Abstract
Rationale: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma.
Objectives: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)β (a splicing variant, and dominant negative inhibitor of, the classic GCRα) in controlling GCRα nuclear translocation and transactivation at a molecular level.
Methods and Measurements: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRα cellular shuttling in response to 10−6 M dexamethasone treatment and GCRβ expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRβ mRNA on GCRα function were assessed.
Main Results: Significantly reduced nuclear translocation of GCRα in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRβ. It was demonstrated that GCRα nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRβ gene into the DO-11.10 cell line. RNA silencing of GCRβ mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRα transactivation.
Conclusions: GC insensitivity is associated with loss of GCRα nuclear translocation in BAL cells and elevated GCRβ, which may inhibit GCRα transactivation in response to steroids.
Glucocorticoids (GCs) are currently the most effective agents for the treatment of inflammation (1). Although the majority of patients respond to GC therapy, up to 25% of patients demonstrate persistent tissue inflammation despite treatment with high doses of GCs (2, 3). GC insensitivity has been widely recognized as complicating the management of chronic inflammatory diseases, such as asthma, inflammatory bowel disease, and autoimmune diseases (1, 4).
The antiinflammatory effects of GCs are mediated through GC receptor α (GCRα), which acts as a ligand-dependent transcription factor (4, 5). GCs interact with GCRα in the cytoplasm. Under GC-responsive conditions, this results in translocation of the hormone–receptor complex into the cell nucleus, and binding of the GCRα to specific DNA response elements within the promoter region of GC-responsive genes to enhance transcription of antiinflammatory genes (transactivation).
Identification of the markers of GC insensitivity is important to be able to minimize side effects from high-dose steroid therapy and prospectively to provide alternative therapeutic approaches to such patients for better treatment outcomes. In humans, alternative splicing of the ninth exon of GCR pre-mRNA results in GCRα and GCRβ proteins that are divergent at the carboxyl terminus (5). The two proteins are 94% identical, but the GCRβ isoform fails to bind hormone or to activate gene expression. Thus, GCRβ functions as a dominant negative inhibitor of GCRα (6). GCRβ has a longer half-life than that of GCRα (7), and its expression is enhanced by proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) (8), combination IL-2 and IL-4 (9), and IL-13 (10). Increased expression of the GCRβ isoform relative to the ligand-binding isoform, GCRα, has been previously reported to be associated with GC insensitivity in several inflammatory cell types (10–12), making GCRβ a potentially attractive marker of GC insensitivity. However, the precise physiological role of GCRβ has been controversial. Several studies have found elevated GCRβ levels in association with GC insensitivity in a variety of the diseases (12–16). Some investigators have argued, however, that under physiological conditions the number of GCRα copies in the cell predominates over the number of copies of GCRβ, making it unlikely that it could have any functional inhibitory effect (17, 18).
Most studies on GC-insensitive asthma have used peripheral blood mononuclear cells (PBMCs) or cell lines to demonstrate potential mechanisms of GC insensitivity in asthma (8, 19–21). The current study examines the functional response to corticosteroids of bronchoalveolar lavage (BAL) airway cells from patients with GC-insensitive and GC-sensitive asthma, thus allowing us to investigate GC insensitivity at the target organ level. Because the initial step in the classic GC signaling pathway is translocation of GCRα from the cytoplasm to the nucleus, decreased nuclear translocation is a plausible molecular mechanism of GC insensitivity. Our current study was designed to address the hypothesis that individuals with GC-insensitive asthma as compared with those with GC-sensitive asthma have reduced GCRα nuclear translocation in response to GCs in BAL cells from sites of airway inflammation. As well, we attempt to define the functional role of GCRβ in controlling GCRα nuclear translocation and transactivation at a molecular level.
Patients with a diagnosis of asthma according to American Thoracic Society criteria (22) were selected for evaluation. Patients with asthma had a baseline FEV1 of 55 to 85% of predicted, a β2-adrenergic response of at least 12% of baseline FEV1, and/or a provocative concentration of methacholine causing a 20% fall in FEV1 not exceeding 8 mg/ml. None of the subjects had received systemic GC therapy for at least 1 mo before bronchoscopy (Table 1). The corticosteroid response of patients with asthma was classified on the basis of their prebronchodilator morning FEV1% predicted response to a 1-wk course of oral prednisone (40 mg/d). Patients with asthma were defined as GC insensitive if they had less than 15% improvement in FEV1, and as GC sensitive if they showed significant improvement (> 20%). None of the subjects with asthma had evidence of other types of lung diseases. All patients who were recruited for the study were not smoking for at least 1 yr before this study. Disease severity was characterized in both groups on the basis of baseline and postbronchodilator FEV1% predicted, number of nocturnal events per month, rescue short-acting β-agonist use, and controller medication use (Table 1). Informed consent was obtained from all patients before enrollment in this study. The Institutional Review Board of the National Jewish Medical and Research Center (Denver, CO) approved this study.
