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1 as an Enhancer of Fas-Mediated Apoptosis of Lung Epithelial Cells
* Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and
Research and Development Planning and Management Department, Mochida Pharmaceutical Company, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (TGF-1) has important roles in
lung fibrosis and the potential to induce apoptosis in several types of
cells. We previously demonstrated that apoptosis of lung epithelial
cells induced by Fas ligation may be involved in the development of
pulmonary fibrosis. In this study, we show that TGF-1 induces
apoptosis of primary cultured bronchiolar epithelial cells via
caspase-3 activation and down-regulation of cyclin-dependent kinase
inhibitor p21. Concentrations of TGF-1 that were not sufficient to
induce apoptosis alone could enhance agonistic anti-Fas Ab or rFas
ligand-mediated apoptosis of cultured bronchiolar epithelial cells.
Soluble Fas ligand in the bronchoalveolar lavage fluid (BALF) from
patients with idiopathic pulmonary fibrosis (IPF) also induced
apoptosis of cultured bronchiolar epithelial cells that was
significantly attenuated by anti-TGF- Ab. Otherwise, BALF from
patients with hypersensitivity pneumonitis (HP) could not induce
apoptosis on bronchiolar epithelial cells, despite its comparable
amounts of soluble Fas ligand. The concentrations of TGF-1 in BALF
from patients with IPF were significantly higher compared with those in
BALF from patients with HP or controls. Furthermore, coincubation with
the low concentration of TGF-1 and HP BALF created proapoptotic
effects comparable with the IPF BALF. In vivo, the administration of
TGF-1 could enhance Fas-mediated epithelial cell apoptosis and lung
injury via caspase-3 activation in mice. Our results demonstrate a
novel role of TGF-1 in the pathophysiology of pulmonary fibrosis as
an enhancer of Fas-mediated apoptosis of lung epithelial
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-1 is important in the fibrotic processes of IPF (4)
and bleomycin-induced pulmonary fibrosis in rodents (5).
This cytokine has multiple effects that may exacerbate fibrosis. It is
a strong extracellular matrix inducer and is chemotactic for
fibroblasts (6) and polymorphonuclear neutrophils
(7). In addition, it can induce apoptosis in gastric
carcinoma cells (8), primary hepatocytes, hepatoma cells
(9), and human lung epithelial cell lines
(10). The mechanism of TGF-1-mediated apoptosis most
likely varies among cell types, although caspase activation (11, 12), up-regulation of p21 (13), and down-regulation
of Bcl-xL expression (14) are
commonly observed. However, TGF-1 induces apoptosis of retinal
endothelial cells with decreased expression of p21 (15).
Recently, it was demonstrated that p21 regulates the activation of
caspase-3 through procaspase-3-p21 complex formation and protects human
hepatoma cells from Fas-mediated apoptosis (16, 17).
Fas Ag (Fas), a type I membrane receptor protein and a member of the TNFR family (18), induces apoptosis after engaging with Fas ligand (FasL) (19). Fas is expressed in various cells and tissues, including the thymus, liver, skin, heart, and lung (20, 21). Loss of epithelial cells through the Fas-mediated pathway may have an important role in tissue injury and organ dysfunction (20, 22). Damage to and loss of epithelial cells are also commonly seen in acute lung injury and in chronic fibrosing alveolitis. Furthermore, it is increasingly apparent that Fas ligation can lead to the release of proinflammatory cytokines and can mediate inflammation and tissue injury (23, 24, 25). We demonstrated that apoptosis and up-regulation of Fas and FasL expression occur in bronchiolar and alveolar epithelial cells and in infiltrating inflammatory cells, respectively, in lung tissue from patients with IPF (26, 27). We also showed that excessive apoptosis of lung epithelial cells induced by the Fas-FasL pathway is essential for the development of bleomycin-induced pulmonary fibrosis in mice (28).
