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Published ahead of print on June 7, 2004, doi:10.1164/rccm.200308-1143OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 420-425, (2004)
© 2004 American Thoracic Society

Original Article

Glucocorticoid Receptor Isoforms {alpha} and ß in in Vitro Cytokine-induced Glucocorticoid Insensitivity

Alfons Torrego, Laura Pujols, Jordi Roca-Ferrer, Joaquim Mullol, Antoni Xaubet and César Picado

Servei de Pneumologia i Al·lèrgia Respiratòria, Institut Clínic de Pneumologia i Cirurgia Toràcica, and Servei d'Otorinolaringologia, Hospital Clínic; and Institut d'Investigacions Biomèdiques August Pi i Sunyer, Departament de Medicina, Universitat de Barcelona, Barcelona, Spain

Correspondence and requests for reprints should be addressed to C. Picado, M.D., Servei de Pneumologia, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. E-mail: cpicado{at}ub.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We stimulated peripheral blood mononuclear cells from 14 healthy subjects, 14 patients with stable asthma, and 13 patients with unstable asthma with interleukin (IL)-2 and IL-4 to induce glucocorticoid insensitivity and we examined the relationship between insensitivity and the expression of glucocorticoid receptor (GR) isoforms. Results are expressed as IC50 (nanomolar) values (means ± SD) in proliferation assays and as 103 cDNA molecules per microgram of total RNA (means ± SD) in real-time polymerase chain reaction analysis. Cells from patients with unstable asthma were less sensitive (316 ± 7 nM) to dexamethasone antiproliferative effects than those from healthy control subjects (102 ± 4 nM, p < 0.05) and patients with stable asthma (107 ± 2 nM, p < 0.05). Coincubation with IL-2 and IL-4 repressed the inhibitory effect of dexamethasone on proliferation in all groups (unstable: 851 ± 47 nM, p < 0.01; stable: 912 ± 52 nM, p = 0.001; control subjects: 537 ± 45 nM, p = 0.001). GR-{alpha} mRNA baseline expression was higher in patients with unstable asthma [(1.95 ± 0.40) x 103 cDNA molecules/µg total RNA, p < 0.05] than in patients with stable asthma [(1.46 ± 0.35) x 103 cDNA molecules/µg total RNA] and healthy subjects [(1.35 ± 0.25) x 103 cDNA molecules/µg total RNA]. GR-ß mRNA was 600 times lower than GR-{alpha} in the three groups. Coincubation with IL-2 and IL-4 significantly increased GR-{alpha} mRNA expression in the three groups (p < 0.01), but caused no significant change in GR-ß mRNA. GR-{alpha}, but not GR-ß, protein was detected at baseline and after cytokine exposure. Our data do not support the hypothesis that increased GR-ß expression can contribute to cytokine-induced glucocorticoid insensitivity.

Key Words: asthma • interleukin-2 • interleukin-4 • peripheral blood mononuclear cell

The response of patients with asthma to glucocorticoids is highly variable: some patients can be controlled with low doses of glucocorticoids, whereas others may require higher doses and some rare patients appear to be resistant to glucocorticoids (1–6). Glucocorticoid insensitivity has also been reported in other inflammatory diseases such as nasal polyposis (7, 8) and ulcerative colitis (9, 10).

Glucocorticoids work by binding to the cytosolic glucocorticoid receptor (GR), inducing the formation of a dimer that is translocated into the nucleus and acts as a transcription factor (11). Cloning of the human GR has identified two isoforms, termed GR-{alpha} and GR-ß, which originate from alternative splicing of the GR primary transcript (12). Cotransfection assays have shown GR-ß to function as a dominant-negative inhibitor of GR-{alpha}–mediated transcriptional activation through a mechanism that involves the formation of GR-{alpha}/GR-ß heterodimers (13, 14). Such heterodimer formation may account for the reduced effectiveness of glucocorticoid action in cells overexpressing GR-ß (15, 16). This inhibitory effect of GR-ß on GR-{alpha} activity led to the hypothesis that excessive expression of the GR-ß isoform with respect to GR-{alpha} might play a role in the regulation of a cell's sensitivity to glucocorticoids in various inflammatory diseases (5, 7, 9, 10).

