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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Elevated levels of exhaled nitric oxide are seen in inflammatory airway diseases such as asthma, but the cellular source remains unknown. This study investigated whether human airway epithelial cells express inducible nitric oxide synthase
(iNOS). Human bronchial epithelial cells stimulated with 50 ng/ml interleukin-1
, tumor necrosis factor-
, and interferon-
express iNOS mRNA, protein and increased nitrite in the cell
culture media, which was inhibited by the selective iNOS inhibitor 1400W. Cells derived from subjects with asthma produced
less nitrite than cells from normal subjects (6.59 ± 0.99 µM nitrite, n = 15 versus 3.89 ± 0.42 µM nitrite, n = 20; P < 0.05).
This was not attributed to steroid treatment of subjects with
asthma because there was no difference in the amount of
nitrite released from steroid-naive and steroid-treated cells
(3.51 ± 0.46 versus 4.27 ± 0.7 µM nitrite, n = 10). Neither dexamethasone nor budesonide inhibited iNOS mRNA induction, protein expression, or nitrite accumulation. The cells
were not steroid insensitive because steroids inhibited GM-CSF release. Therefore, although these cells express iNOS under inflammatory conditions, they do not appear to be regulated directly by glucocorticosteroids.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nitric oxide (NO) is a highly reactive gaseous mediator involved in many physiologic processes. NO is synthesized by a family of enzymes termed nitric oxide synthases (NOS) (EC 1.14.13.39). The oxidation of the guanido nitrogen of L-arginine by these enzymes produces L-citrulline and NO. There are three distinct isoforms of NOS: NOS 1 or nNOS, NOS2 or iNOS, and NOS 3 or eNOS. NOS 1 and NOS 3 each have an absolute requirement for Ca2+ and as such release distinct quanta of NO reflecting the role of NOS1 and NOS3 in regulatory mechanisms such as neurotransmission and smooth muscle contraction. In contrast, NOS2 or iNOS does not have a requirement for Ca2+ but is an inducible protein and can be induced at sites of inflammation by mediators such as cytokines and lipopolysaccharide (1). This mechanism allows large amounts of NO to be produced at sites of inflammation, thus reflecting the putative role of inducible NOS (iNOS) in disease (2).
NO is an important mediator in the lung (3) and has
been shown to be associated with inflammatory lung diseases, such as asthma, where levels of exhaled NO are elevated (4, 5). The source of exhaled NO is unclear, although
increased levels of iNOS have been reported in the asthmatic airway (6). This expression of iNOS has been localized to the airway epithelium and infiltrating inflammatory cells; however, iNOS is also expressed in normal
human epithelium (9). Furthermore, in vitro studies have
demonstrated inducible expression of iNOS in both human
and rodent airway epithelial cells (7, 10). Much of the work regarding the regulation of iNOS expression has been performed in murine models (11). In these systems iNOS is
readily induced and produces large quantities of NO. However, studies of human iNOS have proved to be less successful. This may be due to differences in the promoters of
the respective genes (14). The transcription factor, nuclear
factor 
B (NF-B), is clearly important in the induction of
murine iNOS, whereas it appears to be less important in
the regulation of the human gene (12, 14). Human iNOS
regulation appears to require interferon- (IFN-) and the
activation of the transcription factor signal transducers and
activators of transcription (STAT)-1, (15, 16).
In asthma, exhaled NO levels are reduced by corticosteroid therapy (17), suggesting that human iNOS is regulated by steroids. Murine iNOS expression is readily inhibited by steroids (13, 18), and many studies have demonstrated the inhibitory effects of steroids on rat models of iNOS induction (19). However, the effect of steroids on the regulation of the human isoform of iNOS is more ambiguous. A recent study examining the effect of fluticasone in patients with chronic bronchitis demonstrated no effect of this steroid on iNOS expression in inflammatory cells of the airway (22). In inflammatory bowel disease, corticosteroids failed to reduce the expression of iNOS (23), whereas in cells derived from the human joint, dexamethasone inhibited nitrite production by ~ 30% (24). Therefore, this study investigated the induction of iNOS in human primary airway epithelial cells from normal and asthmatic subjects, and, for the first time, whether it can be regulated by steroids at both the transcriptional and protein level in human primary airway epithelial cells.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A549 and BEAS-2B cell lines were obtained from the American
Type Culture Collection (Rockville Pike, MD). Keratinocyte serum-free medium, epidermal growth factor (EGF), bovine pituitary extract, Dulbecco's modified Eagle's medium (DMEM), Ham's F12
nutrient media, and Hanks' balanced salt solution (HBSS) were purchased from Gibco (Paisley, Scotland). 2,3-Diaminonaphthalene was
purchased from Alexis (Nottingham, UK). Interleukin (IL)-1, tumor necrosis factor (TNF)-, IFN-, and IL-8 enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems
Europe (Abingdon, Oxon, UK). The 3-8% Tris acetate sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels
were purchased from Novex (San Diego, CA). Anti-human iNOS antibody was a kind gift from Dr. P. T. Manning, Monsanto-Searle (St.
