INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Inhaled corticosteroid therapy is the most effective therapy for patients with chronic asthma with significant effects in improving lung function and bronchial hyperresponsiveness, and in controlling asthma symptoms and exacerbations (1). Corticosteroids have potent anti-inflammatory effects in inhibiting the influx of inflammatory cells such as eosinophils, T cells, in reducing the number of mast cells, and in improving the integrity of the airway epithelium (2). The precise molecular mechanisms by which these effects occur are unclear. Recent evidence points to an important effect of corticosteroids in inhibiting the expression of several key proteins including proinflammatory cytokines involved in the control of inflammation. Thus, inhaled corticosteroids reduce the overexpression of several cytokines such as interleukin-4 (IL-4), IL-5, granulocyte-macrophage colony stimulating factor (GM-CSF), and RANTES (regulated upon activation, normal T-cell expressed and secreted) and enzymes such as inducible nitric oxide synthase iNOS (5), which are known to be upregulated in the airways of patients with asthma (5, 6, 8). The direct effect of corticosteroids in inhibiting the transcription of certain cytokine genes such as IL-1, RANTES, IL-8, and IL-5 has also been demonstrated in vitro (9).

Corticosteroids (or glucocorticoids) bind to specific cytoplasmic corticosteroid receptors, which lead to their activation and their translocation to the nucleus where they bind to glucocorticoid-responsive elements located on the 5'-promoter region of many genes, leading to modulation of gene transcription (13). The activated glucocorticoid receptor may also bind directly to other transcription factors, particularly nuclear factor kappa B (NF-B) and activating protein-1 (AP-1) (14), which may themselves activate transcription on binding to specific sequences of the promoters of certain proinflammatory genes. Binding of these factors to the glucocorticoid receptor prevents such gene activation (17). Interactions between the glucocorticoid receptor and NF-B are of particular interest as a potential mechanism of action of corticosteroids in asthma (18).

NF-B consists of heterodimers or homodimers of related proteins belonging to the Rel family of transcription factors, which consists of five family members: p65 (Rel A), Rel B, c-Rel, NF-B1 (consisting of p50 and its precursor p105), and NF-B2 (p52 and its precursor p100) (19). Generally, NF-B consists of p65/p50 heterodimers, usually the most abundant of the transactivating complexes in most cells (20), including airway epithelial cells (21). p65 does not bind DNA efficiently but has potent transcriptional activation, whereas p50 is mainly a DNA-binding subunit (22). There are one or more sites for binding of NF-B on the 5'-promoter regions of the genes of several proinflammatory cytokines/proteins which are upregulated in asthma such as IL-1, monocyte chemotactic protein-1 (MCP-1), RANTES, and iNOS. Transcriptional regulation of some of these genes is at least partly dependent on NF-B binding (23), and NF-B expression and activation are increased in the airways of patients with asthma (21). Corticosteroids may interfere with NF-B activity by increasing the expression of the endogenous inhibitor of NF-B, I-B, in certain cell types (27). Of greater interest is the possibility that corticosteroids may reduce DNA-binding activity of NF-B in vitro in human lung and in mononuclear cells (16, 28). However, other in vitro studies have reported either no effect or an enhanced effect of corticosteroids on NF-B DNA-binding activity on epithelial A549 cells or vascular endothelial cells (29). The reasons behind these disparate findings are unclear, but may relate to the cell type studied or the mechanism by which NF-B is activated.

Because NF-B expression and activation are increased in the airways of patients with asthma, and because corticosteroids are the most effective anti-inflammatory treatment for asthma, we determined whether the mechanisms of corticosteroid effects could be mediated through the modulation of NF-B expression and activation. We therefore examined the effect of 1-mo treatment of mild asthmatic patients with an inhaled corticosteroid, fluticasone propionate, on NF-B DNA- binding activity in alveolar macrophages and bronchial biopsies, and immunohistological expression of NF-B in bronchial biopsies. In addition, because the in vitro studies reported disparate results and because there are no in vitro data on the effect of corticosteroids on the protein expression of NF-B, we also examined the effects of dexamethasone on NF-B expression in an epithelial cell line in vitro.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Fifteen stable subjects with mild asthma (Table 1) receiving treatment with only the inhaled ₂-adrenergic agonist aerosol, albuterol, for intermittent relief of wheeze were recruited. All patients demonstrated a greater than 15% improvement in FEV₁ after 200 µg of albuterol and airway hyperresponsiveness to methacholine with a provocative concentration required to produce a 20% fall in FEV₁ (PC₂₀) < 4 mg/ ml. All patients were atopic as defined by the presence of two or more positive skin prick tests to common allergens. None of the subjects studied had received oral or inhaled corticosteroids for the preceding 6 mo, or any other treatment apart from short-acting inhaled ₂-agonists. Current smokers or ex-smokers of more than 5 pack-years and patients with FEV₁ less than 80% predicted were excluded.

