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Am J Physiol Lung Cell Mol Physiol 290: L790-L796, 2006. First published November 23, 2005; doi:10.1152/ajplung.00300.2004
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Bleomycin initiates apoptosis of lung epithelial cells by ROS but not by Fas/FasL pathway

Shulamit B. Wallach-Dayan,1,* Gabriel Izbicki,1,* Pazit Y. Cohen,1 Regina Gerstl-Golan,1 Alan Fine,2 and Raphael Breuer1,2,3

1Lung Cellular and Molecular Biology Laboratory, Institute of Pulmonology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; and 2Pulmonary Center and 3Department of Pathology, Boston University School of Medicine, Boston, Massachusetts

Submitted 9 August 2004 ; accepted in final form 15 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial cells are considered to be a main target of bleomycin-induced lung injury, which leads to fibrosis in vivo. We studied the characteristics of in vitro bleomycin-induced apoptosis in a mouse lung epithelial (MLE) cell line. Bleomycin caused an increase of reactive oxygen species (ROS) resulting in oxidative stress, mitochondrial leakage, and apoptosis. These were associated with elevated caspase-8 and resultant caspase-9 activity and with upregulation of Fas expression. Glutathione and inhibitors of caspase-8 or caspase-9, but not of FasL, inhibited these effects, suggesting their dependence on ROS, caspase-8 and -9, in a Fas/FasL-independent pathway. However, postbleomycin-exposed MLE cells were more sensitive to Fas-mediated apoptosis. These results demonstrate that the initial bleomycin-induced oxidative stress causes a direct apoptotic effect in lung epithelial cells involving a regulatory role of caspase-8 on caspase-9. Fas represents an amplification mechanism, and not a direct trigger of bleomycin-induced epithelial cell apoptosis.

Fas ligand; reactive oxygen species

BLEOMYCIN IS A NEOPLASTIC drug known to produce interstitial pulmonary fibrosis in humans (46) as well as in experimental models (38). This redox active agent is capable of producing electron-deficient substances such as hydroxyl radicals, superoxide, and hydrogen peroxide (29). These reactive oxygen species (ROS) are capable of damaging important biological molecules and changing the redox state of cells (29), including lung cells (19), and have been implicated in apoptotic cell death (21, 30).

Apoptosis is implemented by molecular pathways that ultimately result in activation of a family of cysteine proteases termed caspases (14). In mammals, two main caspase cascades have been delineated. The first pathway links death receptors (e.g., Fas) and caspase-8 (34), whereas the second pathway, the mitochondrial pathway, occurs through diverse extracellular stresses, such as hypoxia, and involves procaspase-9 activation (15), which in turn cleaves and activates downstream effector caspases (27). However, caspase-8 has also been shown to be activated independently of death receptor induction (51). The role of Fas/Fas ligand (Fas/FasL) pathway in lung fibrosis is controversial (4, 7, 17, 18, 23–25). Fas and FasL are upregulated in fibrosing lung diseases in humans (7, 25) and in murine bleomycin-induced fibrosis (7, 18, 24). Lung epithelial cells are the main target of in vivo bleomycin- or Fas/FasL-induced apoptosis, which terminates in fibrosis (17, 18, 23, 37). To further investigate the pathways involved in bleomycin-induced apoptosis, we studied apoptotic signaling in a murine lung epithelial (MLE) cell line in vitro.

We report that bleomycin causes an increase in ROS levels with sequential activation of caspase-8, mitochondrial leakage, activation of caspase-9, and subsequent cell apoptosis. Moreover, bleomycin upregulates Fas and sensitizes cells to Fas-mediated apoptosis. However, the Fas/FasL pathway is probably not directly involved, since the initiation of apoptosis was inhibited in the presence of reduced glutathione (GSH) and caspase-8 inhibitors, but not by antibodies interfering with the Fas/FasL pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Monoclonal antibodies and other reagents. We used the following monoclonal antibodies (MAbs) and reagents: 1 mg/ml propidium iodide (PI) stock solution (Calbiochem, La Jolla, CA) in phosphate-buffered saline (PBS); annexin V-FITC (PharMingen, San Diego, CA); 1 mCi/ml sterile [3H]methylthymidline (Amersham Biosciences, Rehovot, Israel); GSH (Sigma, St. Louis, MO); 2',7'-dichlorofluorescin diacetate (DCFH/DA) and MitoTracker RedCMXRos mitochondrial membrane potential (MTP) tracker (Molecular Probes, Eugene, OR); bleomycin (ASTA Medica) and caspase-8 [z-IETD-fluoromethyl ketone (fmk)] and caspase-9 (z-LEHD-fmk) inhibitors (R&D Systems, Minneapolis, MN); FasL antagonist, anti-mouse FasL MAb, clone-MFL3, and Jo2-Fas agonist (PharMingen); IgG control, clone-RGE53 (Chemicon International, Temecula, CA); caspase-8 and -9 activity assays kit (BioVision, Mountain View, CA).

