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Published ahead of print on September 21, 2006, doi:10.1165/rcmb.2006-0133OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 270-275, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0133OC

Epithelial Cell Apoptosis by Fas Ligand–Positive Myofibroblasts in Lung Fibrosis

Regina Golan-Gerstl, Shulamit B. Wallach-Dayan, Gail Amir and Raphael Breuer

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

Correspondence and requests for reprints should be addressed to Raphael Breuer, M.D., Institute of Pulmonary Medicine, Hadassah–Hebrew University Medical Center, P.O.B. 12,000, Jerusalem, Israel 91120. E-mail: raffi{at}hadassah.org.il


   Abstract
Top
 Abstract
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The Fas/Fas ligand (FasL) apoptotic pathway has been shown to be involved in bleomycin-induced lung fibrosis. We examined the hypothesis that myofibroblasts from fibrotic lungs possess a cytotoxic phenotype that causes apoptosis of epithelial cells via the Fas/FasL pathway. We show in vivo epithelial cell apoptosis and associated upregulation of Fas and apoptotic Fas pathway genes in epithelial cells of lungs with bleomycin-induced fibrosis. In addition, we show that FasL surface molecules are overexpressed on {alpha}-SMA–positive cells in mice with bleomycin-induced fibrosis, and in humans with idiopathic pulmonary fibrosis. This enables the molecules to kill Fas-positive epithelial cells. In contrast, FasL-deficient myofibroblasts lose this myofibroblast cytotoxic phenotype, both in vivo and in vitro. In vivo, there was no bleomycin-induced epithelial cell apoptosis, as assessed by specific M30 staining in chimeric FasL-deficient mice that lacked FasL-positive myofibroblasts. In vitro, FasL-positive, but not FasL-negative myofibroblasts, induce mouse lung epithelial cell apoptosis. Thus myofibroblast cytotoxicity may underlie the absence of re-epithelialization, resulting in persistent lung fibrosis.

Key Words: apoptosis • epithelial cell • Fas ligand • lung fibrosis • myofibroblast

The intratracheal instillation (IT) of bleomycin into rodents is widely used as an in vivo experimental model to study inflammatory and fibrotic changes that are present in the lung interstitium to varying degrees in human fibrotic lung diseases (1–3). A variety of studies in human lung fibrosis and bleomycin-induced fibrosis in mice involve apoptosis of excessive lung cells, mediated by activation of Fas/Fas ligand (FasL) pathway.

FasL, a type-II membrane protein belonging to the TNF family, induces apoptosis by binding to FasL receptors on target cells. The Fas death receptors in these cells trigger apoptosis when engaged by FasL. Both a Fas-soluble form of Fas antigen and anti-FasL antibody (Ab) (4) have been shown to prevent development of bleomycin-induced fibrosis (5), and Fas- and FasL-deficient mice were resistant to the induction of pulmonary fibrosis (6). Moreover, repeated inhalation of anti-Fas Ab (mimicking Fas–FasL cross linking) induced pulmonary fibrosis in mice (7, 8).

In this model, Fas is upregulated in bronchiolar and alveolar epithelial cells. Excessive apoptosis is also detected in these cells (9). The primary target of Fas-induced apoptosis during active lung fibrogenesis has been shown to be type-II epithelial cells (10), whereas the cytotoxic effector cells were shown to be FasL-positive (FasL+) lymphocytes (9, 11, 12) or transforming growth factor-beta–secreting cells (13).

We show increased FasL expression in myofibroblasts of lung tissue sections in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in lung tissue sections of mice with bleomycin-induced fibrosis. In addition, we demonstrate that epithelial cell apoptosis is decreased in chimeric FasL-deficient mice with no FasL+ myofibroblasts, and prove that myofibroblasts act as effector cells by inducing apoptosis of Fas-positive (Fas+) epithelial cells. The results of this study suggest that the absence of sufficient re-epithelialization in lung fibrosis occurs due to myofibroblasts that function as effector cells, persistently inducing epithelial cell apoptosis.