Subjects with Asthma | |||
|---|---|---|---|
| Parameter | GC Insensitive | GC Sensitive | |
| Number of subjects | 8 | 7 | |
| Age, yr (mean ± SD) | 40.1 ± 9.3 | 35.7 ± 4.0 | |
| Sex, M/F | 2/6 | 5/2 | |
| Atopic subjects* | 8/8 | 7/7 | |
| Active smokers | 0 | 0 | |
| Prior smokers, no. (pack-years) | 1 (2.5) | 1 (< 1.0) | |
| Prebronchodilator FEV1% predicted before prednisone burst (mean ± SEM) | 66.0 ± 3.3 | 68.1 ± 2.9 | |
| Prebronchodilator FEV1% predicted after prednisone burst (mean ± SEM) | 64.7 ± 3.5† | 83.6 ± 3.6 | |
| Postbronchodilator (albuterol) FEV1% predicted (mean ± SEM) | 73.4 ± 4.2 | 82.8 ± 5.2 | |
| Age at asthma onset, yr (mean ± SEM) | 21.6 ± 5.0 | 10.9 ± 4.1 | |
| Duration of asthma, yr (mean ± SEM) | 18.5 ± 6.3 | 24.6 ± 9.3 | |
| Nocturnal symptoms, events/mo (mean ± SEM) | 2.5 ± 1.7 | 5.7 ± 4.1 | |
| Medications | |||
| Rescue β2-agonists, puffs/d (mean ± SEM) | 1.7 ± 0.4 | 2.3 ± 0.9 | |
| ICS | 3/8 | 3/7 | |
| ICS + LABA | 2/8 | 0/7 | |
| Systemic CS | 0 | 0 | |
| Other maintenance medications | None | None | |
PBMCs were isolated from heparinized blood by Ficoll-Hypaque (Pharmacia Biotech, Piscataway, NJ) gradient centrifugation as previously described (23). Fiberoptic bronchoscopies with BAL were performed according to the guidelines of the American Thoracic Society (24). BAL cells were filtered through a 70-μm Nylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ), spun at 200 × g for 10 min, washed two times, and resuspended in Hanks' balanced salt solution. Cytospin preparations were made, and differential counts of BAL cells were performed after staining with Diff-Quik (Scientific Products, McGraw Park, IL), counting a minimum of 500 cells.
For this study, cells were resuspended in RPMI 1640 (BioWhittaker) containing 10% heat-inactivated, charcoal-filtered, GC-free fetal bovine serum (FBS; Gemini Bio-Products, Calabasas, CA), l-glutamine (40 μmol/L), penicillin (100 U/ml), streptomycin (100 U/ml), and N-2- hydroxyethylpiperazine-N′-ethane sulfonic acid (20 mmol/L) buffer solution (GIBCO-BRL/Life Technologies, Rockville, MD).
In these experiments, human BAL cells (1 × 105/ml) were stimulated with LPS (10 ng/ml) or zymosan (100 particles/cell) in the absence or presence of 10−9 to 10−6 M dexamethasone (DEX) for 24 h. Supernatants were collected and stored at −80°C before analysis. The ability of DEX to inhibit production of IL-6 and TNF-α in culture supernatants was measured by ELISA according to the manufacturer's recommendations (R&D Systems, Minneapolis, MN).
GCRα intracellular shuttling in response to 10−6 M DEX (Sigma, St. Louis, MO) treatment was analyzed by immunostaining. BAL cells were seeded at 1×106 cells/ml on poly-d-lysine–coated coverslips. The cells were treated with 10−6 M DEX or cultured in medium alone for 3 h, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized for 15 min at room temperature in permeabilization solution (PBS containing 0.1% [vol/vol] Tween 20, 0.1% [wt/vol] bovine serum albumin [Sigma], and 0.01% [wt/vol] saponin [Sigma]), and blocked with a commercial blocking solution (Super Block; ScyTek Laboratories, Logan, UT) for 15 min at room temperature. The cells were then incubated with an affinity-purified polyclonal antibody to GCRα (Affinity BioReagents, Golden, CO) diluted in permeabilization solution (1:250) overnight at 4°C, washed, and then incubated with secondary antibody (donkey anti-rabbit IgG, F[ab′]2 fragment, Cy3 conjugated, diluted 1:200; Jackson Laboratories, West Grove, PA). Nuclei were counterstained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 1 h at room temperature. The cells were then washed and mounted on slides. Purified nonimmune rabbit IgG (SouthernBiotech, Inc., Birmingham, AL) was used as an isotype control. The slides were analyzed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany) with imaging software (SlideBook; Intelligent Imaging Innovations, Denver, CO) and expressed as a nuclear:cytoplasmic ratio of the mean fluorescence intensity (MFI) of Cy3 staining (GCR) of BAL cells as described previously (20).
