Skip Navigation


Rheumatology Advance Access originally published online on October 13, 2006
Rheumatology 2007 46(4):579-585; doi:10.1093/rheumatology/kel276
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/4/579    most recent
kel276v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kozaci, D. L.
Right arrow Articles by Chikanza, I. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kozaci, D. L.
Right arrow Articles by Chikanza, I. C.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The differential expression of corticosteroid receptor isoforms in corticosteroid-resistant and -sensitive patients with rheumatoid arthritis

D. L. Kozaci1,*, Y. Chernajovsky1 and I. C. Chikanza1,2

1Bone & Joint Research Unit, Barts and The London, Queen Mary's School of Medicine & Dentistry, University of London, Charterhouse Square, London EC1M 6BQ and 2Department of Rheumatology, Barts & The Royal London and Newham University Hospitals, London, UK.

Correspondence to: I. C. Chikanza, Newham University Hospital, Glen Road, London E13 8SL, UK. E-mail: icchikanza{at}sachicorp.net


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Objective. A proportion of patients with rheumatoid arthritis (RA) fail to respond adequately to corticosteroid (CS) therapy. Using an in vitro CS sensitivity bioassay, we have subdivided RA patients into steroid-sensitive (SS) and -resistant (SR) subgroups and this correlates with clinical responses to CS therapy. CSs exert their effects via the CS receptor (CR), which exists as two main isoforms, CR{alpha} and CRß. CRß can function as a negative inhibitor of CR{alpha}. We have hypothesized that steroid resistance in RA patients is due in part to a relative over-expression of the CRß.

Methods. Peripheral blood mononuclear cells (PBMCs) were isolated from SS and SR RA patients. CR{alpha} and CRß mRNA expression was determined by quantitative real time polymerase chain reaction (qRT–PCR). The ratio of CRß/CR{alpha} mRNA expression was determined. CR{alpha} and CRß protein expression by PBMCs was analysed by flow cytometry.

Results. qRT–PCR analysis showed a trend towards higher expression of both CRß and basal CRß/CR{alpha} ratio in SR RA patients. Stimulation of PBMCs in vitro with concanavalin-A induced a significantly higher CRß mRNA expression, and CRß/CR{alpha} ratio in SR RA patients compared with SS patients, which was not inhibited by hydrocortisone. Flow cytometry showed that the percentage of PBMCs staining for CRß protein was significantly lower in the SS RA group (SS 43.3 ± 14.8% vs SR 88.6 ± 8.6%; P < 0.0010). The mean intensity of fluorescence CRß staining was higher in the SR RA patients (P < 0.001).

Conclusion. We show for the first time that CRß is over-expressed in SR RA patients and that hydrocortisone fails to inhibit concanavalin-A stimulated increase in CRß mRNA in SR RA patients. This mechanism may contribute in part to the CS hyporesponsiveness seen in some RA patients.

KEY WORDS: Rheumatoid arthritis, Inflammation, Corticosteroid resistance, Corticosteroid receptor, Corticosteroids


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that is often treated with corticosteroids (CSs) [1]. A proportion of patients with RA fail to respond adequately to CS therapy [2]. RA patients can be subdivided into CS-sensitive (SS) and -resistant (SR) subgroups on the basis of the in vitro inhibition by CSs of T-cell proliferation induced by concanavalin-A (con-A), which correlates with the clinical observations [2]. The IC50 of hydrocortisone on cell proliferation in SS subjects is ≤10–6 M, whilst the value is ≥10–5 M in the majority of SR patients [2]. Patients with asthma, an allergic condition associated with bronchial spasm and eosinophilic infiltration of the bronchial mucosa, have also been divided into similar subgroups that correlate with clinical responses to CS therapy [3, 4]. The underlying mechanisms involved in the SS and SR phenomenon in patients with RA remain unknown but may be related in part to the functional status of the CS receptor (CR).

We have gained more insight into the molecular basis of the anti-inflammatory actions of CS. CSs inhibit T-cell proliferation via the inhibition of interleukin (IL)-2 production and other cytokines via the CR [5]. The promoter region of the IL-2 gene contains cis-acting elements required for the maximal induction of IL-2 production that are recognized by the nuclear transcription factors AP-1, NF-{kappa}B, NF-AT and OCT-1 [6, 7]. When CS binds to CR, the CS/CR complex is released from the heat shock protein 90 containing multi-protein complex, it dimerizes, transactivates and translocates to the nucleus to bind to specific DNA motifs, the glucocorticosteroid response elements (GREs) [8]. The GREs can mediate both positive and negative effects [8, 9]. The CS/CR complex also interferes with the binding of AP-1 to its site on DNA (trans-repression) [6, 7, 10], and CSs up-regulate I{kappa}B production, which sequesters NF-{kappa}B in the cytoplasm [10,11]. NF-{kappa}B exists in the cytoplasm as a heterodimer (most commonly p50 and p65 subunits) bound to an inhibitor I{kappa}B that exists in a number of isoforms [12]. Activation of cells by a variety of stimuli, leads to the degradation of I{kappa}Bs and the released NF-{kappa}B then translocates to the nucleus to initiate transcription of target genes [12–14]. The binding of AP-1 and NF-{kappa}B to DNA sites on target genes induces the production of pro-inflammatory cytokines such as IL-1ß, IL-2, IL-3, IL-6, IL-8, tumour necrosis factor {alpha} (TNF{alpha}) and adhesion molecules [7, 15, 16].