Study Design

The study was a 14-wk double-blind, randomized, crossover study comparing the effects of the inhaled corticosteroid, fluticasone propionate (500 µg twice daily) to that of a matched placebo, both administered via a metered dose inhaler (MDI). After a 2-wk run-in period, each treatment was administered for 4 wk, separated by a 4-wk wash-out phase. Patients were asked to chart their peak flow rates twice daily. Spirometry and airway responsiveness to methacholine were performed at 2 wk during the run-in, and during the wash-out period, and at Day 26 of each treatment phase. Fiberoptic bronchoscopy was performed at the end of both treatment phases, when bronchoalveolar lavage (BAL) and bronchial biopsies were obtained. The study was approved by the Royal Brompton Hospital ethics committee, and all patients gave their informed consent.

Bronchial Responsiveness to Methacholine

Baseline spirometric parameters (FEV₁ and FVC) were recorded from the best of three attempts using a dry-wedge spirometer (Vitalograph, Buckingham, UK). All patients abstained from using inhaled ₂-agonist therapy and from caffeine-containing foods for 12 h before the test. A standardized bronchial provocation protocol was performed using nebulized buffered isotonic methacholine solution. After an initial nebulized 0.9% NaCl challenge, 5 breaths of doubling concentrations of methacholine were administered from a nebulizer connected to a breath-actuated dosimeter, starting at a dose of 0.0625 mg/ml up to 64 mg/ml. FEV₁ was recorded 2 min after administration of each concentration. The challenge was stopped when a 20% fall in FEV₁ was achieved. The PC₂₀ was then calculated by linear interpolation. The results were analyzed as the logarithm to base 10 of PC₂₀ (log PC₂₀).

Fiberoptic Bronchoscopy

After an overnight fast, subjects were sedated with intravenous midazolam (5 to 10 mg). Oxygen (3 L/min) was administered via nasal prongs, and oxygen saturation was monitored by digital pulse oximetry. Using local anesthesia with lidocaine (2% wt/vol) to the upper airways and larynx, a fiberoptic bronchoscope was passed through the nasal passages into the trachea. The bronchoscope was wedged in the right middle lobe and 4 × 60-ml aliquots of prewarmed sterile 0.9% NaCl solution were instilled. This solution was aspirated through the bronchoscope, collected in prechilled glass bottles, and stored on ice. BAL recovery was between 120 and 200 ml. Four bronchial mucosal biopsies were taken from segmental and subsegmental airways of the right lower and upper lobes using a size 19 cupped forceps. Two bronchial biopsies were mounted in optimal cutting temperature (OCT) embedding medium, snap-frozen in isopentane, and kept in liquid nitrogen before storing at 70° C. These were subsequently used for immunohistochemistry. The other two bronchial biopsies were placed in sterile Eppendorf tubes and kept on ice until they were prepared for nuclear protein extraction.

Separation of Alveolar Macrophages from BAL

The BAL fluid (BALF) was filtered through sterile gauze to exclude mucus plugs and was then centrifuged at 1,000 g for 10 min at 4° C to obtain a cell pellet. The cell pellet was washed once in 50 ml of Ca²⁺/ Mg²⁺ free Hanks' balanced salt solution (HBSS). The cells were counted on a hemocytometer slide using a Kimura counterstain and viability assessed by trypan blue exclusion. Cytospins were performed, using 10⁴ cells per slide, and stained with May-Grunwald-Giemsa in order to obtain differential cell counts. The remaining cells were resuspended at a concentration of 2 × 10⁶ macrophages per milliliter in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. 2 × 10⁶ macrophages/well were plated onto 6-well plates and allowed to adhere for 90 min in a humidified incubator in 95% air, 5% CO₂ (vol/vol), at 37° C. Nonadherent cells were removed by washing three times with RPMI 1640 medium, leaving the adherent macrophages, which were > 99% pure, as assessed by staining and morphologic analysis. The macrophages were harvested with a cell scraper or were stimulated, cultured and then recovered.