GCRβ expression by BAL cells was analyzed by immunofluorescence staining. Cells were fixed, permeabilized, and blocked as described above. The cells were then incubated overnight at 4°C with an affinity-purified polyclonal antibody to GCRβ (Affinity BioReagents) diluted in permeabilization solution (1:750), washed, and then incubated with secondary antibody (donkey anti-rabbit IgG, F[ab′]2 fragment, Cy3 conjugated, diluted 1:200). Nuclei were counterstained with 300 nM DAPI for 1 h at room temperature. The cells were then washed and mounted on slides. Purified nonimmune rabbit IgG (SouthernBiotech, Inc.) or synthetic GCRβ antibody–neutralizing peptide N(728)VMWLKPESTSHTLI(742)C (Affinity BioReagents) was used to control the specificity of staining.
BAL cells were preserved in RLT buffer (provided with RNeasy mini kit; Qiagen, Valencia, CA) immediately after isolation. RNA was extracted according to the guidelines of the manufacturer (Qiagen), transcribed into cDNA, and analyzed by real-time polymerase chain reaction (PCR), using the dual-labeled fluorigenic probe method and an ABI PRISM 7000 sequence detector (Applied Biosystems, Foster City, CA) as described (25). Primers and probes for human mitogen-activated protein kinase phosphatase-1 (MKP-1) mRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S RNA were purchased from Applied Biosystems. GCRα and GCRβ primers based on the sequences published by DeRijk and coworkers (26) were custom ordered from Applied Biosystems. Standard curves for MKP-1, GAPDH, and 18S RNA were generated on the basis of fluorescence data from twofold serial dilutions of total RNA from the sample providing the highest expression level. GCRα and GCRβ standard curves were generated from 10-fold serial dilutions of the GCR plasmids. Quantities of each target gene in test samples were normalized to the corresponding levels of the housekeeping genes (18S RNA and GAPDH) in each sample.
cDNA encoding the human GCRβ isoform (base pairs 23–2296) was subcloned into the replication-defective murine stem cell virus (MSCV) retroviral vector as a bicistronic coding unit containing the gene encoding green fluorescent protein (GFP), followed by the encephalomyelitis virus internal ribosome entry site and the GCRβ-coding region, as described (27). Phoenix packaging cells were transiently transfected with the expression vectors for GCRβ and GFP, using calcium phosphate precipitation. Culture supernatants from transfected Phoenix cells producing recombinant MSCV were used to transduce DO-11.10 hybridoma cells by spinfection as described (27). DO-11.10 hybridoma cells transduced with human GCRβ/GFP were sorted for GFP+ cells (MoFlo cell sorter; Dako, Fort Collins, CO). For further experiments, the resulting GFP+ cell populations were sorted for GFPdim and GFPbright cells. After gating on live DO-11.10 hybridoma cells according to forward and side scatter and doublet exclusion, GFPdim cells were defined as the 3 to 4% of gated live cells expressing GFP at the lowest fluorescence intensity. Accordingly, GFPbright cells were defined as the 3 to 4% of gated live cells expressing GFP at the highest fluorescence intensity. The sorted cells were then cultured in 10% FBS–RPMI medium. GCRα nuclear translocation in response to DEX and MKP-1 induction by DEX were evaluated in wild-type and transgenic GCRβdim and GCRβbright DO-11.10 cells.
Analysis of GCRα nuclear translocation and GCRβ localization in DO-11.10 cells was performed by Western blot. Western blot was also used to analyze GCRβ expression in fractionated BAL macrophages from patients with asthma. Nuclear and cytoplasmic extracts from cells were prepared with an NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce Biotechnology, Rockford, IL).
Western blotting was performed as previously described (20). Membranes were blotted with anti-GCRα (P-20; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-GCRβ (Abcam, Cambridge, MA) antibodies. To control the quality of nuclear and cytoplasmic protein preparation the membranes were stripped and reprobed with anti–NF-1 and anti–β-tubulin antibodies (Santa Cruz Biotechnology) as nuclear and cytoplasmic proteins, respectively.
Small interfering RNA (siRNA, targeting GCRβ, annealed) was custom designed and synthesized by Ambion, Inc. (Austin, TX). The sequence targeting human GCRβ was as follows: sense, 5′-GGCUUUUCAUUA AAUGGGAtt-3′; antisense, 5′-UCCCAUUUAAUGAAAAGCCtc-3′. To be able to estimate the efficiency of transfection, siRNA was labeled with a Cy5 siRNA labeling kit (Ambion) in accordance with the manufacturer's recommendations. The specificity of silencing was controlled by nonsilencing control siRNA (Ambion).
Freshly isolated BAL cells from patients with GC-insensitive asthma were transfected with siRNA in a Nucleofector device (Amaxa, Cologne, Germany), using a Nucleofector human monocyte kit (Amaxa). Briefly, 1 μg of GCRβ siRNA was added to 3 × 106 cells that had been previously washed with PBS and resuspended in 100 μl of human monocyte kit transfection solution. Cells were subjected to nucleofection, using the Y001 program (Amaxa). Control cells were either mock transfected or transfected with 1 μg of the nonsilencing control siRNA (Ambion) (negative control). Transfected cells were immediately diluted with prewarmed monocyte growth medium (Amaxa) supplemented with 10% heat-inactivated, charcoal-filtered, GC-free FBS with l-glutamine (40 μmol/L) and cultured in 24-well plates or on poly-d-lysine–coated round coverslips in 24-well plates (1 ml/well). After 20 h, transfection efficiency was estimated by fluorescence microscopy. The cells were treated with or without 10−6 M DEX for an additional 4 h and harvested for RNA isolation or fixed on slides. GCRβ knockdown was ascertained by quantitative real-time PCR and immunostaining as described above. To estimate the effect of the GCRβ silencing on steroid responses, MKP-1 induction by DEX in all test groups was analyzed as well.