Louis, MO). Protein assay reagent was purchased from Bio-Rad
Laboratories Ltd. (Hemel Hempsted, Herts, UK). AMV-reverse
transcriptase, RNAsin, and random primers were all purchased
from Promega (Southampton, UK). Taq polymerase, deoxynucleotide triphosphate (dNTP), and KCl buffer were purchased from Bioline (London, UK). RNA extraction kit was purchased from Qiagen
(Crawley, UK). Anti-rabbit IgG conjugated to HRP was purchased from Dako (High Wycombe, UK). Hybond-ECL and ECL reagent
were purchased from Amersham (Amersham, UK). Sigma, Poole
(Dorset, UK), or BDH (Dorset, UK) supplied all other reagents.
Bronchoscopy
Patients with mild asthma (mean age 26 ± 1.3 yr; mean FEV1% predicted 76.8 ± 3.5) and normal subjects (mean age 26 ± 0.5 yr; mean FEV1% predicted 102 ± 3.5) underwent fiberoptic bronchoscopy. All subjects were nonsmokers. Patients with asthma were stable at the time of bronchoscopy. Subjects attended the bronchoscopy suite after having fasted for 12 h and were pretreated with atropine (0.6 mg intravenously) and midazolam (5- 10 mg intravenously). Oxygen (3 liters/min) was administered via nasal prongs throughout the procedure, and oxygen saturation was monitored with a digital oximeter. Using local anesthesia with lidocaine (2% wt/vol) to the upper airways and larynx, a fiberoptic bronchoscope (Olympus BF10; Key-Med, Southend-on-Sea, Essex, UK) was passed through the nasal passages into the trachea. Brushing of the airway was performed essentially as reported by Kelsen and colleagues (25). Briefly, a 2-mm channel cytology brush (1.8 mm insertion diameter) (Olympus BC-16C; Key-Med) was inserted via the sampling channel of the bronchoscope and rubbed against the epithelial surface. The brush was retracted and cells dissociated by vortexing in ice-cold Ham's F12 nutrient medium. This brushing procedure was repeated 4-6 times. The Royal Brompton Hospital Ethics Committee approved the experimental protocol for fiberoptic bronchoscopy, and all subjects gave their informed consent to participate.
Isolation and Culture of Epithelial Cells
The cell suspension was treated for 20 min with 50 µg/ml DNase
at room temperature and then filtered through a 100-µM-cell strainer. The cells were centrifuged for 5 min at 200 × g and washed once with HBSS. Cells were suspended in Ham's F12 nutrient media containing 5% (vol/vol) fetal calf serum, 1 µM hydrocortisone, 5 ng/ml EGF, 10 µg/ml insulin, 10 nM retinoic
acid, 0.5 µg/ml transferrin, 2 µg/ml triiodothyronine, 1.5 mg/ml
NaHCO3, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B and seeded onto 24-well plates coated in
1% (wt/vol) collagen at a density of 5 × 105 cells/well. The cells
were incubated at 37°C in a humidified atmosphere containing
95% (vol/vol) air 5% (vol/vol) CO2. The cells were cultured until
confluence (~ 5 d) and then cultured for 24 h in additive-free media prior to experimental treatments. For cytokine treatments,
cells were treated with equal quantities of IL-1, TNF-, and
IFN-. For IFN-, 1 ng = 10 units.