   MATERIALS AND METHODS
Top
 Abstract
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Male 11- to 12–wk-old C57BL/6J wild-type (WT) mice, (Harlan, Ltd., Jerusalem, Israel), and 5- to 6-wk-old C57BL/6J GLD FasL-deficient (GLD) mice (Jackson Laboratory, Bar Harbor, ME), were used. All procedures involving animals were approved by the Hebrew University–Hadassah Medical School Committee of Animal Care.

Chimeric GLD Mice
One mo before IT, GLD or WT mice were subjected to sublethal total body irradiation (700 cGy) delivered by a linear accelerator (G6, dose rate 179 cGy/min, source-to-skin distance 80 cm) (Clinac; Varian, Palo Alto, CA). One day after irradiation, mice received intravenous instillation of syngeneic splenocytes (50–100 x 106) obtained from WT donor mice, creating chimeric FasL-deficient (lymphocyte wt/GLD) and chimeric WT (lymphocyte wt/WT) mice.

To confirm chimerism, irradiated WT mice received 0.5 ml of syngeneic splenocytes (50–100 x 106) obtained from GFP mice (generously provided by Dr. R. Gorodosky, Hebrew University, Jerusalem, Israel), creating chimeric GFP mice (lymphocyte gfp/WT). Chimeric GFP mice were killed 7 d after IT instillation. Lung cells from lymphocyte gfp/WT mice were extracted and stained. FACS analysis showed 84% lymphocytes and 66.5% macrophages, and was GFP positive.

IT
Mice were anesthetized by intraperitoneal injection of 0.05–0.07 ml of 40 mg/ml Ketalar (Parke-Davis, Pontypool, Gwent, UK) and 0.5 mg/ml Droperidol (Janssen Pharmaceutica, Beerse, Belgium). The skin and subcutaneous tissues overlying the proximal portion of the trachea were exposed by a 5-mm transverse incision to allow for direct external visualization of the trachea. A metal cannula fitted to a tuberculin syringe was carefully passed into the trachea. A dose of 0.08 U bleomycin (H. Lundbeck, Copenhagen, Denmark) dissolved in 0.1 ml saline solution, or 0.1 ml saline alone, was slowly injected.

Mice were killed with a lethal dose of pentobarbital at 1, 3, 7, or 14 d after IT. The abdominal aorta was transected, and the animal was exsanguinated. To eliminate blood, lungs were perfused with normal saline through the right ventricle and bronchoalveolar lavage was performed. A polyethylene cannula (PE 205; Clay Adams, Parsippany, NJ) was placed in the trachea, and 3 ml of normal saline was slowly injected and withdrawn.

Total Lung Cell Isolation and Myofibroblast Cell Culture
Lung cells were isolated as follows: lungs were removed, minced, and incubated (37°C, 5% CO2 air) for 45 min in PBS containing 1 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO). After enzyme treatment, lung tissue was gently passed through a cell dissociation sieve (Sigma-Aldrich), and washed twice in PBS. For myofibroblast culture experiments, lung cells were resuspended in fibroblast culture medium RPMI 1640 (Sigma-Aldrich) supplemented with 10% FCS, 100 µg/ml penicillin, 100 mg/ml streptomycin, 25 mM HEPES buffer, and 2 mM L-glutamine (Biological Industries Ltd., Kibbut Beit Haemek, Israel).