Parametric-based statistical procedures (namely, t tests and linear mixed models) were used for analysis of outcome variables, which tended to be symmetrically distributed and without extreme outliers. Regarding two-sample t tests: in cases with extreme differences in variance between asthmatic groups, the unequal variance test was used. Regarding linear mixed model analyses: a spatial exponential covariance structure was used to model within-subject repeated measures over time (because time points were unequally spaced), and a compound symmetric covariance structure was used to model within-subject repeated measures over various treatments or between sites. In the linear mixed models, two-way interactions between predictors were examined. Select intergroup comparisons (e.g., comparing patients with GC-insensitive asthma and patients with GC-sensitive asthma at specific DEX concentrations) were conducted if related main effect or interaction terms were significant (p < 0.05). All reported p values are related to two-sided tests. SAS software (version 9.1; SAS Institute, Cary, NC) was used to carry out mixed model analyses. Data are expressed as means ± SEM.
The characteristics of patients who enrolled into this study are shown in Table 1. Patients were divided into GC-insensitive and GC-sensitive groups based on FEV1% predicted responses after a 1-wk burst with oral prednisone. Patients in the GC-insensitive group did not show any improvement in FEV1 after exposure to prednisone (p = 0.431, as compared with FEV1% predicted before steroid burst); in contrast, patients in the GC-sensitive group showed significant improvement in their lung function after steroid burst (p = 0.00008, as compared with FEV1% predicted before steroid burst; Table 1). During the study, the patients continued to use inhaled steroids, but were asked to withdraw them 24 h before bronchoscopy.
In terms of asthma severity both groups were equivalent (Table 1), with one ex-smoker per group. Baseline FEV1% predicted was 66.0 ± 3.2 in the GC-insensitive group and 68.1 ± 2.9 in the GC-sensitive group (p = 0.24). Postbronchodilator FEV1% predicted was 73.4 ± 4.2 and 82.8 ± 5.2%, respectively (p = 0.09). Symptom severity measured by nocturnal events were 2.5 ± 1.7/mo in the GC-insensitive group and 5.7 ± 4.1/mo in the GC-sensitive group (p = 0.24). Group similarities were also reflected in rescue, short-acting β2-agonist use, 1.7 ± 0.4 versus 2.3 ± 0.9 puffs/d, respectively (p = 0.26). Controller medication in the GC-insensitive group occurred in five of eight patients, with three using inhaled corticosteroid alone and two using inhaled corticosteroid and a long-acting β2-agonist. In the GC-sensitive group, controller medication occurred in three of seven patients and consisted only of inhaled corticosteroid.
The number of total white cells in BAL samples varied between patients (mean total white cell counts for GC-insensitive and GC-sensitive groups were [11.9 ± 3.1] × 106 and [17.9 ± 1.5] × 106, respectively). However, the percentages of macrophages, lymphocytes, neutrophils, and eosinophils did not differ between the two groups (Table 2). Macrophages composed a mean percentage of 89.1 ± 3.6 and 91.9 ± 1.5%, lymphocytes composed 9.4 ± 3.6 and 6.2 ± 1.2% in BAL samples from GC-insensitive and GC-sensitive asthma study groups, respectively.
Subjects with Asthma | |||
|---|---|---|---|
| Cell Type | GC Insensitive | GC Sensitive | |
| Total white blood cells, × 106, median (range) | 9.36 (1.88–26.72) | 18.22 (12.73–19.88) | |
| Macrophages/monocytes, % (mean ± SEM) | 89.1 ± 3.6 | 91.9 ± 1.5 | |
| Polymorphonuclear cells, % (mean ± SEM) | 0.5 ± 0.2 | 0.5 ± 0.2 | |
| Lymphocytes, % (mean ± SEM) | 9.4 ± 3.6 | 6.2 ± 1.2 | |
| Eosinophils, % (mean ± SEM) | 0.9 ± 0.3 | 1.4 ± 0.7 | |
The production and suppression by DEX of proinflammatory cytokines by BAL cells from GC-insensitive and GC-sensitive patients after 24 h of stimulation with LPS or zymosan was analyzed. The presence of IL-6 and TNF-α in culture supernatants was measured by ELISA. It was found that within the GC-insensitive group production of IL-6 and TNF-α by BAL macrophages was not suppressed as effectively by DEX as compared with the GC-sensitive group (Figure 1). The reduced inhibitory effect of DEX on cytokine release was observed in BAL cells of GC-insensitive patients both after LPS and zymosan stimulation (Figure 1). Thus, suppression of cytokine production by GCs paralleled the clinical responsiveness of patients to GCs.
