intravenously. A further group, designated Group C' (n = 5), received 1 mg/kg/d of PC-SOD alone. A third group, Group F (n = 5), received 1 mg/kg/d PC-SOD and 15 mg/kg/d mPSL. In each group, the lungs were extracted 29 d after BLM administration, as described for Experiment 1. Histologic features were examined and hydroxyproline content was measured to evaluate the severity of fibrosis. In Experiment 2, hydroxyproline was measured through high-performance liquid chromatography.

Statistical Analysis

Data are expressed as means ± SEM. Statistical analysis was done with StatView-J IV software (Brainpower, Inc., Calabasas, CA) on a Macintosh computer (Apple, Inc., Cupertino, CA). One-way analysis of variance, followed by Fisher's least significant difference test, was used to detect differences among groups, and a value of p < 0.05 was considered significant.

	RESULTS

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Experiment 1

Lung wet weight. Both lung wet weights and the lung wet weight-to-body weight ratio on Day 29 are shown in Table 1. Lung wet weight in Group E was significantly higher than that in Group A. Lung wet weight-to-body weight ratios in Groups B and E were significantly higher than that in Group A. 

Total cell number and cell differentiation of BAL. The recovery rates of BALF were greater than 85% in all groups. On Day 1 after treatment with BLM, total cell numbers and the percentage of neutrophils and lymphocytes in Group B were significantly higher than those in Group A (p < 0.01) (Table 2). Total cell numbers in Groups C, D, and E were significantly lower than that in Group B, and the percentages of neutrophils and lymphocytes in Groups C and D were significantly lower than those in Group B. On Day 29, total cell numbers and the proportion of lymphocytes in Group B were significantly greater than those in Group A. Total cell numbers in Groups C and D were significantly lower than those in Group B, but there were no significant differences in the proportions of neutrophils and lymphocytes in BALF in any group from those in any other except Group A. 

Pathological evaluation. On Day 29, multifocal fibrosis, located mainly in the subpleural regions, was observed in Group B (Figure 2b). In Group C, mild infiltration of inflammatory cells into the alveolar and interstitial regions, and slightly thickened and edematous alveolar walls, were observed, but there were no apparent fibrotic regions (Figure 2c). In Group D, alveolar walls were thickened and edematous, with some inflammatory cells, and minimum fibrotic changes were observed (Figure 2d). In Group E, fibrotic changes as severe as those in Group B occurred (Figure 2e). Throughout the experimental period, there were no abnormal histologic alterations in Group A (Figure 2a).

View larger version (82K): [in this window] [in a new window]   Figure 2.   Histologic analysis of right lung 29 d after last administration of drugs. (a) Group A (control); there were no abnormal histologic findings. (b) Group B (BLM); multifocal fibrosis is seen, located mainly in the subpleural regions. (c) Group C (BLM + 1 mg/kg PC-SOD); mild infiltration of inflammatory cells into the alveolar and interstitial regions and slightly thickened and edematous alveolar walls are observed, without apparent fibrotic regions. (d ) Group D (BLM + 10 mg/kg PC-SOD); the alveolar wall is thickened and edematous, with some inflammatory cells and minimum fibrotic changes. (e) Group E (BLM + mPSL); fibrotic changes as severe as those in Group B are observed.

The grades of fibrosis at 29 d are presented in Table 3. All BLM-treated groups (B, C, D, and E) had significantly higher scores than that for Group A. Among the first four groups, the scores in Group C were significantly lower than those in Groups B, D, and E (p < 0.01). These findings were confirmed by a blinded pathologist.

Lung hydroxyproline content. A comparison of the lung hydroxyproline contents in Groups A through E is presented in Table 3. The lung hydroxyproline content in Group B on Day 29 was higher than that in Group A (p < 0.05). The lung hydroxyproline content in Group C was significantly lower than those in Groups B, D, and E (p < 0.05).

Semiquantification of cytokine mRNA. Cytokine mRNA levels in BALF cells were analyzed and compared among all groups on Day 1 and Day 29. In all samples examined, the reverse transcription (RT)-PCR products amplified from -actin and from each cytokine mRNA could be detected after staining with ethidium bromide. On both Days 1 and 29, the relative amounts of IL-1 and PDGF-A mRNA were higher in Group B than in Group A (Figure 3). On Day 1, the expression levels of IL-1 and PDGF-A mRNA in Groups C, D, and E were significantly lower than the respective levels in Group B. In addition, the suppression of PDGF-A expression was significantly greater in Group C than in Groups D and E. On Day 29, there was no significant difference between any group and any other group except Group A. 

View larger version (20K): [in this window] [in a new window]   Figure 3.   Relative amounts of cytokine mRNA in BALF cells. The relative amounts of IL-1 and PDGF-A mRNA were higher in Group B than in Group A on Days 1 and 29, respectively. On Day 1, the expression levels of IL-1 and PDGF-A mRNA in Groups C, D, and E were significantly lower than those in Group B. Additionally, the suppression of PDGF-A mRNA expression was significantly greater in Group C than in Groups D and E. On Day 29, there was no significant difference in IL-1 or PDGF mRNA among any of the groups except Group A. *p < 0.01.

