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Am J Physiol Lung Cell Mol Physiol 287: L374-L381, 2004. First published April 23, 2004; doi:10.1152/ajplung.00039.2004
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Impaired nitric oxide synthase-2 signaling pathway in cystic fibrosis airway epithelium

Shuo Zheng,1 Weiling Xu,1 Santanu Bose,2 Amiya K. Banerjee,2 S. Jaharul Haque,1 and Serpil C. Erzurum1,2

1Departments of Pulmonary and Critical Care Medicine, Cancer Biology, and 2Virology, Cleveland Clinic Foundation, Lerner Research Institute, Cleveland, Ohio 44195

Submitted 9 February 2004 ; accepted in final form 21 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cystic fibrosis (CF) airway epithelial cells are more susceptible to viral infection due to impairment of the innate host defense pathway of nitric oxide (NO). NO synthase-2 (NOS2) expression is absent, and signal transducer and activator of transcription (STAT) 1 activation is reduced in CF. We hypothesized that the IFN-{gamma} signaling pathway, which leads to NOS2 gene induction in CF airway epithelial cells, is defective. In contrast to a lack of NOS2 induction, the major histocompatibility complex class 2, an IFN-{gamma}-regulated delayed-responsive gene, is similarly induced in CF and non-CF airway epithelial (NL) cells, suggesting an NOS2-specific defect in the IFN-{gamma} signaling pathway. STAT1 and activator protein-1, both required for NOS2 gene expression, interact normally in CF cells. Protein inhibitor of activated STAT1 is not increased in CF cells. IFN-{gamma} induces NOS2 expression in airway epithelial cells through an autocrine mechanism involving synthesis and secretion of IFN-{gamma}-inducible mediator(s), which activates STAT1. Here, CF cells secrete IFN-{gamma}-inducible factor(s), which stimulate NOS2 expression in NL cells, but not in CF cells. In contrast, IFN-{gamma}-inducible factor(s) similarly inhibit virus in CF and NL cells. Thus autocrine activation of NOS2 is defective in CF cells, but IFN-{gamma} induction of antiviral host defense is intact.

IFN-{gamma}-inducible factor; antiviral host defense; signal transducer and activator of transcription 1

LUNG DISEASE IS THE LEADING CAUSE of morbidity and mortality in cystic fibrosis (CF) patients. Compared with other lung diseases, CF lung disease is striking in its relatively restricted bacteria flora, particularly with Pseudomonas aeruginosa. Once bacterial colonization is established, it cannot be eradicated. Airway obstruction, chronic bacterial infection, and excessive inflammation progressively destroy the lung and eventually lead to death. Successful therapies include early intervention to prevent bacterial colonization, delay disease progression, and improve the life expectancy of CF patients. As the largest epithelial surface area exposed to microorganisms and particles in the environment, human lungs have an elaborate array of pulmonary defense mechanisms to keep the lung free of infections (10). Increased mucus secretion and airway obstruction cannot solely account for the increased susceptibility to bacterial infection or the selection of the particular flora associated with CF. Furthermore, there does not appear to be a systemic immune defect in CF, rather susceptibility seems to reside within the respiratory tract itself (28). Our previous study showed that CF lung epithelial cells are more susceptible to viral infection, which may predispose CF airways to subsequent bacterial colonization. Loss of nitric oxide (NO) synthase (NOS)-2 expression was identified as one defect in the innate host defense of CF airway epithelial cells (44). High-level production of NO in human airways through the continuous expression of NOS2 is an important component of host defense of the respiratory epithelium (15, 34). Exhaled NO and airway NOS2 are elevated in individuals with inflammatory lung diseases, such as asthma and bronchiolitis compared with healthy controls (4, 18, 25). Despite the inflammatory nature of CF lung disease, NO levels in exhaled breath from CF patients are lower than those from healthy controls and individuals with other inflammatory lung diseases (2, 30). Infants with CF before the onset of respiratory symptoms have mean levels of exhaled NO threefold lower than healthy control infants, suggesting that lower NO in CF is not the result but, rather, a precursor of pulmonary disease (12). Significant reduction of NOS2 immunostaining in CF epithelium, but not in inflammatory cells (33), suggests that NOS2 gene is normal but the signaling pathway regulating its expression is abnormal in CF lung epithelium.