Epithelial Cell Isolation
Lung epithelial cells were isolated according to a previously published method (14). Briefly, 3 ml dispase (Roche, Mannheim, Germany) and, subsequently, 0.45 ml of 1% of low-melting agarose (Sigma-Aldrich) were instilled intratracheally into the lungs of WT mice. Crushed ice was placed over the lungs for 2 min to allow the agarose to polymerize. Lungs were removed to a tube with 1 ml dispase and incubated at room temperature for 45 min. Lung tissue was separated from the main airways and teased apart in 4 ml Dulbecco's modified Eagle's medium (Sigma-Aldrich) with 0.01% type-II DNase (Sigma-Aldrich). The cell isolate was successively filtered through 100, 40, and, finally, 25-µm nylon mesh filters (Falcon; Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged at 130 x g for 12 min to collect the cell pellet.

Apoptotic Gene Array of Isolated Lung Epithelial Cells
Total cellular RNA was extracted from lung epithelial cells using the Tri-Reagent kit and instructions (Molecular Research, Inc., Cincinnati, OH). A mouse apoptosis pathway gene array kit (GEArray; SuperArray, Inc., Bethesda, MD) was used to determine the expression levels of multiple genes involved in epithelial cell apoptosis. Briefly, 1 µg RNA was used as a template to generate Biotin-16-dUTP–labeled cDNA probes, according to the manufacturer's instructions. The cDNA probes were denatured and hybridized at 60°C with the SuperArray membrane, which was washed and exposed to a chemiluminescent substrate. To analyze the SuperArray membrane, we scanned the X-ray film and imported it into Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) as a "TIFF" file. The image file was inverted. A pool of four cDNA spots for each gene was used and digitized using ScanAlyze software (shareware, available at http://rana.lbl.gov/EisenSoftware.htm), and normalized by subtraction of the background average intensity value of three spots containing plasmid DNA (PUC18). The average of two RPL13A spots was used as a positive control, and set as the baseline value with which the signal intensity of other spots was compared. Using these normalized data, signal intensities were compared using the GEarray analyzer program (available at http://www.superarray.com).

Flow Cytometry of Fas and FasL Expression in Lung Cells
Lung cells (0.5 x 106) were incubated in FACS buffer (PBS, 1% FCS) with anti-Fas or FasL phycoerythrin (PE) conjugated Abs (1 µg/ml) (BD Biosciences, Franklin Lakes, NJ). After fixation with 4% paraformaldehyde, cells were washed with saponin buffer (PBS, 1% BSA, 1% saponin, 1% HEPES) and incubated for 30 min with anti–{alpha}-SMA–FITC conjugate (1 µg/ml) (Sigma-Aldrich), or with goat anti-AQ5 (1 µg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-goat–FITC conjugate (Jackson Immunoresearch Laboratories, Baltimore, MD). The double-stained cells were analyzed by flow cytometry using a FACStar analyzer (Becton Dickinson, Franklin Lakes, NJ).

Tissue Sections
The left lung was fixed by IT infusion through the cannula with 4% paraformaldehyde maintained at 25 cm of hydrostatic pressure for 5 min and then immersed in fixative for an additional 24 h. Human lung sections were obtained from formalin-fixed lung biopsies; 5-µm-thick paraffin-embedded lung sections were deparaffinized in xylene and rehydrated through a series of alcohols.

Immunofluorescence Staining of Tissue Sections
For antigen retrieval, deparaffinized sections underwent microwave pretreatment in citrate buffer 10 mM, pH 6.0, for 10 min. Nonspecific reactions were blocked with blocking solution of the Zymed kit (Zymed Laboratories, San Francisco, CA). The sections were then incubated, first with mouse monoclonal anti–{alpha}-SMA and rabbit anti-FasL for 1 h at room temperature, and then with anti-mouse–Cy5 conjugate (Jackson Laboratories) or goat anti-rabbit–FITC conjugate Abs (Jackson Laboratories) for 30 min at room temperature.