Figure 1. Suppression of bronchoalveolar lavage (BAL) macrophage production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by dexamethasone (DEX) in patients with glucocorticoid (GC)-insensitive asthma (n = 8; solid triangles) and patients with GC-sensitive asthma (n = 7; solid squares) 24 h after LPS (A and B) or zymosan (C and D) stimulation. *p < 0.05, **p < 0.01, ***p < 0.001.
[More] [Minimize]To gain insights into why GCs did not suppress cytokine production by airway macrophages from GC-insensitive groups, GCRα cellular shuttling in response to GCs was analyzed in the BAL cells of patients with asthma. GCRα nuclear translocation in BAL cells from patients with asthma was assessed in response to 10−6 M DEX (3 h) by immunofluorescence staining (Figure 2). In the absence of DEX, GCRα was localized mainly to the cell cytoplasm of BAL cells. This corresponded to a GCRα MFI nuclear:cytoplasmic ratio lower than 1. BAL cells from GC-sensitive patients demonstrated GCRα translocation in response to DEX (n = 7) (statistically significant change in the GCRα nuclear:cytoplasmic ratio was observed; p < 0.001 as compared with the absence of DEX). However, in BAL cells from patients with GC-insensitive asthma, GCRα failed to translocate and was still localized to the cytoplasm of studied cells (n = 6 patients; no difference in the GCRα MFI nuclear:cytoplasmic ratio was found; p = 0.82 as compared with the absence of DEX). The GCRα MFI nuclear:cytoplasmic ratio after 3 h of DEX treatment was (mean ± SD; 0.88 ± 0.10 for the GC-insensitive group, p < 0.001 compared with the GC-sensitive asthma group) and 1.31 ± 0.12 for the GC-sensitive patients (Figure 2).
Figure 2. Impaired GCRα nuclear translocation in BAL macrophages from GC-insensitive patients (original magnification, ×400; blue [DAPI], nuclear staining; red [Cy3], GCRα). Representative images of GCRα cellular shuttling in BAL macrophages from patients with GC-insensitive asthma (A) and patients with GC-sensitive asthma (B) in response to 3 h of 10−6 M DEX treatment in vitro are shown. Note the cytoplasmic localization of GCRα in the cells after DEX treatment of the patient with GC-insensitive asthma versus nuclear localization of GCRα in the cells from the patient with GC-sensitive asthma, respectively (arrows). The mean fluorescence intensity (MFI) of Cy3 staining (GCRα) of BAL cells from GC-insensitive patients (C) and GC-sensitive patients (D) was assessed by analysis software within the computer-generated masks for nuclear and cytoplasmic regions of the cells and is expressed as the GCRα (Cy3) nuclear:cytoplasmic ratio. Fifty to 100 cells were analyzed for each patient studied.
[More] [Minimize]Previous literature on cell lines has reported that GCRβ is localized primarily to the nucleus (28, 29). However, our immunostaining studies revealed that this is not true for human monocytes/macrophages, where GCRβ is distributed equally between the cytoplasm and cell nuclei (Figure 3A). To validate the fluorescence methods regarding intracellular distribution of GCRβ nuclear and cytoplasmic fractions were prepared from freshly isolated BAL macrophages. GCRβ expression in these fractions was examined by Western blot. The quality of fractionation was confirmed by stripping and reprobing membranes for β-tubulin, which is known to have only a cytoplasmic distribution (Figure 3C). These experiments confirmed fluorescence microscopy data showing nuclear and cytoplasmic expression of GCRβ in human macrophages. Because cell staining in BAL samples indicated that macrophages were the predominant cell type in BAL samples (Table 2), we analyzed whether GCRβ can influence steroid responses of BAL macrophages.
Expression of GCRα and GCRβ isoforms by BAL samples was analyzed by real-time PCR. A significant increase in GCRβ mRNA expression by BAL cells from patients with GC-insensitive asthma as compared with the GC-sensitive group was found (Figure 3D). In contrast, there was no difference in GCRα expression by BAL cells from the two asthma groups (Figure 3E). This effect appeared to be BAL specific because no difference in GCRβ and GCRα mRNA expression was observed in purified peripheral blood monocytes from GC-insensitive and GC sensitive asthma groups (GCRβ, 0.0015 ± 0.003 and 0.0011 ± 0.003 fg/ng 18S RNA; GCRα, 0.0005 ± 0.0001 and 0.0010 ± 0.0003 pg/ng 18S RNA for monocytes from patients with GC-insensitive asthma [n = 3] and patients with GC-sensitive asthma [n = 3], respectively).