CR exists in two main isoforms, CR{alpha} and CRß, produced by alternative splicing [17–18]. Only CR{alpha} binds to CS [18]. Other receptor isoforms such as CR-{gamma}, CR-A and CR-P have been described but their functions are not fully characterized. Transfection experiments in COS-1, CV-1, HeLa S3 and COS-7 cells by two groups of investigators showed that the CRß isoform can act as a negative inhibitor of CR{alpha}-mediated effects [18–20]. Bamberger et al. [19] showed that the CRß isoform was able to inhibit the effects of hormone-activated human CR{alpha} (CS/CR{alpha} complex) on GREs in a dose dependent manner. [3H]-dexamethasone binding studies indicated that the CRß isoform did not alter the binding affinity of CR{alpha} for CS [19]. The presence of the CRß isoform in nuclear extracts and its ability to bind to radio-labelled GRE oligonucleotides suggested that its inhibitory effects were due to competition for the GRE target site [19]. Similar observations were made by Oakley et al. [20]. However, Brogan et al. using COS-7 and HEK-293 cells, showed that over-expression of the CRß isoform did not interfere in a specific way with the interaction of CR{alpha} with AP-1 and NF-{kappa}B (transrepression) and they suggested that the effects in COS-7 cells could be due to non-specific transcriptional squelching [21]. Transfection studies in Jurkat cells showed that co-expression of CR{alpha} and CRß did not interfere with CR{alpha}-mediated trans-activation or transrepression [22]. In contrast, CRß from the same plasmid preparation caused a significant inhibition of transcriptional activity in COS-7 cells, indicating a cell type-specific pattern of CRß-mediated inhibition of CR{alpha}-mediated CS effects [22].

Immunohistochemistry studies in asthma patients by Leung et al. [23] have recently shown that the CRß isoform is over-expressed by a significantly higher percentage of broncho-alveolar lavage airway cells from SR asthmatic patients than in SS asthmatics. Furthermore, treatment of the broncho-alveolar lavage airway cells from SR asthmatics with a combination of IL-2 and IL-4 induced a further increase in the over-expression of the CRß isoform above baseline, suggesting that cytokines could induce its expression [23, 24]. This observation in SR asthmatic patients suggests a potential role of CRß in the SR phenomenon seen in asthma patients. Whether a similar situation exists in RA patients who are SR has not been determined.

We have previously shown that CSs fail to inhibit IL-2 and IL-4 production by peripheral blood mononuclear cells (PBMCs) from SR RA patients [2]. This failure to inhibit IL-2 and IL-4 production in SR could potentially result in CRß over-expression. It can thus be hypothesized that the over-expression of the CRß isoform relative to CR{alpha} could potentially contribute to a state of reduced responsiveness to CS therapy in patients with RA. We have investigated the hypothesis that the differences in the sensitivity to CS seen in patients with RA, may be a consequence, in part, of differences in the differential expression of the CR{alpha} and CRß. Thus, we assessed the levels of both CR{alpha} and CRß isoforms at mRNA and protein levels using quantitative real time polymerase chain reaction (qRT–PCR) and by immunocytofluorographic analysis of the cellular protein expression in vitro-defined SS and SR RA patients.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusions
 Acknowledgements
 References
 
Patient selection
We studied a group of patients (n = 22) who were subdivided into SS (n = 10) and SR (n = 12) subgroups using a previously described CS sensitivity in vitro bioassay [2]. In order to control for the potential confounding effects of inflammation on CS sensitivity, only patients with similar levels of disease activity as measured by erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and number of active joints (i.e. tender and/or swollen) were selected. The demographics of these patients were not significantly different and are shown in Table 1. All the patients fulfilled the 1987 American College of Rheumatology diagnostic criteria for RA [25], had erosive joint disease, and had not received CS therapy within the past 3 months. All patients were on methotrexate and similar disease duration. None of the patients had another chronic inflammatory disorder other than RA. Patients with hypothalamic pituitary adrenal disorders, chronic obstructive pulmonary disease or on psychotropic drugs were excluded. Cells were isolated from each RA patient studied and then cultured; mRNA isolated from a portion of cells and cells were also immunocytofluorographically labelled for analysis of CR{alpha} and CRß isoforms expression. The study was approved by our local research ethics committee and all patients gave informed and written consent.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Demographics of patients studied for their CS responsiveness (mean ± SE)

 
Cell preparation
Peripheral blood (20 ml) was collected into heparinized tubes for all the studies between 10:00 and 11:00 a.m. in all the subjects to avoid variations due to diurnal rhythm of hypothalamic pituitary adrenal axis activity, which could potentially affect CR expression. PBMCs were isolated by density gradient centrifugation using LymphoprepTM (Nycomed, Oslo, Norway), washed three times in Hank's buffered saline solution and resuspended in tissue culture medium (TCM) as previously described [2]. The TCM consisted of RPMI 1640 (Life Technologies, UK) containing 10% fetal bovine serum, 100 000 IU/l penicillin and 100 mg/l streptomycin (GIBCO, Paisley, UK). Some of the resuspended cells were aliquotted and distributed equally into 1.8 ml sterile tubes, mixed with freezing medium [20% DMSO in fetal bovine serum (FBS)] 1: 1 and immediately frozen and stored in liquid nitrogen.

