Glucocorticoid Receptor Nuclear Translocation in Airway Cells after Inhaled Combination Therapy
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美国呼吸和危急护理医学 2005年第9期
Airways Disease Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom
ABSTRACT
Clinical evidence is accumulating for the efficacy of adding inhaled long-acting 2-agonists (LABAs) to corticosteroids in asthma. Corticosteroids bind to cytoplasmic glucocorticoid receptors (GRs), which then translocate to the nucleus where they regulate gene expression. This article reports the first evidence in vivo of an interaction between inhaled LABA and corticosteroid on GR nuclear translocation in human airway cells using immunocytochemistry. We initially demonstrated significant GR activation 60 minutes after inhalation of 800 e beclomethasone dipropionate in six healthy subjects. Subsequently, we determined the effects of salmeterol and fluticasone propionate (FP) in seven steroid-naive patients with asthma. We observed dose-dependent GR activation with 100- and 500-e doses of FP, and to a lesser extent with 50 e salmeterol alone. However, combination therapy with 100 e FP and salmeterol augmented the action of FP on GR nuclear localization. In vitro, salmeterol enhanced FP effects on GR nuclear translocation in epithelial and macrophage-like airway cell lines. In addition, salmeterol in combination with FP enhanced glucocorticoid response element (GRE)eCluciferase reporter gene activity and mitogen-activated protein kinase phosphatase 1 (MKP-1) and secretory leuko-proteinase inhibitor (SLPI) gene induction. Together, our data confirm that GR nuclear translocation may underlie the complementary interactions between LABAs and corticosteroids, although the precise signal transduction mechanisms remain to be determined.
Key Words: asthma; human; lung; therapy; transcription factors
Corticosteroids are potent antiinflammatory drugs that are highly effective in the control of chronic inflammatory diseases such as asthma, where they have become the mainstay of therapy (1). Glucocorticoid receptors (GRs) are specific cytoplasmic transcription factors that mediate the biological actions of corticosteroids (2). On ligand binding, GR translocates into the nucleus and binds to DNA at glucocorticoid response elements (GREs) in the promoter region of corticosteroid-responsive genes that induce transcription (3). GR activation may also influence antiinflammatory events by nongenomic pathways, forming inhibitory interactions within the nucleus with proinflammatory DNA-binding transcription factors, such as activator protein (AP)-1 or nuclear factor (NF)eCB, or by recruitment of corepressors, and thereby repressing the actions of these important inflammatory proteins (4, 5). GR nuclear translocation is, therefore, essential and necessary for corticosteroid action.
Several clinical studies have shown that the combination of a long-acting 2-agonist (LABA) with a low dose of inhaled corticosteroid achieves better asthma control than either drug alone, or a higher dose of inhaled corticosteroid (6eC11). Patients have improved lung function (9), better symptom control (6, 7), reduction of exacerbations (10), and improvement in health status (11). Together, LABAs and corticosteroids have complementary modes of action targeting different aspects of the underlying pathophysiology of asthma, but the molecular mechanisms underlying these effects remain to be fully elucidated (12, 13).
LABAs have recently been shown to induce ligand-independent activation of GR nuclear translocation in vitro (14). Using primary human lung fibroblasts and vascular smooth muscle cells, Eickelberg and colleagues (14) found that dexamethasone and fluticasone propionate (FP; 10eC9 M) induced almost complete nuclear translocation of GR. Importantly, it was observed that salmeterol (10eC8 M) was able to cause ligand-independent GR translocation, although to a lesser extent than FP. Similar results have been achieved in cultured primary human bronchial airway smooth muscle cells (15). In a pilot in vivo study, four patients with asthma separately inhaled a single dose of budesonide (800 e) and formoterol (48 e), and GR expression in peripheral blood mononuclear cells was analyzed using immunoblots (16). Formoterol was able to cause GR activation, albeit to a lesser degree and at a later time point compared with inhaled budesonide.
This led us to investigate these complementary effects in vivo, in cells relevant to the acute inflammation of asthma. In this study, we tested the effect of combination treatment, on the intracellular partitioning of GR in cells isolated from induced sputum of patients with asthma. We performed immunocytochemical and Western blot analysis of GR expression and subcellular localization. Our aims were to determine whether GR activation occurred in vivo after clinically relevant inhaled drug concentrations, and to identify if the LABA salmeterol was able to modulate GR nuclear translocation.
METHODS
Subjects
In the first clinical study, six healthy nonsmoking subjects (mean age [SD], 31.8 ± 7.0 years; 6 women; mean FEV1 [SD], 2.84 ± 0.66 L; percentage predicted FEV1, 108.7%) inhaled a single 800-e dose of beclomethasone dipropionate (BDP) from a metered dose inhaler and spacer, on four study visits separated by a minimum washout period of 6 days. At each visit, sputum was induced either at 0, 30, 60, or 120 minutes in a randomized sequence after inhalation of BDP to determine a time-course for GR nuclear translocation. The second clinical study was a randomized, double-blind, placebo-controlled, crossover design, in which seven steroid-naive patients with mild asthma, defined according to the American Thoracic Society criteria (17), participated (mean age [SD], 34.0 ± 7.3 years; 4 women; mean FEV1 [SD], 2.99 ± 0.97 L; percentage predicted FEV1, 87.5%). Single metered dose inhaler doses of 100 e FP, 500 e FP, 50 e salmeterol, 100 e/50 e combination FP/salmeterol (Seretide; GlaxoSmithKline, Greenford, UK), and placebo dummy inhaler were delivered via a spacer at separate visits, with a minimum washout period of 7 days. For each treatment, sputum was induced at 60 and 120 minutes postinhalation on separate visits. Subjects were required to have a baseline FEV1 within 15% of their screening FEV1 value to control for differences in airway function and inflammation between visits. Both studies were approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals National Health Service Trust, and all subjects gave written, informed consent.
Sputum Induction and Processing
Sputum induction was performed exactly as previously described (18), and samples were kept on ice and processed within 1 hour of collection. Total cell count (Kimura stain) and viability (Trypan blue exclusion) were determined before cytospins were undertaken (18). Cytospins were fixed with 4% paraformaldehyde (BDH Ltd., Poole, UK) and stored at eC20°C. Samples with a cell viability of greater than 70% and less than 30% squamous cell contamination were considered adequate.
