Dexamethasone inhibits lung epithelial cell apoptosis induced
by IFN-
and Fas
Long-Ping
Wen1,
Kamyar
Madani1,
Jimothy A.
Fahrni1,
Steven R.
Duncan2, and
Glenn D.
Rosen1
1 Department of Pulmonary and
Critical Care Medicine, Stanford University, Stanford 94305-5236; and
2 Department of Immunology,
Scripps Research Institute, La Jolla, California 92037
 |
ABSTRACT |
Lung epithelium
plays a central role in modulation of the inflammatory response and in
lung repair. Airway epithelial cells are targets in asthma, viral
infection, acute lung injury, and fibrotic lung disease. Activated T
lymphocytes release cytokines such as interferon-
(IFN-
) that can
cooperate with apoptotic signaling pathways such as the Fas-APO-1
pathway to induce apoptosis of damaged epithelial cells. We report that
IFN-
alone and in combination with activation of the Fas pathway
induced apoptosis in A549 lung epithelial cells. Interestingly, the
corticosteroid dexamethasone was the most potent inhibitor of IFN-
-
and IFN-
plus anti-Fas-induced apoptosis. IFN-
induced expression
of an effector of apoptosis, the cysteine protease
interleukin-1
-converting enzyme, in A549 cells. Dexamethasone, in
contrast, induced expression of an inhibitor of apoptosis, human
inhibitor of apoptosis (hIAP-1), also known as cIAP2. We suggest that
the inhibition of epithelial cell apoptosis by corticosteroids may be
one mechanism by which they suppress the inflammatory response.
glucocorticoid; programmed cell death; interferon-
 |
INTRODUCTION |
INTERFERON (IFN)-
is a cytokine that plays an
important role in host defense against viral infections and in tumor
surveillance in the lung (39). It is a type II IFN that is structurally
unrelated to the type I IFNs IFN-
or -
, is synthesized by
activated T cells and cytotoxic T cells, and recognizes cell surface
receptors distinct from those recognized by type I IFNs. Activated
cytotoxic T cells release cytokines, such as IFN-
and tumor necrosis
factor (TNF)-
, that can cooperate with apoptotic signaling pathways to induce apoptosis and clearance of damaged epithelial cells. One of
the apoptotic pathways utilized by cytotoxic T cells involves Fas/APO-1
(referred to as Fas) (21, 26). Fas is a 36-kDa transmembrane receptor
that is a member of the TNF receptor superfamily (54). The endogenous
Fas ligand is a cytotoxic cytokine with significant homology to TNF-
(3, 40, 41). Binding of Fas to its ligand or to an agonistic anti-Fas
antibody activates the Fas apoptotic pathway and induces apoptosis of
the Fas-expressing cell (40). The Fas pathway has been shown to be an
important modulator of the immune response. It is utilized, for
example, by T cells to eliminate self-antigen-expressing T cells,
thereby preventing the development of autoimmune disease (53).
Several recent studies demonstrated that IFN-
alone and in
combination with activation of the Fas pathway induce apoptosis in
different cell types. For example, several reports from a single laboratory have focused on the characterization of genes that are
activated upon IFN-
-induced apoptosis in HeLa epithelial cells (9).
In other reports, IFN-
was shown to induce oligodendrocyte cell
death, which may play a role in the pathogenesis of multiple sclerosis
(52), and, in another study, IFN-
induced programmed cell death in
human salivary gland cells, which may be important in the pathogenesis
of Sjogren's syndrome (56). There are also studies that demonstrate
that IFN-
cooperates with the Fas pathway to induce apoptosis in
retinal pigment epithelial cells, breast cancer cells, and U-937
monocytic cells (11, 23, 44).
A recent study by Hagimoto et al. (15) suggests a role for the Fas
pathway in bleomycin-induced lung injury and fibrosis. In this report,
alveolar type II epithelial cells were apoptotic within 24 h of
exposure to bleomycin, which then resolved, and apoptosis recurred
after 7 days and continued for 14 days. The investigators observed that
Fas was expressed on alveolar epithelium, and Fas ligand was expressed
on infiltrating T lymphocytes (15). The expression of both Fas and Fas
ligand was induced after exposure to bleomycin. Lung injury and the
induction of both Fas and Fas ligand were almost completely abrogated
by the steroid hormone dexamethasone. The data of Hagimoto et al. (15)
suggest that persistent activation of the Fas pathway after bleomycin
exposure may promote epithelial cell damage and fibrosis.
To evaluate the effects of IFN-
and the Fas apoptotic pathway on
lung epithelial cells, we utilized the lung epithelial A549 cell line.
