Dexamethasone inhibits lung epithelial cell apoptosis induced by IFN-gamma 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma (IFN-gamma ) 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-gamma 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-gamma - and IFN-gamma plus anti-Fas-induced apoptosis. IFN-gamma induced expression of an effector of apoptosis, the cysteine protease interleukin-1beta -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-gamma

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

INTERFERON (IFN)-gamma 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-alpha or -beta , 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-gamma and tumor necrosis factor (TNF)-alpha , 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-alpha (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-gamma 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-gamma -induced apoptosis in HeLa epithelial cells (9). In other reports, IFN-gamma was shown to induce oligodendrocyte cell death, which may play a role in the pathogenesis of multiple sclerosis (52), and, in another study, IFN-gamma 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-gamma 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-gamma 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-gamma cooperates with the Fas pathway to induce apoptosis in A549 cells. We also show that IFN-gamma alone induces apoptosis in A549 cells. Additionally, we report that the steroid hormone dexamethasone is a potent inhibitor of apoptosis induced by IFN-gamma alone and IFN-gamma plus an agonistic anti-Fas antibody.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma 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)-1beta -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-alpha and -beta 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-gamma 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-gamma alone, 3) IFN-gamma plus anti-Fas, 4) 1 mM dexamethasone, 5) IFN-gamma plus dexamethasone, or 6) IFN-gamma plus anti-Fas plus dexamethasone. In experiments with IFN-gamma , A549 cells were treated with 250 U/ml IFN-gamma 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-gamma . The dexamethasone was added to the IFN-gamma -treated cells at the same time as IFN-gamma . 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-gamma . 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-gamma -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-gamma plus anti-Fas was harvested 6 h after the addition of anti-Fas because IFN-gamma 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(beta -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-gamma 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-gamma , dexamethasone or anti-Fas alone, or IFN-gamma 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A549 cells express Fas and undergo apoptosis in response to IFN-gamma 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-gamma or TNF-alpha can sensitize cells to Fas-triggered apoptosis (29, 49, 55). We examined, therefore, if a combination of IFN-gamma or TNF-alpha and anti-Fas would trigger apoptosis in the A549 cell line. IFN-gamma 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-gamma 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)-gamma -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-gamma for 24 h and then were stained with a mouse IgM isotype control (black-lozenge ) or the anti-Fas IgM MAb (bullet ) followed by fluorescence-activated cell sorting (FACS) analysis. Fas expression on A549 cells is not affected by IFN-gamma . These data are representative of results from 3 experiments.


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Fig. 2.   IFN-gamma 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 (black-lozenge ) are shown. A549 cells were treated with IFN-gamma (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-gamma ; 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-gamma . Percentages of the number of apoptotic cells are as follows: 2-5% of unstimulated cells were apoptotic after 72 h (A); IFN-gamma 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-gamma ; IFN-gamma 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.

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-gamma alone, and in 74% of cells treated with anti-Fas and IFN-gamma (data not shown).

Pretreatment of the cells with IFN-gamma 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-gamma for as little as 30 min. TNF-alpha alone (10 ng/ml) or in combination with anti-Fas did not induce apoptosis in A549 cells (data not shown). IFN-alpha or -beta 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-gamma and Fas-mediated apoptosis (data not shown).

Some studies suggest that IFN-gamma 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-gamma stimulation of A549 cells. There was no significant increase in Fas receptor or Fas ligand expression on A549 cells after IFN-gamma treatment (Fig. 1 and data not shown).

Dexamethasone and a cysteine protease inhibitor block IFN-gamma - and IFN-gamma plus Fas-induced apoptosis. We postulated that inhibitor studies would provide valuable information about signaling molecules in the IFN-gamma and Fas apoptotic pathway in A549 cells. It is known that IFN-gamma 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-gamma 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-gamma alone and IFN-gamma plus anti-Fas. Dexamethasone inhibited apoptosis in A549 cells induced by IFN-gamma alone from 38 to 7% (Fig. 3, A and C) and by IFN-gamma 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-gamma and IFN-gamma plus anti-Fas. Cells were harvested for analysis of apoptosis by the TUNEL assay. In A-D, unstimulated A549 cells (black-lozenge ) are shown. In A, IFN-gamma -treated cells (solid line) were harvested at 72 h; in B, A549 cells treated with IFN-gamma plus anti-Fas were harvested at 72 h; in C, A549 cells treated with 1 mM dexamethasone at the same time as IFN-gamma were harvested 72 h later; and in D, A549 cells were treated with dexamethasone, IFN-gamma , and anti-Fas and then harvested at 72 h. Percentages of the number of apoptotic cells are as follows: IFN-gamma 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-gamma 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.

We then tested the ability of the tetrapeptide ZVAD-fmk to inhibit cell death induced by IFN-gamma and IFN-gamma 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-gamma -treated cells from 62 to 84% as determined by the MTT assay (Fig. 4). ZVAD-fmk also increased cell viability after IFN-gamma plus anti-Fas treatment from 18 to 82% (Fig. 4). Dexamethasone increased cell viability in IFN-gamma -treated cells from 62 to 95% and from 18 to 93% in IFN-gamma plus anti-Fas-treated cells (Fig. 4).