Immunohistochemical Staining of Tissue Sections
Endogenous peroxidase activity was blocked with immersion of deparaffinized sections in 5% H2O2 methanol for 10 min. For antigen retrieval, the sections were placed in 10 mmol/liter citrate buffer, pH 6.0, and treated with microwaves for 10 min. Nonspecific reactions were blocked with blocking solution of the Zymed kit. The sections were then incubated (30 min, 37°C) with 1:50 mouse monoclonal anti-M30 (15, 16) (Roche Pharmaceuticals, Basel, Switzerland) or anti–caspase-3 monoclonal Ab (mAb) (R&D Systems, Minneapolis, MN). Sections were reincubated (30 min, room temp) with biotinylated rabbit anti-mouse Ab (1 µg/ml) or anti-rabbit Ab (1 µg/ml), reincubated in streptavidin–biotin complex (35 min), and reincubated for a 10 min in peroxidase substrate (Zymed kit). The sections were counterstained with hematoxylin.

Upregulated Fas in Lung Epithelial Mouse Lung Epithelial Cells
Earlier studies in our laboratory, using a mouse line of lung epithelial cells (MLE), showed that 0.005 U bleomycin induces overexpression of the Fas receptor without inducing apoptosis (17). Accordingly, 0.3 x 106 MLE cells were plated in tissue culture dishes and incubated in HEPES 2% FBS medium overnight. Cultured MLE cells were then incubated with bleomycin (5 mU/ml) overnight, and Fas expression was assessed by flow cytometry.

Coculture
Lung myofibroblasts were isolated from bleomycin-treated mice and assayed for their ability to induce apoptosis of Fas+ MLE cells as follows: 3 x 105 Fas+ MLE cells were cocultured for 24 h with FasL+ or FasL-negative (Fas–) myofibroblasts, which were growing adherent to the bottom of a 25-cm2 culture flask after extraction from lungs of bleomycin-treated mice. MLE cells were also cultured alone as controls under the same conditions. After the culture, cells were collected, and cell apoptosis was detected.

In Vitro Apoptosis Detection in Cocultured Epithelial Cells
Epithelial cell apoptosis was detected with flow cytometry using Annexin-V (Becton Dickinson) and {alpha}-SMA double staining. Biotin-conjugated Annexin-V (5 µg/ml) was added to cocultured cells for 30 min. After centrifugation (400 x g, 5 min), streptavidin–PE was added. Cells were then centrifuged for 10 min, resuspended in PBS, paraformaldehyde (4%), and saponin buffer, and then incubated for 30 min with anti–{alpha}-SMA–FITC conjugate (1 µg/ml). Flow cytometry analysis was performed by plotting FL2/Annexin-V versus FL1/{alpha}-SMA–positive cells with a FACScan analyser (Becton Dickinson).

Detection of Epithelial Cell Apoptosis by M30
Epithelial cell apoptosis was detected with flow cytometry using M30 staining (15, 16). Cells were pelleted (400 x g, 5 min), washed with PBS, centrifuged twice (400 x g, 5 min, 2x), and fixed at –20°C for 30 min in methanol (Merck and Co., Whitehouse Station, NJ). Fixed cells were washed twice with washing buffer (PBS, 0.1% Tween 20), and further incubated for 1 h at room temperature with FITC-conjugated M30 CytoDEATH mAb (Roche Pharmaceuticals) in incubation buffer (1:200) (PBS, 1% BSA, 0.1% Tween). Cells were then washed and analyzed by flow cytometry using a FACScan analyzer. Data were processed using CellQuest software (Becton Dickenson).


   RESULTS
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Bleomycin-Induced Apoptosis of Lung Epithelial Cells
To confirm and identify the time kinetics of bleomycin-induced epithelial cell apoptosis, epithelial cells were isolated from bleomycin- or saline-treated lungs 1–14 d after lung instillation and subjected to array analysis of genes related to apoptosis. Expression of the genes of DNA fragmentation factor (DFFA), as well as caspase-2, -3, -7, and -8, which are known to be involved in apoptosis pathways, were upregulated in lung epithelial cells of bleomycin- compared with saline-treated mice at Days 1, 3, and 7, but not at Day 14 (Figure 1A). Increased caspase-3 activity was confirmed in vivo at Day 7 by positive staining with anticleaved caspase-3 mAb (Figure 1B). Apoptosis of lung epithelial cells in bleomycin-treated mice was also confirmed by M30 staining, a specific marker for apoptotic epithelial cells (Figure 1C). Quantification of M30-positive cells further revealed an increased percentage of apoptotic epithelial cells in bleomycin-treated mice (23 cells) compared with saline-treated mice (3 cells) (Figure 1D). These data directly indicate that bleomycin induces increased epithelial cell apoptosis.