Thus, apoptosis of lung epithelial cells by Fas ligation may be
involved in the pathogenesis of pulmonary fibrosis, but the role of
TGF-1 in this process is unclear. Therefore, the first purpose of
this study was to investigate whether TGF-1 can induce apoptosis of
lung epithelial cells and modulates Fas-mediated apoptosis in vitro and
in vivo. Second, we investigated whether bronchoalveolar lavage fluid
(BALF) from patients with IPF can induce Fas-mediated apoptosis of
small airway epithelial cells (SAEC) in vitro, and whether TGF-1 has
some role in this response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cryopreserved primary human bronchiolar epithelial cells (Clonetics, San Diego, CA) were used between passages 2 and 5. Cells were grown in small airway epithelial cell growth medium (Clonetics) supplemented with hydrocortisone (0.5 µg/ml), bovine pituitary extract (30 µg/ml), epidermal growth factor (0.5 ng/ml), epinephrine (500 ng/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamicin (50 µg/ml), amphotericin B (50 ng/ml), and 5% BSA. Cultures were incubated at 37°C in a humidified, 95% air/5% CO2 atmosphere. After trypsinization, cells were subcultured at densities of 1 x 103 cells/cm2. When cells were 50–60% confluent, the medium was changed to small airway epithelial cell growth medium without hydrocortisone, and the cells were incubated for an additional 24–48 h, until they reached 70–80% confluence.
Reagents
Human rTGF-1 and mouse anti-human TGF- mAb were
obtained from R&D Systems (Minneapolis, MN). Anti-human Fas mAbs for
the induction of apoptosis (clone CH-11) and the neutralization of Fas
(clone ZB-4) were purchased from MBL (Nagoya, Japan).
Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone
(Z-VAD.FMK) was purchased from Kamiya (Thousand Oaks, CA). Human
rFasL was prepared and purified, as described previously
(29).
Quantification and detection of apoptosis
Cells (106) were washed in PBS and resuspended in incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) and Annexin V-FITC (Roche Diagnostics, Indianapolis, IN). After 15 min of incubation at 4°C, fluorescence was analyzed by a Coulter EPICS XL flow cytometer (Coulter, Miami, FL). Apoptotic cells with phosphatidylserine on the outer leaflet of the cell membrane and positively stained by annexin V were determined by setting the analytical region to a brighter fluorescent population that exceeded the upper limit of fluorescence with nonapoptotic specimens. Electron microscopy was performed as previously described (25).
Caspase activity analysis
Briefly, cells were washed once in PBS and resuspended at 108 cells/ml in hypotonic lysis buffer (25 mM/L HEPES, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 1 mg/ml leupeptin, and aprotinin). The lysate was then subjected to four freeze-thaw cycles before centrifugation at 10,000 x g for 10 min, and the supernatants were collected. A total of 20 µg extracted proteins was incubated with the fluorescent substrates DEVD-AMC (Ac-Asp-Gul-Val-Asp aminomethyl-coumarin). The fluorescence of cleaved substrates was determined spectrofluorometrically at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Western blot analysis
Cells were washed three times with PBS, collected, and lysed in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1% Nonidet P-40, 0.5% sodium deoxycholate) for 10 min at 4°C. Insoluble material was removed by centrifugation at 14,000 rpm for 30 min at 4°C. Frozen lung tissues were homogenized in buffer solution (25 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) using polytron homogenizer. Extracted protein was used for Western blot analysis for cleaved caspase-3. The protein concentration was determined using a Bio-Rad (Melville, NY) protein assay kit. Identical amounts of protein from each lysate (20 µg/well) were subjected to 15% SDS-PAGE. Proteins were transferred onto nitrocellulose filters, which were then blocked for 1.5 h using 5% nonfat dried milk in PBS containing 0.1% Tween 20 (PBS-T), washed with PBS-T, and incubated at room temperature for 1 h in the presence of each Ab (mouse monoclonal anti-human p21, rabbit polyclonal anti-human p53, mouse monoclonal anti-human p27, goat polyclonal anti-human Bcl-2, rabbit polyclonal anti-human Bcl-xL, Santa Cruz Biotechnology, Santa Cruz, CA; rabbit polyclonal anti-human X-linked mammalian inhibitor of apoptosis protein (XIAP), R&D Systems; rabbit polyclonal anti-human cleaved caspase-3 Ab, Cell Signaling Technology, Beverly, MA). Filters were washed with PBS-T and incubated with a 1/1000 dilution of sheep anti-mouse IgG, anti-goat IgG, or donkey anti-rabbit IgG, coupled with HRP. The ECL system (Amersham Life Science, Buckinghamshire, U.K.) was used for detection.