Culture of Cell Lines
The human lung epithelial cell line A549 was cultured in DMEM containing 10% (vol/vol) fetal calf serum (FCS), 100 µg/ml penicillin, and 100 U/ml streptomycin. Cells were maintained in a humidified incubator at 37°C with 95% (vol/vol) air and 5% (vol/vol) CO2. The cells were replenished with fresh media every 2-3 d. The human bronchial epithelial cell line BEAS-2B was cultured on collagen-coated plasticware in keratinocyte serum-free media supplemented with 5 ng/ml EGF, 50 µg/ml bovine pituitary extract, 100 mg/ml penicillin, and 100 U/ml streptomycin. The cells were replenished with fresh media every 2-3 d.
Cell Viability
Cells were treated with 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in HBSS for 30 min at 37°C. The MTT solution was removed from the surface of the cells, and dimethyl sulfoximine was added. The absorbance of the resultant solution was measured at 550 nm, and treated cells were compared with control cells. None of the treatments used in these studies altered cell viability when compared with untreated cells.
Measurement of Nitrite and Nitrate+Nitrite in Cell Culture Media
The amount of nitrite in cell culture media was measured using a modification of the method of Misko and colleagues (26). Briefly, 200 µl of media or nitrite standard solution was mixed with 100 µl of 2% (wt/vol) charcoal in 0.2% (wt/vol) dextran. The suspension was centrifuged at 10,000 × g for 10 min, and the cleared supernatant was mixed with 10 µl of 0.05 mg/ml 2,3-diaminonaphthalene in 0.625 M HCl and incubated in the dark for 10 min. The reaction was stopped by the addition of 10 µl of 1.4 M NaOH, and fluorescence was measured using a Biolite F1 plate fluorimeter (Labtech, Uckfield, UK) with the excitation wavelength set at 360 nm and the emission wavelength set at 460 nm with the sensitivity of the fluorimeter set between 40 and 50%. The amount of nitrite in the sample was calculated using a standard curve of known nitrite concentrations, and the assay was sensitive to 0.1 µM. The amount of nitrite+nitrate (NOx) was measured by incubating a 100-µl sample in the presence of 0.14 U of nitrite reductase, 10 µM NADPH for 10 at room temperature. The sample was then assayed as above. To test for reproducibility of this method, duplicated samples of cell media were assayed and subjected to the Bland-Altman test (27) and found to be reproducible (P < 0.05).
GM-CSF Measurement by ELISA
GM-CSF was measured by sandwich ELISA. Ninety-six-well plates were coated overnight at 4°C with rat monoclonal capture antibody against GM-CSF diluted 1:250 in 0.1 M NaHCO3 containing 15 mM NaN3. Plates were washed in wash buffer (145 mM NaCl, 4 mM KCl, 10 mM NaH2PO4, 0.05% [vol/vol] Tween-20 [pH 7.4]) and then blocked for 2 h at room temperature with 10% (vol/vol) FCS in wash buffer. The plate was washed, and samples and standards were added to the wells and incubated at 4°C overnight. The plates were washed extensively and then incubated for 45 min with a biotinylated rat anti-human monoclonal GM-CSF antibody diluted 1:500 in 10% (vol/vol) FCS in wash buffer. Plates were then incubated for 30 min with a 1:400 dilution of avidin-peroxidase in 10% (vol/vol) FCS in wash buffer. The plates were then developed with ABTS substrate solution (0.547 mM 2,2'azino-bis[3-ethylbenzthiazoline-6-sulphonic acid], 0.1 M citric acid [pH 4.35], 0.03% [vol/vol] H2O2). The plate was then measured spectrophotometrically at 405 nm, and the amount of GM-CSF in each sample was calculated from a standard curve. The detection limit of this assay is 16 pg GM-CSF/ml.
IL-8 Measurement by ELISA
IL-8 was measured in cell culture media using a kit according to the manufacturer's instructions (IL-8, DuoSet; R&D Systems Europe; Abingdon, Oxon, UK).
RNA Isolation
RNA was isolated from primary epithelial cells using the Qiagen RNeasy mini kit (Qiagen, Crawley, UK) according to the manufacturer's instructions.