Figure 1
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  		Figure 1. Epithelial cell apoptosis in lung tissue of bleomycin-treated mice. (A) Lung epithelial cells from groups of 3–5 mice were isolated at 1, 3, 7, and 14 d after bleomycin or saline instillation. Total RNA samples (1 µg) from isolated lung epithelial cells were subjected to a gene SuperArray analysis. The relative expression level of genes relevant to apoptosis was estimated by comparing signal intensities of four spots of cDNA for each relevant gene with the intensity of four spots of RPLA-housekeeping gene, and then quantified by densitometry after background subtraction. The degree of gene expression after bleomycin and saline treatment, as indicated by fold changes, was calculated by raw densitometry values. (B) Representative immunohistochemical staining with anti–caspase-3 mAb in lung tissue sections of mice at 7 d after bleomycin or saline instillation. Arrows indicate positive caspase-3 staining (red) in epithelial cells. (C) Representative immunohistochemical M30 staining of epithelial cell apoptosis in lung tissues of mice 7 d after bleomycin or saline instillation. Red staining represents early apoptotic changes in lung epithelial cells stained with M30 Ab, an epithelial cell–specific indicator of caspase activation. (D) Quantitation of apoptotic epithelial cells after bleomycin or saline treatment. A total of 16 fields (x200) were counted. Results are presented as median ± SE; n = 4.

 
Bleomycin-Induced Overexpression of Fas Molecule
Lung cells were isolated and double stained using PE-conjugated anti-Fas mAb, together with FITC-conjugated mAbs and one of several specific cell markers (AQ5 for type I epithelial cells, CD8 for lymphocytes, or F4/80 for macrophages) to identify the bleomycin-induced increase in Fas expression. Flow cytometry analysis demonstrated that, compared with saline, bleomycin causes significant overexpression of Fas on AQ5+ cells at Days 3 and 7 (Figure 2A).There were no differences in Fas expression on F4/80+ cells and CD8+ cells at the time points studied (data not shown).


Figure 2
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  		Figure 2. Expression of Fas/FasL apoptotic pathway–associated genes in epithelial cells of bleomycin-treated mice. (A) Isolated lung cells were double stained with fluorescent anti-Fas and anti-AQ5 Abs 1, 3, 7, and 14 d after bleomycin (closed circles) or saline (open squares) instillation, and analyzed by flow cytometry. At each time point, comparisons of double-stained cells (%) were made between bleomycin and saline groups (* P < 0.05). (B) Lung epithelial cells from groups of 3–5 mice were isolated 1, 3, 7, and 14 d after bleomycin or saline instillation. Total RNA samples (1 µg) from isolated lung epithelial cells were subjected to SuperArray apoptosis analysis. The relative expression level of relevant genes was estimated by comparing signal intensities of each gene with the housekeeping gene intensity (RPLA 13A), and then quantified by densitometry after background subtraction. The degree of gene expression after bleomycin and saline treatment, as indicated by fold changes, was calculated by raw densitometry values (optical density ratio).

 
Expression of specific Fas apoptotic pathway–related genes in lung epithelial cells of bleomycin-treated mice was also assessed by gene array. In comparison with epithelial cells from saline-treated mice, epithelial cells from bleomycin-treated mice showed increased expression of Fas apoptotic pathway–related genes Rip, FADD, Flash, and Fas at Days 1, 3, and 7, but not at Day 14 (Figure 2B).