Immunoalkaline Phosphatase Staining
Indirect staining of sputum cells was performed using the alkaline phosphataseeCanti-alkaline phosphatase (APAAP) method using a commercial kit (Vectastain Laboratories, Peterborough, UK) (19). Cells were permeabilized for 10 minutes with 0.5% Nonidet P-40 (NP-40; BDH Ltd.) and blocked in 20% normal swine nonimmune serum (Dako Ltd., Cambridge, UK) for 30 minutes at room temperature. GR was detected with polyclonal antirabbit antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubating with the secondary biotinylated goat antirabbit antibody (1:100; Vectastain Laboratories), the immunoreaction was detected using the APAAP system to produce red staining. Slides were counterstained with hematoxylin for cellular identification, and examined under both modalities of light and fluorescent microscopy (Olympus Optical Co. Ltd., London, UK; Soft Imaging System GmbH, Munster, Germany). The advantage of the APAAP complex is that the red precipitate observed under the light microscope will also immunofluoresce red when viewed under a red fluorescent microscopic filter. Immunoreactive cells from induced sputum were each resolved at the single-cell level by dual light and fluorescent microscopy to determine the subcellular localization of GR. We used methods that others have previously demonstrated in vitro that allow semiquantification of the changes in the intracellular distribution of GR over time (20). Immunopositive cells on sputum cytospins were classified into two categories according to whether the signal intensity was predominately localized to the cytoplasm (cytoplasmic GR) or distributed between both cytoplasmic and nuclear compartments reflecting translocation of activated GR (nuclear GR). No cells exclusively localized GR to the cytoplasm or nucleus alone. An experienced observer blind to the clinical characteristics of the subject performed cell counting on 300 cells within each cell population.
Confocal Laser Immunofluorescence Staining
Separate cytospin preparations were used to detail the magnitude of difference in GR subcellular localization using confocal laser imaging of indirect fluorescent-labeled slides, but were not used in the count analysis. Using a modification of a method previously described (21), cells were permeabilized, blocked, and then incubated with GR antibody for 1 hour at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanateeCconjugated goat antirabbit antibody (Dako Ltd.). Cytospins were counterstained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50:50). The immunopositive signal was characterized using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibody, rabbit serum immunoglobulin (Dako Ltd.) and PBS were used as isotype negative controls for immunocytochemistry (immunoalkaline phosphatase) and confocal laser images.
Isolation of Cytosolic and Nuclear Fractions and Western Blot Analysis
Nuclear and cytosolic fractions of cells were isolated as previously described (22), and Western blot analysis performed (5). For in vivo sputum protein analysis, membranes were washed and incubated with antieCGR-specific monoclonal antibody (Becton Dickinson Biosciences, Oxford, UK) followed by an alkaline phosphataseeCconjugated secondary antibody (Invitrogen Ltd., Paisley, UK). For in vitro cell line experiments, membranes were washed and incubated with polyclonal GR antibody (Santa Cruz Biotechnology, Inc.) followed by horseradish peroxidaseeCconjugated secondary antibody (Dako Ltd.). Bound antibodies were visualized by use of the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Little Chalfont, UK). The membranes were reprobed with antieCmitogen-activated protein kinase kinase 1 (MEK-1) and/or antihistone H1 antibodies (Santa Cruz Biotechnology, Inc.) to confirm equal cytoplasmic and nuclear protein loading, respectively.
Transfection and Luciferase Assays
Cells (BEAS-2B and U937) were cultured according to the supplier's instructions (American Type Culture Collection, Rockville, MD) until 90% confluent. Cells were transiently transfected with a 2 x GRE-luciferase and pSV--galactosidase (Promega Corp., Madison, WI) using lipofectamine, as previously described (22). Eight hours after transfection, cells were treated with salmeterol (10eC7 M) and/or with FP (10eC8 or 10eC12 M) for a further 8 hours, and luciferase and -galactosidase activity were measured (22). Luciferase values were divided by galactosidase values to normalize for variations in transfection efficiency.
Reverse TranscriptioneCReal-Time Polymerase Chain Reaction
Cells were treated with salmeterol (10eC7 M) and/or with FP (10eC8 or 10eC12 M) for 2 hours, and 1 x 106 cells/treatment were harvested for total RNA isolation. Commercially available kits were used to extract total cellular RNA (Rneasy; Qiagene, Crawley, UK) and to perform reverse transcription (Omniscript RT; Qiagene). Gene transcript level of mitogen-activated protein kinase phosphatase 1 (MKP-1/CL-100) and the housekeeping gene GAPDH (glyceraldehyde phosphate dehydrogenase) were quantified by real-time polymerase chain reaction using a QuantiTect SYBR Green polymerase chain reaction kit (Qiagene) on a Rotor-Gene 3000 polymerase chain reaction apparatus (Corbett Research, Cambridge, UK). The primer pairs of GAPDH and MKP-1 were designed as previously described (23). Variations in complementary DNA (cDNA) concentration between different samples were corrected using the housekeeping gene, where the GAPDH concentration in each cDNA sample was calculated, and the cDNA diluted to contain equal amounts of GAPDH. Standard curves for GAPDH were generated by performing a dilution series of the untreated control cDNA. The relative amount of gene transcript present after different treatments was calculated and normalized by dividing the calculated value for the gene of interest by the housekeeping gene value.
Statistical Analysis
Data are expressed as mean ± SEM. Because sputum cells are not normally distributed, nonparametric statistical analyses were performed using the PC analysis package SPSS 10.0 (SPSS, Inc., Chicago, IL). Results were analyzed using Friedman analysis of variance, and comparisons between time points were made using the Wilcoxon matched-pairs signed rank sum test. The difference between treatment groups in the in vitro data was analyzed by Welch's t test using Graph Pad Prism software (Graph Pad Prism, San Diego, CA). A p value less than 0.05 was considered statistically significant.
RESULTS
Characterization of GR Nuclear and Cytoplasmic Staining in Sputum Cells
In initial studies, immunoreactive cells from induced sputum were each resolved at the single-cell level by dual light and fluorescent microscopy to determine the subcellular localization of GR in both epithelial cells and macrophages (Figure 1). In subsequent analysis, the immunopositive signal was characterized using laser scanning confocal microscopy in epithelial cells (Figure 2) and macrophages (Figure 3). In all cases, the green background autofluorescence is low and the blue DAPI staining outlines the nuclear boundary, and helps define the distribution of GR protein (red) within the respective subcellular compartments. Finally, the specificity of the antibody used was determined using an isotype control, which gave no staining (Figures 4A and 4C) compared with that seen with the anti-GR antibody (Figures 4B and 4D) using both light microscopic and confocal laser images.