The potential role of increased expression of GR-ß in patients with glucocorticoid-insensitive asthma has been a matter of controversy (5, 6, 17, 18). Increased expression of the GR-ß isoform has been reported in patients with asthma that responds poorly to glucocorticoids (5, 19, 20). Corticosteroid-resistant asthma was associated with a significantly higher number of GR-ß–immunoreactive peripheral blood mononuclear cells (PBMCs) (5, 20). However, other authors were unable to find increased GR-ß expression in patients with glucocorticoid insensitivity (6). Methodologic deficiencies were suggested as an explanation for the negative results of this study (17). The lack of quantification standards in Western blot analysis, differences in the sensitivity of antibodies, the degradation of proteins, and the use of nonquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) are some of the methodologic aspects that have been questioned in this study (17).

Glucocorticoid insensitivity has been documented in vitro in monocytes and lymphocytes from glucocorticoid-resistant patients with asthma (2–4). Glucocorticoid insensitivity may be induced in these cells by in vitro exposure to a combination of two cytokines, interleukin (IL)-2 and IL-4 (21, 22). The combination of IL-2 and IL-4 has been reported to upregulate GR-ß in peripheral blood T lymphocytes, suggesting that immune activation drives the increased expression of GR-ß and thereby induces glucocorticoid insensitivity (20).

In our study we have used the reported capacity of PBMCs to undergo glucocorticoid insensitivity after in vitro exposure to IL-2 and IL-4 to gain insight into the potential role of GR-ß in the regulation of the glucocorticoid response.

Our objective was to help clarify the controversy concerning the role of both GR isoforms, GR-{alpha} and GR-ß, in glucocorticoid-insensitive asthma. Because the techniques used to assess the GR appear to be crucial to explain the controversy between researchers, we have taken special care to develop sensitive and specific methods (quantitative RT-PCR and Western blot) for measuring the level of expression of GR isoforms at mRNA and protein levels.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The characteristics of the 27 patients with asthma and 14 healthy subjects selected for evaluation are presented in Table 1 . All patients were required to demonstrate a reversibility of at least 12% in their FEV1. On entering the study all subjects were nonsmokers or exsmokers (one in each group) with a smoking history of less than 10 pack-years. Obstructive bronchial diseases other than asthma were also excluded. Fourteen patients were classified as having mild stable asthma on the basis of the presence of mild symptoms, prebronchodilator FEV1 higher than 80% predicted, and treatment consisting of inhaled ß-agonist as needed for relief of symptoms. Thirteen patients were classified as having unstable persistent moderate/severe asthma with an FEV1 value lower than 80% predicted, frequent symptoms, and regular use of rescue medication. Nine patients from this group were treated with budesonide (400 µg/day); five of them were also receiving formoterol (18 µg/day). None of the patients were receiving xanthines or antileukotriene drug therapy. None of the patients had received oral corticosteroids for the preceding 6 months. Informed consent was obtained from all subjects and the study protocol was approved by the Ethics Committee of the Hospital Clinic (Barcelona, Spain).


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  		TABLE 1. Characteristics of the study population

 
Lymphocyte Proliferation Assay
Blood samples were obtained in heparinized syringes between 8:00 and 9:00 A.M., and PBMCs were isolated by Ficoll-Hypaque gradient centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in serum-free and steroid-free X-VIVO 10 cell culture medium (Cambrex Bio Science Walkersville, Walkersville, MD). Cell viability was 95% or greater, as measured by trypan blue dye exclusion. PBMCs were stimulated with phytohemagglutinin (PHA, 10 µg/ml) in the absence or presence of IL-2 and IL-4 (10 ng/ml each) and with or without dexamethasone (10–5 to 10–11 M). Dexamethasone dilutions with medium were made each day from the original stock preparation. Cells were incubated for 72 hours at 37°C in 5% CO2. Four hours before harvesting, tritiated thymidine was added at a final concentration of 1 µCi per well. Cells were then harvested onto glass fiber filter disks and tritiated thymidine incorporation was assessed in a liquid scintillation counter.