FasL Surface Molecule Overexpression in Myofibroblasts from Bleomycin-Treated Mice
There was significant overexpression of FasL on myofibroblasts isolated on Days 3, 7, and 14 after bleomycin instillation (Figure 3A). FasL overexpression in myofibroblasts was confirmed at Day 7 by immunofluorescence of lung sections. Colocalization of FasL and {alpha}-SMA was seen in bleomycin- but not in saline-treated lungs (Figure 3B). FasL overexpression was also observed in AQ5+ epithelial cells at Day 3, and in lymphocytes and macrophages at Day 14 (data not shown).


Figure 3
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  		Figure 3. FasL overexpression in myofibroblasts of bleomycin- and saline-treated mice. (A) Mouse lung cells were isolated at 1, 3, 7, and 14 d after bleomycin (closed circles) or saline (open squares) treatment. Isolated lung cells were double stained with fluorescent anti–{alpha}-SMA and with anti-FasL Abs, and analyzed by flow cytometry. At each time point, comparisons of double-stained cells (%) were made between bleomycin and saline groups (* P < 0.05). (B) Paraffin-embedded sections of mouse lung tissue were examined 7 d after bleomycin or saline treatment. Immunofluorescent staining using Cy5-conjugated anti–{alpha}-SMA and FITC-conjugated anti-FasL mAbs, with confocal microscopy analysis, were performed. Colocalization of anti–{alpha}-SMA (green) and anti-FasL (red) is indicated by yellow regions.

 
Decreased Apoptosis of Lung Epithelial Cells in Bleomycin-Treated Chimeric FasL-Deficient Mice
To exclude FasL+ lymphocytes as the effectors of Fas-induced apoptosis, chimeric GLD FasL-deficient mice were used. These mice were created by transplantation of FasL+ lymphocytes to FasL-deficient mice (lymphocyte wt/GLD). WT mice that received wild FasL+ splenocytes were used as controls (lymphocyte wt/WT). Epithelial lung cells were isolated 7 d after bleomycin or saline instillation in these mice. Apoptotic epithelial cells were identified by the specific marker M30 mAb, and nonapoptotic epithelial cells were identified by staining with the epithelial cell marker cytokeratin 18 (15, 16). The percentage of apoptotic epithelial cells was quantitated by the following formula:

Formula
There was no difference in epithelial cell apoptosis in bleomycin- compared with saline-treated lymphocyte wt/GLD mice (Figure 4). Control lymphocyte wt/WT mice showed a significant bleomycin-induced increase of epithelial cell apoptosis when compared with saline-treated lymphocyte wt/WT mice (Figure 4).


Figure 4
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  		Figure 4. Decreased epithelial cell apoptosis in lymphocytes of wt/GLD mice. In vivo mouse lung cells were isolated from the lymphocytes of wt/GLD chimeric and wt/WT (control) mice 7 d after bleomycin (closed circles) or saline (open circles) instillation. Nonapoptotic epithelial cells were identified by cytokeratin 18, and apoptotic epithelial cells were detected by staining with the specific marker M30. Epithelial cell apoptosis (%) was quantitated (formula shown above). Comparisons were made between bleomycin- and saline-treated mice (* P < 0.05).

 
FasL+, but Not FasL– Myofibroblasts, Cause Apoptosis of Cocultured Fas+ MLE Cells
To determine whether FasL expression enables myofibroblasts to function as effector cells that cause apoptosis of Fas+ epithelial cells via the Fas/FasL pathway, FasL+ myofibroblasts (0.3 x 106) were cocultured with Fas+ MLE cells (0.5 x 106). FasL+ and FasL– myofibroblasts were isolated from either bleomycin-treated WT mice after cell sorting (Figure 5A) or unsorted myofibroblasts isolated from bleomycin-treated WT control and GLD FasL-deficient mice (Figures 5B and 5C).