We have previously shown that IFN-{gamma} and virus do not induce NOS2 expression at both the RNA and protein level in CF airway epithelial cells (44). Thus we hypothesize that the absence of NOS2 in CF epithelial cells is due to decreased gene expression as a result of an impaired signaling pathway. Here, we study the IFN-{gamma}-stimulated signaling pathway that regulates NOS2 expression, as well as other IFN-stimulated genes (ISG), using primary human airway epithelial cells (HAEC) derived from CF and non-CF lungs.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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Extraction of HAEC and cell culture. Segments of bronchus were obtained at surgery. Fat and connective tissue were removed as much as possible, and bronchus segments were washed with HBSS (GIBCO Laboratories, Grand Island, NY) several times to remove all blood and connective tissue followed by incubation in 0.1% protease-DMEM (GIBCO) solution with 1% penicillin-streptomycin-fungizone, 20 µg/ml ciprofloxacin, and 200 µg/ml tobramycin at 4°C overnight. Protease was neutralized with 1 ml of heat-inactivated FCS. Tracheal segments were then incubated in HBSS containing 10 mM EDTA, 1% penicillin-streptomycin-fungizone, 20 µg/ml ciprofloxacin, and 200 µg/ml tobramycin at 37°C for 15 min. The epithelial side of the trachea was scraped with a sterile glass slide to remove remaining adherent cells. Cells were also collected from all previous supernatants and seeded on tissue culture plates precoated with coating medium containing 29 µg/ml collagen (vitrogen; Collagen, Palo Alto, CA), 10 µg/ml BSA (Biofluids), and 10 µg/ml fibronectin (Calbiochem, La Jolla, CA), in serum-free bronchial epithelial basal medium (Clonetics, San Diego, CA) with 1% penicillin-streptomycin-fungizone, 20 µg/ml ciprofloxacin, and 200 µg/ml tobramycin. Some HAEC were also obtained through bronchoscopy with a flexible fiber optic bronchoscope (Olympus BS-IT10; Olympus Optical, Tokyo, Japan) from normal volunteers with no history of lung disease and on no medications. Informed consent was obtained under a protocol approved by the Institutional Review Board at the Cleveland Clinic Foundation. All cells were genotyped for CF mutations by Genzyme Genetics (Boston, MA). In addition, an aliquot of cultured cells was immunostained to confirm epithelial phenotype. In total, seven samples were collected from CF lungs and confirmed to have {Delta}F508/{Delta}F508 mutations; these cells are referred to as CF cells. All five samples collected from non-CF explanted lungs and four samples collected from bronchoscopic brushing of healthy volunteers were confirmed to have no known CFTR mutation; these cells are referred to as non-CF airway epithelial (NL) cells. All experiments were performed in CF and NL cells, replicated a minimum of two times, using a minimum of cells from two different donors each for NL and CF cells.

A549 cells, an epithelial cell line derived from lung adenocarcinoma [American Type Culture Collection (ATCC), Rockville, MD], were cultured in MEM (GIBCO) with 10% heat-inactivated FCS. Human IFN-{gamma} was a gift from InterMune (Brisbane, CA). CV-1 cells (CCL 70, ATCC) were maintained in DMEM (GIBCO-BRL) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine. Recombinant human IL-1{beta} and TNF-{alpha} were from Genzyme. Human parainfluenza virus (HPIV) 3 was a gift from Dr. Bishnu De.

RNA extraction and Northern analysis. Total RNA was extracted by GTC-CsCl gradient method and evaluated by Northern analysis using a 32P-labeled major histocompatibility complex class II (MHCII) (DR{alpha}) (pCCF52) probe (ATCC) (36) or, as a control, GAPDH cDNA probe (6), and then subjected to autoradiography. Expression of MHCII mRNA relative to GAPDH mRNA was accomplished with a PhosphorImager (Molecular Dynamics) to quantitate relative units.

NOS2 promoter luciferase reporter constructs, transient transfection, and luciferase assay. Full-length human NOS2 promoter luciferase reporter construct, a kind gift from Dr. Joel Moss (5), was transiently transfected into cells at 90% confluence with LipofectAMINE PLUS reagent (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, cells were treated with cytokines, and cell lysate was collected to evaluate luciferase activity with the Dual-Luciferase Reporter Assay (Promega, Madison, WI). Cotransfection with Renilla luciferase (Promega) was used to normalize transfection efficiency of the luciferase reporter construct.