Here we demonstrate that A549 cells express significant levels of Fas
and that IFN-
cooperates with the Fas pathway to induce apoptosis in
A549 cells. We also show that IFN-
alone induces apoptosis in A549
cells. Additionally, we report that the steroid hormone dexamethasone
is a potent inhibitor of apoptosis induced by IFN-
alone and IFN-
plus an agonistic anti-Fas antibody.
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MATERIALS AND METHODS |
Cell culture and cell lines.
The A549 cell line was obtained from American Type Culture Collection
(Rockville, MD) and was cultured in F-12K media with heat-inactivated
10% fetal calf serum (FCS; GIBCO-BRL, Gaithersburg, MD),
L-glutamine, penicillin, and
streptomycin.
Cytokines, antibodies, and inhibitors.
IFN-
with a specific activity of 1 × 107 IU/ml was purchased from
Biosource International (Camarillo, CA). Anti-Fas [immunoglobulin (Ig) M] monoclonal antibody (MAb; referred to as
anti-Fas) was purchased from Kamiya Biomedical (Tukwila, WA).
Monoclonal anti-interleukin (IL)-1
-converting enzyme (ICE) hybridoma
supernatant was provided by Dr. Junying Yuan (University of Michigan,
Ann Arbor, MI; see Ref. 5). Anti-Fas ligand MAb (NOK-2) was provided by
Dr. Hideo Yagita (Juntendo University, Tokyo, Japan; see Ref. 22).
Dexamethasone and cycloheximide were purchased from Sigma Chemical (St.
Louis, MO). ZVAD-fmk was purchased from Enzyme Systems Products
(Dublin, CA). Anti-IFN regulatory factor-1 (IRF-1) polyclonal rabbit
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IFN-
and -
were provided by Dr. Peter Kao (Stanford University, Stanford, CA).
Surface expression of Fas.
For analysis of Fas receptor and Fas ligand surface expression on A549
cells, unstimulated cells and cells treated with 250 U/ml IFN-
for
24 h were removed from the plate with 5 mM EDTA, pelleted, and then
resuspended in a staining solution containing phosphate-buffered saline
(PBS) with 3% FCS. Anti-Fas MAb (20 mg/ml) or anti-Fas ligand MAb (10 mg/ml) was then incubated with the cells on ice for 30 min. The cells
were washed three times with the staining solution, and the pellet was
then resuspended in staining solution containing 3.5 mg/ml of a
fluorescein isothiocyanate (FITC)-conjugated sheep anti-mouse secondary
antibody, at a dilution of 1:2,000 (Caltag Laboratories, San Francisco,
CA). Cells were incubated in the dark for 30 min, and the cells were
then washed four times with staining solution and resuspended in 1%
paraformaldehyde before analysis by fluorescence-activated cell sorting
(FACS).
Cell death assay.
The following conditions were tested on A549 cells:
1) 100 ng/ml anti-Fas alone,
2) 250 U/ml IFN-
alone,
3) IFN-
plus anti-Fas, 4) 1 mM dexamethasone,
5) IFN-
plus dexamethasone, or
6) IFN-
plus anti-Fas plus
dexamethasone. In experiments with IFN-
, A549 cells were treated
with 250 U/ml IFN-
for 12 h, then one-half of the cultures were
treated with 100 ng/ml anti-Fas dissolved in PBS, and the other half
were treated with PBS alone. For the dexamethasone treatments, A549
cells were treated with 1 mM dexamethasone dissolved in dimethyl
sulfoxide (DMSO), DMSO alone, or dexamethasone plus IFN-
. The
dexamethasone was added to the IFN-
-treated cells at the same time
as IFN-
. Cells were harvested for analysis of apoptosis by the
terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end
labeling (TUNEL) assay with 2.5 mg/ml propidium iodide
using the protocol provided by the manufacturer (Boehringer Mannheim,
Indianapolis, IN; see Ref. 13). Cell death was quantified by FACS
analysis, and cells that were both TUNEL and propidium iodide positive
were excluded. Cell death was also assayed with the ApoAlert Annexin V
Apoptosis Kit from Clontech (Palo Alto, CA), according to the
manufacturer's protocol. After the cells were incubated with 1 mg/ml
annexin V-FITC and 2.5 mg/ml propidium iodide, they were analyzed by
FACS analysis. Cells were harvested at 12, 24, 48, and 72 h after the
addition of IFN-
. Cells treated with anti-Fas alone were harvested
at 12, 24, and 48 h after the addition of anti-Fas.
Northern blot analysis.