Adenoviral vector construction and purification
The recombinant adenoviral vector, AdV-p21, purchased from the
RIKEN gene bank (Tsukuba, Japan), was constructed by homologous
recombination between sub360 genomic DNA, an adenovirus type 5
derivative with a deletion in the E3 region, and a p21 expression
plasmid, pAd-p21, which had the left-hand sequence of adenovirus type 5
genome, but not E1A and E1B. An adenoviral vector encoding
lacZ was used for control experiments. The structure of
these replication-defective viruses was confirmed by Southern blot
analysis. All were propagated in 293 cells, cesium chloride purified,
sterilized with a 0.45-µm filter, and diluted for storage in 10%
glycerol-PBS solution to yield a final concentration of 1–3 x
109 viral particles/ml (0.8–5 x
108 PFU/ml). For infection, 5 x
105 cells were grown to log phase and infected
with adenoviruses at 10 PFU/cell. Forty-eight hours postinfection, the
cells were treated with TGF-1 for 24 h and analyzed by flow
cytometry to quantify apoptosis.
Patient selection
The diagnosis of IPF was made using previously described criteria (2, 30, 31). All control subjects were healthy volunteers. The diagnosis of hypersensitivity pneumonitis (HP) was based on previously described criteria (32). BALF were obtained at the time of diagnosis and before steroid therapy. In all patients, current infection with bacteria, mycobacteria, or fungi was excluded by negative cultures of BALF and biopsies.
IPF patients were divided by the clinical indication for steroid therapy at the time of bronchoalveolar lavage (BAL). BALF were obtained before treatment. Patients with acute exacerbation were treated with prednisolone. Acute exacerbation was defined as an increased dyspnea (an increase of >1 grade in the Hugh-Johns classification system), or a decrease in PaO2 (>10 torr in the same condition), accompanied with remarkable exacerbation of the findings on chest x-ray or computed tomography during the prior 2 mo. Predictive vital capacities (mean ± SD) were 57.9 ± 2.8% in IPF with indication of steroid therapy, 67.2 ± 16.9% in IPF without indication of steroid therapy, 92.3 ± 18.4% in HP, and 97.25 ± 8.78% in controls.
BAL procedure and treatment
BAL was performed as previously described (33). Briefly, BAL was performed using a total of 150 ml sterile physiologic saline solution. The recovered fluid was filtered through a single layer of gauze to remove mucus. Cells in the lavage fluid were counted using a hemocytometer. Differential counts were performed on a total of 100 cells stained with Wright and Giemsa stain. The lavage fluid supernatant was stored at -70°C until the measurement of soluble FasL (sFasL).
SAEC was incubated in medium supplemented at a 50% concentration with either BALF from patients with IPF, HP, and normal volunteers. The cells were incubated for 24 h at 37°C, 5% CO2, then analyzed by flow cytometry to detect apoptosis.
sFasL and TGF-1 measurement by ELISA
The BALF sFasL levels were measured with a newly developed ELISA
system (34). In brief, microtiter plates were coated with
an anti-human FasL Ab (F918-20-2 Ab). The purified human rFasL
produced in pichia pastoris was used as a standard. After an incubation
for 2 h at 37°C, the wells were washed with saline containing
0.05% Tween 20. The wells were incubated with the poly
peroxidase-labeled anti-human FasL Ab F919-9-18 for 1 h at
37°C, and washed with saline containing 0.05% Tween 20 and distilled
water. The wells were incubated with tetramethylbenzidine for 20 min.