BDP Effect on GR Translocation in Healthy Subjects
The first clinical study investigated the time course of GR redistribution in cells from induced sputum after therapeutic concentrations of inhaled corticosteroid to determine the optimal time point for GR translocation (Figure 5). Healthy subjects were used to avoid confounding effects of disease or medication. All cells in sputum (macrophages, epithelial cells, neutrophils, and eosinophils) expressed GR protein, which confirms the ubiquitous expression of GR in airway cells (24). GR depletion from the cytoplasm was associated with a corresponding increase in GR accumulation into the nucleus. At baseline, approximately 30% of GR was localized to the nucleus in all cell types. We found a significant increase in nuclear translocation (73 ± 8%) in macrophages 30 minutes after BDP inhalation, which was sustained for the period of the study (p < 0.05; Figure 5A). For epithelial cells, this effect occurred at 60 minutes, with 71 ± 3% GR nuclear localization (p < 0.05; Figure 5B). There was no significant difference in the response of neutrophils when compared with baseline values. Neutrophils were relatively corticosteroid insensitive when GR translocation was used as a marker of response because there was less GR translocation with the same corticosteroid dose compared with the other cells.
LABA and Corticosteroid Effect on GR Activation in Subjects with Asthma
Having determined the optimum time point for in vivo GR activation as 60 minutes after corticosteroid inhalation, we studied the effect of a combination of inhaled LABA and corticosteroid on GR nuclear translocation and expression in subjects with asthma, using corticosteroid as the positive control. Macrophages and epithelial cells were studied, because the remaining cellular counts are low in patients with asthma (25). In keeping with data in our healthy subjects, after placebo inhalation, approximately 30% of GR was localized to the nuclear compartment in both cell types (Figures 6A and 6B). In epithelial cells, we observed a clear dose-dependent increase in GR nuclear translocation with 100 e FP and 500 e FP (45 ± 2.2 vs. 64 ± 1.6% positively stained nuclei, p < 0.05). The effects of both doses of FP were significant compared with placebo (Figure 6A; p < 0.05). Salmeterol (50 e) alone significantly induced GR nuclear translocation in sputum epithelial cells (38 ± 2.2%) compared with placebo (p < 0.05), and furthermore, in combination with 100 e FP, salmeterol was able to significantly enhance GR intracellular partitioning by FP (57 ± 1.9%) to a greater extent than either drug alone (p < 0.05; Figure 6A). Similar results were seen with sputum macrophages (Figure 6B).
Immunoblots on pooled sputum samples confirmed the magnitude of the cellular response within the nuclear compartment (Figure 7). The results were consistent with the immunocytochemistry data and showed a clear doseeCresponse relationship to FP with respect to GR nuclear translocation after 60 minutes. In addition, the results showed a lesser effect of salmeterol alone on GR activation, but when salmeterol was combined with FP, there was enhanced GR nuclear translocation compared with low-dose FP alone, reaching a similar level as that seen with 500 e FP (Figure 7). Because of the short time course of study (60 minutes), we were unable to investigate changes in mediator expression from these same cells using reverse transcriptioneCpolymerase chain reaction.
GR Nuclear Translocation after Combination Therapy In Vitro
To corroborate the in vivo data from airway cells on GR nuclear translocation, the effect of FP and salmeterol both singly and in combination were studied in an in vitro epithelial and macrophage-like cell line model, BEAS-2B and phorbol 12-myristate 13-acetate (PMA)-treated U-937 cells, respectively (Figure 8). FP (10eC12, 10eC8 M) induced GR nuclear translocation in a concentration-dependent manner both in PMA-treated U-937 and BEAS-2B cells. Salmeterol in combination with FP (10eC12 M) significantly enhanced FP-induced GR nuclear translocation compared with FP (10eC12 M) alone in both cell lines (p < 0.05). There was no significant difference in GR nuclear translocation between the high concentration of FP and low-concentration combination treatment. Concurrently, salmeterol enhanced FP-induced GR reduction from the cytoplasm in both cell lines.
Induction of GRE-dependent Transcription
Having confirmed the ability to induce nuclear GR translocation in an in vitro model, we examined whether this translocation was able to produce a functional readout. Transient transfection of the GRE-dependent reporter, pGL3.2GRE.luc, into PMA-treated U-937 cells showed that FP (10eC12, 10eC8 M) activated GRE-dependent transcription in a concentration-dependent manner (Figure 9A). Salmeterol (10eC7 M) alone also activated GRE-dependent transcription, although this was not statistically significant. However, in combination with low-concentration FP (10eC12 M), salmeterol significantly enhanced FP-dependent GRE transcription in both cell lines compared with FP (10eC12 M) alone (p < 0.05). Similar results were obtained in BEAS-2B cells (Figure 9B).
MKP-1 Expression in U-937 Cells and SLPI Expression in BEAS-2B Cells
Finally, we determined whether salmeterol enhanced the glucocorticoid-genomic functional readout using the corticosteroid-inducible genes MKP-1 and secretory leuko-proteinase inhibitor (SLPI) expression as readouts in PMA-treated U-937 and BEAS-2B cells, respectively. FP (10eC12, 10eC8 M) induced MKP-1 mRNA expression in PMA-treated U-937 cells in a concentration-dependent manner (Figure 10A). Salmeterol in combination with FP (10eC12 M) significantly enhanced FP-induced MKP-1 mRNA expression compared with FP (10eC12 M) alone (p < 0.05). FP also induced SLPI mRNA expression in a concentration-dependent manner compared with GAPDH expression in BEAS-2B cells (Figure 10B). Salmeterol (10eC7 M) had no effect on SLPI mRNA expression on its own, but significantly enhanced FP (10eC12 M)-induced SLPI mRNA expression in combination when compared with FP (10eC12 M) alone (p < 0.05).
DISCUSSION
This study evaluated the effects of inhaled combination therapy using LABA and corticosteroid on GR nuclear translocation in airway cells isolated in vivo and in relevant cell lines in vitro. We initially found that, after 800 e of inhaled BDP in healthy subjects, ligand-induced GR nuclear translocation occurred with distinct kinetics in all cell types. This was not an all-or-nothing event but rather resulted in redistribution of the dynamic balance of GR between the subcellular compartments. We observed significant GR nuclear translocation within 60 minutes of inhaled corticosteroid therapy, and nuclear retention was maintained for at least 2 hours. It is well established that inhaled corticosteroids target the underlying inflammatory pathophysiology of asthma, but most of their effect is obtained from low doses because there is a relatively flat doseeCresponse relationship (26). Conversely, there is the potential for dose-dependent systemic side effects with higher doses. LABAs, on the other hand, principally relax airway smooth muscle, resulting in prolonged bronchodilation and bronchoprotection, but they have an independent action, which complements the effects of corticosteroids. We demonstrated a significant increase in GR nuclear translocation in sputum epithelial cells and macrophages with salmeterol alone compared with placebo, and furthermore, in combination with FP, salmeterol was significantly better than low-dose FP alone at enhancing GR nuclear translocation.