RNA was harvested from A549 cells with RNA STAT-60, a solution
containing guanidine isothiocyanate and phenol (Tel-Test B, Friendswood, TX), and Northern blot analysis was done as previously described (34). The IRF-1 and ICE human cDNAs were generated by reverse
transcriptase (RT)-polymerase chain reaction (PCR) with RNA from
unstimulated and IFN-
-treated A549 cells. The cDNA for human
inhibitor of apoptosis (hIAP-1) was generated by RT-PCR with RNA from
dexamethasone-treated A549 cells. In Fig. 6, the sample that was
treated with IFN-
plus anti-Fas was harvested 6 h after the addition
of anti-Fas because IFN-
plus anti-Fas induces apoptosis 8-12 h
after the addition of anti-Fas.
Western blot analysis.
Adherent and floating cells were treated under the conditions described
above and then were lysed in HNET buffer [50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5), 100 mM NaCl, 1 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 1% Triton X-100, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride]. Samples were centrifuged for 10 min followed by measurement of protein concentration by the Bradford
method (Bio-Rad Laboratories, Hercules, CA). Samples containing equal
protein concentrations were denatured by boiling, analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, and then
transferred to nitrocellulose. The blot was placed in blocking buffer
containing 4% milk, 10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween
20 for 1 h at room temperature or overnight at 4°C. The blot was
then incubated in blocking buffer with the polyclonal IRF-1 antisera,
at a dilution of 1:1,000, or monoclonal ICE hybridoma supernatant, at a
dilution of 1:3, washed in a solution containing 10 mM
Tris · HCl (pH 7.5), 100 mM NaCl, and 0.1% Tween 20, followed by incubation in blocking buffer containing a horseradish
peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Caltag
Laboratories, San Francisco, CA) at a dilution of 1:2,000, and detected
by enhanced chemiluminescence (Amersham, Arlington Heights, IL)
followed by autoradiography.
Inhibitor and cell viability assays.
The tetrapeptide caspase inhibitor ZVAD-fmk was added to A549 cells 1 h
before the addition of IFN-
and was added to the media each day
during the course of the experiment. Cell viability was assessed by the
thiazolyl blue (MTT) dye reduction assay. MTT (25 mg) was added to a
96-well microtiter plate under conditions described in the legend for
Fig. 4. The plate was incubated at 37°C for 4 h. The media were
removed, and then 0.04 M HCl was added to stop the reaction. The
samples were then analyzed on a micro-enzyme-linked immunosorbent assay
reader at a wavelength of 595 nm.
Cell growth and cell cycle analysis.
To study the incorporation of
[3H]thymidine, cells
were grown in six-well dishes to 50% confluency and were treated with
IFN-
, dexamethasone or anti-Fas alone, or IFN-
plus
dexamethasone. Twenty-four hours later, 1 mCi of
[3H]thymidine, diluted
in PBS, was added to each well, and cells were labeled for 8 h. The
media were removed, the cells were treated with 2 ml of 5%
trichloroacetic acid for 5 min, then washed in 2 ml of distilled water,
and solubilized with 0.4 ml of 0.1% NaOH, and incorporation of
radioactivity was measured with a scintillation counter.
To assess the cell cycle distribution of the A549 cells, the cells were
stained with propidium iodide 24 and 48 h after the same treatment
conditions as described above. A549 cells were pelleted and were washed
two times with PBS plus 5 mM EDTA. The cells were fixed and lysed by
slowly adding 100% ethanol while they were vortexed and incubating at
room temperature for 30 min. Cells were pelleted, resuspended in 0.5 ml
of PBS with 5 mM EDTA plus 20 mg of ribonuclease A, and incubated for
an additional 30 min at room temperature. Then 0.5 ml of 100 mg/ml
propidium iodide was added to the cells followed by FACS analysis.
 |
RESULTS |
A549 cells express Fas and undergo apoptosis in response to
IFN-
and activation of the Fas pathway.
Significant basal levels of Fas receptor but minimal surface levels of
Fas ligand were expressed by A549 cells (Fig.
1 and data not shown). To determine whether
A549 cells were susceptible to Fas-mediated apoptosis, a cross-linking
Fas antibody was added to the cultures. This treatment alone induced
apoptosis in 6% of A549 cells as determined by the TUNEL assay
followed by FACS analysis (Fig.
2D).