Real-Time RT-PCR
Extraction of total RNA and reverse transcription were performed as previously reported (23). GR-{alpha} and GR-ß cDNAs were amplified with specific antisense primers that shared the same sense primer. Sequences were as follows: 5'-CTTACTGCTTCTCTCTTCAGTTCCT-3' (GR-{alpha}/ß sense, nucleotides 2136–2160), 5'-GCAATAGTTAAGGAGATTTTCAACC-3' (GR-{alpha} antisense, nucleotides 2316–2340), and 5'-AGTGCACATAATCTTCTTTTTC TCA-3' (GR-ß antisense, nucleotides 2404–2428). Quantification of GR transcripts was achieved by extrapolation to a plasmid double-stranded DNA external standard curve added in each PCR run. See the online data supplement for the real-time PCR protocol and validation of the method.

Western Blot Analysis
GR isoforms protein expression was determined by Western blot as previously reported (23). Blots were probed with an anti-{alpha}-tubulin antibody (Sigma, St. Louis, MO), an antibody raised against epitopes common to both GR isoforms (antibody 57), and specific antibodies for GR-{alpha} (AShGR) and GR-ß (BShGR), generously provided by J. A. Cidlowski (Laboratory of Signal Transduction, NIH, Research Triangle Park, NC). See the online supplement for further details.

Statistical Analysis
Data are presented as means and SD or SEM. IC50 values were calculated with the GraphPad Prism3 program (GraphPad Software, San Diego, CA). The IC50 is the concentration of dexamethasone that results in 50% suppression of PHA-induced cell proliferation. Differences between the three groups were determined by nonparametric analysis (Kruskal–Wallis test). Individual differences between groups were determined by paired t test, Mann–Whitney U test, or Wilcoxon paired test when appropriate. All reported p values are two-tailed. A p value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Dexamethasone and of IL-2 and IL-4 on Lymphocyte Proliferation
We assessed the sensitivity of PHA-stimulated PBMCs to increasing concentrations of dexamethasone (10–5 to 10–11 M) in the absence or presence of IL-2 and IL-4 (10 ng/ml each) (Figure 1) .



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  		Figure 1. Dose–response curves for inhibition of PHA-induced proliferation of PBMCs in the absence (open columns) or presence (black columns) of IL-2 plus IL-4 and increasing concentrations of dexamethasone. PBMCs were isolated from healthy subjects (n = 11), patients with untreated stable asthma (n = 14), and patients with unstable asthma (n = 13). The ordinate shows uptake of tritiated thymidine expressed as a percentage of that observed in the absence of PHA. The IC50 values (means ± SD) before and after coincubation with IL-2 and IL-4 were as follows: healthy control subjects, 102 ± 4 versus 537 ± 45 nM; patients with stable asthma, 107 ± 2 versus 912 ± 52 nM; and patients with unstable asthma, 316 ± 7 versus 851 ± 47 nM. *p < 0.05, compared with non–cytokine-stimulated PBMCs (open columns); {dagger}p < 0.05, compared with non–cytokine-stimulated PBMCs (open columns) from healthy subjects and patients with stable asthma.