In vitro CS sensitivity assay
The in vitro CS sensitivity assay measured the inhibitory effect of hydrocortisone on con-A-stimulated PBMCs proliferation [2]. PBMCs (1 x 105 cells in a final volume of 100 µl/well) were plated in triplicate in flat-bottomed Nunclon® 96-Well MicroWell® plates (Nunclon, Life Technologies, UK) and cultured at 37°C, 5% CO2 and 95% air in a humidified atmosphere. Con-A 10 µg/ml (dose established by preliminary experiments) was used to stimulate the cells in the presence of different concentrations of hydrocortisone (10–8, 10–7, 10–6, 10–5, 10–4 M). Hydrocortisone was always prepared fresh at the start of each experiment.

After 72 h of culture, 100 µl of Cell Titer-Glo reagent was added to each well according to manufacturer's instructions, and inhibitory effects of hydrocortisone were determined on cell proliferation/viability (Promega G7570, Southampton, UK) by measuring the relative light units (RLU) using a MLX microtitre plate luminometer (Dynex Technologies, Chantilly, VA, USA). The mean RLU of triplicate of each sample were calculated. The percentage inhibition of cell proliferation by hydrocortisone was calculated using the following formula:


Formula

where, x, RLU in hydrocortisone and Con-A; n, RLU in TCM alone; y, RLU in con-A alone.

The IC50 of hydrocortisone on cell proliferation was determined from the dose–response curves. The IC50 was defined as the concentration of hydrocortisone that caused a 50% inhibition of cell proliferation; by this calculation the variations in con-A-induced proliferation that occur between individuals and in the same individual were compensated for. Subjects with an IC50 of >10–6 M were considered to be SR resistant as previously described [2].

Determination of CR isoform expression
Cell culture
PBMCs, collected as described above, were re-suspended at a concentration of 1 x 106 cells/ml in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (GIBCO), 100 000 IU/l penicillin, and 100 mg/l streptomycin. Cells were pretreated with hydrocortisone (10–6 M) or TCM for 5 min. and then incubated in the absence or presence of con-A (10 µg/ml) for 1 h at 37°C in 5% CO2. Subsequently, cells were centrifuged at 400 g/15 min at 4°C, supernatant was discarded and RNA extracted as described below.

RNA isolation and cDNA synthesis
Total RNA from PBMCs were isolated using RNA STAT-60 Reagent (Biogenesis, Poole, UK) according to manufacturer's instructions. After washing, the mRNA was air dried and suspended in DEPC-treated water after which the optical density (OD) 260/280 was determined (>1.7), and used for calculation of the concentration. cDNA was prepared using PROMEGA A3500 kit (Southampton, UK). 1.0 µg of sample RNA was denatured for 10 min at 70°C, and then mixed with a buffer containing 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 5 mM MgCl2, 1 mM each dGTP, dATP, dTTP, dCTP, 0.5 µg oligo dT primer, 20 U RNase inhibitor, 15 U AMV reverse transcriptase, in a final volume of 20 µl. This mixture was incubated for 15 min at 42°C, heated at 99°C for 5 min, and then incubated at 0–5°C for 5 min.

Quantitative real time PCR (qRT–PCR)
For qRT–PCR analysis, TaqMan technology was applied, according to the manufacturer's instructions. Taqman qRT–PCR involves the continuous detection of newly formed amplicons during PCR cycling, using a forward primer, a reverse primer and a FAM–TAMRA probe, which anneals between the two other primers. qRT–PCR was performed in triplicate for each sample using the ABI PRISM 7900HT Sequence Detector (Perkin–Elmer Applied Biosystems) and the TaqMan EZ RT–PCR Core Reagents Kit (Perkin–Elmer Applied Biosystems). The qRT–PCR was performed for CR{alpha}, CRß and ß-actin as a control housekeeping gene. Sequences for forward and reverse oligonucleotide primers as well as the TaqMan probes used in this study are detailed in Table 2. These oligonucleotides were designed using Primer Express software (PE-Applied Biosystems, Warrington, UK) and primers spanning intron sequences were selected to differentiate amplification from genomic DNA. All primers and probes were synthesized by Sigma (UK). The qRT–PCR primers and probes are shown in Table 2.