Cell culture systems. We used the murine type II lung epithelial cell line (ATCC, MLE-15), an extensively used model (5, 6, 10, 16, 20, 35, 48, 49, 52). Cells were maintained in hydrocortisone insulin transferrin estradiol selenium (HITES) medium (Industrial Laboratories, Beit HaEmek, Israel) that was supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS (Sigma, St. Louis, MO), and 5 mM Pen-Strep (Industrial Laboratories, Beit HaEmek, Israel). Cultures were incubated at 37°C in a humidified atmosphere with 5% CO2. MLE cells (0.5 x 106) were cultured in HITES medium, with or without 100 mU/ml of bleomycin and with or without 10 mM reduced glutathione, or 30–60 µM caspase inhibitors, and were assayed at 0.5, 1, 24, or 48 h. ROS levels, caspase activities, and MTP disruption were assayed at 24 h. To trigger Fas-induced apoptosis, Fas was upregulated in MLE cells by pretreatment with 5 mU/ml of bleomycin. At 24 h, this medium was replaced by medium containing 1 µg/ml agonist Fas and/or 20 µM antagonist anti-MFL3 FasL MAb. For fluorescence-activated cell sorting (FACS) analysis, cells were maintained in 3% FCS in PBS (FACS buffer).

Trypan blue exclusion. Cells were exposed to trypan blue dye (0.04% in 1x PBS), placed on a hemocytometer, and examined under light microscopy. We counted 200 random cells.

[3H]thymidine-labeled DNA release by dead cells. Cells (3–5 x 105/ml) were labeled with 5 µCi/ml (final concentration) at their exponential phase of growth for 4–6 h after treatment with different bleomycin concentrations; cells were harvested and counted in a beta-counter.

DNA fragmentation analysis. DNA fragmentation assay was described previously (40). In brief, 5 x 105 cells were lysed in 30 µl of lysis buffer [80 mM EDTA, 200 mM Tris (pH 8.0), 1.6% sodium lauril sarcosinate, and 1 mg/ml proteinase K], incubated at 50°C for 4 h followed by incubation with RNase A (0.2 mg/ml), at 37°C for 4.5 h, fractionated by agarose gel electrophoresis, visualized under UV, and confirmed by digitized densitometry using a tabletop scanner (Bio-Rad Multi-Analyst/PC version 1.1). Data was analyzed with Fluon-S-Multiimager (Bio-Rad).

DNA content and cell cycle flow cytometry analysis. Genomic DNA integrity was also analyzed by determining the DNA content in the nuclei with flow cytometry as described previously (36). Cells were fixed with 70% ethanol for 30 min at 4°C, washed, and incubated in PBS containing PI (50 µg/ml) and 0.1 mg/ml RNase (Boehringer Mannheim, Indianapolis, IN) for 30 min at room temperature. DNA content was determined by flow cytometry on FACStar (Becton Dickinson, Mountain View, CA).

In vitro detection of apoptotic cells by annexin V affinity labeling. FITC-conjugated annexin V (1 µg) was added to 0.5 x 106 MLE adherent cells in the culture flask and incubated for 30 min. Cells were pelleted (400 g, 5 min) and resuspended in FACS buffer. PI (5 µg/ml) was added for 15 min incubation on ice. Flow cytometry analysis by plotting green fluorescence (FL1)/FITC-annexin V vs. red fluorescence (FL2)/PI-positive cells was performed with a FACStar (Becton Dickinson, Mountain View, CA). PI- and PI/annexin V-positive cells were excluded from the presented data.

ROS measurement. MLE cells were treated with bleomycin (100 mU) at different time points, and the peroxide anion levels were estimated by staining with DCFH/DA, final concentration 5 µM (Eastman Kodak, Rochester, NY), for 50 min at 37°C. Cells were harvested and immediately underwent flow cytometric analysis with excitation and emission settings of 488 and 530 nm, respectively.

Assay for caspase activation. Caspase-8 and caspase-9 activities were measured using the BioVision FLICE/Caspase-8/9 Colorimetric Assay Kit (BioVision). Cells (0.3–1 x 106) were lysed on ice for 10 min and centrifuged (10,000 g, 1 min). Cytosolic extracts (supernatant) were incubated for 1–2 h at 37°C with IETD-p-nitroanilide (pNA) or LEHD-pNA color substrates (200 µM), respectively. Samples were analyzed by spectrophotometer (400 nm).

Monitoring inner MTP. MitoTracker RedCMXRos was added to the cells for the last 30 min of bleomycin incubation. Cells were washed in PBS, and their fluorescence was measured using the FL-3 channel and analyzed by flow cytometry.

Fas and FasL expression assayed by flow cytometry. MLE cell surface expression of Fas and FasL were assessed by indirect immunofluorescence and analyzed by flow cytometry. In brief, 0.5 x 106 cells were washed with FACS buffer, incubated on ice for 45 min with 1 µg PE-conjugated anti-Fas or FasL MAbs, washed, and analyzed by flow cytometry using a FACStar (Becton Dickinson).