Figure 3. Increased expression of GCRβ in BAL macrophages of patients with GC-insensitive asthma. GCRβ expression in BAL macrophages from patients with asthma was analyzed by immunofluorescence staining (A and B) (original magnification, ×400; blue [DAPI], nuclear staining; red [Cy3], GCRβ) and revealed significantly higher GCRβ in BAL macrophages from GC-insensitive patients. The MFI of Cy3 staining (GCRβ) in BAL cells from patients with GC-insensitive asthma and from patients with GC-sensitive asthma was assessed by analysis software within the computer-generated masks for nuclear and cytoplasmic regions of the cells. Forty to 60 cells were analyzed for each patient studied. Note the nuclear and cytoplasmic expression of GCRβ in human macrophages (A). The nuclear and cytoplasmic distribution of GCRβ in BAL macrophages was confirmed by Western blot (C). Compared with the GC-sensitive group, increased GCRβ mRNA expression (D), but not GCRα mRNA expression (E), was observed by real-time polymerase chain reaction (PCR) in freshly isolated BAL cells from patients with GC-insensitive asthma.
[More] [Minimize]In addition, our data indicated that BAL macrophages from patients with GC-insensitive asthma have significantly higher levels of both nuclear and cytoplasmic GCRβ protein expression as compared with BAL macrophages from GC-sensitive patients (GCRβ MFI was 562.5 ± 53.1*vs. 167.3 ± 18.6 and 429.9 ± 45.5*vs. 104.2 ± 7.6 in the nuclear and cytoplasmic compartments of BAL macrophages in GC-insensitive [n = 7] and GC-sensitive asthma [n = 6] groups, respectively; *p < 0.0001 compared with the GC-sensitive asthma group; Figure 3B).
Because GCRβ can form heterodimers with GCRα, but does not engage GCs because of an absent ligand-binding site (6, 7), we explored the possibility that cytoplasmic GCRβ controls GCRα nuclear translocation in response to GCs, thus influencing GCRα transactivation capacity. To more precisely demonstrate the effect of GCRβ on steroid responsiveness, we compared GCRα nuclear translocation in murine DO-11.10 cells that expressed different levels of GCRβ (Figure 4A). Mice have just one isoform of the GCR, that is, GCRα. Unlike humans, they do not express GCRβ. Therefore mice may be considered natural GCRβ knockouts (30). Murine DO-11.10 cells that were virally transduced with a GFP-GCRβ bicistronic construct (as described in Hauck and coworkers [27]) had both cytoplasmic and nuclear expression of GCRβ, with a majority of the GCRβ accumulating in the cytoplasm (Figure 4B). As shown by Western blot, we found that overexpression of GCRβ inhibits GCRα nuclear translocation in response to steroids (Figure 4C).
Figure 4. Overexpression of GCRβ controls GCRα nuclear translocation and its transactivation properties. (A) Distribution of green fluorescent protein (GFP) expression in GFP-GCRβ retrovirally transduced DO-11.10 cell lines. As shown previously (27), the level of GFP expression in transgenic DO-11.10 cells correlates with GCRβ expression, because GCRβ cDNA was subcloned into the viral vector as a bicistronic coding unit containing GFP. (B) GCRα has cytoplasmic localization in wild-type (WT) and GCRβ-expressing DO-11.10 cells. GFP GCRβdim transgenic cells have nuclear and cytoplasmic localization of GCRβ, with the majority of protein residing in the cell cytoplasm. To control the quality of nuclear and cytoplasmic protein preparations, the membranes were reprobed with anti–NF-1 and anti–β-tubulin antibodies as nuclear and cytoplasmic proteins, respectively. (C) GCRα nuclear shuttling in response to DEX is impaired in the cells expressing GCRβ. (D) GCRα transactivation properties are proportionately reduced by GCRβ expression. DO-11.10 wild-type, GFP GCRβdim, and GFP GCRβbright cells were collected at various time points after DEX treatment. MKP-1 mRNA induction by DEX in the cell isolates as compared with medium-treated cells was analyzed by real-time PCR.
[More] [Minimize]Induction of MKP-1 by corticosteroids, measured by real-time PCR, was used to confirm these data because MKP-1 is one of the early markers induced by steroids via binding of the GCRα to glucocorticoid response element (GRE) sequences in the promoter region of the MKP-1 gene (31), and therefore reflects GC transactivation responses. We demonstrated that GCRα transactivation properties are proportionately reduced by GCRβ expression: MKP-1 induction by DEX was significantly reduced in the cells that express GCRβ (MKP-1 mRNA fold induction after 2 h of 10−7 M DEX treatment: 1.58 ± 0.05, 1.16 ± 0.11,*and 0.59 ± 0.33*in wild-type DO-11.10 cells, DO-11.10 GCRβ GFPdim cells, and DO-11.10 GCRβ GFPbright cells, respectively, n = 3; *p < 0.05 vs. wild-type cells; Figure 4D). This suggests that elevated cytoplasmic GCRβ inhibits GCRα nuclear translocation in response to DEX and reduces GCR transactivation.