Reverse Transcription Polymerase Chain Reaction
Reverse transcription was performed on 0.5 µg of RNA. RNA was heated to 70°C for 5 min and then mixed with 0.01 µg/µl random primers, 1.0 mM dNTP, 1 U/µl RNAsin, and 0.25 U/µl AMV reverse transcriptase in 1× reverse transcriptase buffer (Promega, Southampton, UK) and incubated at 42°C for 1 h followed by denaturation at 90°C for 4 min. The cDNA was then diluted by the addition of 80 µl of water.
For PCR, 5 µl of cDNA was incubated in a final volume of 25 µl containing 1× KCl buffer (Bioline, London, UK), 2 mM dNTP, 5 ng/µl specific primers, and 0.02 U/µl Taq polymerase (Bioline). Specific primers for iNOS PCR gave a PCR product of 312 bp. The forward primer was 5'-GAGCTTCTACCTCAAGCTATC-3', and the reverse primer was 5'-CCTGATGTTGCCATTGTTGGT-3'. The cycles used were 94°C for 45 s, 56°C for 45 s, and 72°C for 60 s for 32 cycles followed by 72°C for 10 min. Polymerase chain reaction (PCR) of GAPDH was performed to act as an internal control to give a PCR product of 571 bp. The forward primer was 5'-ATTCCATGGCACCGTCAAGGCT-3', and the reverse primer 5'-TCAGGTCCACCACTGACACGT-3'. Cycles used were 94°C for 45 s, 56°C for 45s, and 72°C for 60 s for 26 cycles followed by 72°C for 10 min. The number of PCR cycles was determined empirically, and the cycle numbers used were within the linear range for each primer set. PCR products were identified on 2% (wt/vol) agarose gels. Samples that did not contain reverse transcriptase were used as negative controls.
Western Blotting
The anti-human iNOS primary antibody was a kind gift from Dr.
Pamela T. Manning (Monsanto). The antibody was raised in a
rabbit against recombinant human iNOS, and the resultant antisera was purified using affinity chromatography. Cells were lysed
in 50 mM Tris/HCl (pH 7.4) containing 0.25 mM ethylenediamine
tetraacetic acid, 0.5 mM phenylmethylsulphonyl fluoride, 5 µg/ml
antipain, 5 µg/ml leupeptin, and 5 µg/ml benzaminidine. Protein
concentration was determined using the Bio-Rad protein assay
kit according to the manufacturer's instructions. Cell proteins
were solubilized in SDS-PAGE sample buffer (0.0625 mM Tris/
HCl [pH 6.8] containing 10% vol/vol glycerol, 1% wt/vol SDS,
1% wt/vol -mercaptoethanol, and 0.01% wt/vol bromophenol
blue). The proteins (15 µg per lane) were resolved by electrophoresis in 3-8% (wt/vol) Tris-acetate SDS-polyacrylamide gels
and transferred to Hybond-Enhanced Chemiluminescence (ECL)
nitrocellulose membranes. Equal protein loading was determined by staining the blot with 0.1% (wt/vol) Ponceau S in 5% (vol/vol) acetic acid. The nitro-cellulose was blocked overnight at 4°C in
0.5 M Tris-HCl (pH 7.4) containing 3% (wt/vol) normal goat serum, 1% (wt/vol) bovine serum albumin, and 0.05% (vol/vol)
Tween-20. The blots were washed in phosphate-buffered saline
containing 0.05% (vol/vol) Tween-20 and incubated for 1 h in the
presence of primary antibody (1:1,000). The blots were washed
extensively and then incubated for 1 h with anti-rabbit IgG conjugated to horseradish peroxidase (1:4,000). The blots were
washed extensively again, and the bands were visualized using
ECL reagent.