Western analysis. Adherent cells were scraped off culture plates in lysis buffer [3 mM dithiothreitol (DTT), 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 0.1 mM PMSF, 1% Nonidet P-40 (NP-40), and 40 mM HEPES, pH 7.5]. Cell lysate was prepared by three cycles of freeze-thaw and centrifugation at 14,000 g for 20 min at 4°C. The supernatant protein concentration was measured by Coomassie Plus protein assay (Pierce, Rockford, IL). Whole cell lysate protein was denatured and reduced in buffer containing 0.05 M Tris, pH 6.8, 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.00125% bromphenol blue, and 0.5% 2-mercaptoethanol for 2 min at 95°C. Total protein (50 µg/lane) was separated by electrophoresis on 8% or 10% SDS-polyacrylamide gel and then eletrophoretically transferred onto nitrocellulose membrane (Osmonics, Minnetonka, MN) for 1.5 h at 4°C. The membrane was blocked with 5% nonfat milk in 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.0 at room temperature for 1 h with shaking and then with primary antibody overnight at 4°C. After washing, a peroxidase-conjugated secondary antibody was incubated with membrane for 1 h at room temperature followed by washing again. The primary antibodies used for protein detection included a rabbit polyclonal antibody against NOS2 protein (NO53; Merck, Rahway, NJ), a goat polyclonal antibody against protein inhibitor of activated STAT1 (PIAS1; Santa Cruz Biotechnology, Santa Cruz, CA), and a rabbit polyclonal antibody against N protein of HPIV3. The secondary antibodies used were peroxidase-linked donkey anti-rabbit antibody (Amersham, Arlington Heights, IL) or anti-goat antibody (Santa Cruz Biotechnology). As a control of equal protein loading, Western analysis for {beta}-actin was performed with a primary monoclonal anti-{beta}-actin antibody (A-5316; Sigma, St. Louis, MO) followed by a sheep secondary anti-mouse immunoglobulin antibody (Sigma). Enhanced chemiluminescent system (Amersham Laboratories) was used to detect signals and expose films. The image was scanned and relative intensity to {beta}-actin quantitated by ImageQuant 1.2 (Molecular Dynamics).

Nuclear extract and EMSA. Adherent cells (90–100% confluent), treated with reagents as indicated, were washed twice with ice-cold PBS and harvested with a cell lifter. The cell suspensions were centrifuged and resuspended in 0.4 ml of ice-cold buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, and 1 mM DTT] by gentle pipetting in a yellow tip, and then cells were allowed to swell on ice for 15 min. Subsequently, 25 µl of 10% solution of NP-40 were added, vigorously vortexed for 10 s, and then spun for 30 s in a microfuge. The nuclear pellet was resuspended in 50 µl of ice-cold buffer [20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, and 1 mM DTT].

The duplex oligonucleotide corresponding to IFN-{gamma} activation site [{gamma}-activation sequence (GAS)] in NOS2 promoter region (5'-CGGGCGTTTCCAGTAAAAATC-3') was synthesized by Operon (Alameda, CA) and then end-labeled with [{gamma}-32P]ATP by polynucleotide kinase. For binding reactions, nuclear extract (NE) containing 4 µg of protein was incubated in 20 µl of total reaction volume containing 20 mM HEPES (pH 7.9), 5% glycerol, 50 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 100 µg/ml BSA, and 2 µg poly(dI-dC) for 15 min at room temperature. The 32P-labeled oligonucleotide (2 x 105 counts/min) was added to the reaction mixture and incubated for 20 min at room temperature. The reaction mixture was analyzed by electrophoresis on a 4% polyacrylamide gel in 0.25x TBE (12.5 mM Tris, 12.5 mM borate, and 0.5 mM EDTA). The gels were dried and analyzed by autoradiography. To demonstrate specificity of binding, we performed competition by adding unlabeled wild-type and mutated oligonucleotide at a 100-fold molar excess of 32P-labeled oligonucleotide probe in the binding reaction. To specifically identify activator protein (AP)-1, STAT1 proteins in binding complexes, 4 µg of rabbit anti-c-fos, c-jun, STAT1 antibody (Santa Cruz Biotechnology), or nonimmune rabbit IgG (Biodesign, Saco, ME) were added to the binding reaction mixture and incubated for 30 min at room temperature before the 32P-labeled oligonucleotide was added.