Data analysis and statistics. Flow cytometry and colorimetric assayed experiments were repeated at least two or three times. Data were analyzed according to mean fluorescence intensity or optical density, respectively, presented as means ± SD, and plotted on graphs or on dot plots. Statistical analysis was performed by simple t-test for experiments with single comparisons and ANOVA with Bonferroni test for experiments with multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bleomycin induces MLE cell death and apoptosis in a dose-response manner. MLE cells were cultured with increasing doses of bleomycin (5–1,000 mU) and assayed at 24 h. Cell viability decreased with increasing doses of bleomycin as assayed by trypan blue exclusion and [3H]thymidine release (Fig. 1A). To further test whether the bleomycin-induced MLE cell death was due to apoptosis induction, three assays were used to detect apoptosis: DNA ladder (Fig. 1B), annexin V staining (Fig. 1C), and DNA content analysis (Fig. 1D). As demonstrated, bleomycin caused apoptosis of MLE cells in a dose-response manner.


Figure 1
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  		Fig. 1. Decreased viability and increased apoptosis in murine lung epithelial (MLE) cells after bleomycin (Bleo) exposure. MLE cells (0.5 x 106), cultured for 24 h with different doses of Bleo (5–1,000 mU), show a dose-dependent decrease in cell viability as shown in A, when assayed by trypan blue exclusion and [3H]thymidine release. Dose-dependent Bleo induction of apoptosis was assayed by DNA ladder (B), annexin V staining (C), and increases in pre-G1 phase, by DNA content (D). Data presented are representative of 3 different experiments. RFI, relative fluorescence intensity.

 
Bleomycin increases ROS levels, altering mitochondrial membrane potential with sequential apoptosis. The kinetics of bleomycin (100 mU)-induced MLE cell apoptosis was studied by annexin V staining, followed by flow cytometric analysis, as presented in Fig. 2. Apoptosis increased at 24 h, and at 48 h no viable cells were found.


Figure 2
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  		Fig. 2. Elevated ROS levels appeared before mitochondrial dysfunction and apoptosis. MLE cells (0.5 x 106) were incubated with Bleo (100 mU) and assayed at 0.5, 1, 24, and 48 h for oxidative stress and apoptosis parameters; intracellular peroxide (ROS), mitochondrial membrane potential changes (MTP disruption), and apoptosis. Fold ratios were determined by dividing the value of Bleo-treated cells by the average of untreated controls at each time point. ROS levels were increased within 30 min, whereas apoptosis and MTP changes were seen at 24 h or later. Statistical comparison of Bleo and untreated control was performed at each time point. *P < 0.05; **P < 0.01.

 
To clarify the association of bleomycin-induced apoptosis and the oxidative stress, peroxide (ROS) levels (using DCFH staining followed by flow cytometry analysis) were determined over time, as demonstrated in Fig. 2. ROS was seen to increase at 0.5 h and at all other time points.

Bleomycin-induced apoptosis was attenuated by preincubation with GSH (see Fig. 7). This also supports the idea that bleomycin-induced apoptosis occurs via increased ROS.


Figure 7
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  		Fig. 7. GSH but not antagonist anti-FasL MAb decrease bleomycin-induced MLE cell apoptosis. MLE cells (0.5 x 106) were preincubated for 1 h with MFL3 anti-FasL (20 µM) or GSH (10 mM) and then for 24 h with Bleo (100 mU). The percentage of apoptotic cells was determined by FACS annexin V staining. GSH decreased Bleo-induced apoptosis (Bleo + GSH vs. Bleo). In contrast, addition of anti-FasL (Bleo + {alpha}-FasL) resulted in no change from cells treated with Bleo alone. To confirm the inhibitory effect of antagonist MFL3 anti-FasL MAb, 0.5 x 106 Jurkat cells (Ctrl-Jurkat) were treated with agonist Jo2 anti-Fas (1 µg/ml) to induce apoptosis, which was inhibited by MFL3 anti FasL MAb (inset: segregated histograms, Bleo vs. Untr). *P < 0.05.

 
The kinetics of change in the collapse of the inner MTP was studied to assess whether the bleomycin-induced rise in ROS levels occurs due to MTP disruption. MitoTracker Red CMXRos, whose accumulation in the mitochondria depends on the membrane potential, was used. A collapse in MTP was detected by the decrease of a cell population with high MitoTracker Red staining. Changes in MTP are presented in Fig. 2. The percentage of disrupted MTP in cells exposed to bleomycin and assayed at 0.5 or 1 h was not affected, mildly affected at 24 h, and considerably affected (11x) at 48 h.

As demonstrated in Fig. 2, changes in ROS levels occurred earlier (0.5 h) than in MTP or apoptosis (24 h).