Consistent with our in vivo data, we observed that salmeterol was able to enhance low-dose FP effects on GR nuclear translocation in both epithelial and macrophage airway cell lines treated in vitro. The magnitude and trend of the response in vitro was comparable to that seen in vivo, and our results support those of previous authors that show LABAs are able to promote GR nuclear translocation in cultured fibroblasts and smooth muscle cells (14, 15). Taken together, our results are the first evidence in vivo to demonstrate that the LABA salmeterol, in combination with the corticosteroid fluitcasone propionate, at clinically relevant inhaled concentrations in subjects with asthma, is able to influence the intracellular partitioning of GR in both macrophages and epithelial airway cells to a greater extent than either drug alone.
Although nuclear translocation signifies that the drug is able to reach the target cell at a sufficient concentration to promote GR subcellular nuclear movement, it does not imply per se that functional antiinflammatory events will follow. The final biological outcome of GR transcription factor modulation depends on the net balance of both transactivation and transrepression effects, and will differ between cells, and under different stimuli. We therefore investigated the biological relevance of GR activation in vitro, and demonstrated that salmeterol modulation of GR translocation had functional effects on GRE-luciferase activity and MKP-1 and SLP1 gene induction. LABAs have been shown previously to augment the antiinflammatory effects of corticosteroids (13), particularly via inhibitory effects on cytokine release from a variety of cells involved in the pathogenesis of asthma. Korn and Brattsand (27) observed the combination of formoterol and budesonide inhibited granulocyte-macrophage colonyeCstimulating factor release from tumor necrosis factor eCstimulated human airway epithelial cells more than either drug alone. Similarly, Pang and Knox reported that salmeterol enhanced the inhibitory actions of FP on tumor necrosis factor eCinduced interleukin 8 (28) and eotaxin (29) release from human airway smooth muscle cells. We chose to examine the ability of FP to induce GR-GRE DNA binding, and induce antiinflammatory genes such as SLPI and the dual mitogen-activating protein kinase inhibitor MKP-1. We found that salmeterol was able to augment the ability of FP to enhance both GR-GRE binding and SLPI and MKP-1 expression in both macrophage-like and epithelial cell lines. The effect was at least additive and clearly synergistic with respect to SLPI induction in BEAS-2B cells. The effects on GR-GRE activation and gene expression were not totally superimposable due to the more complex promoter structure of the native MKP-1 and SLPI promoters and the requirement for other factors for full gene expression.
In vivo studies using bronchial biopsies generate further supporting evidence for complementary interactions on airway inflammation and pathology in subjects with asthma. Li and colleagues (30) found the addition of salmeterol in patients receiving inhaled corticosteroid led to a reduction in the total number of activated eosinophils within the lamina propria, together with a concurrent improvement in clinical status. Orsida and coworkers (31) demonstrated a significant reduction in the vascularity of the lamina propria in patients with asthma receiving salmeterol and inhaled corticosteroid, whereas no effect was observed after high-dose inhaled corticosteroid treatment. We used established noninvasive techniques of sputum induction to obtain airway cellular samples to investigate the interaction of inhaled LABA and corticosteroid in human subjects. We applied immunocytochemistry techniques to investigate the subcellular localization of GR in a semiquantitative manner similar to that described by previous authors in vitro (20). Although bronchoscopy with airway brushing may have provided sufficient cellular material to undertake quantitative protein analysis, it would not have been ethical to undertake this invasive procedure on the number of occasions outlined in our study design. In vitro experiments clearly demonstrate that these effects of LABA on corticosteroid actions are mediated through the 2-receptor (14, 27eC29). However, this has not been formally shown in this study in vivo due to the prolonged time required to examine cytokine profiles such as interleukin 6 or granulocyte-macrophage colonyeCstimulating factor (32), and the potential risk of giving subjects with asthma -blockers. Studies examining the effect of chronic combination treatment on cytokine profiles released from airway cells in vivo will need to be performed to address, in part, these issues.
GRs are phosphoproteins (33) and altered phosphorylation of GR and components of its signal transduction pathway have been implicated in the regulation of corticosteroid binding to GR (34), GR subcellular localization (35), and GR nuclear cytoplasmic trafficking through the nuclear pore complex (36, 37). Receptor phosphorylation may also influence the interactions of GR with other transcription factors, such as NF-B, required for transactivation (38). Many of the kinases that catalyze the phosphorylation of GR have been identified (39) and mitogen-activated protein kinaseeCdependent phosphorylation may play a role (40). Interestingly, phosphorylation of inactive GR may block subsequent hormone binding and nuclear translocation (41). LABAs may prime GR for activation directly by kinase-dependent phosphorylation and this may account for the additional effects of LABA on corticosteroid effects on NF-B (42). We postulate that phosphorylation of GR and its change in protein structure may alter function such that there are more enhanced responses to combination treatment.
In vitro experiments show that GR is entirely localized to the cytoplasm before corticosteroid treatment (43). However, we observed basal nuclear GR translocation of approximately 30% within airway cells in both healthy subjects and in subjects with asthma, which may reflect responses to endogenous levels of circulating cortisol. This may also account, at least in part, for the effect seen with salmeterol alone in vivo. Although not a primary aspect of our study, we found a degree of corticosteroid insensitivity to GR nuclear translocation within neutrophils. Glucocorticoid sensitivity varies between cell types and on the stage of the cell cycle (24, 44) and often parallels changes in receptor density (45, 46), and low GR levels have been associated with a poor response to corticosteroid treatment (45, 47). Decreased GR sensitivity of neutrophils to corticosteroids has been shown in vitro (48), and this has been postulated to be the result of enhanced GR expression (49). The differential response of the cells to inhaled corticosteroids observed within our healthy volunteers suggests that the decreased sensitivity of neutrophils to glucocorticoids may also be due to reduced GR nuclear translocation.
In conclusion, our data show that salmeterol, in combination with fluticasone propionate, can enhance GR nuclear translocation in vivo, as well as in vitro, and the data suggest that salmeterol may play an important role in the additional benefits seen with combination therapy. In vitro, the enhanced GR nuclear translocation is associated with an amplified GR functional response. Further studies are needed to address whether these differences are maintained after chronic treatment with inhaled LABA and corticosteroids, and whether this results in an alteration in cytokine profiles in vivo.