Statistical Analysis
The data are presented as mean of n experiments using cells derived from separate subjects ± standard error of the mean (SEM). Differences were analyzed using one-way ANOVA or Mann-Whitney tests as appropriate. P < 0.05 was considered significant.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect of IL-1, TNF-, and INF- in Combination on the
Production of Nitrite and GM-CSF from Human Primary
Epithelial Cells
Culture of primary epithelial cells with differing concentrations of IL-1, TNF-, and IFN- led to a dose-dependent increase in GM-CSF release. The increase in GM-CSF
production reached a significant difference from baseline
at a IL-1, TNF-, and IFN- concentration of 5 ng/ml
(Figure 1A). Similar results were obtained with nitrite release; however, 50 ng/ml of IL-1, TNF-, and IFN- was
required to show the greatest increase in the release of nitrite above baseline (Figure 1B). Therefore, a concentration of 50 ng/ml of IL-1, TNF-, and IFN- was selected
for future experiments. The effect of 50 ng/ml IL-1, TNF-,
and IFN- on both nitrite and GM-CSF release from epithelial cells derived from both asthmatic patients and normal subjects was examined. Epithelial cells were derived
from brushings attached to the cell plates and were cultured until confluence. There was no difference in the morphology or cell viability as measured using MTT between
cells derived from asthmatic or normal subjects. IL-1,
TNF, and IFN- together increased the production of nitrite in cells derived from both asthmatic and normal subjects; however, the levels of nitrite released from normal
subjects was significantly greater than the levels from asthmatics (Figures 2A and 2B and Table 1). There was no
difference in the levels of GM-CSF released from cells derived from either normal subjects or asthmatic patients either at baseline (normal: 0.1 ± 0.04 ng/ml, n = 15; asthmatic: 0.1 ± 0.05 ng/ml, n = 20) or following stimulation
with 50 ng/ml IL-1, TNF-, and IFN- (normal: 0.8 ± 0.2 ng/ml, n = 15; asthmatic: 0.7 ± 0.1 ng/ml, n = 20). This
would suggest that the difference in nitrite release from
cells derived from normal and asthmatic subjects is not a
general effect seen with all inflammatory mediators. To investigate this difference further, the level of iNOS mRNA in these cells following stimulation was compared. RT-PCR for human iNOS showed that incubation of cells
with IL-1, TNF-, and IFN- led to an increase in iNOS
mRNA expression (Figures 2C and 2D). There was a significant decrease in the level of iNOS mRNA expression
in epithelial cells derived from asthmatic patients compared with cells from normal subjects. This reduced level of iNOS expression in cells derived from asthmatic subjects
may explain the reduced level of nitrite released from
these cells upon stimulation with IL-1, TNF-, and IFN-.
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The level of nitrite released from primary epithelial
cells following IL-1, TNF-, and IFN- stimulation was
compared with that from the cell lines A549 and BEAS-2B.
A549 cells and BEAS-2B cells released significantly less
nitrite at both basal and stimulated conditions (Table 1).
Similarly, primary cells that had been passaged once also
showed reduced levels of nitrite release at both the basal
level and following stimulation with 50 ng/ml IL-1, TNF-,
and IFN- (Table 1).
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Effect of a Specific iNOS Inhibitor, 1400W, on Nitrite Release from Primary Epithelial Cells
To determine the source of nitrite release from primary
epithelial cells, the effect of a specific iNOS inhibitor,
1400W, was examined. Cells were cultured for 24 h in the
absence or presence of 50 ng/ml IL-1, TNF-, and IFN-
in the absence or presence of 5 µM 1400W. 1400W had no
effect on basal release of nitrite from primary epithelial
cells but did inhibit the release of nitrite from cells exposed to IL-1, TNF-, and IFN- (Figure 3A). There was
no effect of 1400W on the release of GM-CSF from these
cells (Figure 3B).
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Effect of Corticosteroids on Nitrite, GM-CSF, and IL-8 Release
The observation that cells derived from patients with
asthma released lower amounts of nitrite when compared
with the levels released from cells derived from normal
subjects (Figure 2B) could not be explained by the fact
that some of the patients with asthma were taking glucocorticosteroids as part of their therapy. When the subjects
with asthma were subdivided into two groups comprising of steroid-naive patients and those taking steroids, there
was no difference in the amount of nitrite released from
each group either at basal (1.98 ± 0.22 versus 2.28 ± 0.52 µM nitrite, n = 10) or following stimulation with IL-1,
TNF-, and IFN- (3.51 ± 0.46 versus 4.27 ± 0.7 µM nitrite, n = 10). This would suggest that there is no residual
effect of steroid administration on primary epithelial cells
after 5 d in culture.