Preparation of Nuclear Proteins

Nuclear proteins were extracted from alveolar macrophages or bronchial biopsies by detergent lysis according to a method modified from Gough (32) and Osborn (33). The samples were lysed in 200 µl of cold Gough 1 solution (TRIS-HCl 10 mM [pH 7.5], 0.15 M NaCl, 1.5 mM MgCl₂, 0.65% (vol/vol) Nonidet P-40 [NP-40], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM dithiothreitol [DTT]) for 3 min. After centrifugation at 1,000 g for 1 min at 4° C, the nuclear pellet was lysed with 20 µl of Buffer B (20 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 0.42 M NaCl, 0.5 mM DTT, 0.2 mM ethylenediaminetetraacetic acid [EDTA], 0.5 mM PMSF, 25% [vol/vol] glycerol), incubated on ice for 30 min and underwent one freeze-thaw cycle. After brief centrifugation, the subsequent soluble fraction was mixed with 50 µl of Buffer C (20 mM HEPES at pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) and stored at 20° C.

Electrophoretic Mobility Shift Assay (EMSA)

Mobility shift assays were performed as described originally by Dignan and coworkers (34). Double-stranded oligonucleotides encoding the consensus target sequence of NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') were end-labeled using [-³²P]-ATP and T4 polynucleotide kinase. Nuclear protein (2.5 µg) from each sample was incubated with 5 µl binding buffer (20% [vol/vol] glycerol, 250 mM NaCl, 2.5 mM EDTA, 2.5 mM DTT, 50 mM TRIS-HCl [pH 7.5], 5 mM MgCl₂, 0.4 mg/ml sonicated salmon sperm DNA) for 10 min. 0.0175 pmol of radiolabeled oligonucleotide was added and the volume made up to 25 µl with Buffer C. This was incubated for 45 min on ice. Specificity was determined by the prior addition of 100-fold excess of unlabeled competitor consensus oligonucleotide. Protein-DNA complexes were resolved on 7% polyacrylamide gels (37:1 acrylamide:bis acrylamide) using 0.25× TRIS-borate-EDTA running buffer. Gels were vacuum dried and autoradiographed at 70° C using Kodak X-OMAT film. The retarded bands were quantified by laser densitometry. Band density measurements were expressed as NF-B per microgram of protein loaded.

Immunohistochemistry

The frozen bronchial biopsies were cut on a cryostat into 6-µm sections and placed on poly-L-lysine-coated microscope slides (Sigma, Poole, UK). The sections were fixed in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) at 4° C, for 10 min, washed twice with PBS, and air-dried for 20 min. Endogenous peroxidase activity was blocked by incubating the slides in 3% (wt/vol) hydrogen peroxide and 0.02% (wt/vol) sodium azide in PBS for 1 h. The slides were washed 3 × 5 min in PBS and then blocked with normal swine serum (5% vol/vol) in PBS for 20 min. The sections were then incubated with primary rabbit polyclonal antibody: anti-p65 (1:200) overnight at 4° C. For negative control, sections were incubated with normal rabbit immunoglobulin at the same concentration as the primary antibody. The slides were then washed 2 × 5 min with PBS and then incubated with biotinylated swine anti-rabbit antibody (1:200) for 45 min. After two further washes, the sections were incubated for 45 min in peroxidase-conjugated avidin (1:500) and then washed twice again. Chromogen fast diaminobenzidine was used for 5 min and the slides counterstained in 20% (wt/vol) hematoxylin, dehydrated in alcohol and afterwards in xylene, and then mounted in mountant medium. In order to stain for the presence of eosinophils, a mouse monoclonal anti- human major basic protein (MBP) antibody (Clone BMK-13; Monosan, Uden, The Netherlands) was used at a concentration of 1:80 for 1 h at room temperature. After labeling with a biotinylated horse anti-mouse monoclonal antibody, positive cells were visualized by using an avidin-biotin complex reagent conjugated to alkaline phosphatase (Vector; Vector Labs, Peterborough, UK), and 3,3-diaminobenzidine tetrachloride solution (Sigma) with glucose oxidase-nickel enhancement to give a black staining (35).