Figure 5
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  		Figure 5. FasL+, but not FasL– myofibroblasts, cause apoptosis in cocultured Fas+ MLE cells. (A) Fas+ MLE cells were cocultured for 24 h with FasL+- or FasL–-sorted myofibroblasts isolated from bleomycin-treated mice 7 d after instillation. Lone Fas+ MLE cells served as the control. Apoptosis was detected by Annexin-V affinity labeling. To exclude apoptotic myofibroblasts from the coculture, {alpha}-SMA and Annexin-V staining were also performed. (B) Fas+ MLE cells were cocultured with myofibroblasts extracted from bleomycin-treated FasL-deficient or WT mice 7 d after instillation. Lone Fas+ MLE cells served as control. Apoptosis was detected by Annexin-V affinity labeling. {alpha}-SMA and Annexin-V staining were also performed to exclude apoptotic myofibroblasts from the coculture. (C) Fas+ MLE cells were cocultured with myofibroblasts extracted from bleomycin-treated FasL-deficient and WT mice 7 d after instillation. Epithelial cells apoptosis was detected by M30 staining.

 
FasL+, but not FasL–, myofibroblasts induced MLE cell apoptosis, as assessed by the percentage of cells stained by Annexin-V. One experiment representative of two is shown in Figure 5A. Apoptosis increased from 11.8 to 22.1% in MLE cells cocultured with FasL+ myofibroblasts, but was unchanged (12.5%) in MLE cells cocultured with FasL– myofibroblasts (Figure 5A). Similarly, when MLE cells were cocultured with FasL+ myofibroblasts isolated from bleomycin-treated WT mice, MLE cell apoptosis increased from 6.5 to 15% in an experiment using Annexin-V (Figures 5A and 5B) and M30 staining (Figure 5C). In contrast, MLE cell apoptosis was unchanged in cocultures with FasL– myofibroblasts in all experiments (Figures 5A–5C).

To exclude the possibility that MLE cell apoptosis was due to substances secreted into the coculture media by myofibroblasts, MLE cells were also cultured in myofibroblast medium, and showed no change in cell apoptosis (data not shown).

FasL Expression in Lung Myofibroblasts of Patients with Idiopathic Pulmonary Fibrosis
FasL expression was studied in human fibrotic lung myofibroblasts by immunofluorescence of lung sections from patients with IPF. Double staining with anti-FasL and anti–{alpha}-SMA mAbs revealed dual expression of FasL and {alpha}-SMA molecules (Figure 6).


Figure 6
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  		Figure 6. FasL expression in human lung myofibroblasts from a patient with IPF. Immunofluorescent staining of paraffin-embedded sections of lung tissue from patients with IPF was performed using Cy5-conjugated anti–{alpha}-SMA, and FITC-conjugated anti-FasL mAbs. Confocal microscopy analysis was then performed. Localization of anti–{alpha}-SMA (green) and anti-FasL (red) in the same cells is indicted by arrows.

 

   DISCUSSION
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Epithelial cell injury and apoptosis are recognized as early features in the pathogenesis of lung fibrosis (9, 11, 18). Epithelial cell apoptosis has been demonstrated by electron microscopy or by light microscopy using terminal deoxyribonucleotidyl transferase–mediated dUTP-biotin nick-end label staining of cells that appear epithelial by morphologic criteria in lung biopsies of patients with IPF (18, 19), and in fibrotic lungs of bleomycin-treated C57BL/6 mice (9). In experiments using a specific epithelial cell apoptosis M30 marker, we have seen evidence of epithelial cell apoptosis in lung tissues of mice at 7 d after bleomycin instillation (Figure 1A). Furthermore, epithelial cells in these bleomycin-treated mice showed upregulation of Fas-related genes and Fas protein expression (Figures 2A and 2B). Taken together, our data show direct evidence of epithelial cell apoptosis that may be mediated through the Fas/FasL pathway.