Acknowledgments
Plasmids containing 2x glucocorticoid response element (GRE)eCluciferase were kindly donated by Dr. J. Bloom (Tucson, AZ).
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ABSTRACT
Clinical evidence is accumulating for the efficacy of adding inhaled long-acting 2-agonists (LABAs) to corticosteroids in asthma. Corticosteroids bind to cytoplasmic glucocorticoid receptors (GRs), which then translocate to the nucleus where they regulate gene expression. This article reports the first evidence in vivo of an interaction between inhaled LABA and corticosteroid on GR nuclear translocation in human airway cells using immunocytochemistry. We initially demonstrated significant GR activation 60 minutes after inhalation of 800 e beclomethasone dipropionate in six healthy subjects. Subsequently, we determined the effects of salmeterol and fluticasone propionate (FP) in seven steroid-naive patients with asthma. We observed dose-dependent GR activation with 100- and 500-e doses of FP, and to a lesser extent with 50 e salmeterol alone. However, combination therapy with 100 e FP and salmeterol augmented the action of FP on GR nuclear localization. In vitro, salmeterol enhanced FP effects on GR nuclear translocation in epithelial and macrophage-like airway cell lines. In addition, salmeterol in combination with FP enhanced glucocorticoid response element (GRE)eCluciferase reporter gene activity and mitogen-activated protein kinase phosphatase 1 (MKP-1) and secretory leuko-proteinase inhibitor (SLPI) gene induction. Together, our data confirm that GR nuclear translocation may underlie the complementary interactions between LABAs and corticosteroids, although the precise signal transduction mechanisms remain to be determined.
Key Words: asthma; human; lung; therapy; transcription factors
Corticosteroids are potent antiinflammatory drugs that are highly effective in the control of chronic inflammatory diseases such as asthma, where they have become the mainstay of therapy (1). Glucocorticoid receptors (GRs) are specific cytoplasmic transcription factors that mediate the biological actions of corticosteroids (2). On ligand binding, GR translocates into the nucleus and binds to DNA at glucocorticoid response elements (GREs) in the promoter region of corticosteroid-responsive genes that induce transcription (3). GR activation may also influence antiinflammatory events by nongenomic pathways, forming inhibitory interactions within the nucleus with proinflammatory DNA-binding transcription factors, such as activator protein (AP)-1 or nuclear factor (NF)eCB, or by recruitment of corepressors, and thereby repressing the actions of these important inflammatory proteins (4, 5). GR nuclear translocation is, therefore, essential and necessary for corticosteroid action.
Several clinical studies have shown that the combination of a long-acting 2-agonist (LABA) with a low dose of inhaled corticosteroid achieves better asthma control than either drug alone, or a higher dose of inhaled corticosteroid (6eC11). Patients have improved lung function (9), better symptom control (6, 7), reduction of exacerbations (10), and improvement in health status (11). Together, LABAs and corticosteroids have complementary modes of action targeting different aspects of the underlying pathophysiology of asthma, but the molecular mechanisms underlying these effects remain to be fully elucidated (12, 13).
LABAs have recently been shown to induce ligand-independent activation of GR nuclear translocation in vitro (14). Using primary human lung fibroblasts and vascular smooth muscle cells, Eickelberg and colleagues (14) found that dexamethasone and fluticasone propionate (FP; 10eC9 M) induced almost complete nuclear translocation of GR. Importantly, it was observed that salmeterol (10eC8 M) was able to cause ligand-independent GR translocation, although to a lesser extent than FP. Similar results have been achieved in cultured primary human bronchial airway smooth muscle cells (15). In a pilot in vivo study, four patients with asthma separately inhaled a single dose of budesonide (800 e) and formoterol (48 e), and GR expression in peripheral blood mononuclear cells was analyzed using immunoblots (16). Formoterol was able to cause GR activation, albeit to a lesser degree and at a later time point compared with inhaled budesonide.
This led us to investigate these complementary effects in vivo, in cells relevant to the acute inflammation of asthma. In this study, we tested the effect of combination treatment, on the intracellular partitioning of GR in cells isolated from induced sputum of patients with asthma. We performed immunocytochemical and Western blot analysis of GR expression and subcellular localization. Our aims were to determine whether GR activation occurred in vivo after clinically relevant inhaled drug concentrations, and to identify if the LABA salmeterol was able to modulate GR nuclear translocation.
METHODS
Subjects
In the first clinical study, six healthy nonsmoking subjects (mean age [SD], 31.8 ± 7.0 years; 6 women; mean FEV1 [SD], 2.84 ± 0.66 L; percentage predicted FEV1, 108.7%) inhaled a single 800-e dose of beclomethasone dipropionate (BDP) from a metered dose inhaler and spacer, on four study visits separated by a minimum washout period of 6 days. At each visit, sputum was induced either at 0, 30, 60, or 120 minutes in a randomized sequence after inhalation of BDP to determine a time-course for GR nuclear translocation. The second clinical study was a randomized, double-blind, placebo-controlled, crossover design, in which seven steroid-naive patients with mild asthma, defined according to the American Thoracic Society criteria (17), participated (mean age [SD], 34.0 ± 7.3 years; 4 women; mean FEV1 [SD], 2.99 ± 0.97 L; percentage predicted FEV1, 87.5%). Single metered dose inhaler doses of 100 e FP, 500 e FP, 50 e salmeterol, 100 e/50 e combination FP/salmeterol (Seretide; GlaxoSmithKline, Greenford, UK), and placebo dummy inhaler were delivered via a spacer at separate visits, with a minimum washout period of 7 days. For each treatment, sputum was induced at 60 and 120 minutes postinhalation on separate visits. Subjects were required to have a baseline FEV1 within 15% of their screening FEV1 value to control for differences in airway function and inflammation between visits. Both studies were approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals National Health Service Trust, and all subjects gave written, informed consent.
Sputum Induction and Processing
Sputum induction was performed exactly as previously described (18), and samples were kept on ice and processed within 1 hour of collection. Total cell count (Kimura stain) and viability (Trypan blue exclusion) were determined before cytospins were undertaken (18). Cytospins were fixed with 4% paraformaldehyde (BDH Ltd., Poole, UK) and stored at eC20°C. Samples with a cell viability of greater than 70% and less than 30% squamous cell contamination were considered adequate.