Bleomycin induces caspase-8 and caspase-9 activation, and their inhibition decreases bleomycin-induced apoptosis. Caspase-8 and -9 activities were increased in MLE cells after exposure to bleomycin (100 mU, 24 h) as demonstrated in Fig. 3A. To determine whether a mitochondrial (caspase-9) or death receptor pathway (caspase-8) is required for bleomycin-induced apoptosis, MLE cells were cultured in RPMI (B0) and then were exposed to bleomycin for 24 h, following preincubation with 30 µM caspase-8 inhibitor, z-LEHD-fmk, and/or 60 µM caspase-9 inhibitor, z-IETD-fmk. These caspase-8 and -9 inhibitors, when used separately, significantly suppressed apoptosis, as detected by annexin V flow cytometry analysis (Fig. 3B), from average values of 60 ± 8.1% in bleomycin-treated cells (B1) to 36 ± 11.2% (B2) and 45 ± 6.8% (B3), respectively. However, when these inhibitors were used together, there was no additive effect on bleomycin-induced apoptosis (data not shown).


Figure 3
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  		Fig. 3. Bleo activates caspase-8 and caspase-9, and their specific inhibitors decrease Bleo-induced apoptosis in MLE cells. A: 0.5 x 106 MLE cells were treated with Bleo (100 mU, 24 h). Caspase-8 and caspase-9 activities were determined by a colorimetric kit. Both caspase-8 and -9 were increased [Bleo vs. untreated (Untr)]. OD, optical density. *P < 0.05, **P < 0.01. B: 0.5 x 106 MLE cells were preincubated for 1 h, with or without caspase inhibitors, and cultured with Bleo (100 mU, 24 h), stained with annexin V, and analyzed by flow cytometry. MLE cells cultured in RPMI (B0), cultured with Bleo (B1), with Bleo and 30 µM caspase-8 inhibitor (z-IETD-fmk) (B2), or with Bleo and 60 µM caspase-9 inhibitor (z-LEHD-fmk) (B3). Both caspase inhibitors decreased the MLE cell apoptosis from 60 ± 8.1% (B1) to 36 ± 11.2% (B2) and 45 ± 6.8% (B3), respectively. Casp., caspase. All panels depict 1 of 2 independent experiments showing similar results, presented by dot plots. The number in each box represents means ± SD of 2 independent experiments.

 
Sequential activation of caspase-8 and -9 in bleomycin-treated MLE cells. To examine the regulatory relationship between caspase-8 and -9 activities, bleomycin-treated MLE cells were pretreated with 30 µM caspase-8 inhibitor (z-IETD-fmk; Fig. 4A) or 60 µM of caspase-9 inhibitor (z-LEHD-fmk; Fig. 4B). Pretreatment with caspase-8 inhibitor markedly suppressed caspase-9 activity and attenuated MTP-disruption. In contrast, caspase-9 inhibitor had a marked effect on MTP but only a mild effect on caspase-8 activity. Therefore, caspase-8 is required for bleomycin-induced MTP-disruption and caspase-9 activation.


Figure 4
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  		Fig. 4. Dependence of caspase-9 and MTP on caspase-8 activation. We treated 0.5 x 106 MLE cells with Bleo (100 mU, 24 h), with Bleo and preincubation with 30 µM caspase-8 inhibitor (A) or with 60 µM caspase-9 inhibitor (B). Caspase activities were determined by using the colorimetric caspase activity assay kit. MTP changes were determined by MitoTracker assay. Caspase activities and MTP changes are presented as fold ratios, determined by dividing each experimental value by the average of control untreated cells. Caspase-9 (A) and caspase-8 (B) activities were increased in MLE cells treated by Bleo. Caspase-9 was inactivated by caspase-8 inhibitor (A, Bleo + inh8), whereas caspase-8 was slightly inactivated by caspase-9 inhibitor (B). MTP was reduced by both caspase inhibitors (A and B). Inhibition of increased Bleo-induced caspase-8 and -9 activities by their specific inhibitors was confirmed (insets: Bleo vs. Bleo + inh8 or inh9). *P < 0.05, **P < 0.01.

 
ROS upregulates caspase-8 activity. GSH was used to determine a regulatory effect of ROS-oxidative stress on caspase-8 activity. MLE cells were pretreated with GSH (10 mM) and then 1 h later with bleomycin (100 mU, 24 h). GSH decreased the bleomycin-induced caspase-8 activation as shown in Fig. 5.


Figure 5
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  		Fig. 5. Bleo-induced upregulation of caspase-8 activation is inhibited by GSH. Caspase-8 activity, determined by a caspase activity assay kit, was increased after being cultured with 100 mU Bleo for 24 h compared with untreated MLE cells (Untr). Addition of 10 mM glutathione 1 h before (Bleo + GSH) inhibited Bleo-induced caspase-8 activation. *P < 0.05.

