INTRODUCTION

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Fas is a cell-surface receptor whose binding with Fas ligand (FasL) mediates apoptosis in various types of cells, including pulmonary epithelial cells. Although the etiology of pulmonary fibrosis is still unknown, recent evidence implicates excessive apoptosis mediated by upregulated Fas/FasL systems as the pathogenetic mechanism of both lung injury and fibrosis. For example, Hagimoto and colleagues found excessive apoptosis and Fas overexpression in alveolar epithelial cells and FasL overexpression in infiltrating lymphocytes in bleomycin-induced pulmonary fibrosis in mice (1). They also observed that repeated inhalation of agonistic anti-Fas antibody induces apoptosis of bronchial and alveolar epithelial cells, followed by the development of pulmonary fibrosis (2). In addition, Kuwano and coworkers have shown that Fas and FasL are overexpressed in the lungs of humans with pulmonary fibrosis (3). Although these observations suggest a role of the Fas/FasL system in the pathogenesis of pulmonary fibrosis, evidence to support this concept is still indirect. The present study was designed to determine whether bleomycin-induced pulmonary fibrosis depends on the Fas/FasL system. We found that Fas-deficient (lpr) mice and FasL-deficient (gld) mice develop pulmonary fibrosis after bleomycin administration, just as do wild-type mice. This suggests that the Fas/FasL system is not a prerequisite for bleomycin-induced pulmonary fibrosis in mice.

	METHODS

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Animals

Six-week-old male C57BL/6 wild-type, lpr/lpr (lpr), and gld/gld (gld) mice were purchased from SLC (Shizuoka, Japan). Mice bearing the lpr mutation have a defect in the expression of Fas caused by insertion of a retroviral transposon into the second intron of Fas, resulting in a marked reduction of Fas messenger RNA (mRNA) and protein (4, 5). In gld mice, a point mutation in the COOH-terminal region of FasL results in the expression of a non-functional FasL (6). It should be noted that lpr and gld mice used at 6 wk of age did not exhibit any lymphoproliferative disease. Animals were handled in accordance with institutional animal care and use committee protocols approved by the animal facility of Tokyo Women's Medical University. The animals were maintained under standard conditions, with a dark period from 8:00 P.M. to 8:00 A.M., and with water and food provided ad libitum.

Animal Model of Bleomycin-Induced Pulmonary Fibrosis

An animal model of bleomycin-induced pulmonary fibrosis was created as described previously (1, 7). Briefly, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (Abbott Laboratories, North Chicago, IL), and this was followed by intratracheal administration of 50 µl of bleomycin hydrochloride (Nippon Kayaku, Co., Tokyo, Japan) solution containing bleomycin (5 U/kg B.W.) dissolved in sterile saline. Mice were killed by intraperitoneal injection of an overdose of pentobarbital at 1, 3, 7, 10, 30 d after bleomycin instillation, and thoracotomy was performed. Bronchoalveolar lavage (BAL) was done by cannulating the trachea and lavaging the lungs with three 1-ml aliquots of phosphate-buffered saline (PBS). The cells recovered were counted in a hemocytometer. Differential cell counts were made in May-Grünwald-Giemsa-stained cytocentrifuge preparations of cells recovered by BAL. The right lungs were reserved for hydroxyproline assay and frozen in liquid nitrogen. The left lungs were used for histologic studies by inflating them with 10% formalin solution instilled through the trachea, and fixing them for 24 h. After embedding the lungs in paraffin, sections were prepared and stained with hematoxylin and eosin (H&E) or Masson's-trichrome.