Experiment 2

On Day 29 after the last administration of BLM, multifocal fibrosis was observed mainly in the subpleural regions in Group B'. In Groups C' and F there were no apparent fibrotic regions. These findings were similar to those observed in Experiment 1.

A comparison of the grades of fibrosis on Day 29 and the lung hydroxyproline contents in Groups B', C', and F is given in Table 4. Both the fibrosis scores and the hydroxyproline contents in Groups C' and F were significantly lower than those in Group B'. However, there was no significant difference between the fibrosis scores and lung hydroxyproline contents in Groups C' and F. 

	DISCUSSION

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In this study, we demonstrated the inhibitory effects of PC-SOD on the development of BLM-induced pulmonary fibrosis, using the pathologic evaluation and measurement of lung hydroxyproline content. Development of BLM-induced pulmonary fibrosis was inhibited with a low dose of PC-SOD (1 mg/kg/d, Group C). In BALF of Group C, total cell number and the percentages of lymphocytes and neutrophils were lower than those in the BLM-treated group (Group B). These BALF findings suggest that PC-SOD inhibited migration of neutrophils and AM caused by active oxygen and proteinase, preventing injury to alveolar structure and interstitium, since it has been shown that neither lecithin, a modifier, nor xylitol, which was used as vehicle, influences various responses related to BLM administration (18). However, development of fibrosis was not attenuated with a high dose (10 mg/kg/d) of PC-SOD. One possible explanation for this finding is that administration of an excessive dose of PC-SOD might have caused tissue injury related to another species of active oxygen. Generally, it has been reported that in vivo, various kinds of SOD exhibit maximal protective actions at a certain dose, but show opposite effects at massive doses and that this difference does not completely disappear even in the presence of the hydrogen peroxide (H₂O₂) scavenger catalase (21). Yim and colleagues indicated that SOD changed H₂O₂ into hydroxyl radicals and that hydroxyl radicals caused tissue injury (22).

Alternatively, the loss of protection at high PC-SOD concentrations may reflect a hypersensitivity reaction. Since the SOD used in our study was of human origin, and the animals received repeat dosages of this foreign protein, neutralizing antibodies may have abrogated the protective effects of high-dose PC-SOD. Thus, it is possible that a dose-dependent effect will be seen in humans. However, BLM-induced pulmonary fibrosis and IPF are not the same disease. It may be that the protective effect of PC-SOD is specific to BLM-induced fibrosis, since BLM directly generates superoxide. Further study, including establishment of the optimal dose of PC-SOD, will be required before its clinical application. Taken together, however, our findings suggest that oxidant/antioxidant imbalance plays an important role in fibrogenesis.

The effects of corticosteroids on acute lung injury, and especially inhibitory effects of these agents on the expression of cytokines and adhesion molecules, have been extensively discussed (23, 24). In the present study, mPSL showed antiinflammatory effects but not inhibitory effects against pulmonary fibrosis. BLM-induced increases in total cell number and in the expression of IL-1 and PDGF-A by BALF cells were inhibited in the group of mice (Group E) treated with mPSL on Day 1 after the last administration of BLM. This may indicate that a dose of mPSL sufficient to inhibit acute injury has little effect on the development of pulmonary fibrosis in the model used in our study. The reason for this remains unclear. The BALF findings showed only that the increase in total cell number was suppressed whereas the ratios of lymphocytes and neutrophils remained high. These neutrophils could release various kinds of chemical mediators, resulting in lung injury. At present, corticosteroids are standard therapeutic agents for IPF. However, their effectiveness remains equivocal, which is compatible with the findings of the present study. Regarding the effects of combination therapy with PC-SOD and mPSL, neither beneficial nor harmful effects were observed during the experimental period. Since our findings indicate that these two agents would exert antiinflammatory effects through different mechanisms, further investigation is needed to elucidate the additive effects.

There are many reports about the relationship between cytokines and oxidant/antioxidant imbalance (25, 26). However, few reports describe cytokine expression levels in BALF cells during the treatment of pulmonary fibrosis with antioxidants. In the present study, expression levels of IL-1 and PDGF-A mRNA in BALF cells on Day 1 in the PC-SOD- or mPSL-treated groups of animals (Groups C, D, and E) were significantly lower than those in mice treated only with BLM (Group B). Interestingly, the expression levels of PDGF-A in Group C were significantly lower than those in Group D or Group E. IL-1 is known as a proinflammatory cytokine that promotes accumulation of inflammatory cells. PDGF is known to be involved in the activation of fibroblasts and proliferation of the extracellular matrix (27). In our previous study, some of the fibroblast proliferative activity in BLM-induced pulmonary fibrosis was considered to be due to the increased release of PDGF-A from AM (17). Therefore, part of the inhibition of development of pulmonary fibrosis in mice treated with low-dose PC-SOD (Group C) in the present study may have been due to the strong suppression of PDGF-A.

In summary, the present study showed inhibitory effects of PC-SOD on BLM-induced pulmonary fibrosis. This suggests that oxidant/antioxidant imbalance may play an important role in fibrogenesis, and that PC-SOD may be promising as a new treatment agent for IPF, or at least for preventing the development of BLM-induced pulmonary fibrosis during antineoplastic therapy.