 
Bleomycin upregulates Fas/FasL expression. Fas receptor, which functions to initiate a signal, causing apoptosis, was shown to be expressed in lung epithelial cells (13). Flow cytometry analysis was performed to determine whether bleomycin caused an upregulation of Fas or FasL expression and to determine their dependence on oxidative stress. MLE cells were treated with bleomycin (100 mU, 24 h), followed by FACS staining using phycoerythrin-conjugated anti-Fas or FasL MAb. Fas and FasL expression was increased (Fig. 6, A and B, respectively). To define ROS dependency, cells were treated with the ROS scavenger GSH. GSH decreased bleomycin-induced Fas upregulation, as shown in Fig. 6A, but not FasL upregulation (data not shown).


Figure 6
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  		Fig. 6. Bleo causes an upregulation of Fas/FasL expression. CD95 (Fas, A) and CD95L (FasL, B) receptor expression, using 1 µM of CD95-PE- or CD95L-PE-conjugated MAbs, respectively, were analyzed by flow cytometry and are presented by segregated histograms. Fas and FasL expression was increased in Bleo-treated (100 mU, 24 h) 0.5 x 106 MLE cells compared with untreated cells. Glutathione (10 mM) inhibited the increased Bleo-induced Fas expression (Bleo + GSH), as shown in A. There was no inhibition of FasL expression by GSH (data not shown). Data presented are representative of 3 different experiments.

 
Bleomycin-induced apoptosis is decreased by glutathione but not by Fas/FasL pathway antagonist. We have shown that Fas/FasL upregulation (Fig. 6) and caspase-8 activity (Fig. 5) are associated with ROS upregulation. To determine whether bleomycin-induced MLE cell apoptosis is due to oxidative stress or Fas/FasL pathway, GSH or antagonistic anti-FasL MAbs were used, respectively. MLE cells were preincubated for 1 h with MFL3 (20 µM) or GSH (10 mM) and then for 24 h with bleomycin (100 mU).

The percentage of apoptotic cells was determined by FACS annexin V staining. GSH decreased bleomycin-induced apoptosis (Fig. 7). In contrast, FasL antagonist MFL3 did not decrease bleomycin-induced MLE cell apoptosis (Fig. 7).

Bleomycin sensitizes MLE cells to Fas-induced apoptosis, with ROS dependency. Because bleomycin increases Fas expression on MLE cells (Fig. 6A), and inhibitory FasL antibodies had no effect on apoptosis of bleomycin-treated cells (Fig. 7), we wanted to determine whether the postbleomycin Fas overexpression regulates hypersensitivity of cells to Fas agonists and whether this effect is ROS dependent. To upregulate Fas expression (see inset in Fig. 8), without induction of apoptosis, cells were incubated with bleomycin (5 mU, 24 h), followed by medium replacement with a medium containing a Fas agonist (1 µg/ml). As demonstrated in Fig. 8, the exposure to bleomycin increased Fas-induced apoptosis. This effect was decreased with GSH (10 mM), thereby demonstrating that bleomycin causes sensitization to Fas-induced apoptosis, which is also ROS dependent.


Figure 8
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  		Fig. 8. Increased sensitivity to Fas-induced apoptosis of Bleo-treated MLE cells. MLE cells (0.5 x 106) were cultured in medium containing 0 (Untr) and 5 mU of Bleo, and Fas upregulation is presented (inset). In parallel, at 24 h, the media of untreated or Bleo-treated cells were substituted with media containing 1 µg/ml of Jo2 Fas agonist ({alpha}-Fas) or (Bleo + {alpha}-Fas), respectively. Flow cytometry analysis of annexin V staining was performed at 24 h, demonstrating that MLE cells exposed to Bleo are more sensitive to Fas-induced apoptosis (Bleo + {alpha}-Fas) than those exposed to Fas agonist alone ({alpha}-Fas). Glutathione (10 mM) decreased this post-Bleo sensitivity to Fas-induced apoptosis (Bleo + {alpha}-Fas + GSH vs. Bleo + {alpha}-Fas). *P < 0.05, **P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Apoptosis of alveolar epithelial cells has been implicated as a mechanism leading to pulmonary fibrosis (18, 23, 37). We used an MLE cell line to investigate bleomycin-induced apoptosis of lung epithelial cells. Bleomycin caused ROS accumulation, MTP disruption, apoptosis, increased caspase-8 and -9 activity, and increased expression of cell surface Fas and FasL. The onset of ROS occurred earlier than that of MTP or apoptosis, indicating that the increased ROS evolves directly from the bleomycin effect and not secondary to cell apoptosis and/or a subsequent mitochondrial leak.

Our data show inhibition of apoptosis by glutathione and not by Fas/FasL pathway antagonists. This confirms that bleomycin functions via ROS-induced mitochondrial cell death (1, 28, 42) and that the Fas/FasL pathway is not predominant. Fas/FasL antagonists in hepatocytes were shown to interfere with bleomycin-induced apoptosis (32, 33). However, results from other systems, which are consistent with ours, indicate that apoptosis is independent of this pathway (9, 11, 12, 22, 31, 47).

Although the Fas/FasL pathway was not involved, bleomycin caused an activation of caspase-8. Inhibitors of caspase-8 also inhibited bleomycin-induced caspase-9 and apoptosis, with ROS dependency, demonstrating that bleomycin triggered apoptosis through caspases regulated by oxidative stress.