Immunoalkaline Phosphatase Staining
Indirect staining of sputum cells was performed using the alkaline phosphataseeCanti-alkaline phosphatase (APAAP) method using a commercial kit (Vectastain Laboratories, Peterborough, UK) (19). Cells were permeabilized for 10 minutes with 0.5% Nonidet P-40 (NP-40; BDH Ltd.) and blocked in 20% normal swine nonimmune serum (Dako Ltd., Cambridge, UK) for 30 minutes at room temperature. GR was detected with polyclonal antirabbit antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubating with the secondary biotinylated goat antirabbit antibody (1:100; Vectastain Laboratories), the immunoreaction was detected using the APAAP system to produce red staining. Slides were counterstained with hematoxylin for cellular identification, and examined under both modalities of light and fluorescent microscopy (Olympus Optical Co. Ltd., London, UK; Soft Imaging System GmbH, Munster, Germany). The advantage of the APAAP complex is that the red precipitate observed under the light microscope will also immunofluoresce red when viewed under a red fluorescent microscopic filter. Immunoreactive cells from induced sputum were each resolved at the single-cell level by dual light and fluorescent microscopy to determine the subcellular localization of GR. We used methods that others have previously demonstrated in vitro that allow semiquantification of the changes in the intracellular distribution of GR over time (20). Immunopositive cells on sputum cytospins were classified into two categories according to whether the signal intensity was predominately localized to the cytoplasm (cytoplasmic GR) or distributed between both cytoplasmic and nuclear compartments reflecting translocation of activated GR (nuclear GR). No cells exclusively localized GR to the cytoplasm or nucleus alone. An experienced observer blind to the clinical characteristics of the subject performed cell counting on 300 cells within each cell population.
Confocal Laser Immunofluorescence Staining
Separate cytospin preparations were used to detail the magnitude of difference in GR subcellular localization using confocal laser imaging of indirect fluorescent-labeled slides, but were not used in the count analysis. Using a modification of a method previously described (21), cells were permeabilized, blocked, and then incubated with GR antibody for 1 hour at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanateeCconjugated goat antirabbit antibody (Dako Ltd.). Cytospins were counterstained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50:50). The immunopositive signal was characterized using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibody, rabbit serum immunoglobulin (Dako Ltd.) and PBS were used as isotype negative controls for immunocytochemistry (immunoalkaline phosphatase) and confocal laser images.
Isolation of Cytosolic and Nuclear Fractions and Western Blot Analysis
Nuclear and cytosolic fractions of cells were isolated as previously described (22), and Western blot analysis performed (5). For in vivo sputum protein analysis, membranes were washed and incubated with antieCGR-specific monoclonal antibody (Becton Dickinson Biosciences, Oxford, UK) followed by an alkaline phosphataseeCconjugated secondary antibody (Invitrogen Ltd., Paisley, UK). For in vitro cell line experiments, membranes were washed and incubated with polyclonal GR antibody (Santa Cruz Biotechnology, Inc.) followed by horseradish peroxidaseeCconjugated secondary antibody (Dako Ltd.). Bound antibodies were visualized by use of the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Little Chalfont, UK). The membranes were reprobed with antieCmitogen-activated protein kinase kinase 1 (MEK-1) and/or antihistone H1 antibodies (Santa Cruz Biotechnology, Inc.) to confirm equal cytoplasmic and nuclear protein loading, respectively.
Transfection and Luciferase Assays
Cells (BEAS-2B and U937) were cultured according to the supplier's instructions (American Type Culture Collection, Rockville, MD) until 90% confluent. Cells were transiently transfected with a 2 x GRE-luciferase and pSV--galactosidase (Promega Corp., Madison, WI) using lipofectamine, as previously described (22). Eight hours after transfection, cells were treated with salmeterol (10eC7 M) and/or with FP (10eC8 or 10eC12 M) for a further 8 hours, and luciferase and -galactosidase activity were measured (22). Luciferase values were divided by galactosidase values to normalize for variations in transfection efficiency.
Reverse TranscriptioneCReal-Time Polymerase Chain Reaction
Cells were treated with salmeterol (10eC7 M) and/or with FP (10eC8 or 10eC12 M) for 2 hours, and 1 x 106 cells/treatment were harvested for total RNA isolation. Commercially available kits were used to extract total cellular RNA (Rneasy; Qiagene, Crawley, UK) and to perform reverse transcription (Omniscript RT; Qiagene). Gene transcript level of mitogen-activated protein kinase phosphatase 1 (MKP-1/CL-100) and the housekeeping gene GAPDH (glyceraldehyde phosphate dehydrogenase) were quantified by real-time polymerase chain reaction using a QuantiTect SYBR Green polymerase chain reaction kit (Qiagene) on a Rotor-Gene 3000 polymerase chain reaction apparatus (Corbett Research, Cambridge, UK). The primer pairs of GAPDH and MKP-1 were designed as previously described (23). Variations in complementary DNA (cDNA) concentration between different samples were corrected using the housekeeping gene, where the GAPDH concentration in each cDNA sample was calculated, and the cDNA diluted to contain equal amounts of GAPDH. Standard curves for GAPDH were generated by performing a dilution series of the untreated control cDNA. The relative amount of gene transcript present after different treatments was calculated and normalized by dividing the calculated value for the gene of interest by the housekeeping gene value.
Statistical Analysis
Data are expressed as mean ± SEM. Because sputum cells are not normally distributed, nonparametric statistical analyses were performed using the PC analysis package SPSS 10.0 (SPSS, Inc., Chicago, IL). Results were analyzed using Friedman analysis of variance, and comparisons between time points were made using the Wilcoxon matched-pairs signed rank sum test. The difference between treatment groups in the in vitro data was analyzed by Welch's t test using Graph Pad Prism software (Graph Pad Prism, San Diego, CA). A p value less than 0.05 was considered statistically significant.
RESULTS
Characterization of GR Nuclear and Cytoplasmic Staining in Sputum Cells
In initial studies, immunoreactive cells from induced sputum were each resolved at the single-cell level by dual light and fluorescent microscopy to determine the subcellular localization of GR in both epithelial cells and macrophages (Figure 1). In subsequent analysis, the immunopositive signal was characterized using laser scanning confocal microscopy in epithelial cells (Figure 2) and macrophages (Figure 3). In all cases, the green background autofluorescence is low and the blue DAPI staining outlines the nuclear boundary, and helps define the distribution of GR protein (red) within the respective subcellular compartments. Finally, the specificity of the antibody used was determined using an isotype control, which gave no staining (Figures 4A and 4C) compared with that seen with the anti-GR antibody (Figures 4B and 4D) using both light microscopic and confocal laser images.