Previous studies report that IFN-
or TNF-
can sensitize cells to
Fas-triggered apoptosis (29, 49, 55). We examined, therefore, if a
combination of IFN-
or TNF-
and anti-Fas would trigger apoptosis
in the A549 cell line. IFN-
plus anti-Fas induced apoptosis in 60%
of A549 cells at 48 h and 81% of the cells at 72 h as determined by
TUNEL staining followed by FACS analysis (Fig. 2,
C and
F). Interestingly, IFN-
alone induced apoptosis in 20% of the cells at 48 h and in 35% of the cells
at 72 h, as determined by TUNEL staining (Fig. 2,
B and E).

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Fig. 1.
Surface expression of Fas on unstimulated and interferon
(IFN)- -treated A549 cells. Unstimulated A549 cells were harvested
and were stained with an anti-Fas immunoglobulin (Ig) M monoclonal
antibody (MAb; 20 mg/ml; solid line). A549 cells were treated with 250 U/ml IFN- for 24 h and then were stained with a mouse IgM isotype
control ( ) or the anti-Fas IgM MAb ( ) followed by
fluorescence-activated cell sorting (FACS) analysis. Fas expression on
A549 cells is not affected by IFN- . These data are representative of
results from 3 experiments.
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Fig. 2.
IFN- alone and in synergy with anti-Fas induces apoptosis in A549
cells. Cells were treated under conditions described in
MATERIALS AND METHODS and were
harvested for analysis of apoptosis by the terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL) assay. Apoptotic
cells were stained with fluorescein-dUTP, which is used to label DNA
strand breaks, and were detected here by FACS analysis. In
A-F,
unstimulated A549 cells ( ) are shown. A549 cells were treated with
IFN- (solid line) in B,
C, E,
and F and anti-Fas (solid line) in
C, D,
and F. In
B and
C, cells were harvested 48 h after the
addition of IFN- ; in D, cells were
harvested 48 h after the addition of anti-Fas; and in
E and
F, cells were harvested 72 h after the
addition of IFN- . Percentages of the number of apoptotic cells are
as follows: 2-5% of unstimulated cells were apoptotic after 72 h
(A); IFN- alone induced apoptosis
in 20% of the cells at 48 h (B) and
35% of the cells at 72 h (E) after
the addition of IFN- ; IFN- plus anti-Fas induced apoptosis in
60% of the cells at 48 h (C) and
80% of the cells at 72 h (F);
anti-Fas alone added in solution induced apoptosis in 6% of the cells
(D). This analysis was repeated 5 times with similar results.
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To confirm that the cell death was apoptotic, A549 cells were treated
under the same conditions described above and were stained with annexin
followed by FACS analysis. Anti-Fas alone induced apoptosis in 9% of
A549 cells at 72 h after the addition of anti-Fas, in 37% of cells
treated with IFN-
alone, and in 74% of cells treated with anti-Fas
and IFN-
(data not shown).
Pretreatment of the cells with IFN-
for at least 6-8
h before the addition of anti-Fas was necessary to observe maximal
cell death with anti-Fas, but the cells could be exposed to IFN-
for as little as 30 min. TNF-
alone (10 ng/ml) or in combination with
anti-Fas did not induce apoptosis in A549 cells (data not shown).
IFN-
or -
alone or in combination with anti-Fas did not induce
apoptosis (data not shown). Also, maintaining the cells in
serum-depleted media (0.1% FCS) did not affect the susceptibility to
IFN-
and Fas-mediated apoptosis (data not shown).
Some studies suggest that IFN-
sensitizes lymphocytes and adherent
cells to Fas-mediated apoptosis by inducing Fas receptor or Fas ligand
expression (36, 38, 42, 55). Therefore, we examined Fas receptor and
Fas ligand surface expression after IFN-
stimulation of A549 cells.
There was no significant increase in Fas receptor or Fas ligand
expression on A549 cells after IFN-
treatment (Fig. 1 and data not
shown).
Dexamethasone and a cysteine protease inhibitor block
IFN-
- and IFN-
plus Fas-induced
apoptosis.
We postulated that inhibitor studies would provide valuable information
about signaling molecules in the IFN-
and Fas apoptotic pathway in
A549 cells. It is known that IFN-
produces pleiotropic effects on
cell growth and activity by inducing transcription of a diversity of
genes through the Jak-STAT (signal transducers and activators of
transcription) pathway (8, 19). Several reports document that many
apoptotic stimuli such as irradiation, Fas activation, and chemotherapy
induce apoptosis by activating cysteine proteases (now called
caspases), which leads to cleavage of death substrates (reviewed in
Ref. 20). Dexamethasone has been shown to induce apoptosis in resting
thymocytes, but it inhibits T-cell activation-induced apoptosis. A
recent study reports that dexamethasone inhibits apoptosis triggered by
IFN-
plus anti-Fas in a glioma cell line; the mechanism, however, is
unknown (55).