View this table:
[in this window]
[in a new window]

  		TABLE 2. The details of primers used for the quantitative real time PCR

 
Before application of Taqman qRT–PCR to determine CR{alpha} and CRß ratio, the capacity of the technique to detect differences in ratios of synthesized RNAs was validated. Amplicons presenting parts of exon 9{alpha} or exon 9ß were generated from HeLaS3 cDNA, mixed in different ratios and analysed by Taqman qRT–PCR. Cycle conditions were as follows: initial denaturizing at 95°C for 10 min, 50 cycles of 95°C for 15 s, 60°C for 60 s, elongating at 72°C for 60 s and one final elongation step of 5 min at 72°C. Tenfold serial dilutions (spanning four orders of magnitude) of HeLa S3 cDNA of known concentrations were used to construct standard curves of cycle threshold (Ct) values for CR{alpha}, CRß and ß-actin.

Analysis of cellular expression of CR protein isoforms by flow cytometry
PBMCs were isolated from blood collected between 10:00 and 11:00 A.M. from RA patients as described above. Cells were stained for CR{alpha} and CRß using a modification of the flow-cytometry method for intracellular proteins as previously described [26] and analysed by fluorescent activated cell sorter (FACS). HeLaS3 cells, which over-express CRß, were used as a positive test control.

Antibodies for immunofluorescence staining
The irrelevant non-specific SimultestTM IgG control was a mixture of FITC- and PE-conjugated mouse antibody isotype IgG2a (Becton Dickinson, San Jose, California). The first layer antibodies were an affinity purified protein rabbit polyclonal antibody GR(P-20) (Autogen Bioclear UK Ltd, Devizes, UK) and/or GCR-ßPA3-514 (Affinity Bioreagents, Golden, USA) directed against CR{alpha} and CRß, respectively, as indicated. The affinity purified protein antibodies only bind to their target protein molecules. The second antibody was FITC-conjugated polyclonal goat anti-rabbit (GAR) (BD Biosciences, San Diego, CA, USA). In order to reduce non-specific binding to other intracellular proteins, GAR-FITC was serially pre-adsorbed as previously described [25].

PBMCs isolated by density gradient centrifugation were washed twice with a wash solution [WS; phosphate-buffered saline (PBS) containing 0.01% azide and 0.1% bovine serum albumin (BSA)] by resuspension of the cell pellet in the solution and centrifugation at 500 g. Cells were then fixed by incubation for 10 min on ice, in 1 ml of a 1% paraformaldehyde solution in PBS containing 0.1% BSA and then washed twice. Cells were then placed in each tube and treated as detailed below.

Permeabilization was performed with 1.2% n-octyl glucoside (NOG) for 7.5 min at 4°C. After permeabilization the cells were washed twice with WS and then stained. Cells were incubated with 20% normal human serum (NHS) for 10 min, washed twice. The first antibody was added (5 µl) and cells were incubated at 25°C for 20 min then washed twice. The second antibody pre-adsorbed GAR-FITC (5 µl) was added and cells were incubated at 25°C for 20 min then washed twice again with WS. Normal mouse serum (10 µl) was added, and cells were incubated at 25°C for 20 min then washed twice before adding the third final layer of antibodies. Finally, cells were washed twice and then resuspended in 500 µl of PBS/1% paraformaldehyde/0.1% BSA solution before FACS analysis.

Flow-cytometric analysis was carried out using a Becton Dickinson FACscan Calibur and results were analysed using WinMDI V2.8 software. The cells were gated for forward and side scatter. The total number and percentage of positive PBMCs and the mean intensity of fluorescence (MIF) of CRß and CR{alpha} staining over background were determined.

Statistical analysis
The results are expressed as mean ± SEM. The results were analysed using SYSTAT 11 software. Non-parametric tests were used to analyse the differences between the observations seen in the SS and SR RA subjects with Wilcoxon matched-pair test for two-group comparisons with exact, two-tailed significance levels and analysis of variance. A value of P ≤ 0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusions
 Acknowledgements
 References
 
Steroid sensitivity status of patients
There were no significant differences in the degree of inflammation between the two subgroups of patients as measured by the levels of ESR, CRP and number of active joints (Table 1). The distribution of RF seropositivity status, gender ratios and disease duration were similar between the SS and SR RA subgroups. Subjects were subdivided using the hydrocortisone IC50 of 10–6M into SS and SR subgroups as previously described [2]. The mean dose–response curves of the subgroups are shown in Fig. 1. As can be seen, there are significant differences in the percentage inhibition of cell proliferation at each dose point between the SS and SR subjects.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Inhibition of cell proliferation by hydrocortisone in PBMCs from SS and SR RA patients. The mean of the in vitro doses to hydrocortisone treatment on con-A stimulated PBMCs cell proliferation are shown. Subjects with an IC50 of less 10–6 M hydrocortisone are considered to be SS as previously described [2]. X axis notation: (con-A = concanavalin-A alone; For hydyrocortisone treatment of con-A stimulated PBMCs - 10–8 M = 10–8M, 10–7 M = 10–7M, 10–6 M = 10–6M, 10–5 M = 10–5M, 10–4 M = 10–4 M).