Cell Counts

At least 4 separate fields at ×400 magnification were examined by each of two observers. On each field, a length of epithelium 175 µm long, and an area of subepithelium of 175 × 175 µM² were counted. Immunoreactivity for MBP was graded from 0 to 10 depending on the presence of positive staining within one row of the counting grid. Zero was defined as no positive staining within any rows of the counting grid; 1 was defined as positive staining within one row of the counting grid; and 10 was defined as staining within 10 grid rows. Counts of positive immunoreactive cells for MBP were divided according to whether the immunoreactivity was in the airway epithelium or beneath the epithelium to a depth of 175 µm. All counts were made by two experienced observers unaware of the origin of the sections. The coefficient of variation between two observers was less than 10%, and the mean of the counts obtained by the two observers was used.

p65-positive cell counts were made on epithelial cells and submucosal cells; however, the majority of positive staining was observed in the epithelium with little positivity in submucosal cells. As noted previously (21), staining in the airway epithelial cells was usually nuclear, indicating the presence of activated p65. The counts were expressed as percent of all nucleated cells in the airway epithelium or per mm² area occupied by the epithelium, using a computerized image analysis system attached to the microscope (Seescan plc, Cambridge, UK). At least 500 cells were counted.

Effect of Dexamethasone and IL-1 on Epithelial A549 Cells

A549 cells (American Type Culture Collection, Rockville, MD), derived from lung alveolar cell carcinoma, were grown in 175 cm² culture flasks (Costar, Bucks, UK) in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Paisley, UK) supplemented with 10% (vol/vol) fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B (Gibco BRL) in a humidified incubator in 95% air, 5% CO₂ (vol/vol), at 37° C. Once confluent, the A549 cells were washed with HBSS and the monolayer detached with 0.05% (wt/vol) trypsin, 1 mM EDTA (Sigma Chemicals, Poole, UK). The cells were subcultured on 6-well plates until confluent. They were then incubated overnight in supplemented serum-free media prior to treatment with IL-1 (R&D Systems, Oxon, UK), dexamethasone (Sigma Chemicals, Poole, UK) or pretreated for 1 h with dexamethasone before IL-1 treatment. After treatment for 0.5, 1, 2, 6, or 24 h the cells were then harvested and total protein extracted by the addition of 10 µl Gough 1 solution to the cell pellet and subjected to two freeze/thaw cycles in order to fracture nuclear membranes.

Before loading onto 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, samples were denatured by boiling for 5 min. Total protein (30 µg) was then size fractionated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Colored molecular weight markers were also run to assess product size. After electrophoresis, proteins were transblotted onto Hybond-ECL nitrocellulose membranes (Amersham Life Sciences, Amersham, UK) in blotting buffer (200 mM glycine, 50 mM TRIS.Base, 20% [vol/vol] methanol) at 400 mA for 1 h. Equality of protein loading and transfer was confirmed by Ponceau red staining.

For immunoblotting, the membranes were blocked for 1 h with a 10% (wt/vol) nonfat milk solution in PBS/T (PBS, 0.05% [vol/vol] Tween 20) before incubating for 1 h with rabbit anti-human p65 (1: 2,500) (Santa Cruz Biotechnologies, Santa Cruz, CA). Membranes were washed for 6 × 5 min in PBS/T and incubated for a further hour with goat anti-rabbit horseradish peroxidase-linked IgG (1:4,000) (Dako, High Wycombe, UK). The filters were then washed again for 6 × 5 min. Finally, antibody-labeled proteins were detected by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham Life Sciences). Quantification of band density was assessed by laser densitometry.