BDP Effect on GR Translocation in Healthy Subjects
The first clinical study investigated the time course of GR redistribution in cells from induced sputum after therapeutic concentrations of inhaled corticosteroid to determine the optimal time point for GR translocation (Figure 5). Healthy subjects were used to avoid confounding effects of disease or medication. All cells in sputum (macrophages, epithelial cells, neutrophils, and eosinophils) expressed GR protein, which confirms the ubiquitous expression of GR in airway cells (24). GR depletion from the cytoplasm was associated with a corresponding increase in GR accumulation into the nucleus. At baseline, approximately 30% of GR was localized to the nucleus in all cell types. We found a significant increase in nuclear translocation (73 ± 8%) in macrophages 30 minutes after BDP inhalation, which was sustained for the period of the study (p < 0.05; Figure 5A). For epithelial cells, this effect occurred at 60 minutes, with 71 ± 3% GR nuclear localization (p < 0.05; Figure 5B). There was no significant difference in the response of neutrophils when compared with baseline values. Neutrophils were relatively corticosteroid insensitive when GR translocation was used as a marker of response because there was less GR translocation with the same corticosteroid dose compared with the other cells.
LABA and Corticosteroid Effect on GR Activation in Subjects with Asthma
Having determined the optimum time point for in vivo GR activation as 60 minutes after corticosteroid inhalation, we studied the effect of a combination of inhaled LABA and corticosteroid on GR nuclear translocation and expression in subjects with asthma, using corticosteroid as the positive control. Macrophages and epithelial cells were studied, because the remaining cellular counts are low in patients with asthma (25). In keeping with data in our healthy subjects, after placebo inhalation, approximately 30% of GR was localized to the nuclear compartment in both cell types (Figures 6A and 6B). In epithelial cells, we observed a clear dose-dependent increase in GR nuclear translocation with 100 e FP and 500 e FP (45 ± 2.2 vs. 64 ± 1.6% positively stained nuclei, p < 0.05). The effects of both doses of FP were significant compared with placebo (Figure 6A; p < 0.05). Salmeterol (50 e) alone significantly induced GR nuclear translocation in sputum epithelial cells (38 ± 2.2%) compared with placebo (p < 0.05), and furthermore, in combination with 100 e FP, salmeterol was able to significantly enhance GR intracellular partitioning by FP (57 ± 1.9%) to a greater extent than either drug alone (p < 0.05; Figure 6A). Similar results were seen with sputum macrophages (Figure 6B).
Immunoblots on pooled sputum samples confirmed the magnitude of the cellular response within the nuclear compartment (Figure 7). The results were consistent with the immunocytochemistry data and showed a clear doseeCresponse relationship to FP with respect to GR nuclear translocation after 60 minutes. In addition, the results showed a lesser effect of salmeterol alone on GR activation, but when salmeterol was combined with FP, there was enhanced GR nuclear translocation compared with low-dose FP alone, reaching a similar level as that seen with 500 e FP (Figure 7). Because of the short time course of study (60 minutes), we were unable to investigate changes in mediator expression from these same cells using reverse transcriptioneCpolymerase chain reaction.
GR Nuclear Translocation after Combination Therapy In Vitro
To corroborate the in vivo data from airway cells on GR nuclear translocation, the effect of FP and salmeterol both singly and in combination were studied in an in vitro epithelial and macrophage-like cell line model, BEAS-2B and phorbol 12-myristate 13-acetate (PMA)-treated U-937 cells, respectively (Figure 8). FP (10eC12, 10eC8 M) induced GR nuclear translocation in a concentration-dependent manner both in PMA-treated U-937 and BEAS-2B cells. Salmeterol in combination with FP (10eC12 M) significantly enhanced FP-induced GR nuclear translocation compared with FP (10eC12 M) alone in both cell lines (p < 0.05). There was no significant difference in GR nuclear translocation between the high concentration of FP and low-concentration combination treatment. Concurrently, salmeterol enhanced FP-induced GR reduction from the cytoplasm in both cell lines.
Induction of GRE-dependent Transcription
Having confirmed the ability to induce nuclear GR translocation in an in vitro model, we examined whether this translocation was able to produce a functional readout. Transient transfection of the GRE-dependent reporter, pGL3.2GRE.luc, into PMA-treated U-937 cells showed that FP (10eC12, 10eC8 M) activated GRE-dependent transcription in a concentration-dependent manner (Figure 9A). Salmeterol (10eC7 M) alone also activated GRE-dependent transcription, although this was not statistically significant. However, in combination with low-concentration FP (10eC12 M), salmeterol significantly enhanced FP-dependent GRE transcription in both cell lines compared with FP (10eC12 M) alone (p < 0.05). Similar results were obtained in BEAS-2B cells (Figure 9B).
MKP-1 Expression in U-937 Cells and SLPI Expression in BEAS-2B Cells
Finally, we determined whether salmeterol enhanced the glucocorticoid-genomic functional readout using the corticosteroid-inducible genes MKP-1 and secretory leuko-proteinase inhibitor (SLPI) expression as readouts in PMA-treated U-937 and BEAS-2B cells, respectively. FP (10eC12, 10eC8 M) induced MKP-1 mRNA expression in PMA-treated U-937 cells in a concentration-dependent manner (Figure 10A). Salmeterol in combination with FP (10eC12 M) significantly enhanced FP-induced MKP-1 mRNA expression compared with FP (10eC12 M) alone (p < 0.05). FP also induced SLPI mRNA expression in a concentration-dependent manner compared with GAPDH expression in BEAS-2B cells (Figure 10B). Salmeterol (10eC7 M) had no effect on SLPI mRNA expression on its own, but significantly enhanced FP (10eC12 M)-induced SLPI mRNA expression in combination when compared with FP (10eC12 M) alone (p < 0.05).
DISCUSSION
This study evaluated the effects of inhaled combination therapy using LABA and corticosteroid on GR nuclear translocation in airway cells isolated in vivo and in relevant cell lines in vitro. We initially found that, after 800 e of inhaled BDP in healthy subjects, ligand-induced GR nuclear translocation occurred with distinct kinetics in all cell types. This was not an all-or-nothing event but rather resulted in redistribution of the dynamic balance of GR between the subcellular compartments. We observed significant GR nuclear translocation within 60 minutes of inhaled corticosteroid therapy, and nuclear retention was maintained for at least 2 hours. It is well established that inhaled corticosteroids target the underlying inflammatory pathophysiology of asthma, but most of their effect is obtained from low doses because there is a relatively flat doseeCresponse relationship (26). Conversely, there is the potential for dose-dependent systemic side effects with higher doses. LABAs, on the other hand, principally relax airway smooth muscle, resulting in prolonged bronchodilation and bronchoprotection, but they have an independent action, which complements the effects of corticosteroids. We demonstrated a significant increase in GR nuclear translocation in sputum epithelial cells and macrophages with salmeterol alone compared with placebo, and furthermore, in combination with FP, salmeterol was significantly better than low-dose FP alone at enhancing GR nuclear translocation.