 
When compared with unstimulated cells (without PHA), the incubation with PHA alone provoked a 100 (± 5)-fold induction of PBMC proliferation both in patients with stable asthma and healthy control subjects, and a significantly greater induction of PBMC proliferation [145 (± 6)-fold, p < 0.05] in patients with unstable asthma. Dexamethasone provoked a dose-dependent decrease in PHA-induced proliferation of PBMCs, particularly between 10–7 and 10–5 M, in the three groups (Figure 1, open columns). Interestingly, the cells from patients with unstable persistent asthma were less sensitive (IC50 = 316 ± 7 nM) to the antiproliferative effects of dexamethasone than were those from healthy control subjects (IC50 = 102 ± 4 nM, p < 0.05) and patients with stable asthma (IC50 = 107 ± 2 nM, p < 0.05). No differences were found between healthy control subjects and patients with stable asthma.

Coincubation with IL-2 and IL-4 significantly increased the PHA-induced proliferation of PBMCs in the three groups (Figure 1, solid columns). However, the effect of IL-2 and IL-4 on PHA-induced cell proliferation was significantly higher in healthy subjects (73 ± 5% increase) than in patients with stable asthma (55 ± 4% increase, p < 0.01) and in patients with unstable asthma (30 ± 5% increase, p < 0.001). In all three groups, dexamethasone was less efficient in inhibiting the proliferation of PBMCs incubated with IL-2 and IL-4 than in inhibiting the proliferation of non–cytokine-stimulated cells (Figure 1, compare solid columns with open columns). Thus, the IC50 values in the absence or presence of cytokines were as follows: patients with unstable persistent asthma, 316 ± 7 versus 851 ± 47 nM (p < 0.01); patients with stable asthma, 107 ± 2 versus 912 ± 52 nM (p = 0.001); and healthy control subjects, 102 ± 4 versus 537 ± 45 nM (p = 0.001). These findings suggest that these cytokines had decreased the sensitivity of PBMCs to the antiproliferative effect of dexamethasone.

There were no significant differences in the response to dexamethasone in cytokine-stimulated PBMCs between healthy subjects (IC50 = 537 ± 45 nM) and patients with stable (IC50 = 912 ± 52 nM) or unstable asthma (IC50 = 851 ± 47 nM) (Figure 1, solid columns).

GR-{alpha} and GR-ß Expression before and after Stimulation with Cytokines
Real-time RT-PCR.
Baseline expression of GR-{alpha} mRNA in PBMCs was significantly higher (p < 0.05) in patients with unstable persistent asthma [(1.95 ± 0.40) x 103 cDNA molecules/µg total RNA] than in healthy control subjects [(1.35 ± 0.25) x 103 cDNA molecules/µg total RNA] and patients with stable asthma [(1.46 ± 0.35) x 103 cDNA molecules/µg total RNA] (Figure 2) . After stimulation with cytokines there was a small, but significant, increase in the expression of GR-{alpha} mRNA in the three groups (p < 0.05) (Figure 2).



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  		Figure 2. GR-{alpha} mRNA (top) and GR-ß mRNA expression (bottom) in PBMCs from healthy subjects (n = 11), patients with stable asthma (n = 14), and patients with unstable asthma (n = 13) before (0 hours, open columns) and after 72 hours of incubation with medium (shaded columns) or with IL-2 plus IL-4 (solid columns). *p < 0.05, with respect to non–cytokine-stimulated PBMCs; {dagger}p < 0.05, with respect to healthy subjects and patients with stable asthma. Data are expressed as means ± SEM.

 
Although GR-ß mRNA was detected in PBMCs from the three groups of subjects, the abundance of GR-ß mRNA was at least 600 times lower than that of GR-{alpha} in all the groups. There were no significant differences in the expression of GR-ß isoform between healthy subjects and patients with asthma (Figure 2). Because high GR-ß expression levels have been associated with cytokine-induced glucocorticoid insensitivity we investigated whether coexposure to IL-2 and IL-4 induced the expression of GR-ß. As shown in Figure 2, coincubation with these two cytokines caused no increase in GR-ß mRNA expression in any of the three groups.