Statistical Analysis

Results are expressed as means ± SEM. Statistical analysis was performed using the Wilcoxon matched paired test and a p value less than 0.05 was taken as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Lung Function and Eosinophils in BALF and Biopsies

There were no significant differences in FEV₁ between the placebo- and fluticasone-treated periods but mean log PC₂₀ increased significantly from 0.49 ± 0.11 to 0.13 ± 0.20 (p < 0.05) after fluticasone treatment but not after placebo. No carry-over effect was observed. There was no difference in the total cell count between the two treatments (11.7 ± 1.4 versus 11.9 ± 1.3 × 10⁴/ml BALF). There was a decrease in the percentage of eosinophils in BALF after treatment with fluticasone (3.1 ± 0.8%) compared with placebo (0.8 ± 0.3%; p = 0.002; Figure 1), together with an increase in the percentage of macrophages following fluticasone (89.5 ± 1.6%) compared with placebo (95.0 ± 1.3%, p = 0.002). No differences in percentages of neutrophils and lymphocytes were observed. MBP immunoreactivity in the submucosa of biopsies after the fluticasone treatment was reduced compared with that after placebo treatment (1.6 ± 0.5 versus 5.4 ± 0.7, p = 0.002; Figure 1); a reduction in MBP-immunoreactive cells in the airway epithelium after fluticasone was also observed (0.5 ± 0.2 versus 1.5 ± 0.4, p = 0.04; Figure 2). Examples of immunoreactive MBP⁺ cells in the epithelium and submucosa during placebo and fluticasone treatment are shown on Figure 3.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Bronchial responsiveness to methacholine expressed as log PC20, the concentration of methacholine needed to cause a 20% fall in baseline FEV1, before and after either placebo or fluticasone treatment. Placebo had no significant effect, but fluticasone caused a significant increase in log PC20. There were no significant changes in FEV1 either after placebo or fluticasone. *p = 0.03 compared with prefluticasone measurement.

View larger version (19K): [in this window] [in a new window]   Figure 2.   BAL cells ( panel A) and eosinophil immunoreactivity in bronchial biopsies ( panel B) after treatment with placebo or fluticasone. There was a significant decrease in the percentage of eosinophils following fluticasone with no significant change in the total cell counts. After fluticasone, the immunoreactivity scores for MBP-staining cells were reduced in both the submucosa and in the epithelium. *p < 0.02, **p < 0.002.

View larger version (96K): [in this window] [in a new window]   Figure 3.   High-power photomicrographs (×200 original magnification) of eosinophils as measured by staining with the anti-MBP antibody selected from two asthmatic patients, obtained during placebo treatment ( panels A1 and A2), and during treatment with fluticasone ( panels B1 and B2). Eosinophils are stained black using the glucose oxidase-nickel enhancement technique. After treatment with fluticasone, there was a dramatic reduction in the number of MBP+ cells both in the airway epithelium and submucosa. Arrows indicate the presence of eosinophils in the airway epithelium.

NF-B DNA-binding in Alveolar Macrophages and Bronchial Biopsies

EMSAs were not performed in one subject for alveolar macrophages and two subjects for bronchial biopsies, owing to inadequate nuclear protein yields. All EMSAs showed a specific retarded band because excess cold NF-B oligonucleotide but not Oct-1 oligonucleotide competed out the NF-B DNA-binding in these nuclear extracts from alveolar macrophages or bronchial biopsies (Figure 4). There were no significant differences in NF-B DNA-binding of nuclear protein (2 µg) obtained from alveolar macrophages after fluticasone compared with placebo for alveolar macrophages (1.5 ± 0.3 versus 2.8 ± 1.0 in 14 subjects) or bronchial biopsies (0.8 ± 0.2 versus 0.7 ± 0.1 in 13 subjects; Figure 4).