The mechanisms of caspase-8 activation by ROS, in a death receptor-independent pathway, remain to be defined. Caspase-8 activation that induces mitochondria-mediated downstream events (43) can be achieved independently from death receptor induction (26, 51, 53). Caspase-8 may help amplify mitochondria-dependent apoptosis through a feed-forward loop on which initial mitochondrial damage by ROS causes caspase-3 activation. This caspase then activates caspase-8, which further activates mitochondrial release of cytochrome c and caspase-3 (44). These studies and our results demonstrate a role for ROS in regulating caspases of the extrinsic apoptotic pathway.

The Fas/FasL pathway in our system is not directly responsible for apoptosis. Nevertheless, bleomycin-exposed MLE cells were found to be more sensitive to further apoptosis induction by agonistic anti-Fas MAb in an ROS-dependent manner (Fig. 8). The observation of a later Fas-induced apoptosis presented in this study relates to a specific epithelial cell line (MLE cells). However, Fas receptor upregulation was also tested on primary epithelial cells, isolated from lungs, at 3 but not 1 day after bleomycin treatment of C57BL/6 mice (unpublished data from our laboratory). Fas upregulation and in vivo Fas-associated apoptosis were also reported to appear in lung sections of bleomycin treated mice by day 7 (18). Fas was also overexpressed in alveolar epithelial cells in lung tissues of human idiopathic pulmonary fibrosis (25). In addition, a study by Aoshiba et al. (4) showed no change of bleomycin-induced apoptosis in alveolar epithelial cells of Fas- or FasL-deficient mice compared with wild type when cells were assayed at day 1. Moreover, administrations of a soluble form of Fas (hFas-Fc) prevented apoptosis of these cells in bleomycin-treated mice before day 7 (23). The absence of Fas/FasL involvement in the first days of bleomycin-induced murine fibrosis is also in accordance with in vivo studies showing ROS as the initial apoptotic trigger in the fibrotic process (1–3, 27, 42). These in vivo findings are consistent with our in vitro findings and imply that two types of epithelial cell apoptosis appear in vivo after bleomycin exposure.

The extent of alveolar fibrosis is often related to the severity of epithelial cell injury (41). The importance of a first rapid apoptosis of epithelial cells in the fibrotic process may be due to their involvement in mesenchymal cell proliferation (39, 45) and their initiation and modulation of inflammatory reactions. The Fas/FasL apoptotic pathway, which appears to take place at a later time, seems essential to clear intra-alveolar granulated tissue, without induction of an acute inflammatory response (8, 50).