Whole cell extract and EMSA. The cell suspensions were centrifuged at 1,000 g for 5 min at 4°C. The cell pellet was washed in ice-cold PBS and resuspended in 1 ml of ice-cold buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 20 µM Na-orthovanadate, 0.5 mM DTT, and 0.5 mM PMSF], incubated on ice for 5–10 min, and centrifuged. The cell pellet was resuspended in equal volume of buffer [20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM Na-orthovanadate, 0.5 mM DTT, 0.5 mM PMSF, 5 µg/ml leupeptin, 200 µg/ml aprotinin, and 10 µg/ml pepstatin A] and incubated on ice for 30 min. Extract was centrifuged at 14,000 g for 20 min at 4°C, and the supernatant was used for EMSA. Protein concentration was measured by the Coomassie Plus protein assay (Pierce).

The IFN-{gamma} activation site (GAS) (5'-TCGAGCCTGATTTCCCCGAAATGACG GC-3'), corresponding to the inverted repeat element of human interferon regulatory factor (IRF)-1 gene with a SalI linker at the 5'-end and the complementary strand, were synthesized by BRL Laboratories (Grand Island, NY). The annealed duplex oligonucleotides were end-labeled with [{gamma}-32P]ATP by polynucleotide kinase. Whole cell extract (WCE) containing 5 µg of protein was incubated at room temperature with total volume of 19 µl of binding reaction buffer [20 mM HEPES (pH 7.9), 10% glycerol, 80 mM NaCl, 1 mM DTT, 0.6 mM EDTA, pH 8.0, 4 mM Tris·HCl, pH 7.9, 5 mM MgCl2, and 3 µg poly(dI-dC)] and 200 µg of end-labeled GAS duplex probe for 20 min. The protein-DNA complex was analyzed by electrophoresis on a 6% polyacrylamide (acrylamide/bis, 75:2) gel with 0.5x TBE (44.5 mM boric acid, 2 mM EDTA, pH 8.0) at room temperature for 1.5–2.0 h. Gels were dried and exposed to X-ray film at –70°C. To specifically identify STAT1 protein in the protein-DNA complex, we added STAT1 antibody (Santa Cruz Biotechnology) to the binding reaction mixture and incubated it for 30 min at room temperature before adding the 32P-labeled oligonucleotide.

Conditioned medium transfer. Cells were stimulated with IFN-{gamma} (1,000 U/ml) for 1 h and washed vigorously with HEPES-buffered saline to remove residual IFN-{gamma}. Fresh culture media were added to the cells and incubated for 72 h. The overlying media [{gamma}-conditioned medium ({gamma}CM)] were then transferred to unstimulated fresh cells as indicated. Cell lysates were collected 24 h later for Western analysis. WCE was collected 30 min after conditioned media transfer for EMSA.

Plaque assay. Culture supernatants overlying cells that underwent virus infection were collected, and the yield of infectious HPIV3 in the supernatants was measured by plaque assay as previously described (8). Briefly, samples to be tested were diluted from 1:102 to 1:107 in opti-MEM (GIBCO-BRL) and placed on CV-1 cells (CCL 70, ATCC). After 1 h at 37°C, the supernatants were removed, and cells were overlaid with freshly made MEM-agar that contains 1% agar, 10% FCS, penicillin, streptomycin, and glutamine. Cells were cultured for 24 h, and the plaques were counted visually under a microscope.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Lack of NOS2 expression and decreased NOS2 promoter activity in CF cells. To confirm that CF cells do not express NOS2 protein, we exposed CF and NL cells to IFN-{gamma}, and cell lysates were collected at 8, 24, and 48 h. NL cells had NOS2 expression at 24 h, and the protein level increased further at 48 h, whereas CF cells did not have detectable NOS2 even at 48 h (Fig. 1A). To evaluate NOS2 gene transcription, we cotransfected a 8,296-bp human NOS2 promoter luciferase reporter construct and Renilla luciferase construct (RL-TK) into NL and CF cells at 90% confluence. Twenty-four hours after transfection, the cells were treated with 104 U/ml IFN-{gamma} or cytokine mix (CK; 104 U/ml IFN-{gamma}, 0.5 ng/ml IL-1{beta}, and 10 ng/ml TNF-{alpha}) or left untreated. Cell lysates were collected for luciferase assay, and the NOS2 promoter luciferase activity was normalized to Renilla luciferase activity as a control for transfection efficiency. CF cells had significantly decreased NOS2 promoter activity upon IFN-{gamma} or CK stimulation compared with NL cells (P < 0.05) (Fig. 1B), indicating a defect in NOS2 expression in CF cells at the transcriptional level.