To determine whether steroids could regulate iNOS activity and GM-CSF release in human primary epithelial
cells, the effect of culturing cells in the presence of 1 µM
dexamethasone was examined. Over a time course of up to
24 h, dexamethasone had no effect on the release of nitrite
from human primary epithelial cells (Figure 4A). However, GM-CSF release was inhibited by 24 h treatment of
dexamethasone by ~ 40% (Figure 4B). This lack of effect
of dexamethasone on iNOS expression was confirmed by
RT-PCR (Figures 4C and 4D). Over the same time course,
dexamethasone did not inhibit iNOS mRNA expression in
human primary epithelial cells. In these experiments, the
cells were stimulated with 50 ng/ml IL-1, TNF-, and
IFN-; because this is the maximal dose required for nitrite and GM-CSF release, it was possible that this dose
may be supramaximal for dexamethasone inhibition to occur. To investigate this, cells were stimulated with different doses of IL-1, TNF-, and IFN- in the absence and
presence of 1 µM dexamethasone, and both nitrite and
GM-CSF release were measured. Dexamethasone had no
effect on nitrite release at any of the concentrations of IL-1,
TNF-, and IFN- (Figure 5A); however, dexamethasone
did inhibit GM-CSF release at all concentrations of IL-1,
TNF-, and IFN- examined (Figure 5B). To ensure that
these cells could respond to steroids, cells were incubated
with 1 µM dexamethasone and 50 ng/ml IL-1, TNF-,
and IFN- for 24 h. Both IL-8 (basal 3.8 ± 2.4 ng/ml, stimulated 23.5 ± 1.8 ng/ml, n = 3) and GM-CSF release were
inhibited by dexamethasone with EC50 values of 6 × 10
8
M and 3.5 × 109 M, respectively (Figures 5C and 5D).
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To exclude the possibility that iNOS expression was insensitive to dexamethasone, a second steroid, budesonide,
was also examined. In a similar experiment, budesonide
had no effect on iNOS expression as measured by RT-PCR (Figures 6A and 6B) or nitrite release (Figure 6C)
but did inhibit GM-CSF release with an EC50 of 9.04 × 109 M (Figure 6D) and budesonide with an EC50 of 1.1 × 1012 M (Figure 6E).
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Effect of Dexamethasone on iNOS Protein Expression
The possibility that the lack of effect of dexamethasone on
nitrite accumulation might be due to the possibility that
the product of iNOS was predominantly nitrate was addressed by measuring NOx in the samples. Dexamethasone failed to inhibit either IL-1, TNF-, and IFN- induced nitrite or NOx induction in these cells (Figures 7A
and 7B). Together with the data regarding iNOS mRNA expression, this result would suggest that steroids also had
no effect on the expression of iNOS protein. To confirm
this, cells were cultured for 24 h in the absence or presence
of 50 ng/ml IL-1, TNF-, and IFN- in the absence or
presence of 1 µM dexamethasone. However, dexamethasone had no effect on the expression of IL-1, TNF-, and
IFN- induced iNOS protein (Figure 7C).
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Human airway epithelial cells can be induced to express
iNOS in vitro. However, induction of iNOS required culture in the presence of IL-1, TNF-, and IFN- because
unstimulated cells did not express iNOS mRNA or protein. This finding is similar to other studies that have reported that iNOS expression in airway epithelial cells is
lost within 24 h of removal from the airway (9) but can be
stimulated in the presence of IL-1, TNF-, and IFN-
(14, 16). The present study also demonstrated that two of
the most commonly used epithelial cell lines, namely A549 and BEAS-2B, express very little iNOS, as reflected in
their lack of production of nitrite following stimulation with
IL-1, TNF-, and IFN-. Therefore, A549 and BEAS-2B
cells are not good models for human airway epithelial
iNOS expression.
This study also demonstrated that epithelial cells derived from subjects with asthma produced lower levels of nitrite when compared with cells from normal individuals. This appeared to be specific to nitrite release because there was no difference in the level of GM-CSF released from cells derived from these subjects. This discrepancy could not be attributed to effects of steroid treatment of asthmatic patients because there was no difference in the levels of either nitrite or GM-CSF release from steroid-naive compared with steroid-treated asthmatic subjects. This is in contrast to studies demonstrating increased levels of NO in the exhaled breath of patients with asthma compared with normal subjects (5). Because inflammatory cytokines are required for the induction of iNOS, there might be greater levels of inflammatory cytokines in the asthmatic airway compared with the normal airway (28, 29), hence increased iNOS expression in the asthmatic epithelium.