View larger version (44K): [in this window] [in a new window]   Figure 4.   EMSA of nuclear proteins extracted from alveolar macrophages ( panel A) and from bronchial biopsies ( panel B) obtained after placebo (P) or fluticasone (F). For each panel, representative EMSAs are shown for five separate subjects (1 to 5) in panel A and from three subjects (1 to 3) in panel B on the right. Individual optical density data of the NF-B-retarded band expressed as a ratio of µg protein loaded (with mean ± SEM) obtained during fluticasone or placebo treatment are on the left panels. NF-B binding is demonstrated by the intensity of the retarded band (right) and specificity of the binding was confirmed by the addition of 100-fold excess of cold double-stranded NF-B oligonucleotide obtained from Subject 1 (XS NF-B) which inhibited binding as compared with a sample with excess cold double-stranded Oct-1 oligonucleotide also from Subject 1 (XS Oct-1). There was no significant difference in NF-B DNA-binding observed between the two treatments.

p65 Protein Expression in Bronchial Biopsies

There was no background staining when the anti-p65 NF-B antibody was omitted. Staining was observed in the cytoplasm of airway epithelial cells in each subject, and only very occasionally in submucosal cells. Staining was also present in the nuclei of airway epithelial cells representing the activated form of NF-B (Figure 5). After placebo, up to 42.4 ± 5.9% of epithelial cells showed positive nuclear staining compared with 63.1 ± 6.2% after fluticasone treatment (p = 0.04; Figure 5). A similar increase was observed following fluticasone when the number of positive nuclear-staining cells were expressed per mm² of epithelial area (fluticasone = 64.1 ± 8.0 versus placebo = 34.1 ± 5.0; p = 0.002). There were no differences in the size of nucleated epithelial cells in biopsies taken after placebo (1.4 ± 0.2 × 10² µm²) or fluticasone (1.2 ± 0.2 × 10² µm²).

View larger version (48K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Figure 5.   (Top panels) Representative high-power photomicrographs of biopsy sections showing immunostaining for anti-p65 within one subject. Using an anti-p65 antibody, positive nuclear staining was observed in a few epithelial cells in a subject after placebo treatment for 1 mo (top left panel ). After 1 mo treatment with inhaled glucocorticoids, there was an increase in p65 nuclear staining (top right panel ). (Bottom panels) Individual numbers of nuclei with mean ± SEM in the airway epithelium staining with the anti-p65 antibody in sections of bronchial biopsies from asthmatic patients during treatment with placebo or fluticasone. There was a significant increase in p65-positive nuclear epithelial cells expressed either as percent of the total number of epithelial cells or as number of cells per mm2 of epithelial cell area. *p < 0.05, **p < 0.002 compared with placebo.

In the submucosal area, there were very few p65-positive staining cells. Thus, in the biopsies obtained during placebo, we found no such cells in 12 of 15 biopsies, whereas 46 cells were counted per 4 high-power fields in the three other biopsies. On average, there were 0.8 ± 0.4 p65-positive cell per high-power field. Biopsies obtained during the corticosteroid treatment period showed 0.6 ± 0.3 p65-positive cells per high-power field. Therefore, eosinophils did not show positive staining for p65.

Effect of Dexamethasone and IL-1 on A549 Cells

Total p65 protein expression was increased after a 24-h treatment with IL-1 and dexamethasone together (p = 0.02), but with IL-1 alone (Figure 6). No differences in p65 protein expression were observed at the earlier time-points.

View larger version (26K): [in this window] [in a new window]   Figure 6.   Effect of IL-1 and dexamethasone (Dex) on the expression of p65 protein as measured by Western blot analysis in pulmonary A549 cells stimulated with IL-1, Dex or pretreated for 1 h with Dex before IL-1 treatment. Total protein was harvested at the times indicated for polyacrylamide gel electrophoresis and immunoblotted for p65. Representative blots are shown ( panel A). After densitometric analysis, data (n = 8) were expressed as fold-increase above untreated cells at 24 h and presented as means ± SEM ( panel B). *p < 0.05 compared with other groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Inhaled fluticasone reduced the number of eosinophils in BALF and in bronchial biopsy specimens obtained from patients with mild asthma. Concomitantly, there was an improvement in the degree of bronchial hyperresponsiveness, although FEV₁ did not improve, which is likely a result of the relatively normal values of FEV₁ in these subjects. Despite these effects of fluticasone, we did not detect any changes in the binding of the NF-B heterodimeric component, p65, in terms of its binding capacity to DNA using electrophoretic migration shift assay of nuclear proteins from alveolar macrophages or bronchial biopsies. In addition, staining of bronchial biopsies with an anti-p65 antibody did not reveal a significant decrease in epithelial nuclear staining after fluticasone treatment. On the contrary, there was an increase in p65 staining of nuclei in the epithelium after inhaled steroid therapy. In vitro studies on A549 epithelial cells also showed that corticosteroids increased the expression of p65 when the cells were stimulated with IL-1. Our data do not provide support for the actions of glucocorticoids occurring through the binding of its activated receptor to the transcription factor, NF-B, because no reduction in the binding of NF-B or its activation was apparent after 1 mo of treatment with a potent inhaled corticosteroid.