Western blot.
In an attempt to investigate the relative abundance of GR-{alpha} and GR-ß proteins, Western blot analysis was performed with anti-GR antibody 57, which is raised against the N terminus of the GR, a region shared by both receptor isoforms. This antibody recognized GR-{alpha} in BEAS-2B cells (a bronchial epithelial cell line previously reported to contain GR-{alpha}), GR-ß in GR-ß–transfected COS-7 cells, and GR-{alpha} in all PBMCs, both at baseline and after cytokine stimulation (Figure 3) . There was a significant increase in GR-{alpha} protein expression at 72 hours of incubation with both medium (n = 9, 3 from each group; p < 0.01) and IL-2/IL-4 (n = 9; p < 0.01), compared with basal GR-{alpha} protein levels. There was no difference in the amount of protein detected between cytokine-stimulated and nonstimulated PBMCs (n = 9) (Figure 4) . No GR-ß was detected with antibody 57 in any of the subjects either at baseline or after cytokine exposure.



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  		Figure 3. Relative expression of GR-{alpha} and GR-ß proteins in PBMCs before (basal) and after incubation with medium or with IL-2 plus IL-4 for 72 hours. GR-ß–transfected COS-7 cells (control for GR-ß), BEAS-2B cells (control for GR-{alpha}), and PBMCs were analyzed by Western blotting with antibody 57, which is directed to an epitope common to GR-{alpha} and GR-ß. COS-7 cells (15 µg) and BEAS-2B cells (30 µg) were loaded alone or as a mixture of both protein extracts (BEAS-2B + COS-7). No difference was found in GR-{alpha} protein levels between unstimulated (medium) and cytokine-stimulated PBMCs. No GR-ß was detected either at baseline or after medium or cytokine exposure.

 


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  		Figure 4. Western blot analysis of PBMCs from three healthy subjects (A), three patients with stable intermittent asthma (B), and three patients with unstable persistent asthma (C) before (basal) and after incubation with medium or with IL-2 plus IL-4 for 72 hours. Membranes were stained with either antibody 57 (top lanes) or anti–{alpha}-tubulin (bottom lanes) and the densitometric ratio between GR-{alpha} and {alpha}-tubulin was calculated. Note the increase in GR-{alpha} protein after incubation with medium or with IL-2 plus IL-4 for 72 hours, compared with baseline. The lower band seen in some GR-{alpha} blots most probably represents a GR-{alpha} protein degradation product. Data are expressed as means ± SEM.

 
Similar results were obtained with GR-{alpha}–specific (AShGR) and GR-ß–specific (BShGR) antibodies raised against human GR-{alpha} and GR-ß proteins, respectively. AShGR detected GR-{alpha} in PBMCs from all examined subjects (n = 7; one control subject and three from each asthma group) (data not shown). BShGR detected GR-ß in GR-ß–transfected COS-7 cells, but no GR-ß protein band was found in any of the analyzed PBMCs (n = 7) (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of this study was to help clarify the potential role of glucocorticoid receptor isoforms in glucocorticoid insensitivity. We first evaluated the growth of PBMC colonies stimulated with PHA and the inhibitory effects of dexamethasone. This test has already been validated in the assessment of glucocorticoid sensitivity in asthma (2–4). The effect of PHA on PBMC proliferation was significantly greater in patients with unstable asthma than in patients with stable asthma and control subjects, a finding that has been previously observed in glucocorticoid-insensitive patients with asthma (3, 4). This finding suggests that PBMCs from patients with unstable persistent asthma have an increased production of inflammatory factors that lead to enhanced proliferation of PBMCs.

The suppressive effect of dexamethasone on growth was significantly lower in patients with unstable persistent asthma than in patients with stable intermittent asthma and control subjects. This finding suggests a relative insensitivity of PBMCs from patients with unstable asthma to glucocorticoids, probably resulting from a more active and persistent activation by proinflammatory cytokines in these patients with asthma, compared with the stable ones. As previously reported (21), and supporting the notion that glucocorticoid insensitivity is an acquired phenomenon, we significantly reduced the sensitivity of PBMCs to glucocorticoids in all groups of subjects by incubating the cells with a combination of IL-2 and IL-4.