In summary, bleomycin induces an ROS-dependent, oxidative stress involving caspase-8 activation, terminating in apoptosis and subsequent upregulation of Fas and FasL expression that further sensitizes cells to Fas-induced apoptosis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by the David Shainberg Fund and the Charlotte and Louis Kaitz Boston University School of Medicine-The Hebrew University-Hadassah Medical School Program.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. B. Wallach Dayan, Lung Cellular and Molecular Laboratory, Inst. of Pulmonology, Hadassah Univ. Hospital, POB 12000, Jerusalem, Israel (e-mail: wallach-dayan{at}hadassah.org.il)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* S. B. Wallach-Dayan and G. Izbicki contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alonso MM, Asumendi A, Villar J, Gil MJ, Martinez-Merino V, Encio IJ, and Migliaccio M. New benzo(b)thiophenesulphonamide 1,1-dioxide derivatives induce a reactive oxygen species-mediated process of apoptosis in tumour cells. Oncogene 22: 3759–3769, 2003.[CrossRef][ISI][Medline]
  2. An J and Hsie AW. Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells. Mutat Res 270: 167–175, 1992.[ISI][Medline]
  3. An J and Hsie AW. Polymerase chain reaction-based deletion screening of bleomycin induced 6-thioguanine-resistant mutants in Chinese hamster ovary cells: the effects of an inhibitor and a mimic of superoxide dismutase. Mutat Res 289: 215–222, 1993.[ISI][Medline]
  4. Aoshiba K, Yasui S, Tamaoki J, and Nagai A. The Fas/Fas-ligand system is not required for bleomycin-induced pulmonary fibrosis in mice. Am J Respir Crit Care Med 162: 695–700, 2000.[Abstract/Free Full Text]
  5. Barrett EG, Johnston C, Oberdorster G, and Finkelstein JN. Silica binds serum proteins resulting in a shift of the dose-response for silica-induced chemokine expression in an alveolar type II cell line. Toxicol Appl Pharmacol 161: 111–122, 1999.[CrossRef][ISI][Medline]
  6. Barrett EG, Johnston C, Oberdorster G, and Finkelstein JN. Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-{alpha}-induced oxidant stress. Am J Physiol Lung Cell Mol Physiol 276: L979–L988, 1999.[Abstract/Free Full Text]
  7. Chapman HA. A Fas pathway to pulmonary fibrosis. J Clin Invest 104: 1–2, 1999.[ISI][Medline]
  8. Cohen JJ. Programmed cell death in the immune system. Adv Immunol 50: 55–85, 1991.[ISI][Medline]
  9. Datta R, Banach D, Kojima H, Talanian RV, Alnemri ES, Wong WW, and Kufe DW. Activation of the CPP32 protease in apoptosis induced by 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents. Blood 88: 1936–1943, 1996.[Abstract/Free Full Text]
  10. Duncan JE, Whitsett JA, and Horowitz AD. Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro. Hum Gene Ther 8: 431–438, 1997.[ISI][Medline]
  11. Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS, Earnshaw WC, Leibson PJ, and Kaufmann SH. Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. Blood 90: 935–943, 1997.[Abstract/Free Full Text]
  12. Erhardt P and Cooper GM. Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL. J Biol Chem 271: 17601–17604, 1996.[Abstract/Free Full Text]
  13. Fine A, Anderson NL, Rothstein TL, Williams MC, and Gochuico BR. Fas expression in pulmonary alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 273: L64–L71, 1997.[Abstract/Free Full Text]
  14. Fraser A and Evan G. A license to kill. Cell 85: 781–784, 1996.[CrossRef][ISI][Medline]
  15. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]
  16. Grummer MA and Zachman RD. Retinoic acid and dexamethasone affect RAR-beta and surfactant protein C mRNA in the MLE lung cell line. Am J Physiol Lung Cell Mol Physiol 274: L1–L7, 1998.[Abstract/Free Full Text]
  17. Hagimoto N, Kuwano K, Miyazaki H, Kunitake R, Fujita M, Kawasaki M, Kaneko Y, and Hara N. Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen. Am J Respir Cell Mol Biol 17: 272–278, 1997.[Abstract/Free Full Text]
  18. Hagimoto N, Kuwano K, Nomoto Y, Kunitake R, and Hara N. Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice. Am J Respir Cell Mol Biol 16: 91–101, 1997.[Abstract]
  19. Hay J, Shahzeidi S, and Laurent G. Mechanisms of bleomycin-induced lung damage. Arch Toxicol 65: 81–94, 1991.[CrossRef][ISI][Medline]
  20. Hoffert JD, Leitch V, Agre P, and King LS. Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway. J Biol Chem 275: 9070–9077, 2000.[Abstract/Free Full Text]
  21. Imlay JA and Linn S. DNA damage and oxygen radical toxicity. Science 240: 1302–1309, 1988.[Abstract/Free Full Text]
  22. Kim PK, Mahidhara R, and Seol DW. The role of caspase-8 in resistance to cancer chemotherapy. Drug Resist Updat 4: 293–296, 2001.[CrossRef][ISI][Medline]
  23. Kuwano K, Hagimoto N, Kawasaki M, Yatomi T, Nakamura N, Nagata S, Suda T, Kunitake R, Maeyama T, Miyazaki H, and Hara N. Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis. J Clin Invest 104: 13–19, 1999.[ISI][Medline]
  24. Kuwano K, Kaneko Y, Hagimoto N, Kawasaki M, Kunitake R, Tanaka T, Maeyama T, Miyazaki H, Matsuba T, and Hara N. Expression of B7–1, B7–2, and interleukin-12 in anti-Fas antibody-induced pulmonary fibrosis in mice. Int Arch Allergy Immunol 119: 112–119, 1999.[CrossRef][ISI][Medline]
  25. Kuwano K, Miyazaki H, Hagimoto N, Kawasaki M, Fujita M, Kunitake R, Kaneko Y, and Hara N. The involvement of Fas-Fas ligand pathway in fibrosing lung diseases. Am J Respir Cell Mol Biol 20: 53–60, 1999.[Abstract/Free Full Text]