Activated lymphocytes overexpress FasL molecules, and have been considered the main inducers of epithelial cell apoptosis via the Fas/FasL pathway (9). To date, upregulation of FasL in fibroblasts has been demonstrated in experimental murine scleroderma of the skin (4), but not in lung fibrosis. We show FasL overexpression in lung myofibroblasts of humans with IPF (Figure 6) and during bleomycin-induced fibrosis (Figure 3A). We therefore hypothesize that FasL+ myofibroblasts may play a role in epithelial cell apoptosis.

FasL expression has been shown to be upregulated by proinflammatory cytokines like IFN-{gamma} (20, 21) and TNF (20). During the evolution of lung fibrosis, TNF and IFN-{gamma} expression are upregulated (22–24), and they may be the inducers of FasL upregulation of myofibroblasts during lung fibrosis.

To test the functional impact of bleomycin-induced overexpression of FasL on lung myofibroblasts, we used FasL-deficient GLD mice whose lymphocytes were replaced by wt lymphocytes to create chimeric FasL-deficient mice. FasL+ lymphocytes were thus excluded as inducers of cell apoptosis in these mice. Indeed, bleomycin-induced epithelial cell apoptosis was almost absent in chimeric GLD FasL-deficient mice, but not in WT mice, indicating that epithelial cell apoptosis is induced by FasL on cells other than lymphocytes.

Potential FasL apoptotic effector cells include macrophages, epithelial cells, and myofibroblasts, which all show overexpression of FasL in this model. However, macrophage FasL is unlikely to be responsible for the absence of epithelial cell apoptosis in our chimeric model, as transplanted splenocytes contain a range of hematopoietic cells, including macrophages. Epithelial cells were previously shown to coexpress Fas and FasL without apparent apoptosis due to auto- or paracrine apoptosis (25), and, in our laboratory, we have shown that bleomycin causes FasL overexpression in epithelial cells, with no autocrine apoptosis via the Fas/FasL pathway (17).

Taken together, these findings indicate that FasL+ myofibroblasts are the key inducers of Fas-dependent epithelial cell apoptosis, based on our findings in chimeric FasL-deficient mice together with evidence from studies of ex vivo FasL+ myofibroblast-induced epithelial cell apoptosis. This concept is further supported by in vivo studies in fibrotic lungs showing apoptotic epithelial cells situated adjacent to myofibroblasts (26).

Myofibroblasts were initially thought to be structural cells, but recently other functions have been attributed to them (27, 28). For example, when cocultured with primary mouse hepatocytes, FasL-transfected NIH 3T3 fibroblasts were shown to act as cytotoxic cells (29). In our study, we show that FasL+, but not FasL–, lung myofibroblasts act as effector cells that induce apoptosis of Fas+ epithelial cells (Figure 5). Several other studies have indicated the existence of "cross-talk" between lung epithelium and myofibroblasts, which may be involved in the development of lung fibrosis (30). Epithelial cells overlying fibroblastic foci were found to be apoptotic (26), and fibroblasts isolated from fibrotic human or rat lung were shown to produce factors capable of inducing epithelial cell apoptosis (30). These studies are consistent with our findings implicating Fas-induced epithelial cell apoptosis by FasL+ myofibroblasts.

In summary, we demonstrate direct evidence for bleomycin-induced Fas+ epithelial cell apoptosis during the evolution of lung fibrosis. Based on our results, we propose that FasL+ myofibroblasts induce Fas-dependent epithelial cell apoptosis during lung fibrosis. This finding may underlie the persistent absence of re-epithelialization during irreversible lung fibrosis.


   Footnotes
 
This work was supported by the David Shainberg Fund.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0133OC on September 21, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 4, 2006

Accepted in final form August 21, 2006


   References
Top
 Abstract
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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