Corticosteroids are potent antiviral and anti-inflammatory agents. A
recent study showed that dexamethasone inhibited bleomycin-induced apoptosis of type II epithelial cells, possibly through inhibition of
the Fas pathway (15). We tested, therefore, the ability of dexamethasone to inhibit apoptosis in A549 cells caused by IFN-
alone and IFN-
plus anti-Fas. Dexamethasone inhibited apoptosis in
A549 cells induced by IFN-
alone from 38 to 7% (Fig.
3, A and
C) and by IFN-
plus anti-Fas from
81 to 10% as determined by TUNEL staining followed by FACS analysis
(Fig. 3, B and
D). Dexamethasone did not affect
surface expression of Fas receptor or Fas ligand (data not shown).

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Fig. 3.
Dexamethasone inhibits apoptosis of A549 cells induced by IFN- and
IFN- plus anti-Fas. Cells were harvested for analysis of apoptosis
by the TUNEL assay. In
A-D,
unstimulated A549 cells ( ) are shown. In
A, IFN- -treated cells (solid line)
were harvested at 72 h; in B, A549
cells treated with IFN- plus anti-Fas were harvested at 72 h; in C, A549 cells treated with 1 mM dexamethasone at the same time as IFN- were harvested 72 h later;
and in D, A549 cells were treated with
dexamethasone, IFN- , and anti-Fas and then harvested at 72 h.
Percentages of the number of apoptotic cells are as follows: IFN-
alone induced apoptosis in 38% of the cells at 72 h
(A), and 7% of the cells were
apoptotic in the presence of dexamethasone
(C); IFN- plus anti-Fas induced
apoptosis in 81% of the cells at 72 h
(B), and 10% of the cells were
apoptotic in the presence of dexamethasone
(D). This analysis was repeated 4 times with similar results.
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We then tested the ability of the tetrapeptide ZVAD-fmk to inhibit cell
death induced by IFN-
and IFN-
plus anti-Fas in A549 cells.
ZVAD-fmk is an irreversible inhibitor of ICE-like proteases, and it has
been shown to be a potent inhibitor of Fas-mediated apoptosis (1).
Treatment of A549 cells with 40 mM ZVAD-fmk increased cell viability in
IFN-
-treated cells from 62 to 84% as determined by the MTT assay
(Fig. 4). ZVAD-fmk also increased cell
viability after IFN-
plus anti-Fas treatment from 18 to 82% (Fig.
4). Dexamethasone increased cell viability in IFN-
-treated cells
from 62 to 95% and from 18 to 93% in IFN-
plus anti-Fas-treated cells (Fig. 4).

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Fig. 4.
Inhibition of cell death induced by IFN- and IFN- plus anti-Fas
in A549 cells by an inhibitor of interleukin-1 -converting enzyme
(ICE)-like proteases. A549 cells were treated with IFN- or IFN-
plus anti-Fas as described in Fig. 1. Dexamethasone was added as
described in Fig. 3. ZVAD-fmk (40 mM) was added 1 h before the addition
of IFN- . Cell viability was determined by the thiazolyl blue assay.
Control refers to unstimulated cells. Results are means ± SE of
quadruplicate measurements for a representative experiment. Assay was
repeated 3 times with similar results.
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IFN-
does not significantly affect cell growth or
cell cycle distribution of A549 cells.
Several studies report that IFN-
inhibits cell growth (2, 9, 24, 37,
39). A previous study reported that HeLa cells first growth arrest
and then die slowly by apoptosis in response to IFN-
(9). Therefore,
we examined whether IFN-
affected cell growth and cell cycle
distribution of A549 cells. We found that IFN-
decreased
[3H]thymidine
incorporation slightly in A549 cells by 25% at 24 h after
the addition of IFN-
(Table 1).
Dexamethasone also inhibited [3H]thymidine
incorporation by 25-30%, and the combination of IFN-
plus
dexamethasone inhibited
[3H]thymidine
incorporation by 45%. Anti-Fas alone did not affect incorporation of
[3H]thymidine (Table
1).
To assess cell cycle distribution of the cells, A549 cells were stained
with propidium iodide 24 and 48 h after addition of IFN-
alone or
with anti-Fas alone. No significant difference in cell cycle
distribution was observed between IFN-
-treated, anti-Fas-treated,
and control cells at these time points (data not shown).
IFN-
induces ICE expression in A549 cells.