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  		Fig. 1. A: lack of nitric oxide synthase (NOS)-2 expression in cystic fibrosis (CF) cells. Cell lysate (50 µg protein/lane) from non-CF airway epithelial (NL) and CF cells stimulated with IFN-{gamma} for 8, 24, and 48 h were evaluated for NOS2 expression by Western blot. Lysate from A549 cells stimulated with cytokine mixture (CK; 104 U/ml IFN-{gamma}, 0.5 ng/ml IL-1{beta}, 10 ng/ml TNF-{alpha}) for 24 h was used only as positive control (lane 9). Similar results were obtained from 2 separate experiments using cells from different donors. B: decreased NOS2 promoter activity in CF cells. Cells were cotransfected with human NOS2 promoter luciferase reporter construct (1 ng/ml) and Renilla reporter construct (0.1 ng/ml RL-TK) and then treated with IFN-{gamma} (104 U/ml) or CK. CF cells have less NOS2 promoter activity compared with NL (P < 0.05). NOS2 promoter activity means ± SD are from 6 separate experiments using cells from different donors. NS, nonstimulated.

 
IFN-{gamma}-induced MHCII expression is not altered in CF cells. NOS2 and MHCII expression are induced as a delayed response to IFN-{gamma}, requiring new protein synthesis. To evaluate whether MHCII expression is impaired in CF cells, we exposed CF and NL cells to 104 U/ml IFN-{gamma} and extracted RNA at 2, 8, 24, and 48 h after treatment. Northern analysis revealed MHCII induction at 24 and 48 h after IFN-{gamma} in NL and CF cells (Fig. 2A). CF cells were also exposed to different concentrations of IFN-{gamma} (102-104 U/ml), and RNA was extracted 24 h later. Northern analysis showed a dose-dependent induction of MHCII in CF cells by IFN-{gamma} (Fig. 2B). These results indicate that CF cells have a defect in NOS2 expression, whereas other delayed IFN-{gamma}-responsive ISGs are not affected.



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  		Fig. 2. Major histocompatibility complex class II (MHCII) expression in CF and NL cells. A: Northern analysis of total RNA from primary human airway epithelial cells from NL (lanes 1–5, 10 µg/lane) or CF (lanes 6–10, 10 µg/lane) at indicated hours after IFN-{gamma} stimulation was performed with a 32P-labeled MHCII (DR{alpha}) cDNA probe. B: Northern analysis of total RNA from airway epithelial cells from CF (lanes 1–4, 10 µg/lane) 24 h after IFN-{gamma} stimulation at indicated doses. Human 32P-labeled GAPDH cDNA probe was used as control for RNA loading. Similar results were obtained from 2 separate experiments using cells from different donors.

 
Normal STAT1/c-Fos interaction in CF cells following treatment with IFN-{gamma}. Physical interaction between c-fos, a component of AP-1, and STAT1 plays a role in transcriptional activation of NOS2 gene (43). To investigate the interaction between c-fos and STAT1 in CF cells, we exposed CF and NL cells to IFN-{gamma} for 1 h or unstimulated. The NE were analyzed by EMSA with a 32P-labeled duplex oligonucleotide probe corresponding to the IFN-{gamma} activation site (GAS) sequence present in the NOS2 promoter region (Fig. 3A). The EMSA revealed that GAS-STAT1 interaction was less in CF than in NL cells. Supershift analysis revealed the presence of STAT-1 and c-fos, but not c-Jun, in DNA-protein complexes in CF and NL cells. These results suggest that c-fos interacts with STAT1 in binding to GAS in CF and NL cells. Thus c-fos is not involved in the impairment of STAT1-DNA binding in CF cells following treatment with IFN-{gamma}.