DNA Nick-End Labeling of Tissue Sections

Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) was done with the Takara In Situ Apoptosis Detection Kit (Takara Biomedicals, Tokyo, Japan) according to the manufacturer's instructions. With this kit, fluorescein isothiocyanate (FITC)-labeled nucleotides are incorporated at sites of DNA strand breaks by TdT, reacted with horseradish peroxidase-conjugated anti-FITC antibody, and visualized by an aminoethylcarbazole-substrate reaction. In the counting of TUNEL-positive alveolar epithelial cells, we examined only a single layer of cells in the alveolar walls on Day 1, to avoid bias caused by technical artifacts, such as adjacent alveolar walls or alveolar wall remodeling. Briefly, at a magnification of ×400, we counted the number of TUNEL-positive cells per 200 alveolar epithelial cells. Ten fields randomly distributed across the slides were studied, and the result was expressed as the number of TUNEL-positive cells per 100 alveolar epithelial cells.

Hydoxyproline Assay

The right lungs were homogenized in saline and hydrolyzed in concentrated HCl at 100° C for 20 h. The hydoxyproline content of each sample was determined as described previously (8). Data are expressed as multiples of the baseline values.

Statistical Analysis

Data are expressed as means ± SEM, and were analyzed with Student's t test or analysis of variance (ANOVA), as appropriate. A value of p < 0.05 was considered statistically significant.

	RESULTS

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Histologic Features of Bleomycin-Induced Pulmonary Injury

Before intratracheal instillation of bleomycin (Day 0), the lungs of the Fas-deficient (lpr), FasL-deficient (gld), and wild-type mice were histologically normal, and the histologic changes following bleomycin instillation in all three groups of mice were similar (Figure 1). By Day 3 after bleomycin instillation, the alveolar septa had begun to thicken as a result of edema and infiltration by neutrophils and lymphocytes. By that time, some of the nuclei of the bronchiolar epithelial cells had become small and condensed (Figure 1, arrows), showing features of apoptosis, as described previously (1). By Day 10, infiltration of the thickened alveolar septa by lymphocytes had become more intense, and air spaces had collapsed. After 30 d, we observed thickening of the alveolar septa by fibroblast proliferation and collagen deposition with extensive remodeling of alveolar structures. Positive staining of tissue sections with Masson's-trichrome confirmed collagen deposition in the lungs of all three genotypes of mice. There was also a persistent cellular infiltrate. These changes in infiltration by inflammatory cells, and the development of pulmonary fibrosis following bleomycin administration, are similar to previously reported histologic findings (1, 7, 9, 10).

View larger version (162K): [in this window] [in a new window]   View larger version (59K): [in this window] [in a new window]   Figure 1.   Histologic findings in bleomycin-induced pulmonary injury. Tissue was fixed in 10% formalin and processed for staining with H&E (A through L) or Masson's trichrome (M through O). (A, D, G, J, M) Wild-type mice. (B, E, H, K, N ) lpr mice. (C, F, I, L, O) gld mice. Before bleomycin instillation (A through C ), the lungs were normal in the wild-type, Fas-deficient (lpr), and FasL-deficient (gld ) mice. On Day 1 after bleomycin instillation (D through F ), minimum thickening of alveolar septa was observed, with the appearance of condensed and small nuclei in some bronchial epithelial cells (arrows). By Day 3 (G through I ), alveolar septa had become thicker due to edema and infiltration by neutrophils and lymphocytes. On Day 10 ( J through L), extensive thickening of alveolar septa, predominant infiltration by lymphocytes, and collapse of the alveolar spaces were observed. On Day 30 (M through O), thickening of the alveolar septa by fibroblast proliferation and collagen deposition was seen with extensive remodeling of alveolar structure. These histologic changes following bleomycin instillation were similar in all three groups of mice. (Original magnification: ×100; insets: ×1,000.) Three mice per group were examined at each data point.

BAL Fluid Cell Analysis

Neutrophils appeared early in the inflammatory process, in association with bleomycin exposure, and their appearance was followed by lymphocyte infiltration, as described previously (9, 10). These changes in inflammatory cell populations in BAL fluid (BALF) from Day 1 to Day 7 were similar in the Fas-deficient (lpr), FasL-deficient (gld), and wild-type mice (Figure 2).