Peroxidase activity was developed in proportion to the amount of sFasL.
The reaction was stopped by adding 1 N
H2SO4. The color generated
was determined by measuring the OD at 450 nm in a spectrophotometric
microtiter plate reader. The limit of detection (negative
control (0 pg/ml) + 2 SD) was 5 pg/ml in BALF. Intra- and interassay
coefficient variations were <2.4–11.7% and <8.3–15.7%,
respectively. Total TGF-1 (after acid activation) in the BALF was
analyzed using a sandwich ELISA system (Promega, Madison, WI) with
monoclonal capture and polyclonal detection Abs and visualization with
tetramethylbenzidine, according to the manufacturer’s instructions. We
performed all assays in duplicate, and the mean of two data was
determined for individual sample.
Treatment of animals
The present study protocol was reviewed and approved by the
Committee on Ethics on Animal Experiments, Kyushu University Faculty of
Medicine, and the experiments were conducted according to the
Guidelines for Animal Experiments of Kyushu University Faculty of
Medicine. The 6-wk-old ICR mice (30 g) were divided into five
groups. The first or second group was treated with agonistic
anti-mouse Fas mAb (JO-2; PharMingen, San Diego, CA) dissolved at
10 µg/body (n = 20) or 50 µg/body
(n = 10) in PBS intratracheally. The third group
consisted of control mice, treated with isotype-matched control hamster
IgG Ab (Organon Teknika, Durham, NC) (n = 20) dissolved
at 50 µg/body in PBS. The fourth group (n = 20) was
treated with human TGF-1 dissolved at 500 ng/body in PBS i.v.
through the tail vein. The fifth group (n = 20) was
treated with 500 ng/body TGF-1 i.v. with 10 µg/body anti-Fas
Ab intratracheally. TGF-1 was injected i.v. at 30 min before
anti-Fas Ab instillation intratracheally. Mice were sacrificed at
6, 12, 18, and 24 h after the treatment. Lung tissues were
prepared for the histological examination (H&E staining), the TUNEL
method, and Western blot analysis.
Apoptosis analysis in lung tissues
Apoptosis was detected by the TUNEL method using DeadEnd Colorimetric Apoptosis Detection system (Promega). After proteinase digestion and removing of endogenous peroxidase, the sections were incubated in a mixture containing TdT and FITC-labeled dUTP. The sections were then treated with the peroxidase labeled with anti-FITC Ab. The reaction products were developed with 3,3'-diaminobenzidine tetrahydrochloride and counterstained with methyl green. Ten fields at x200 were randomly selected, and the number of TUNEL-positive cells was calculated.
Immunohistochemistry
A 5-µm paraffin section was adhered to slides pretreated with poly(L-lysine). After the dehydration and the deparaffinization, the tissue sections were autoclaved at 121°C for 5 min in a glass pot filled with distilled water to completely immerse the sections, and washed three times in 0.1 M PBS. Immunohistochemistry was performed using a streptavidin-biotinylated peroxidase technique using a Histofine SAB-PO kit from Nichirei (Tokyo, Japan). The sections were subsequently counterstained with hematoxylin faintly and mounted.
Statistics
Data are expressed as mean ± SD from at least three independent experiments. The data were first analyzed by one-way ANOVA, and statistical significance was determined with multiple comparison methods by Fisher’s protected least significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 induces apoptosis in bronchiolar epithelial cells
(SAEC)
Treatment of SAEC with 25 ng/ml TGF-1 for 24 h resulted in
58% cell death with dramatic morphological changes characteristic of
apoptosis, such as large vacuoles, cell shrinkage, and cytoplasmic
blebbing (Fig. 1
, a–f).
TGF-1 induced apoptosis in a dose-dependent manner (Fig. 1, g and h). The number of apoptotic cells induced
by Fas ligation with anti-human Fas mAb (CH-11) was greater than
that induced by TGF-1 (Fig. 1i). The expression of Fas on
SAEC was not affected by TGF-1 (data not shown).