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  		Fig. 3. Impaired STAT1 activation in CF airway epithelial cells. A: EMSA of nuclear extract from CF (lanes 1–5) or NL (lanes 6–13) was performed using [{gamma}-32P]ATP end-labeled NOS2 {gamma}-activation sequence (GAS) oligonucleotide duplex probe. The binding complexes of both CF and NL cells were supershifted by STAT1 (p91) antibodies (Ab) (lanes 3, 8) and c-fos Ab (lanes 4, 9), but not by c-jun Ab (lanes 5, 10) or rabbit unimmune IgG (lane 15). Competition with unlabeled probe (lane 11) but not NOS2 GAS mutant probe (lane 12) confirmed the specificity of the binding complex. B: Western analysis of cell lysate (20 µg total protein/lane) from CF (lanes 1–4) or NL (lanes 5–8) with IFN-{gamma} (104 U/ml) stimulation for indicated time was performed using protein inhibitor of activated STAT1 (PIAS1) Ab. {beta}-Actin Ab was used as control of protein loading. Similar results were obtained from 2 separate experiments using cells from different donors.

 
STAT1 activation is also impaired in mice lacking CFTR expression (24). A previous report suggests that the level of PIAS1 protein, which binds to and inactivates STAT1, is higher in lung epithelium of CF mice (24). Thus we evaluated the expression of PIAS1 in CF and NL cells. CF cells had PIAS1 expression similar to NL at baseline and a lower PIAS1 level after IFN-{gamma} treatment compared with NL cells (Fig. 3B). Thus unlike mouse lung epithelium, PIAS1 does not play a role in the decreased STAT-1 activation in CF airway epithelial cells in vitro.

CF cells do not respond to IFN-{gamma}-induced soluble mediator(s). Our previous work identified an autocrine mechanism of induction and maintenance of NOS2 in HAEC through the synthesis and secretion of IFN-{gamma}-stimulated soluble mediator(s) (39). To evaluate the ability of CF cells to produce and respond to these soluble mediator(s), we treated CF and NL cells with IFN-{gamma} for 1 h and washed the cells five times with HEPES-buffered saline, then added fresh media, and cultured them for an additional 72 h. The IFN-{gamma}CM were collected and transferred to fresh CF and NL cells, and the cell lysates were collected 24 h later. Western blot analysis showed that, in contrast to the NL cells, CF cells fail to induce NOS2 expression following treatment with {gamma}CM derived from either CF or NL cells (Fig. 4A). The mechanism(s) of {gamma}CM-mediated induction of NOS2 expression is not completely understood but requires activation of STAT1 for NOS2 gene expression (39). To evaluate the STAT1-activating property of {gamma}CM derived from CF cells, WCE collected 30 min after {gamma}CM transfer to fresh CF and NL cells was subjected to EMSA with 32P-labeled IRF-1 GAS probe, which binds activated STAT1. Both CF and NL cells activate STAT1 following treatment with {gamma}CM derived from CF or NL cells (Fig. 4B). Thus although STAT1 activation is required to induce NOS2 in cells, these results suggest that additional factors are necessary for NOS2 expression in airway epithelial cells. Furthermore, the ability of CF-{gamma}CM to induce NOS2 expression in NL cells lessens the likelihood of a NOS-inhibitory or blocking factor in CF.



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  		Fig. 4. CF cells make soluble mediator in {gamma}-conditioned medium ({gamma}CM), which induces NOS2. A: {gamma}CM made from CF (lane 1) and NL (lane 4) cells were transferred to fresh cells. Cell lysates were collected 24 h later, and 20 µg of total protein were subject to Western analysis for NOS2. {gamma}CM from CF (1) and NL (4) both induced NOS2 expression in NL cells (lanes 5, 6), but not in CF cells (lanes 2, 3). A549 stimulated with CK (IFN-{gamma}, IL-1{beta}, TNF-{alpha}) served as positive control for NOS2 expression (lane 7). B: whole cell extracts were collected 30 min after {gamma}CM transfer to cells. Five micrograms of total protein were used in EMSA using 32P-labeled interferon regulatory factor-1 GAS. {gamma}CM from both CF (2) and NL (6) cells activated similar level of STAT1 in CF (lanes 3, 4) and NL (lanes 7–10) cells. Supershift with STAT1 Ab confirmed the specificity of the binding complex (lane 9). Similar results were obtained from 2 separate experiments using cells from different donors.