View larger version (37K): [in this window] [in a new window]   Figure 2.   Analysis of BAL cell differential counts. Data shown are the means ± SEM of the values for three mice.

DNA Fragmentation Analysis by TUNEL

Localization of DNA fragmentation and apoptosis in lung tissue was investigated with TUNEL. TUNEL demonstrated positive signals in bronchiolar and alveolar epithelial cells on Days 1 and 3 after bleomycin instillation (Figure 3). The positive signals were distributed diffusely throughout the pulmonary parenchyma, and no differences were observed among the Fas-deficient (lpr), FasL-deficient (gld), and wild-type mice (Figures 3 and 4). The positive TUNEL signals subsequently disappeared in all three groups of mice. Very few cells in lung tissue were TUNEL-positive on Day 10 and Day 30. 

View larger version (130K): [in this window] [in a new window]   Figure 3.   TUNEL analysis of lung tissue. (A through C ) Day 0. (D through F ) Day 1. (G through I ) Day 3. (A, D, and G) wild-type mice. (B, E, and H ) lpr mice. (C, F, and I ) gld mice. TUNEL yielded positive signals in bronchiolar and alveolar epithelial cells on Days 1 and 3 after bleomycin instillation. No differences were observed among the wild-type, Fas-deficient (lpr), and FasL-deficient (gld ) mice. There were very few signals in the lung tissue on Days 10 and 30 (data not shown). (Original magnification: ×100; insets: ×1,000.)

View larger version (14K): [in this window] [in a new window]   Figure 4.   Quantitative analysis of TUNEL-positive cells in the lung parenchyma on Days 0 and 1 after bleomycin instillation. Data shown are the means ± SEM of the values for three mice. No differences were observed between the wild-type, Fas-deficient (lpr), and FasL-deficient (gld ) mice.

Hydroxyproline Assay

Collagen deposition in the lung was determined quantitatively by measuring the hydroxyproline content of lung tissue (Figure 5). The hydroxyproline content of lungs unexposed to bleomycin (Day 0) was similar in Fas-deficient (lpr), FasL-deficient (gld), and wild-type mice. The hydroxyproline content of lungs was significantly increased at 30 d after bleomycin instillation in all three genotypes of mice. Although the hydroxyproline content in the Fas-deficient mice on Day 30 was higher than in the other two genotypes, the difference was not statistically significant (p = 0.13).

View larger version (15K): [in this window] [in a new window]   Figure 5.   Increases in lung hydroxyproline content after bleomycin instillation. The right lungs were excised from three mice of each genotype, and were assessed for hydroxyproline content. Data are shown as multiples of the hydroxyproline level in lungs unexposed to bleomycin (-fold increases from baseline).

	DISCUSSION

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In the present study, Fas-deficient (lpr) mice and FasL-deficient (gld) mice developed bleomycin-induced pulmonary lesions, including inflammatory cell infiltration and fibrosis, as did also wild-type mice. These results indicate that the Fas/ FasL system is not a prerequisite for the development of bleomycin-induced pulmonary lesions.

Recent studies have implicated excessive apoptosis in alveolar epithelial cells as a potential mechanism of pulmonary injury and fibrosis (1, 3, 11, 12), and very recent studies have shown that caspase inhibitors, which block apoptosis, attenuate the pulmonary injury or fibrosis induced by lipopolysaccharide (13), N-methyl D-aspartate plus L-arginine (14), and bleomycin (15), suggesting a central role of apoptosis in pulmonary injury and fibrosis.

Our observation that bleomycin induced bronchial and alveolar epithelial cell apoptosis even in Fas-deficient (lpr) and FasL-deficient (gld) mice suggests the presence of apoptotic mechanism(s) not based on Fas-FasL interactions. Such apoptotic mechanism(s) may include direct cellular and DNA damage by bleomycin and activated-T-cell-dependent perforin/granzyme systems. Bleomycin-induced pulmonary fibrosis depends on tumor necrosis factor (TNF)- and transforming growth factor (TGF)-, and inflammatory cells and epithelial cells are sources of these cytokines (16). Thus, other pathways leading to apoptosis in the bleomycin model may include proapoptotic cytokines such as TNF-, TGF-, and interferon-, or expression of proapoptotic genes such as p53 and Bax (16).