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1-mediated apoptosis
on SAEC
Because caspase-3 activation is an important element in the
apoptotic signaling pathway induced by TGF- (11, 12), we characterized the role of caspase-3 activation in
TGF-1-induced apoptosis. TGF-1 induced caspase-3 activity in a
dose-dependent manner (Fig. 2a), and this was delayed for
12 h compared with that in anti-Fas Ab-mediated
apoptosis (Fig. 2b). When SAEC were cotreated with the
broad-spectrum caspase inhibitor, Z-VAD.FMK, TGF-1-induced apoptosis
was inhibited in a dose-dependent manner (Fig. 2c).
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1 and CH-11 act synergistically on SAEC
Cells (4% and 12.3%) were induced to apoptosis by a low
concentration of TGF-1 (0.1 ng/ml) or CH-11 (50 ng/ml),
respectively. However, 25.7% of cells were induced with a combination
of them (Fig. 3a).
Caspase-3 activity was also markedly increased by the combination
compared with TGF-1 or CH-11 alone (Fig. 3b).
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1 down-regulates the expression of p21 on SAEC
We also examined the expression of the apoptosis-regulatory
proteins, p53, p21, p27, Bcl-2, Bcl-xL, and XIAP.
The expression of p21 in SAEC was reduced from 18 to 48 h after
TGF-1 treatment, independent of p53 expression (Fig. 4a), and was significantly
decreased in apoptotic detached cells compared with untreated or
attached living cells (Fig. 4b). The expression of Bcl-2,
Bcl-xL, and XIAP was not affected (Fig. 4a). In contrast, the low concentration of CH-11 did not
affect p21 expression. Z-VAD.FMK had no effect on the decrease of p21
expression after treatment with the low concentration of TGF-1 (Fig. 4c). Therefore, down-regulation of p21 by TGF-1 was not
caused by caspase cleavage.
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1-mediated
apoptosis and reduces caspase-3 activity on SAEC
SAEC were infected with an adenoviral vector capable of expressing
high levels of p21 (AdV-p21), resulting in a significant elevation of
p21 expression relative to that in SAEC infected with an adenoviral
vector encoding lacZ (data not shown). AdV-p21-infected
cells were well protected from TGF-1-induced apoptosis (Fig. 5a). Simultaneously, caspase-3
activation induced by TGF-1 was suppressed by AdV-p21 infection
(Fig. 5b).
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Recent results suggest that sFasL is relatively ineffective in
inducing apoptosis, and release of sFasL may actually down-regulate the
apoptotic activity of the membrane-bound form (35).
However, other investigators demonstrated that bioactive sFasL may have
some role in human diseases (33, 36). Thus, the role of
sFasL is controversial. To determine their sensitivity to sFasL, SAEC
were exposed to serial dilutions of rFasL ranging from 0.1 to 1000
ng/ml for 24 h. This caused a linear, dose-dependent increase in
annexin V binding, ranging from 1 to 1000 ng/ml rFasL. Furthermore, the
coincubation with 0.1 ng/ml TGF-1 directly enhances the effect of
sFasL on SAEC apoptosis (Fig. 6a).
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We previously demonstrated that sFasL levels in BALF from patients
with IPF, especially those who required steroid therapy or those with
HP, were elevated compared with those from patients with IPF without
indication for therapy or from healthy volunteers (33, 34). In this study, we measured sFasL concentrations in BALF
from 12 patients with IPF (including 5 patients who required steroid
therapy), 6 patients with active HP, and 4 normal volunteers. BALF
sFasL levels (mean ± SD) were 5.5 ± 3.6 pg/ml in controls.