 
Antiviral property of {gamma}CM in NL and CF cells. Although NOS2 is absent, IFN-{gamma} pretreatment inhibits viral replication in CF cells, likely through induction of other host defense pathways including double-stranded RNA-dependent protein kinase (PKR) and 2',5'-oligoadenylate synthetase (38). Because STAT1 activation is essential for antiviral defense and {gamma}CM from CF or NL cells led to similar levels of STAT1 activation, we evaluated whether or not the antiviral effect of {gamma}CM would be similar in CF and NL cells. {gamma}CM collected from CF or NL cells were transferred to other CF or NL cells. After overnight incubation, cells were exposed to HPIV3. Twenty-four hours later, cell lysates were collected and analyzed for viral N-protein synthesis by Western blot. {gamma}CM inhibited viral replication in both CF and NL cells, although {gamma}CM from CF cells was slightly less effective than {gamma}CM from NL (Fig. 5).



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  		Fig. 5. {gamma}CM inhibits viral replication. Western analyses of viral N protein in lysates (20 µg/lane) of NL and CF cells infected with human parainfluenza virus (HPIV) 3. {gamma}CM derived from NL (NL-{gamma}CM) or CF cells (CF-{gamma}CM) inhibit viral N-protein production and, hence, virus replication in cells (lanes 2, 3, 5, and 6). CF-{gamma}CM may not be as completely effective in antiviral effect as NL-{gamma}CM. Similar results were obtained from 3 separate experiments using cells from different donors.

 
Definitive evidence of antiviral effect of conditioned media from NL cells was provided by quantitative viral titers. The active viral particles in the supernatant overlying HPIV3-infected NL cells pretreated with {gamma}CM or with the supernatant overlying NL HAEC not exposed to IFN-{gamma} (mock-CM) were determined by viral plaque assay. Production of viral particles was inhibited by {gamma}CM compared with mock-CM [{gamma}CM, (3 ± 1) x 104 plaque-forming units (pfu)/ml; mock-CM, (2 ± 0.7) x 106 pfu/ml; n = 3]. Thus components in {gamma}CM from CF and NL HAEC provide antiviral effect. This suggests that CF cells have an inability to express NOS2 in response to IFN-{gamma} or {gamma}CM but have other effective inducible antiviral systems that respond appropriately to IFN or {gamma}CM.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
NO produced by NOS2, following its induction by inflammatory cytokines, acts as a major effector in host defense against virus and bacteria. The expression of NOS2 in healthy airway epithelial cells has been demonstrated at both the protein and mRNA levels in vivo (15, 16, 26). CF airway epithelial cells are more susceptible to viral and bacterial infection because of defective innate host defense mechanisms of NOS2 expression and STAT1 activation (23, 29, 44). Here, the mechanism for lack of NOS2 expression in CF is identified at the transcriptional level with decreased NOS2 promoter activity in CF cells in response to IFN-{gamma}.

When bound to its specific receptor on the cell membrane, IFN-{gamma} initiates a signaling cascade, which leads to immediate and/or delayed expression of ISGs (19, 42). NOS2, along with MHCII, is one of the genes induced by IFN-{gamma} as a delayed response, whereas STAT1 and IRF-1 are immediate-response genes (1, 15–17, 41, 42). MHCII and IRF-1 are similarly induced by IFN-{gamma} in both CF and NL cells (44). However, we previously showed that STAT1 induction and activation are impaired in CF (44). This suggests that the delayed and immediate IFN-{gamma}-signaling pathways are intact but that component(s) involved in the regulation of NOS2 expression may be specifically affected in CF cells.

Studies of the human NOS2 promoter have identified important regulatory components in the promoter, including AP-1 sites, nuclear factor-{kappa}B sites, GAS, and IRF-1 sites (5, 31, 35). Only impairment of STAT1 has been identified in CF; other signal transduction molecules are similarly activated in CF and NL cells (44). Interaction between c-fos, a component of AP-1, and STAT1 is required for NOS2 induction by IFN-{gamma} in airway epithelial cells (43). Here, CF cells exposed to IFN-{gamma} have c-fos and STAT1 interaction and binding to GAS, indicating that AP-1 is not involved in the decreased STAT1-GAS binding. In contrast to previous studies using CF mouse models, PIAS1 is not overexpressed by human CF cells, and IFN-{gamma} does not induce PIAS1 in CF or NL cells. Thus PIAS1 is not the cause of decreased STAT1 activation or lack of NOS2 expression in CF cells.