  26. Lee MW, Park SC, Yang YG, Yim SO, Chae HS, Bach JH, Lee HJ, Kim KY, Lee WB, and Kim SS. The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis. FEBS Lett 512: 313–318, 2002.[CrossRef][ISI][Medline]
  27. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, and Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489, 1997.[CrossRef][ISI][Medline]
  28. Lin PS, Kwock L, and Goodchild NT. Copper, superoxide radical, diethyldithiocarbamate, and bleomycin cytotoxicity. Lancet 1: 777, 1979.[ISI][Medline]
  29. Mahmutoglu I, Scheulen ME, and Kappus H. Oxygen radical formation and DNA damage due to enzymatic reduction of bleomycin-Fe(III). Arch Toxicol 60: 150–153, 1987.[CrossRef][ISI][Medline]
  30. Moraes EC, Keyse SM, Pidoux M, and Tyrrell RM. The spectrum of mutations generated by passage of a hydrogen peroxide damaged shuttle vector plasmid through a mammalian host. Nucleic Acids Res 17: 8301–8312, 1989.[Abstract/Free Full Text]
  31. Moreno MB, Memon SA, and Zacharchuk CM. Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. J Immunol 157: 3845–3849, 1996.[Abstract]
  32. Muller M, Strand S, Hug H, Heinemann EM, Walczak H, Hofmann WJ, Stremmel W, Krammer PH, and Galle PR. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J Clin Invest 99: 403–413, 1997.[ISI][Medline]
  33. Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, and Krammer PH. p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med 188: 2033–2045, 1998.[Abstract/Free Full Text]
  34. Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, and Dixit VM. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817–827, 1996.[CrossRef][ISI][Medline]
  35. Nguyen NM, Bai Y, Mochitate K, and Senior RM. Laminin {alpha}-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells. Am J Physiol Lung Cell Mol Physiol 282: L1004–L1011, 2002.[Abstract/Free Full Text]
  36. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, and Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139: 271–279, 1991.[CrossRef][ISI][Medline]
  37. Nomoto Y, Kuwano K, Hagimoto N, Kunitake R, Kawasaki M, and Hara N. Apoptosis and Fas/Fas ligand mRNA expression in acute immune complex alveolitis in mice. Eur Respir J 10: 2351–2359, 1997.[Abstract]
  38. Phan SH, Thrall RS, and Ward PA. Bleomycin-induced pulmonary fibrosis in rats: biochemical demonstration of increased rate of collagen synthesis. Am Rev Respir Dis 121: 501–506, 1980.[ISI][Medline]
  39. Polunovsky VA, Chen B, Henke C, Snover D, Wendt C, Ingbar DH, and Bitterman PB. Role of mesenchymal cell death in lung remodeling after injury. J Clin Invest 92: 388–397, 1993.[ISI][Medline]
  40. Shi YF, Szalay MG, Paskar L, Sahai BM, Boyer M, Singh B, and Green DR. Activation-induced cell death in T cell hybridomas is due to apoptosis. Morphologic aspects and DNA fragmentation. J Immunol 144: 3326–3333, 1990.[Abstract]
  41. Simon AH. Alveolar epithelial cells in pulmonary fibrosis. In: Pulmonary Fibrosis, edited by Phan SH and Thrall RS. New York: Dekker, 1995, p. 511–540.
  42. Stohs SJ and Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18: 321–336, 1995.[CrossRef][ISI][Medline]
  43. Strasser A, O'Connor L, and Dixit VM. Apoptosis signaling. Annu Rev Biochem 69: 217–245, 2000.[CrossRef][ISI][Medline]
  44. Tang D, Lahti JM, and Kidd VJ. Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis. J Biol Chem 275: 9303–9307, 2000.[Abstract/Free Full Text]
  45. Terzaghi M, Nettesheim P, and Williams ML. Repopulation of denuded tracheal grafts with normal, preneoplastic, and neoplastic epithelial cell populations. Cancer Res 38: 4546–4553, 1978.[Abstract/Free Full Text]
  46. Weiss RB and Muggia FM. Cytotoxic drug-induced pulmonary disease: update 1980. Am J Med 68: 259–266, 1980.[CrossRef][ISI][Medline]
  47. Wesselborg S, Engels IH, Rossmann E, Los M, and Schulze-Osthoff K. Anticancer drugs induce caspase-8/FLICE activation and apoptosis in the absence of CD95 receptor/ligand interaction. Blood 93: 3053–3063, 1999.[Abstract/Free Full Text]
  48. Wikenheiser KA, Vorbroker DK, Rice WR, Clark JC, Bachurski CJ, Oie HK, and Whitsett JA. Production of immortalized distal respiratory epithelial cell lines from surfactant protein C/simian virus 40 large tumor antigen transgenic mice. Proc Natl Acad Sci USA 90: 11029–11033, 1993.[Abstract/Free Full Text]
  49. Wong HR, Ryan M, and Wispe JR. The heat shock response inhibits inducible nitric oxide synthase gene expression by blocking I kappa-B degradation and NF-kappa B nuclear translocation. Biochem Biophys Res Commun 231: 257–263, 1997.[CrossRef][ISI][Medline]
  50. Wyllie AH, Kerr JF, and Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 68: 251–306, 1980.[Medline]
  51. Zhang B, Hirahashi J, Cullere X, and Mayadas TN. Elucidation of molecular events leading to neutrophil apoptosis following phagocytosis: cross-talk between caspase 8, reactive oxygen species, and MAPK/ERK activation. J Biol Chem 278: 28443–28454, 2003.[Abstract/Free Full Text]
  52. Zhang L, Wikenheiser KA, and Whitsett JA. Limitations of retrovirus-mediated HSV-tk gene transfer to pulmonary adenocarcinoma cells in vitro and in vivo. Hum Gene Ther 8: 563–574, 1997.[ISI][Medline]
  53. Zhuang S, Demirs JT, and Kochevar IE. p38 mitogen-activated protein kinase mediates bid cleavage, mitochondrial dysfunction, and caspase-3 activation during apoptosis induced by singlet oxygen but not by hydrogen peroxide. J Biol Chem 275: 25939–25948, 2000.[Abstract/Free Full Text]



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