Consistent with our in vivo data, we observed that salmeterol was able to enhance low-dose FP effects on GR nuclear translocation in both epithelial and macrophage airway cell lines treated in vitro. The magnitude and trend of the response in vitro was comparable to that seen in vivo, and our results support those of previous authors that show LABAs are able to promote GR nuclear translocation in cultured fibroblasts and smooth muscle cells (14, 15). Taken together, our results are the first evidence in vivo to demonstrate that the LABA salmeterol, in combination with the corticosteroid fluitcasone propionate, at clinically relevant inhaled concentrations in subjects with asthma, is able to influence the intracellular partitioning of GR in both macrophages and epithelial airway cells to a greater extent than either drug alone.
Although nuclear translocation signifies that the drug is able to reach the target cell at a sufficient concentration to promote GR subcellular nuclear movement, it does not imply per se that functional antiinflammatory events will follow. The final biological outcome of GR transcription factor modulation depends on the net balance of both transactivation and transrepression effects, and will differ between cells, and under different stimuli. We therefore investigated the biological relevance of GR activation in vitro, and demonstrated that salmeterol modulation of GR translocation had functional effects on GRE-luciferase activity and MKP-1 and SLP1 gene induction. LABAs have been shown previously to augment the antiinflammatory effects of corticosteroids (13), particularly via inhibitory effects on cytokine release from a variety of cells involved in the pathogenesis of asthma. Korn and Brattsand (27) observed the combination of formoterol and budesonide inhibited granulocyte-macrophage colonyeCstimulating factor release from tumor necrosis factor eCstimulated human airway epithelial cells more than either drug alone. Similarly, Pang and Knox reported that salmeterol enhanced the inhibitory actions of FP on tumor necrosis factor eCinduced interleukin 8 (28) and eotaxin (29) release from human airway smooth muscle cells. We chose to examine the ability of FP to induce GR-GRE DNA binding, and induce antiinflammatory genes such as SLPI and the dual mitogen-activating protein kinase inhibitor MKP-1. We found that salmeterol was able to augment the ability of FP to enhance both GR-GRE binding and SLPI and MKP-1 expression in both macrophage-like and epithelial cell lines. The effect was at least additive and clearly synergistic with respect to SLPI induction in BEAS-2B cells. The effects on GR-GRE activation and gene expression were not totally superimposable due to the more complex promoter structure of the native MKP-1 and SLPI promoters and the requirement for other factors for full gene expression.
In vivo studies using bronchial biopsies generate further supporting evidence for complementary interactions on airway inflammation and pathology in subjects with asthma. Li and colleagues (30) found the addition of salmeterol in patients receiving inhaled corticosteroid led to a reduction in the total number of activated eosinophils within the lamina propria, together with a concurrent improvement in clinical status. Orsida and coworkers (31) demonstrated a significant reduction in the vascularity of the lamina propria in patients with asthma receiving salmeterol and inhaled corticosteroid, whereas no effect was observed after high-dose inhaled corticosteroid treatment. We used established noninvasive techniques of sputum induction to obtain airway cellular samples to investigate the interaction of inhaled LABA and corticosteroid in human subjects. We applied immunocytochemistry techniques to investigate the subcellular localization of GR in a semiquantitative manner similar to that described by previous authors in vitro (20). Although bronchoscopy with airway brushing may have provided sufficient cellular material to undertake quantitative protein analysis, it would not have been ethical to undertake this invasive procedure on the number of occasions outlined in our study design. In vitro experiments clearly demonstrate that these effects of LABA on corticosteroid actions are mediated through the 2-receptor (14, 27eC29). However, this has not been formally shown in this study in vivo due to the prolonged time required to examine cytokine profiles such as interleukin 6 or granulocyte-macrophage colonyeCstimulating factor (32), and the potential risk of giving subjects with asthma -blockers. Studies examining the effect of chronic combination treatment on cytokine profiles released from airway cells in vivo will need to be performed to address, in part, these issues.
GRs are phosphoproteins (33) and altered phosphorylation of GR and components of its signal transduction pathway have been implicated in the regulation of corticosteroid binding to GR (34), GR subcellular localization (35), and GR nuclear cytoplasmic trafficking through the nuclear pore complex (36, 37). Receptor phosphorylation may also influence the interactions of GR with other transcription factors, such as NF-B, required for transactivation (38). Many of the kinases that catalyze the phosphorylation of GR have been identified (39) and mitogen-activated protein kinaseeCdependent phosphorylation may play a role (40). Interestingly, phosphorylation of inactive GR may block subsequent hormone binding and nuclear translocation (41). LABAs may prime GR for activation directly by kinase-dependent phosphorylation and this may account for the additional effects of LABA on corticosteroid effects on NF-B (42). We postulate that phosphorylation of GR and its change in protein structure may alter function such that there are more enhanced responses to combination treatment.
In vitro experiments show that GR is entirely localized to the cytoplasm before corticosteroid treatment (43). However, we observed basal nuclear GR translocation of approximately 30% within airway cells in both healthy subjects and in subjects with asthma, which may reflect responses to endogenous levels of circulating cortisol. This may also account, at least in part, for the effect seen with salmeterol alone in vivo. Although not a primary aspect of our study, we found a degree of corticosteroid insensitivity to GR nuclear translocation within neutrophils. Glucocorticoid sensitivity varies between cell types and on the stage of the cell cycle (24, 44) and often parallels changes in receptor density (45, 46), and low GR levels have been associated with a poor response to corticosteroid treatment (45, 47). Decreased GR sensitivity of neutrophils to corticosteroids has been shown in vitro (48), and this has been postulated to be the result of enhanced GR expression (49). The differential response of the cells to inhaled corticosteroids observed within our healthy volunteers suggests that the decreased sensitivity of neutrophils to glucocorticoids may also be due to reduced GR nuclear translocation.
In conclusion, our data show that salmeterol, in combination with fluticasone propionate, can enhance GR nuclear translocation in vivo, as well as in vitro, and the data suggest that salmeterol may play an important role in the additional benefits seen with combination therapy. In vitro, the enhanced GR nuclear translocation is associated with an amplified GR functional response. Further studies are needed to address whether these differences are maintained after chronic treatment with inhaled LABA and corticosteroids, and whether this results in an alteration in cytokine profiles in vivo.
Acknowledgments
Plasmids containing 2x glucocorticoid response element (GRE)eCluciferase were kindly donated by Dr. J. Bloom (Tucson, AZ).
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