 
Levels of CR isoform mRNA expression in PBMCs as determined by qRT–PCR
Taqman RT–PCR was used to quantitate levels of expression of the receptor isoforms. All PCR products were evaluated on the basis of the individual ß-actin housekeeping gene expression. For CR{alpha} and CRß isoform expression, cDNA samples from PBMCs from RA patients were analysed. The expression of mRNA levels of CR{alpha} and CRß isoforms at basal and following stimulation with con-A, in the absence or presence of hydrocortisone were compared between the SS and SR subgroups (Fig. 2). The level of CR{alpha} mRNA expression was the same in both subgroups. A trend towards higher basal expression of CRß mRNA in PBMCs was observed in SR RA patients than in the SS RA subgroup. Con-A stimulation without or with hydrocortisone caused a significant increase in the expression of the CRß isoform in SR RA patients compared with the SS RA subjects (P < 0.05 and P < 0.02, respectively). Con-A stimulation did not cause an increase in CRß mRNA in the SS RA subgroup and hydrocortisone caused a 14.5% downward trend inhibition in expression (Fig. 2B). In contrast, con-A enhanced CRß isoform mRNA expression by 20%, in the SR RA patients, which was not inhibited at all by hydrocortisone (Fig. 2B). However, the differences in the relative over-expression of CRß in SR RA patients become more highly significant when one looks at the CRß/CR{alpha} ratio of mRNA expression (Fig. 2C). The basal CRß/CR{alpha} ratio shows a trend towards higher levels in PBMCs from SR RA subjects than in SS RA patients. Significant changes are observed following stimulation of PBMCs in vitro with con-A alone and treatment of con-A-stimulated PBMCs with hydrocortisone with a significantly higher CRß/CR{alpha} ratio in SR RA patients (P ≤ 0.01 and P ≤ 0.01, respectively). Thus, overall, the qRT–PCR data suggest that CRß mRNA is over-expressed in SR RA patients.


Figure 2
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Levels of mRNA expression of CRß and CR{alpha} by PBMCs. The mean results of the qRT–PCR analysis of CR{alpha} and CRß mRNA expression in arbitrary units (AU) in PBMCs from SR RA and SS RA patients are depicted. Panel A shows the CR{alpha} mRNA data at baseline (cont); following stimulation of PBMCs with con-A (con-A) and treatment of con-A stimulated PBMCs with hydrocortisone (con-A + HC). Panel B shows CRß mRNA data. Notation as in Panel A. Panel C shows the mRNA expression ratio of CRß/CR{alpha} at baseline; following stimulation of PBMCs with con-A, and treatment of con-A stimulated PBMCs with hydrocortisone. Notation as in Panel A. CRß mRNA is overexpressed relative to CR{alpha} in SR RA patients (SS vs SR: *P < 0.02; **P < 0.05; ***P ≤ 0.01).

 
Protein expression levels of the CR isoforms in PBMCs
Since overexpression of CRß protein has been shown to reduce the effects of CS, we analysed the protein expression of the CR isoforms using flow cytometry. Fig. 3 (panels A–D) shows representative immunocytofluorographic data from an SR and SS RA subject. As can be seen, the percentage of PBMCs expressing CRß protein is higher in SR RA subjects. The results from a group of SS and SR patients are summarized in Fig. 4 (panels A and B). For the CR{alpha} isoform, there were no significant differences between the SS and SR RA patients (Fig. 4, panels A and B). However, the percentage of PBMCs staining for CRß was significantly lower in the SS RA group (P < 0.001). The MIF of CRß staining was also higher in the SR RA subgroup of patients than in the SS subjects (P < 0.001). These data support the qRT–PCR observations in SR RA patients that CRß is relatively over-expressed in PBMCs from RA patients who are SR as defined by the in vitro CS bioassay.


Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. Representative FACS analysis of CR{alpha} and CRß protein expression in an SS and SR RA patient. CR{alpha} protein expression by PBMCs from an SR RA (Panel A) and an SS RA patient (Panel B) analysed by flow cytometry. Panels C and D show data for CRß from an SR RA patient (Panel C) and an SS RA patient (Panel D) analysed by flow cytometry. Background staining is shown in clear histogram and that for CR{alpha} or CRß protein is shown in shaded histogram. The percentage of positive cells staining for CR isoform is shown in each panel.

 

Figure 4
View larger version (54K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. The Statistical analysis of CR{alpha} and CRß protein levels in SS and SR RA patients: The mean percentage number of PBMC from SR and SS patients staining positive for CRß and CR{alpha} are shown in Panel A. The mean of the MIF (mean intensity of fluorescence) for CRß and CR{alpha} staining is shown in Panel B. The percentage of PBMC staining for CRß was significantly lower in the SS RA group (p < 0.001) whilst the MIF of CRß staining was significantly higher in the SR RA subgroup of patients than in the SS subjects (p< 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusions
 Acknowledgements
 References
 
A number of molecular mechanisms may be involved in SR and this may be related to perturbations of the cellular pathways used by CSs to exert their biological effects [27, 28]. One of these relates to perturbations of the CR isoform expression. CSs mediate their genomic effects via the CR{alpha} receptor. A number of cell line transfection experiments have shown that the relative over-expression of the CRß isoform induces a state of reduced sensitivity to the inhibitory effects of CS on gene expression [18–20]. Using qRT–PCR analysis and flow cytometry, we show for the first time enhanced CRß mRNA and protein expression in PBMCs from SR RA patients when compared with the SS RA patient subgroup. We also demonstrate that con-A stimulation of PBMCs from SR RA patients enhances overexpression of CRß mRNA, which is not inhibited by hydrocortisone treatment. This observation is of pathophysiological relevance to the molecular basis of the SR phenomenon, since such a relative increase in CRß expression during cell activation may occur during inflammation and could potentially contribute to a reduced state of responsiveness to the biological effects of CS therapy.