Freshly isolated BAL macrophages from patients with GC-insensitive asthma were transfected with GCRβ siRNA or nonsilencing control siRNA, or remained untreated, and were cultured for 20 h, followed by treatment for 4 h with 10−6 M DEX or medium to analyze DEX-induced MKP-1 production. Using Cy5-labeled siRNA oligonucleotides, the efficiency of transfection was estimated to be about 60 to 70%, 20 h after electroporation. Twenty-four hours after transfection GCRβ mRNA expression was significantly suppressed as compared with nonsilencing control siRNA (Figure 5A). This effect was GCRβ specific and introduction of GCRβ siRNA did not alter GCRα mRNA expression (Figure 5B). Significant enhancement of DEX-induced MKP-1 mRNA production was observed when GCRβ expression by the cells was suppressed (Figure 5C) (MKP-1 fold induction by DEX was 2.7 ± 0.5 in the GCRβ siRNA group [p = 0.0168 as compared with 1.2 ± 0.3 in the medium group and 0.9 ± 0.3 in the nonsilencing control siRNA group]).
Because GCRβ silencing efficiently suppressed GCRβ expression in both cytoplasmic and nuclear compartments of the cells, we cannot separate the effect of silencing on the two aspects of GCRα action—nuclear translocation and transactivation—as both these functions of GCRα can be controlled by GCRβ. But the experiments performed demonstrate that inhibition of GCRβ expression enhances GCRα transactivation and overall steroid responsiveness.
Figure 5. Specific GCRβ siRNA enhances GCRα transactivation in BAL macrophages from patients with GC-insensitive asthma. Introduction of GCRβ siRNA into the cells resulted in specific inhibition of GCRβ (A) but not GCRα (B) mRNA expression by the cells as shown by real-time PCR. Data are expressed as the percentage of GCR mRNA expression as compared with such after introduction of nonsilencing control siRNA. (C) Silencing of GCRβ significantly increased DEX-induced MKP-1 mRNA production by BAL macrophages. The cells were transfected with siRNA or remained untreated, and were cultured for 20 h and treated with 10−6 M DEX for 4 h. MKP-1 mRNA induction by DEX as compared with medium-treated cells was analyzed by real-time PCR.
[More] [Minimize]Although the majority of patients with asthma respond favorably to inhaled and systemic steroid therapy, up to 25% of patients with difficult-to-control asthma have poor clinical responses even to high doses of systemic GCs (2, 3). Given the increase in asthma prevalence and severity worldwide, GC insensitivity has become a challenging health problem that is costly to the health care system. Understanding the mechanisms that control GC insensitivity will be important in developing alternative therapies and in minimizing side effects from long-term systemic GC therapy, by allowing early identification of biomarkers that predict GC insensitivity (32).
The current study was performed on patients with CS-insensitive asthma and patients with GC-sensitive asthma as defined by response to a 1-wk course of oral prednisone treatment. The groups were equivalent regarding asthma severity at baseline. The GC-insensitive group was not more symptomatic, did not have worse lung function, and did not use more rescue medication. Interestingly, the two groups were markedly different in age at onset of disease (although the difference observed was not significantly different, p = 0.11). Spirometry studies revealed greater postbronchodilator response in the GC-sensitive group than in the GC-insensitive group, but because of the small sample size the difference observed was not significantly different (p = 0.09). The latter observation was probably not due to recruitment of possible former smokers into the study because only two former smokers were involved in this study (one in each group). These differences in postbronchodilator response suggest that GC-insensitive, as compared with GC-sensitive, asthma is associated with decreased reversibility of airway obstruction. However, future studies powered to examine this aspect of lung function in these two forms of asthma are required before any firm conclusion can be made.
In the current article, we describe for the first time that BAL airway cells from patients with GC-insensitive asthma have reduced GCRα nuclear translocation in response to GCs. Our data in human BAL cells extend other data (Matthews and coworkers [21]) indicating that most patients respond to GCs according to the degree of PBMC GCRα nuclear translocation. Importantly, in vitro studies have demonstrated that there are several potential pathways for induction of GC insensitivity including overexpression of GCRβ, an endogenous inhibitor of GCRα (9, 11, 12), and reduced nuclear translocation of GCRα (20, 33). It has been suggested that T-cell GC insensitivity may be associated with a failure of GCs to control mitogen-activated protein kinase activation (33, 34). In addition, in vitro studies have revealed that these kinases alter phosphorylation status of GCRα, thus negatively regulating its function (19, 35–37). Interaction of GCRα with heat shock proteins (38) and FK506 proteins 51 and 52 (39, 40) has been reported to control GCRα nuclear translocation. The molecular mechanism for GC insensitivity of BAL macrophages, the predominant airway cell in asthma, has not been previously studied nor have the potential relationships between various potential mechanisms of GC insensitivity been explored.
Increased expression of GCRβ has been found in several diseases associated with GC insensitivity, suggesting that an imbalance between GCRα and GCRβ expression is associated with GC insensitivity, leading to a reduction in the ability of GCRα to interact with the GRE. In contrast to GCRα, GCRβ interacts weakly with heat shock proteins, does not bind GCs, and is transcriptionally inactive (6, 7, 28). GCRβ is known to inhibit GCRα-mediated transactivation in a dose-dependent manner (29). It has been reported that proinflammatory cytokines induce GCRβ expression (8). The ability of GCRβ to antagonize the function of GCRα suggests its importance in the regulation of cell sensitivity to GCs. It has been reported that GCRβ competes with GCRα in the nucleus for coactivators, and that GCRα–GCRβ heterodimers are transcriptionally inactive (41).