Induction of NOS2 by IFN-{gamma} in airway epithelial cells occurs through an autocrine mechanism by which IFN-{gamma} induces synthesis and secretion of soluble mediator(s) that activate STAT1 and induce NOS2 expression (15, 17, 39). Here, CF cells also synthesize and secrete soluble mediator(s) that induce NOS2 expression in NL cells. However, {gamma}CM derived from NL or CF cells does not induce NOS2 expression in CF cells, although CF cells respond to {gamma}CM through activation of STAT1. A similar level of STAT-1 activation in NL and CF cells by {gamma}CM suggests that the STAT1 activation in CF is not uniformly impaired for all inducing factors.

Consistent with the central role of STAT1 for antiviral effects, the {gamma}CM has potent antiviral activity, although the CF {gamma}CM is not as complete in antiviral protection as NL {gamma}CM. The fact that {gamma}CM fails to induce NOS2 expression in CF cells but still protects CF cells from viral infection indicates that NO does not mediate the antiviral effects of conditioned media and points out the redundancy in antiviral defenses.

Currently, we have little information about the identity of the IFN-{gamma}-induced soluble mediator(s) that induces NOS2 and/or the antiviral factor(s) (17, 39). There are likely many proteins and mediators present in the {gamma}CM. Researchers have identified >300 ISGs from pooled data sets of HT1080 fibrosarcoma cells stimulated with IFN-{alpha}/{beta}/{gamma}. The ISGs are classified into functional categories, including 51 genes involved in host defense, 8 genes involved in antiviral mechanisms, 12 hormones, 15 growth factors, and 6 chemokines (9). Some of the cytokines and growth factors stimulate STAT1 activation, such as prolactin, colony-stimulating factor-1, and epidermal growth factor (7, 21, 32). Prolactin, a pituitary hormone, also acts as a cytokine at various extrapituitary sites including immune cells (40). Interestingly, prolactin may induce NOS2 expression in granulocytes and in peripheral blood mononuclear cells through at least two different signaling pathways, the STAT and the MAP kinase pathways (11). There are also several specific airway epithelial secreted factors that have antimicrobial activities, including {beta}-defensin, lysozyme, surfactant, secretory phospholipase A2, secretory leukocyte protease inhibitor, and the regulated on activation, normal T cells expressed and secreted factor (3, 14, 20, 27). Further investigation is required to understand in detail the precise mechanism(s) that regulates NOS2 expression in human lung epithelial cells and the role of NOS2 in innate host defense. However, on the basis of prior studies and our data, we suggest a model for NOS2 induction in respiratory epithelial cells and the defect in CF. Upon binding to its specific receptor on the cell membrane, IFN-{gamma} activates the JAK/STAT pathway and induces the expression of a set of ISGs including the soluble mediator(s). The soluble mediator(s) is secreted and stimulates the cells in an autocrine fashion to activate STAT1 and other factors that are required for NOS2 expression (39). STAT1 activation and its downstream targets, as well as other secreted host defense ISGs, provide antiviral protection to cells. The defect in human CF cells appears to be in the response to the secreted soluble mediator(s).

Extension of CF children's life expectancy occurred with the introduction of antibiotics, but also through vaccination against virus. For example, vaccination against measles was one of the earliest interventions that led to improved survival in the 1960s (22). Before vaccination, CF children who contracted measles, a virus like HPIV3 in the paramyxovirus family, had progressive decline in their general condition, worsening of pulmonary infections, and early death (13, 37). These clinical observations, together with the identification of enhanced virus susceptibility of CF airway cells, suggest that therapies aimed at improving antiviral host defense may further delay the onset of bacterial colonization and extend the lives of CF individuals.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Department of Army Medical Research and Development Grant 17-01-C-0065 and National Institutes of Health Grants HL-60917, HL-04265, M01 RR-018390, and AI-70649.


   ACKNOWLEDGMENTS
 
Thanks to C. Bevins and B. De for helpful discussions, B. De for HPIV3 antibody, J. Moss for NOS2 promoter, J. Lang for artwork, J. Foertch for assistance with clinical samples, and InterMune for IFN-{gamma}.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. C. Erzurum, Lung Biology Program, Cleveland Clinic Foundation, Lerner Research Inst., 9500 Euclid Ave/NB40, Cleveland, OH 44195 (E-mail erzurus{at}ccf.org)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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