Our observations in RA patients at the mRNA level were also reflected at the protein level in PBMCs. Our flow-cytometry data are highly significant since the CRß isoform does not undergo ligand-dependent down-regulation and consequently has a much longer half-life than that of CR{alpha} [29]. Thus, even low levels of CRß protein may achieve a significant dominant negative effect on the activity of CR{alpha} isoform [29]. Hence, even the small increases in CRß mRNA expression such as observed at the basal level in the SR RA patients, may translate into significant protein expression and the associated biological effects. Minimal perturbations of the CRß/CR{alpha} mRNA ratio, may lead to prolonged and enhanced CRß mediated inhibitory effects at the protein level. Our observations at the protein level in SR RA patients, are similar to the in vivo observations of Sousa et al. [30] and Hamid et al. [31] in SR asthmatic patients. Using fluorescence microscopy, Hamid et al. [31] examined broncho-alveolar lavage cell cytospins from SS and SR asthmatic patients stained by immunohistochemistry for the expression of CRß. broncho-alveolar lavage cell preparations from SR asthmatic patients had a significantly higher expression of CRß. Sousa et al. [30] measured the CR{alpha} and CRß immunoreactive cells in skin biopsies from tuberculin-induced skin lesions in SS and SR asthmatic patients. The number of cells expressing CRß was higher in skin lesions biopsies of SR asthmatic patients [30]. Similar findings have been seen in PBMCs from patients with ulcerative colitis by Honda et al. [32]. They used real time-polymerase chain reaction (RT–PCR) analysis to detect CR{alpha} and CRß in PBMCs from ulcerative colitis patients and assessed the potential of detecting CRß as a predictor of the likelihood of responding to CS therapy in patients whose disease flared [32]. CRß mRNA was detected in a significantly higher percentage of patients who failed to respond adequately to CS therapy.

The mechanisms responsible for the enhanced expression of CRß in SR RA patients are currently unknown. Alternative splicing of the CR pre-mRNA gives rise to the homologous CRß mRNA. Knowing the exact mechanisms that regulate alternative splicing of CR pre-mRNA could be exploited as a therapeutic application. Such research is currently being done in Duchenne muscular dystrophy to produce a different isoform of dystophin [33].

The physiological and pathophysiological role of the CRß isoform is controversial. Co-transfection experiments with human CRß and CR{alpha} expression vectors and GRE-containing reporter plasmid to COS cells by three independent groups have shown that CRß over-expression inhibits GRE-containing gene expression [18–20]. Others have, however, provided conflicting data [21–22, 34]. The latter may represent a limitation of cell line co-transfection experiments. Human neutrophils are less sensitive than T cells to the apoptotic effects induced by CS and this may explain in part the observation that CSs are less effective at resolving neutrophil-associated inflammation [35]. This may be a consequence of the higher constitutive expression of CRß by neutrophils in comparison with T cells [36]. Mouse neutrophils do not express CRß and are very sensitive to CS-induced apoptosis [37]. Transfection of mouse neutrophils with a pRSh CRß expression vector results in a significant reduction of dexamethasone-induced neutrophil apoptosis [36]. This is the closest indirect ex-vivo experimental evidence supporting the view that the over-expression of CRß in SR RA patients we have observed in our studies has direct pathophysiological relevance to the phenomenon of SR. More recently, Charmandari et al. [37] have studied the mechanisms underlying the dominant negative effects of the CRß isoform on steroid receptor coactivators [38]. They showed that CRß suppressed the transcriptional activity of both activation of function (AF)-1 and AF-2 of CR{alpha} by binding to the p160 coactivators and the CR interacting protein 1 (CRIP1) [38]. This finding supports our hypothesis that in SR RA patients the enhanced CRß expression is of pathophysiological relevance to the SR phenomenon. RA patients have reduced numbers of CRs on their lymphocytes but this does not result in reduced biological effects of CS per se [39]. It therefore remains to be determined whether there are differences in post-receptor mechanisms in the SS and SR RA patients. We are currently investigating this possibility.

It has been suggested that methotrexate may increase CR expression and thus potentially increase susceptibility to CS inhibition. All the SS and SR RA patients were on methotrexate and thus the differences in the expression of CR isoforms are not secondary to methotrexate. Furthermore, the duration of RA disease, levels of inflammation as measured by the levels of ESR, CRP and number of active joints were not significantly different between the SS and SR subjects.