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Fig. 4.   Inhibition of cell death induced by IFN-gamma and IFN-gamma plus anti-Fas in A549 cells by an inhibitor of interleukin-1beta -converting enzyme (ICE)-like proteases. A549 cells were treated with IFN-gamma or IFN-gamma 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-gamma . 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.

IFN-gamma does not significantly affect cell growth or cell cycle distribution of A549 cells. Several studies report that IFN-gamma 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-gamma (9). Therefore, we examined whether IFN-gamma affected cell growth and cell cycle distribution of A549 cells. We found that IFN-gamma decreased [3H]thymidine incorporation slightly in A549 cells by 25% at 24 h after the addition of IFN-gamma (Table 1). Dexamethasone also inhibited [3H]thymidine incorporation by 25-30%, and the combination of IFN-gamma plus dexamethasone inhibited [3H]thymidine incorporation by 45%. Anti-Fas alone did not affect incorporation of [3H]thymidine (Table 1).

                              
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Table 1.   [3H]thymidine incorporation in A549 cells

To assess cell cycle distribution of the cells, A549 cells were stained with propidium iodide 24 and 48 h after addition of IFN-gamma alone or with anti-Fas alone. No significant difference in cell cycle distribution was observed between IFN-gamma -treated, anti-Fas-treated, and control cells at these time points (data not shown).

IFN-gamma induces ICE expression in A549 cells. IFN-gamma 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-gamma , that induces transcription of several genes that modulate the inflammatory response. In that study, gamma -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-gamma 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-gamma induces IRF-1 and ICE expression in A549 cells. IFN-gamma induced de novo IRF-1 expression that was maximal at 5 h and remained at that level at 24 h (Fig. 5A). IFN-gamma also induced de novo expression of ICE mRNA and ICE protein in A549 cells at 24 h after the addition of IFN-gamma , but we did not detect the p20 and p10 ICE cleavage fragments that result from ICE activation after treatment with IFN-gamma or IFN-gamma 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-gamma or by IFN-gamma plus anti-Fas (Fig. 5C).


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Fig. 5.   IFN-gamma induces expression of IFN regulatory factor-1 (IRF-1) and ICE. A: IFN-gamma stimulates de novo expression of IRF-1 protein in A549 cells. Western blot analysis of cellular protein harvested from IFN-gamma -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-gamma induces ICE mRNA. Northern blot analysis of RNA from unstimulated A549 cells and A549 cells treated with IFN-gamma 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-gamma 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.

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-gamma and IFN-gamma 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-gamma -induced and IFN-gamma 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-gamma (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-gamma alone induced hIAP-1 slightly, and IFN-gamma 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-gamma 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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-gamma 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-gamma 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-gamma 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-gamma plus an agonistic anti-Fas antibody induced apoptosis in A549 cells, but IFN-gamma did not affect Fas receptor or Fas ligand expression (Figs. 1 and 2 and data not shown). Interestingly, IFN-gamma alone induced apoptosis in A549 cells, and the combination of IFN-gamma and anti-Fas induced apoptosis in two to three times the number of cells than IFN-gamma alone plus anti-Fas alone, demonstrating that IFN-gamma acts in synergy with the Fas apoptotic pathway.

IFN-gamma 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-gamma signal to the nucleus, but proper regulation of the many genes induced by IFN-gamma 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-gamma 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 gamma -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-1beta and leads to IL-1beta release from cells. IL-1beta 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-gamma -inducing factor was also shown to be activated in response to ICE (14). IL-18 induces Th1 cells to produce IFN-gamma and granulocyte macrophage colony-stimulating factor, and it appears to act as a proinflammatory cytokine in septic shock (30, 51). IFN-gamma 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-gamma and IFN-gamma plus anti-Fas in A549 cells (Fig. 4). It is unclear if the induction of ICE by IFN-gamma 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-gamma and IFN-gamma plus anti-Fas and inhibition of apoptosis by ZVAD-fmk, which demonstrate a caspase pathway is involved in IFN-gamma - and IFN-gamma plus anti-Fas-induced apoptosis in A549 cells.

In our study, the most potent inhibitor of A549 lung epithelial cell apoptosis induced by IFN-gamma alone and IFN-gamma 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-gamma -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-gamma induces maximal expression of hIAP-1 mRNA, but IFN-gamma alone induces hIAP-1 only slightly, suggesting that dexamethasone and IFN-gamma act in synergy to induce hIAP-1 (Fig. 6). A recent study showed that TNF-alpha 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-gamma 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-gamma -induced and IFN-gamma plus anti-Fas-induced apoptosis in A549 cells.

These data suggest that IFN-gamma - and IFN-gamma plus anti-Fas-induced apoptosis in the A549 lung epithelial cell line results from activation of caspases by IFN-gamma 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-gamma -induced and IFN-gamma 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-1beta -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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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