BALF sFasL levels were significantly higher in IPF patients who
required steroids (151 ± 66.0 pg/ml, p < 0.01)
and in patients with HP (85.8 ± 41.6 pg/ml, p <
0.05), but not in IPF patients who did not require therapy,
compared with controls. There was a significant difference in BALF
sFasL levels between the IPF subgroup with and without steroid therapy
(p < 0.05) (Fig. 6
b). BALF from
patients with IPF (with and without steroid therapy), but not HP, could
induce apoptosis of SAEC (Fig. 6c). To demonstrate the
specificity of these effects for sFasL, we incubated SAEC (in
medium supplemented at a 50% concentration with BALF from
patients with IPF who required steroid therapy) with
anti-Fas-neutralizing mAb (ZB-4). Inhibition of Fas ligation with
ZB-4 blocked the proapoptotic effect of BALF (Fig. 6d). To
further examine whether TGF-1 is involved in BALF sFasL-induced
apoptosis, we incubated SAEC in 50% BALF medium with anti-TGF-
mAb. Anti-TGF- mAb significantly inhibited apoptosis of SAEC (Fig. 6d). BALF TGF-1 levels (mean ± SD) were 253.6
± 33.5 pg/ml in controls and 265.0 ± 96.8 pg/ml in HP. BALF
TGF-1 levels were significantly higher in both IPF patients who
required steroid therapy (493.7 ± 105.0 pg/ml, p
< 0.01) and IPF patients who did not require steroid therapy
(451.5 ± 58.5 pg/ml, p < 0.01) compared with
that in BALF from control or HP group. There was no significant
difference in BALF TGF-1 levels between the IPF subgroup with and
without steroid therapy (Fig. 6e). Finally, coincubation
with 0.1 ng/ml TGF-1 and HP BALF created proapoptotic effects
comparable with the IPF BALF (Fig. 6f).
TGF-1 enhanced Fas-mediated epithelial cell apoptosis and lung
injury via caspase-3 activation in vivo
To confirm the direct link of TGF-1 to apoptosis of lung
epithelium in vivo, we used Fas-mediated lung injury model in mice.
Nine of ten mice treated with 50 µg/body agonistic anti-Fas Ab
were dead within 24 h. In these mice, other Fas-susceptible
organs, such as liver, kidney, and heart, were almost intact
pathologically (data not shown). They were very tachypneic and
exhausted. Therefore, it is likely that acute lung injury was the main
cause of death. Although none of mice treated with i.v. injection of
500 ng TGF-1 or intratracheal instillation of 10 µg anti-Fas
Ab was dead, half of mice treated with the combination of these two
treatments were dead (Fig. 7). The i.v.
injection of 500 ng/body TGF-1 or intratracheal injection of 10
µg/body anti-Fas Ab alone induced mild inflammation (Fig. 8, a and b).
However, the treatment of anti-Fas Ab with TGF-1 induced severe
lung inflammation and apoptosis of epithelial cells (Fig. 8, c and d). The number of TUNEL-positive cells was
significantly increased compared with controls at 12–24 h after the
treatment of anti-Fas Ab with TGF-1. However, the TGF-1 or
anti-Fas Ab treatment alone did not significantly increase the
number of apoptotic cells, except at 18 h after the TGF-1
treatment (Fig. 9). The results of
Western blot analysis show that the caspase-3 activation was
significantly increased predominantly at 12–18 h after the treatment
of anti-Fas Ab with TGF-1 compared with that after the treatment
of TGF-1 or anti-Fas Ab alone (Fig. 10a). The results of
immunohistochemistry showed that the expression of cleaved casapase-3
was predominantly detected in alveolar epithelial cells (Fig. 10b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 that can explain amplification of pulmonary fibrosis. TGF-1
is prominently expressed in macrophages and epithelial cells and found
to be associated with the extracellular matrix using
immunohistochemistry in IPF (37). In lungs with early
lesions of IPF and only inflammatory changes, TGF-1 is present in
alveolar macrophages, but not present in epithelial cells
(37). In bleomycin-induced pulmonary fibrosis model in
rat, TGF-1 is expressed in alveolar macrophages at acute phase of
inflammatory cell infiltration, and in epithelial cells at later phase
of pulmonary fibrosis (38). It seems that the expression
of TGF- may associate with inflammation at acute phase, and with
tissue remodeling at late phase. Therefore, the significance of
TGF-1 in IPF and animal models of pulmonary fibrosis is well known;
however, it is unclear whether TGF-1 participates in the epithelial
cell injury and apoptosis.