   Conclusions
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
Acknowledgements
 References
 
In conclusion, we show for the first time that the CRß isoform is over-expressed in SR RA patients at both the mRNA and protein levels. Furthermore, hydrocortisone fails to inhibit the con-A induced increase in CRß mRNA. This over-expression of CRß by PBMCs may in part explain the reduced responsiveness to CS therapy observed in these patients.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
References
 
The authors thank the Arthritis Research Campaign, UK for funding this research and C. Mein of the Genome Centre for technical support in the qRT–PCR studies and Rukshana Patel and Cherma St Claire Ali for logistical and administrative support. We are also most grateful to all the RA patients who volunteered to participate in these studies.

The authors have declared no conflicts of interest.


   Notes
 
*Present address: Adnan Menderes University, School of Medicine, Department of Biochemistry, Aydin 09100, Turkey. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 Acknowledgements
 References
 

  1. Feldmann M. (2001) Pathogenesis of arthritis: recent research progress. Nat Rev Immunol 2:771–3.
  2. Chikanza IC and Panayi GS. (1993) The differential effects of hydrocortisone on in vitro lymphocyte proliferation, IL-2, IL-4 production in corticosteroid sensitivity and resistant rheumatoid patients. Eur J Clin Invest 23:845–50.[Web of Science][Medline]
  3. Carmichael J, Paterson IC, Diaz P. (1981) Corticosteroid resistance in asthma. Br Med J 283:1491.
  4. Poznansky MC, Gordon ACH, Douglas JG. (1984) Resistance to methyl prednisolone in cultures of blood mononuclear cells from glucocorticosteroid resistant asthmatic patients. Clin Sci 67:639.
  5. Arya SK, Staal FW, Gallo RC. (1984) Dexamethasone mediated inhibition of human T cell growth factor and gama interferon mRNA. J. Immunol 133:273.[Abstract]
  6. Palliogianni F, Raptis A, Ahuja SS, Najjar SM, Boumpas DT. (1993) Negative transcriptional regulation of human IL-2 gene by glucocorticosteroids through interference of nuclear transcription factor AP-1 and NF-AT. J Clin Invest 91:1481–9.[Web of Science][Medline]
  7. Beato M. (1989) Gene regulation by steroid hormones. Cell 56:335.[CrossRef][Web of Science][Medline]
  8. Cato ACB and Wade E. (1996) Molecular mechanisms of anti-inflammatory action of glucocorticosteroids. BioEssays 18:371–8.[CrossRef][Web of Science][Medline]
  9. Akerblom IE, Slater EP, Beato M, Baxter JD, Mellon PL. (1988) Negative regulation by glucocorticosteroids through interference with cAMP response enhancer. Science 241:350–7.[Abstract/Free Full Text]
  10. Auphan N, Didonato JA, Rosette C, Helmberg A, Karin M. (1995) Immunosuppression by glucocorticosteroids: inhibition of NF-{kappa}B activity through induction of I{kappa}B{alpha} synthesis. Science 270:286–91.[Abstract/Free Full Text]
  11. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS. (1995) Role of transcriptional activation of I{kappa}B{alpha} in the mediation of immunosuppression by glucocorticosteroids. Science 270:283–6.[Abstract/Free Full Text]
  12. Baeuerle PA and Baltimore D. (1988) I{kappa}B{alpha}: a specific inhibitor of the NF-{kappa}B transcription factor. Science 242:540–6.[Abstract/Free Full Text]
  13. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. (1995) Control of I{kappa}B{alpha} proteolysis by site specific signal induced phosphorylation. Science 267:1485–8.[Abstract/Free Full Text]
  14. Beg AA and Baldwin AS. (1993) I{kappa}B{alpha} proteins: multifactional regulators of Rel/NF-{kappa}B transcription factors. Gene Dev 7:2064–70.[Free Full Text]
  15. Barnes PJ and Adcork I. (1993) Anti-inflammatory actions of steroids: molecular mechanisms. TiPs Reviews 14:436–41.
  16. Cronstein BN, Kimmel SC, Levin R, Martiniuk F. (1992) A mechanism for the antiinflammatory effects of corticosteroids: The glucocorticosteroid receptor regulates leucocyte adhesion to endothelial cells and expression of ELAM-1 and ICAM-1. Proc Natl Acad Sci USA 89:9991–5.[Abstract/Free Full Text]
  17. Encio IJ and Detera-Wadleigh SD. (1991) The genomic structure of the human glucocorticosteroid receptor. J Biol Chem 266:7182–8.[Abstract/Free Full Text]
  18. Castro M, Elliot S, Kino T, et al. (1996) The non-ligand binding ß isoform of the human glucocorticosteroid receptor: tissue levels, mechanisms of action and potential physiologic role. Mol Med 2:597–607.[Web of Science][Medline]
  19. Bamberger CM, Bamberger A, de Castro A, Chrousos G. (1995) Glucocrticosteroid receptor ß, a potential endogenous inhibitor of glucocorticosteroid action in humans. J Clin Invest 95:2435–41.[Web of Science][Medline]