IFN-
did not affect Fas or Fas ligand expression in A549 cells (Fig.
1 and data not shown). A recent study by Tamura et al. (43) showed a
requirement for IRF-1 in DNA damage-induced apoptosis in
mitogen-activated T lymphocytes. IRF-1 is a transcription factor, induced by IFN-
, that induces transcription of several genes that
modulate the inflammatory response. In that study,
-irradiation induced ICE expression in wild-type but not IRF-1
/
splenocytes, suggesting that ICE expression was induced by IRF-1. There
are also recent reports that IFN-
can sensitize breast cancer or U-937 monocytic cells to Fas-mediated apoptosis through the induction of ICE (23, 44). We examined, therefore, if IFN-
induces IRF-1 and
ICE expression in A549 cells. IFN-
induced de novo IRF-1 expression
that was maximal at 5 h and remained at that level at 24 h (Fig.
5A).
IFN-
also induced de novo expression of ICE mRNA and ICE protein in
A549 cells at 24 h after the addition of IFN-
, but we did not detect
the p20 and p10 ICE cleavage fragments that result from ICE activation
after treatment with IFN-
or IFN-
plus anti-Fas (Fig. 5,
B and
C), possibly because the anti-ICE monoclonal antibody does not detect the p20 and p10 ICE cleavage fragments that result from ICE activation in vivo (personal
communication from Dr. Junying Yuan). Anti-Fas did not induce ICE
expression in A549 cells, and dexamethasone did not affect the
induction of ICE by IFN-
or by IFN-
plus anti-Fas (Fig.
5C).

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Fig. 5.
IFN- induces expression of IFN regulatory factor-1 (IRF-1) and ICE.
A: IFN- stimulates de novo
expression of IRF-1 protein in A549 cells. Western blot analysis of
cellular protein harvested from IFN- -treated A549 cells. Equal
amounts of cellular protein were loaded in each lane and were then
analyzed by 12% SDS-polyacrylamide gel electrophoresis (PAGE), and the
blot was probed with a rabbit anti-human IRF-1 polyclonal antibody and
was detected by enhanced chemiluminescence (ECL).
B: IFN- induces ICE mRNA. Northern
blot analysis of RNA from unstimulated A549 cells and A549 cells
treated with IFN- for 24 h. Blot was probed with a 500-bp
32P-labeled human ICE cDNA
fragment. Blot was stripped and was reprobed with a
glyceraldehyde-3-phosphate dehydrogenase (GAP) cDNA to demonstrate
equal loading of RNA. C: IFN-
induces ICE protein expression. Western blot analysis with cellular
protein harvested from A549 cells treated as shown. Equal amounts of
cellular protein were loaded in each lane and were then analyzed by
12% SDS-PAGE, and the blot was probed with a mouse anti-human ICE MAb
and was detected by ECL. DEX, dexamethasone.
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Activation of the Fas apoptotic pathway has been shown to induce
activation of caspase-3 (CPP32/YAMA/Apopain) and caspase-7 (MCH3/ICE-LAP3/CMH-1), which then cleave nuclear and
cytoplasmic proteins (reviewed in Ref. 10). We found that IFN-
and
IFN-
plus anti-Fas induce activation of both caspase-3 and caspase-7 in A549 cells and that dexamethasone inhibits activation of these caspases (Wen and Rosen, unpublished results).
Dexamethasone induces expression of hIAP-1.
We have shown that dexamethasone is a potent inhibitor of
IFN-
-induced and IFN-
plus anti-Fas-induced apoptosis in A549 epithelial cells (Figs. 3 and 4). A recent study reported that dexamethasone inhibits expression of Fas on type II lung epithelial cells and Fas ligand on T lymphocytes after bleomycin-induced lung
injury. We did not observe an effect of dexamethasone on expression of
Fas, Fas ligand, IRF-1, or ICE in A549 cells after stimulation with
IFN-
(Fig. 5 and data not shown). We examined, therefore, if
dexamethasone may induce expression of a suppressor of apoptosis.
Dexamethasone did not affect expression of members of the Bcl-2 family,
such as Bcl-2 or Bcl-x (data not shown), so we examined the effect of
dexamethasone on expression of members of the IAP protein family, which
include hIAP-1, hIAP-2, also known as cIAP1, and X-linked inhibitor of
apoptosis (XIAP). hIAP-1, hIAP-2, and XIAP are mammalian
homologues of the IAP family that was originally identified in
baculovirus (25, 35). We observed that dexamethasone induces expression
of a 6.5-kb mRNA that corresponds to the predicted size of hIAP-1 mRNA
and, to a lesser extent, a 4.5-kb mRNA that corresponds to the
predicted size of hIAP-2 (Fig. 6).