Recently, Hagimoto and colleagues demonstrated upregulation of Fas in alveolar epithelial cells and upregulation of FasL in infiltrating lymphocytes after bleomycin administration, as well as excessive apoptosis of alveolar epithelial cells (1). Again, our results suggest that Fas and FasL are not required for the development of bleomycin-induced pulmonary fibrosis. If that is true, what role do Fas and FasL play? The first possibility is that upregulated Fas and FasL play a role in eliminating injured epithelial cells that need to be replaced by epithelial renewal. In other words, the upregulation of the Fas/ FasL system in bleomycin-treated lungs may be the result, not the cause, of alveolar epithelial cell injury. The second possibility is that upregulated Fas and FasL are responsible for potentiating inflammatory responses in bleomycin-exposed lungs. Fas-FasL binding has been shown to induce colon carcinoma cells to release interleukin-8, a powerful chemoattractant for neutrophils (17), and soluble FasL has been shown to exhibit chemotactic activity toward neutrophils (18, 19). The second possibility, however, seems unlikely, because the neutrophil counts in BALF following bleomycin administration were similar in Fas-deficient (lpr), FasL-deficient (gld), and wild-type mice.

The positive TUNEL signals in bronchial and alveolar epithelium in the present study disappeared by 10 d after bleomycin administration. By contrast, Hagimoto and colleagues reported the persistent appearance of TUNEL-positive signals in alveolar epithelial cells over a period of 14 d, even though they administered bleomycin to mice at the same dose (5 U/kg) and via the same route (intratracheal injection) as we did (1). The reason(s) for the rapid disappearance of TUNEL-positive cells in our study are currently unknown, but it may have been due to the use of different mouse strains: we used C57BL/6 mice, whereas Hagimoto and colleagues used ICR mice. It is well known that mouse strains respond differently to bleomycin-induced lung toxicity because of having different degrees of bleomycin sensitivity (20).

In contrast to our findings, Kuwano and coworkers recently reported that epithelial cell apoptosis and fibrosis after bleomycin instillation are suppressed in Fas-deficient (lpr) and FasL-deficient (gld) C3H mice as compared with wild-type mice (21). This difference between their findings and ours may have been due to the use of different mouse strains or the different timing of evaluation for apoptosis in the two studies. Kuwano and coworkers evaluated apoptosis with TUNEL on Days 7 and 14 after bleomycin instillation, and found that the number of TUNEL-positive cells was decreased in Fas-deficient (lpr) and FasL-deficient (gld) mice as compared with wild-type mice. However, we found very few TUNEL-positive cells on Day 10 in mice of each of these two genotypes. Although the reason(s) for this difference are unclear, Fas-FasL interactions may play a role in epithelial cell apoptosis during the late period in bleomycin-induced pneumopathy (21).

A limitation of our study was that despite the lpr Fas defect, low levels (2% or less) of normally spliced mRNA have been detected in the tissues of lpr mice, although it is unknown whether lpr mice can express functional protein (4, 5). Therefore, we cannot completely exclude the involvement of Fas in development of the bleomycin-induced pulmonary fibrosis in lpr mice.

In conclusion, our results strongly suggest that the Fas/ FasL system is not a prerequisite for the development of bleomycin-induced pulmonary fibrosis in mice. Apoptosis of pulmonary epithelial cells after bleomycin exposure is probably mediated by Fas-independent pathways. However, since the chronic progressive type of lesion seen in patients with idiopathic pulmonary fibrosis does not occur in mouse models of bleomycin-induced pulmonary fibrosis, care must be taken in interpretating it in relation to clinical situations.