  20. Oakley R, Sar M, Codlowski JA. (1996) The human glucocrticosteroid receptor ß isoform. J Biol Chem 271:9550–9.[Abstract/Free Full Text]
  21. Brogan IJ, Murray IA, Cerillo G, Needham M, White A, Davis JRE. (1999) Interaction of glucocorticoid receptor isoforms with transcription factors AP-1 and NF-kB: lack of effect of glucocorticoid receptor ß. Mol Cell Endocrinol 157:95–104.[CrossRef][Web of Science][Medline]
  22. de Lange P, Koper JW, Brinkmann Ao, de Jong FH, Lamberts SW. (1999) Natural variants of the beta isoform of the human glucocorticoid receptor do not alter sensitivity to glucocorticoids. Mol Cell Endocrinol 153:163–8.[CrossRef][Web of Science][Medline]
  23. Leung DY, Hamid Q, Vottero A, et al. (1997) Association of glucocorticosteroid insensitivity with increased expression of glucocorticosteroid receptor ß. J Exp Med 186:1567–74.[Abstract/Free Full Text]
  24. Kam JC, Szefler SJ, Surs W, Sher ER, Leung DYM. (1993) Combination of IL-2 and IL-4 reduces glucocorticoid receptor binding affinity and T cell response to glucocorticoids. J Immunol 151:3460–6.[Abstract]
  25. Arnett FC, Edworthy SM, Bloch DA, et al. (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis & Rheumatism 31:315–24.[Web of Science][Medline]
  26. Chikanza IC, Corrigal V, Kingsley GH, Panayi GS. (1992) Enumeration of intracellular IL-1 beta and alpha in mononuclear cells by flow cytometry. J Immunol Method 154:173–8.[CrossRef][Web of Science][Medline]
  27. Chikanza IC. (2000) Mechanisms of corticosteroid resistance in rheumatoid arthritis: a putative role for the corticosteroid receptor ß isoform. Ann NY Acad Sci 966:39–48.
  28. Leung DY and Bloom JW. (2003) Update on glucocorticoid action and resistance. J Allergy Clin Immunol 111:3–22.[CrossRef][Web of Science][Medline]
  29. Webster JC, Oakley RH, Jewel CM, Cidolowiski JA. (2001) Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance. Proc Natl Acad Sci USA 98:6865–70.[Abstract/Free Full Text]
  30. Sousa AR and Lane SJ, et al. (2000) Glucocorticoid resistance in asthma is associated with elevated in vivo expression of the glucocorticoid receptor beta-isoform. J Allergy Clin Immunol 105:943–50.[CrossRef][Web of Science][Medline]
  31. Hamid QA, Wenzel SE, Hauk PJ, et al. (1999) Increased glucocorticoid receptor ß in airways cells of glucocorticoid-insenstive asthma. Am J Resp Crit Care Med 159:1600–4.[Abstract/Free Full Text]
  32. Honda M, Orii FF, Ayabe T, et al. (2000) Expression of glucocorticoid receptor beta in lymphocytes of patients with glucocorticoid-resistant ulcerative colitis. Gastroenterology 118:859–66.[CrossRef][Web of Science][Medline]
  33. McClorey G, Fletcher S, Wilton S. (2005) Splicing intervention for Duchenne muscular dystrophy. Curr Opin Pharmacol 5:529–34.[CrossRef][Web of Science][Medline]
  34. Hecht K, Carlstedt DJ, Stierna P, Gustafsson J, Bronnegard M, Wikstrom AC. (1997) Evidence that the beta isoform of the human glucocorticoid receptor does not act as a physiologically significant repressor. J Biol Chem 272:26659–64.[Abstract/Free Full Text]
  35. Schleimer RP. (1990) Effects of glucorticosteroids on inflammatory cells relevant to their therapeutic applications in asthma. Am Rev Respir Dis 141:S59–69.[Web of Science][Medline]
  36. Strickland I, Kisich K, Hauk PJ, et al. (2001) High constitutitve glucorticoid receptor ß in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids. J Exp Med 193:585–93.[Abstract/Free Full Text]
  37. Otto C, Reichardt HM, Schutz G. (1997) Absence of glucorticosteroid receptor beta in mice. J Biol Chem 272:26665–8.[Abstract/Free Full Text]
  38. Charmandari E, Chrousos GP, Ichijo T, et al. (2005) The human glucocorticoid receptor (hGR)[beta] isoform suppresses the transcriptional activity of hGR[alpha] by interfering with the formation of active coactivator complexes. Mol Endocrinol 19:52–64.[Abstract/Free Full Text]
  39. Schlaghecke R, Beuscher D, Kornely E, Specker C. (1994) Effects of glucocorticosteroids in rheumatoid arthritis; diminished glucocorticosteroid receptors do not result in glucocorticoid resistance. Arthritis Rheum 37:1127–31.[Web of Science][Medline]
Submitted 25 October 2005; revised version accepted 4 July 2006.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/4/579    most recent
kel276v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Kozaci, D. L.
Right arrow Articles by Chikanza, I. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kozaci, D. L.
Right arrow Articles by Chikanza, I. C.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?