Interestingly, IFN-
alone induced hIAP-1 slightly, and IFN-
in
combination with dexamethasone induced the highest level of hIAP-1 mRNA
(Fig. 6). Anti-Fas did not affect expression of hIAP-1 (Fig. 6).
Dexamethasone did not affect expression of XIAP-1 (data not shown).

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Fig. 6.
DEX induces human inhibitor of apoptosis-1 (hIAP-1) expression.
Northern blot analysis of hIAP-1 with 15 mg of total RNA per lane
probed with a 32P-labeled 1.0-kb
hIAP-1 cDNA probe. Treatment conditions are identical to those
described in Figs. 1 and 3, except that the IFN- plus anti-Fas
sample was harvested 6 h after the addition of anti-Fas.
Right arrow, band of 6.5 kb, the
predicted size of hIAP-1. A lower less prominent band of 4.5 kb likely
corresponds to hIAP-2. Blot was stripped and was reprobed with a GAP
cDNA to demonstrate equal loading of RNA.
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DISCUSSION |
The inflammatory response is an essential component in defense of the
lung against infections and transforming agents, but inappropriate
activation of this response or persistence of the inflammatory response
can result in lung diseases such as asthma and fibrosis. IFN-
is a
cytokine that is secreted by activated and cytotoxic T cells, which is
important for the elimination of viral-infected and transformed cells.
The Fas pathway modulates the immune response, and it may regulate
apoptosis of type II cells after bleomycin-induced lung injury and
fibrosis (15). There are several reports that demonstrate that IFN-
cooperates with the Fas apoptotic pathway to induce apoptosis, but the
mechanism of this synergy is, as yet, not well characterized (29, 49, 55). Some reports suggest that IFN-
functions to sensitize cells to
Fas-mediated apoptosis by upregulating Fas or Fas ligand expression,
but many cell lines expressed significant basal levels of Fas (38, 42,
55). In this study, we show that the lung epithelial-derived A549 cell
line expresses Fas. IFN-
plus an agonistic anti-Fas antibody induced
apoptosis in A549 cells, but IFN-
did not affect Fas receptor or Fas
ligand expression (Figs. 1 and 2 and data not shown). Interestingly,
IFN-
alone induced apoptosis in A549 cells, and the combination of
IFN-
and anti-Fas induced apoptosis in two to three times the number
of cells than IFN-
alone plus anti-Fas alone, demonstrating that
IFN-
acts in synergy with the Fas apoptotic pathway.
IFN-
induces the expression of many early response genes by tyrosine
phosphorylation of Jak kinases and transcription factors called STAT
proteins (8, 19, 39). The Jak-STAT pathway is responsible for initial
transmission of the IFN-
signal to the nucleus, but proper
regulation of the many genes induced by IFN-
involves other
transcription factors such as IRF-1 (reviewed in Refs. 24 and 48).
IRF-1 is a transcriptional activator that binds to a specific sequence
in the promoters of many IFN-inducible genes (32, 47). IRF-1 is not
only a potent transcriptional mediator but is also a critical mediator
of radiation- and chemotherapy-induced apoptosis (43, 45). Previous
studies report that IRF-1 protein has a short half-life of 30 min and
that induction of IRF-1 is transient (17, 48). We show, however, that
in A549 cells IFN-
induces de novo expression of IRF-1 protein and
that IRF-1 levels peak at 5 h and remain at that level for 24 h (Fig.
5).
A recent study demonstrated a requirement for IRF-1 in DNA
damage-induced apoptosis in T lymphocytes (46). Tanaka et al. (46)
showed that
-irradiation and DNA damaging agents such as Adriamycin
or etoposide induced ICE only in wild-type but not IRF-1
/
splenocytes. They proceeded to show that IRF-1
transactivates ICE gene expression. ICE is a caspase that activates
IL-1
and leads to IL-1
release from cells. IL-1
is an
important mediator of the immune and inflammatory response in acute
lung injury and possibly asthma (16, 50). More recently, IL-18- or
IFN-
-inducing factor was also shown to be activated in response to
ICE (14). IL-18 induces Th1 cells to produce IFN-
and granulocyte
macrophage colony-stimulating factor, and it appears to act as a
proinflammatory cytokine in septic shock (30, 51). IFN-
induces ICE
expression in A549 cells, which suggests that IRF-1 also transactivates
ICE in A549 cells (Fig. 5). Overexpression of ICE has been shown to induce apoptosis in some cell types (28, 57).