We performed this study to investigate the effect of TGF-1 on
Fas-mediated apoptosis in lung epithelial cells. Our data show that
TGF-1 can induce apoptosis of SAEC through the caspase-3 activation
and the down-regulation of the p21 expression. In addition, a low
concentration of TGF-1 that could not induce apoptosis alone
enhanced Fas-mediated apoptosis of SAEC. Concomitant with our studies,
others reported that this process involved the activation of caspase-3
(11, 12) or the down-regulation of
Bcl-xL (14). However, we found that
the caspase-3 activation by TGF-1 leads to SAEC apoptosis without
the down-regulation of Bcl-xL expression. We
found the p53-independent down-regulation of p21 expression by
TGF-1, even at the low concentration. Detached apoptotic cells
exhibited significantly reduced levels of p21 expression, whereas
attached living cells exhibited elevated levels. Thus, for SAEC,
induction of p21 appears to be correlated with resistance to
TGF-1-mediated apoptosis. It is also possible that in the presence
of TGF-1, p21 protects SAEC from apoptosis. This hypothesis appears
to be valid for myocyte differentiation in low serum (39),
and similar observations have been reported in neuroblastoma
(40) or colorectal carcinoma cell lines (41).
To confirm this protective effect, we infected SAEC with AdV-p21.
Ectopic expression of p21 protected SAEC from TGF-1-mediated
apoptosis and reduced caspase-3 activity. Interestingly, some
investigators have demonstrated that activation of caspase-3 is
regulated by p21, and procaspase-3-p21 complex formation is an
essential system for cell survival (42, 43). Therefore,
the down-regulation of p21 by TGF-1 may enhance Fas-mediated
apoptosis through caspase-3 activation. Since the down-regulation of
p21 expression by the low concentration of TGF-1 was not affected by
a caspase inhibitor, another regulatory system, such as the ubiquitin
proteasome system (44), may be involved in the
down-regulation of p21.
We previously demonstrated that the concentrations of sFasL in BALF
from patients with IPF and HP were significantly higher than those in
BALF from normal volunteers (33, 34). In addition, sFasL
can be released in BALF from patients with acute respiratory distress
syndrome as a biologically active molecule that can induce apoptosis of
the pulmonary epithelium (45). In this study, we
demonstrated that BALF from patients with IPF, but not HP, could induce
apoptosis of SAEC, despite the fact that BALF from both IPF and HP
contained similar concentrations of sFasL. Anti-Fas-neutralizing Ab
could significantly inhibit SAEC apoptosis induced by BALF from
patients with IPF. However, much higher concentrations of rFasL were
required for the induction of apoptosis in vitro (Fig. 6a).
BALF TGF-1 levels were significantly higher in both
steroid-indicated and not indicated IPF patients compared with that in
BALF from HP patients. Anti-TGF--neutralizing Ab inhibited apoptosis
of SAEC induced by BALF administration. Furthermore, adding TGF-1 to
the HP BALF created proapoptotic effects comparable with the IPF BALF.
Thus, TGF-1 may act synergistically as a cofactor for the
development of a full apoptotic response to sFasL in BALF from patients
with IPF. This may be one explanation for the difference in prognosis
between IPF and HP. Patients with HP usually improve spontaneously by
isolating patients from Ag. As other investigators demonstrated,
fibroblasts are refractory to Fas-mediated apoptosis, and TGF-1 can
abrogate this resistance (46). These opposing responses to
Fas ligation or TGF-1 between epithelial cells and fibroblasts may
be one of a key mechanism of pulmonary fibrosis. Furthermore, it was
demonstrated that the increased production of TGF-1 could not be
suppressed by the high-dose corticosteroids treatment in
bleomycin-induced pulmonary fibrosis in rat (47). This
result may be an explanation for the ineffectiveness of corticosteroids
treatment in patients with IPF.