In asthma, exhaled NO can be reduced by steroid therapy (17), suggesting that iNOS is downregulated by steroids. However, the present study has demonstrated, for
the first time, that iNOS expressed by human primary airway epithelial cells is steroid insensitive. This is similar to
the situation in inflammatory bowel disease where iNOS
expression is not affected by corticosteroid therapy (23).
This data is further supported by the observations that
iNOS expression as measured by NOx accumulation from
the cell media in the human intestinal epithelial cell lines is
also steroid insensitive (30, 31). Small inhibitory effects
(30%) of dexamethasone have also been reported in cells
derived from the human joint (24). However, the degree
of steroid inhibition in the human models of iNOS regulation studied thus far does not approach the levels of inhibition seen in rodent models of iNOS induction (12, 13, 32).
The effect of inhaled corticosteroids on exhaled NO in
asthmatic subjects is therefore likely to be indirectly mediated and may be due to either a suppression of inflammatory cytokines such as IL-1 or TNF- that induce iNOS or to a reduction in inflammatory cell infiltrate in the airways. Indeed, patients with chronic bronchitis do not show
a reduction in iNOS expression in inflammatory airway
cells when treated with inhaled steroids (22). The effects of
steroids in asthma to reduce iNOS expression might therefore reflect the nature of asthmatic inflammation rather
than a direct effect on iNOS itself.
The discrepancy between the levels of induction of
iNOS and regulation by steroids in human and rodent
models might be due to differences in gene regulation.
There are differences in the promoters of human and murine iNOS that are thought to account for the hyporesponsiveness of human iNOS (14). Murine models of iNOS induction produce large quantities of iNOS protein and NO
as measured by NOx accumulation in cell media, whereas
the human gene is less easily induced (33). It would appear
that IFN- is required for expression of human iNOS via
activation of the transcription factor STAT-1 (16). There
is increasing evidence that STAT-1 activation is important
in the induction of iNOS in both human and rodent models (16, 34, 35). Furthermore, in human intestinal epithelial
cell lines, activation of STAT-1 has been demonstrated to
be steroid insensitive (34). A similar mechanism in human
primary epithelial cells would explain the lack of steroid
responsiveness of iNOS following IL-1, TNF-, and IFN- stimulation.
This study confirms the observation that human iNOS and hence NO production is more tightly regulated than murine models. This may reflect the role of NO in human disease to control the regulation of gene expression rather than direct cytotoxicity (2). Therefore, it may be unwise to extrapolate data regarding the role of iNOS in disease from such animal models to human disease.
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Footnotes |
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Address correspondence to: Dr. L. E. Donnelly, Department of Thoracic Medicine, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom. E-mail: l.donnelly{at}ic.ac.uk
(Received in original form November 27, 2000 and in revised form July 30, 2001).
Abbreviations: Dulbecco's modified Eagle's medium, DMEM; deoxynucleotide triphosphate, dNTP; epidermal growth factor, EGF; enzyme-linked immunosorbent assay, ELISA; fetal calf serum, FCS; Hanks' balanced salt solution, HBSS; interferon, IFN; interleukin, IL; inducible nitric oxide synthase, iNOS; nuclear factorB, NF-B; nitric oxide, NO; nitric oxide
synthase, NOS; nitrate+nitrite, NOx; polymerase chain reaction, PCR; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; signal transducers and activators of transcription, STAT; tumor necrosis factor, TNF.
Acknowledgments: The collection of samples by Drs. S. Lim, R. K. R. Russell, and J. V. Collins is acknowledged. This work was supported by grants from National Asthma Campaign (U.K.), the Clinical Research Committee of the Royal Brompton Hospital, Monsanto-Searle, and Pharmacia.
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References |
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Materials and Methods
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Discussion
References
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1. Forstermann, U., and H. Kleinert. 1995. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn Schmiedebergs Arch. Pharmacol. 352: 351-364 [Medline].
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