In a previous study, we examined a similar group of mild asthmatic patients and found that, in comparison to a group of normal nonasthmatic subjects, there was increased binding of nuclear proteins from alveolar macrophages and bronchial biopsies of asthmatics to double-stranded oligonucleotides encoding the consensus target sequence of NF-B (21). In addition, there were more nuclei staining with an anti-p65 antibody in the airway epithelium and in macrophages from induced sputum of these asthmatic patients compared with normal subjects, indicating increased activation of NF-B (21). The mechanisms by which NF-B is activated in asthma are not known. Activation of NF-B may occur through oxidative mechanisms such as exposure to environmental oxidant pollutants (36), or through the release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-) or IL-1 (16, 29, 37), which are expressed in asthmatic airways (38, 39). Other mechanisms may include upper respiratory tract virus infections (40).

The reported effects of corticosteroids on induced NF-B DNA-binding activity in vitro have been diverse. Corticosteroids have been shown to inhibit the activation of NF-B induced by TNF- or IL-1 in blood monocytes or human lung in vitro (16, 28), and in the lungs of rats exposed to ozone (41). However, other studies demonstrated no effect on NF-B DNA-binding activity induced by IL-1 in endothelial cells and epithelial A549 cells (29, 31); enhancement of this activity by corticosteroids has also been reported when cells were activated with the phorbol ester, phorbol myristate acetate (30). These stimuli represent acute increases in NF-B activation rather than the chronic increased NF-B expression; by contrast, chronic activation as observed in asthma was not inhibited by chronic inhaled glucocorticoid therapy. It is also possible that the effect of inhaled glucocorticoids may have only been transient on the initial doses with persistence of activation on continued use of inhaled corticosteroids. Other transcription factors such as c-fos which is a component of the heterodimer, (AP-1), are also overexpressed in asthmatic epithelium (42), and the effect of inhaled corticosteroids on AP-1 expression in chronic asthma is not known. Corticosteroids are known to inhibit AP-1 activation in human lung in vitro (43) and c-fos immunoreactivity was reduced in the mucosa of nasal polyps from subjects with nasal obstruction treated with topical glucocorticoids (44).

Of interest is the immunohistochemical increase in p65 expression in the airway epithelium observed after topical corticosteroid therapy. This increase was not related to changes in size of the epithelial cells after fluticasone treatment because there were no significant changes in airway epithelial cell size with corticosteroid treatment, and therefore no changes in epithelial cell number. We do not have any plausible explanation for the increase in p65 expression. In studies in A549 pulmonary epithelial cell line, dexamethasone had no effect on the expression of NF-B activation induced by IL-1 (29). This is similar to the report in endothelial cells (31). In this study, we also found that p65 protein expression was increased in vitro in A549 pulmonary epithelial cells by corticosteroid treatment in the presence of IL-1. One possibility is that the transcription of NF-B may be increased by corticosteroids. NF-B activity is regulated by sequestration of NF-B heterodimers in the cytoplasm as inactive complexes with the inhibitory molecules, I-B, thereby inhibiting NF-B-DNA binding activation (45). Glucocorticoids may rapidly induce I-B messenger RNA (mRNA) and protein synthesis (27), and therefore I-B may mediate the effects of corticosteroids. We have not yet examined the expression of I-B in our biopsies.

In summary, inhaled corticosteroid therapy of patients with mild asthma led to a reduction in eosinophilic inflammation without a reduction in NF-B expression and activation. On the contrary, there was an increase in p65 expression as determined by immunohistology. This observation was reproduced in vitro in A549 epithelial cell line. The significance of this increase in p65 expression is not known. Our observations indicate that corticosteroids may not mediate its anti-inflammatory actions through inhibition of NF-B, but possibly through interactions with other transcription factors, or through other mechanisms.