However, the measurement of GCRβ expression in various diseases has resulted in contradictory findings (42–46). This may relate to the fact that such studies have focused on mixed cell populations. It is likely that GCRβ can be detected more easily with a homogeneous cell population in the target organ of the disease as opposed to mixed cell populations (such as peripheral blood), in which a potentially positive signal may be hidden by high background signals coming from cells that do not upregulate GCRβ expression.
Our data confirm observation by Hamid and coworkers (12) that BAL cells from patients with GC-insensitive asthma express increased levels of GCRβ as shown by immunocytochemistry. Indeed, our current study found no difference in GCRβ gene expression between monocytes of patients with GC-insensitive asthma and patients with GC-sensitive asthma. To our knowledge, the current report is the first to show elevated GCRβ expression in BAL cells that is confirmed both by real-time PCR and immunofluorescence and to show that GCRβ is evenly distributed between cell cytoplasm and nuclei in human monocytes/macrophages. This suggests, aside from the known ability of GCRβ to inhibit GCRα transactivation, that it can influence GCRα nuclear translocation in response to steroids, most likely by heterodimer formation between GCRα and GCRβ. Several studies have already demonstrated that these two GCR isoforms can interact when found in the same cell compartment, but most studies performed in cell lines have reported the presence of GCRβ in the nucleus, where it has been postulated to interfere with GCRα transactivation (28, 29). At this time, we do not know what factors control GCRβ subcellular localization in monocytes. However, the novel observation that cytoplasmic GCRβ may interfere with GCRα nuclear translocation provides a new dimension by which GCRβ may act as a dominant negative inhibitor of GCRα action independent of its effects on transactivation. Our experiments show that GCRα still remains present in larger quantities than GCRβ in GC-insensitive cells, but siRNA experiments suggest that despite this, GCRβ still plays a role in reduction of steroid responses, indicating that GCRβ is not working solely by competing with GCRα for DNA GRE.
To evaluate how GCRβ can influence GCRα function, two approaches were used: overexpression of GCRβ and silencing of GCRβ mRNA. We reported previously that viral transduction of GCRβ cDNA into mouse hybridoma cells to induce stable expression of GCRβ results in GC insensitivity of these cells (27). Furthermore, using whole cell lysates, we have shown that in such cells GCRα is complexed with GCRβ (27). Using the same system in this article, we have found that retrovirally transduced DO-11.10 cells have a predominance of GCRβ in the cytoplasm. Importantly, our current study demonstrates that no GCRα nuclear translocation was observed in cells that overexpress GCRβ. We hypothesize that GCRα–GCRβ heterodimer formation in GCRβ-overexpressing cells inhibits GCRα nuclear shuttling in response to steroids. As well, we found that overexpression of GCRβ abolishes GCRα transactivation capacity. As shown by real-time PCR, MKP-1 mRNA induction by steroids (as a readout of GCRα transactivation capacity) was proportionally reduced by GCRβ expression.
Previous studies have proposed that a likely mechanism for the dominant negative activity of GCRβ is via the formation of heterodimers with GCRα, however, the precise mechanism and structural basis of this phenomenon are only starting to be understood. Immunoprecipitation experiments from our laboratory have previously demonstrated that murine GCRα can heterodimerize with human GCRβ (27). Sequence comparison of murine GCR (NP_032199) and human GCRα (NM_008173) shows 89% identity between two proteins, including a ligand-binding domain (LBD), which is known to participate in GCR protein dimerization. The human GCRα LBD structure has been determined (47). This structure suggests that the dimerization interface of GCR is distinct from that of other nuclear receptor LBD structures, in that it involves the H4 domain and β-strands 3 and 4, located on the opposite face of the folded domain from H10 to H12 and therefore structural differences in H11 and H12 domains of human GCRβ do not perturb the dimer interface found in human GCRα. On the basis of the crystal structure it is believed that GCR dimerization is ligand independent because the conformational changes that occur on hormone binding are far away from the published dimer interface; the solved structure supports the possibility of heterodimerization of GCRα and GCRβ (29).
To address potential confounding problems with cell lines that express nonphysiologic levels of GCRβ, we investigated whether specific RNA silencing of GCRβ expression in human BAL macrophages from patients with GC-insensitive asthma can enhance the response of these primary cells to steroids. Indeed, we found that GCRβ has nuclear and cytoplasmic localization in human BAL macrophages, and that introduction of a specific GCRβ siRNA, but not control siRNA, results in significant inhibition of both cytoplasmic and nuclear GCRβ expression. Concomitant with the inhibition of GCRβ expression, MKP-1 mRNA induction by steroids was significantly enhanced in such cells (Figure 5D). These data demonstrate that GCRβ does have a physiologic role in modulating steroid responsiveness in cells derived from patients with GC-insensitive asthma and that increased GCRβ expression may be a therapeutic target in restoring steroid responsiveness in GC-insensitive asthma.
The authors thank Maureen Sandoval for help in preparing this manuscript.
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