Finally, to confirm the direct link of TGF-1 to apoptotic
effects in a physiologically relevant system in vivo, we designed the
Fas-mediated lung injury model of mice. Matute-Bello et al.
(48) demonstrated that lung epithelial cell apoptosis and
acute inflammation were caused by intranasal instillation of agonistic
anti-Fas Ab in mice. We also previously demonstrated that repeated
inhalation of anti-Fas Ab induced lung injury and fibrosis in mice
(49). The intratracheal instillation of 50 µg
anti-Fas Ab killed 90% of mice within 24 h. Because the
intratracheal instillation of anti-Fas Ab did not affect other
Fas-susceptible organs, we presumed that the respiratory failure
induced by Fas-mediated lung epithelial cell apoptosis and inflammation
was the cause of death. In this model, TGF-1 enhanced lung
epithelial cell apoptosis and inflammation induced by low dose of
anti-Fas Ab, and increased the mortality of mice. Western blot
analysis using extracts from lung homogenates demonstrated that the
cleaved caspase-3 was significantly increased from 12 to 18 h
after the treatment of anti-Fas Ab with TGF-1. Thus, TGF-1
augments Fas-mediated apoptosis on lung epithelium via the caspase-3
activation in vivo. As seen in the vitro system, TGF-1 alone also
induced the caspase-3 activation and apoptosis on lung epithelial
cells. It is possible that TGF-1 may be a primary factor, which
induces lung injury in the course of pulmonary fibrosis.
We conclude that TGF-1 is a potent inducer of apoptosis through the
caspase-3 activation and the down-regulation of p21 and is also an
enhancer of Fas-mediated apoptosis of lung epithelial cells. This novel
function of TGF-1 in apoptosis of lung epithelial cells should be
considered in the treatment of pulmonary fibrosis, and could be a new
treatment strategy.
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Footnotes |
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2 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; BAL, bronchoalveolar lavage; BALF, BAL fluid; FasL, Fas ligand; HP, hypersensitivity pneumonitis; SAEC, small airway epithelial cell; sFasL, soluble FasL; XIAP, X-linked mammalian inhibitor of apoptosis protein; Z-VAD.FMK, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone.
Received for publication January 25, 2002. Accepted for publication April 17, 2002.
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References |
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in idiopathic pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 5:155.
precede bleomycin-induced pulmonary fibrosis in mice. J. Pharmacol. Exp. Ther. 246:765.. J. Exp. Med. 165:251.1-induced signal transduction in human neutrophils. Biochim. Biophys. Acta 246:55.
1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 52:4042.-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes. J. Hepatol. 26:1079.[Medline]
in human normal lung epithelial cells. Oncogene 17:1743.[Medline]
1 potently activates CPP32-like proteases in human hepatoma cells. Cell. Signal. 10:511.[Medline]
-induced apoptosis. Cell Growth Differ. 8:821.[Abstract]
1 induces apoptosis through down-regulation of c-myc gene and overexpression of p27Kip1 protein in cervical carcinoma. Gynecol. Oncol. 69:230.[Medline]
-mediated apoptosis in the Ramos B- lymphoma cell line is accompanied by caspase activation and Bcl-xL down-regulation. Exp. Cell Res. 242:244.[Medline]
1 induces apoptotic cell death in cultured retinal endothelial cells but not pericytes: association with decreased expression of p21waf1/cip1. J. Cell. Biochem. 70:70.[Medline]
release and inflammation induced by the apoptosis inducer Fas ligand. Nat. Med. 4:1287.[Medline]
1, but not TGF-2 or TGF-3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am. J. Respir. Cell Mol. Biol. 14:131.[Abstract]
and fibroblast collagen synthesis in chronic pulmonary inflammation. J. Exp. Med. 170:727.
subunit in TGF- and activin signaling. Science 271:1120.[Abstract]
1 can abrogate this resistance. Am. J. Pathol. 157:1661. secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat. J. Clin. Invest. 92:1812.
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