ZVAD-fmk is a peptidyl fluoromethylketone that acts as an irreversible
inhibitor of ICE or ICE-like caspases and Fas-mediated apoptosis (1).
ZVAD-fmk inhibited cell death induced by IFN-
and IFN-
plus
anti-Fas in A549 cells (Fig. 4). It is unclear if the induction of ICE
by IFN-
leads to activation of ICE in A549 cells because we did not
detect the active ICE cleavage products. We did observe, however,
activation of caspase-3 and caspase-7 by IFN-
and IFN-
plus
anti-Fas and inhibition of apoptosis by ZVAD-fmk, which demonstrate a
caspase pathway is involved in IFN-
- and IFN-
plus
anti-Fas-induced apoptosis in A549 cells.
In our study, the most potent inhibitor of A549 lung epithelial cell
apoptosis induced by IFN-
alone and IFN-
plus anti-Fas was
dexamethasone (Fig. 3). Dexamethasone is a steroid hormone with
pleiotropic effects on gene activation and cellular activity (12, 27,
33). Corticosteroids are widely used anti-inflammatory agents for lung
disease, but their mechanism of action is not well understood.
Corticosteroids were recently shown to protect against
bleomycin-induced lung injury, possibly through the inhibition of the
Fas pathway (15). We hypothesized that dexamethasone protects cells
from IFN-
-induced cell death through the induction of a suppressor
of apoptosis. We tested this hypothesis by examining the effect of
dexamethasone on expression of members of the Bcl-2 family and its
effect on expression of the IAP family. Dexamethasone did not affect
expression of Bcl-2 or Bcl-x, but it induced expression of hIAP-1 mRNA
and, to a lesser extent, hIAP-2 mRNA. The IAP family was originally
described in baculovirus proteins that were designated IAP proteins
because these proteins inhibit the apoptotic response of insect cells
to viral infection (4, 7). hIAP-1 and hIAP-2 are closely related
members of the IAP family that suppress apoptosis in mammalian cells
(25, 35). Recent studies demonstrate that IAPs inhibit caspase-mediated
apoptosis by blocking activation of caspases (18, 31). We have observed
that dexamethasone inhibits caspase-3 and caspase-7 activation,
suggesting that dexamethasone blocks apoptosis by inhibiting activation
of caspases in A549 cells. Interestingly, we show that dexamethasone in
combination with IFN-
induces maximal expression of hIAP-1 mRNA, but
IFN-
alone induces hIAP-1 only slightly, suggesting that
dexamethasone and IFN-
act in synergy to induce hIAP-1 (Fig. 6). A
recent study showed that TNF-
increased glucocorticoid-induced
transcriptional activity of the glucocorticoid receptor (GR), increased
GR number, and increased GR binding to glucocorticoid responsive
elements (6). It is possible that IFN-
increases GR activity in A549 cells, which would lead to increased expression of the hIAP-1 gene in
response to dexamethasone. Studies are ongoing to evaluate if hIAP-1
protects against IFN-
-induced and IFN-
plus anti-Fas-induced apoptosis in A549 cells.
These data suggest that IFN-
- and IFN-
plus anti-Fas-induced
apoptosis in the A549 lung epithelial cell line results from activation
of caspases by IFN-
and may be mediated, at least in part, by de
novo induction of ICE. In view of our finding that dexamethasone is a
potent inhibitor of IFN-
-induced and IFN-
plus anti-Fas-induced
apoptosis and that dexamethasone induces expression of hIAP-1, we
propose that hIAP-1 may play a role in protection against apoptosis in
A549 cells. The recent study by Hagimoto et al. (15) supports a role
for the Fas pathway in epithelial lung injury in vivo. The protective
effects of dexamethasone in this in vivo lung injury model coupled with
our in vitro data suggest one potential mechanism for how
corticosteroids protect against epithelial lung injury.
 |
ACKNOWLEDGEMENTS |
We thank Sergiu Troie for help with preparation of the manuscript.
We also thank Dr. Hideo Yagita for providing the NOK-2 Fas ligand
monoclonal antibody and Dr. Junying Yuan for providing the
interleukin-1
-converting enzyme monoclonal antibody.
 |
FOOTNOTES |
This work was funded in part by a grant from the Program in Molecular
and Genetic Medicine, Stanford University.
Address for reprint requests: G. D. Rosen, Stanford Univ. School of
Medicine, Pulmonary and Critical Care Medicine, 300 Pasteur Dr.,
Stanford, CA 94305-5236.
Received 26 February 1997; accepted in final form 14 July 1997.
 |
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