Second, we evaluated the relative endogenous levels of both glucocorticoids receptor isoforms before and after stimulation of PBMCs with IL-2 and IL-4. We quantified the mRNA expression of GR-{alpha} and GR-ß by using a highly sensitive and accurate real-time PCR method, and analyzed GR-{alpha} and GR-ß proteins by using an antibody that recognizes both isoforms of the receptor as well as specific antibodies against GR-{alpha} and GR-ß.

Our real-time RT-PCR demonstrated that GR-{alpha} is the predominant isoform in PBMCs, with the GR-ß isoform being almost undetectable. Reinforcing these results, our Western blot analysis demonstrated the presence of GR-{alpha} but not of GR-ß protein.

We found that the mRNA levels of GR-{alpha} were significantly higher in patients with unstable asthma than in patients with stable asthma and healthy control subjects. We also found that stimulation with IL-2/IL-4 increased GR-{alpha} mRNA in all groups of subjects, although no significant changes were detected at the protein level. This discrepancy may be due either to the limited sensitivity of Western blot technique in detecting small changes in protein levels or to a dual effect of cytokines, one stimulating gene transcription and at the same time increasing protein degradation. It is worth noting that upregulation of GR-{alpha} in both patients with unstable asthma and cytokine-stimulated PBMCs was accompanied by an apparently paradoxic decrease in the antiproliferative effect of dexamethasone on PBMCs. Because a direct correlation between sensitivity to glucocorticoids and the concentration of GR-{alpha} is expected, our findings suggest that the IL-2/IL-4–induced increase in GR-{alpha} does not result in an enhanced effect of the agonist dexamethasone. Several mechanisms can account for the impaired function of GR-{alpha}, including the reduction of GR-{alpha} binding activity or other mechanisms interfering in the translocation of activated GR-{alpha} to the nucleus, its binding to the glucocorticoid response elements, or its interaction with transcriptional factors, coactivators, and chromatin. In keeping with this, Irusen and coworkers (22) have reported that IL-2 and IL-4 induced glucocorticoid insensitivity in PBMCs through activation of the p38 mitogen-activated protein kinase, which phosphorylates GR and reduces glucocorticoid binding affinity and glucocorticoid-induced nuclear translocation of the GR.

Increased expression of GR-{alpha} mRNA isoforms has been previously reported in cells exposed to cytokines (15). Because this overexpression may exert inhibitory effects on proinflammatory transcription factors such as AP-1 and nuclear factor-{kappa}B, it has been suggested that an increase in GR isoform expression may contribute to the regulation of inflammatory responses (24).

We found that GR-{alpha} mRNA was much more abundant than GR-ß mRNA in PBMCs from all subjects and that no significant changes were observed in the expression of GR-ß mRNA after stimulation with IL-2/IL-4. Orii and coworkers (10), also using real-time PCR, have reported low expression of GR-ß mRNA in PBMCs of patients with inflammatory bowel disease. Similarly, Gagliardo and coworkers (6), using a nested PCR, were unable to detect GR-ß mRNA in PBMCs of patients with asthma. In agreement with the low levels of the GR-ß transcript, our Western blot analysis failed to demonstrate GR-ß protein in PBMCs from all subjects. Similarly, Irusen and coworkers (22) were unable to detect any GR-ß expression, using the same technique, in IL-2– plus IL-4–stimulated PBMCs from healthy subjects and patients with mild asthma and steroid-dependent severe asthma. Gagliardo and coworkers (6), studying patients with asthma, and Orii and coworkers (10), studying patients with ulcerative colitis, detected little or no GR-ß protein. Western blot studies based on the use of two different antibodies for the detection of each GR isoform may not accurately assess the relative proportion of both isoforms because the antibodies may have different affinities for the epitopes. The use of a single antibody that recognizes a common epitope appears to be more suitable for comparing the relative levels of GR-{alpha} and GR-ß proteins by means of Western blotting. Using this antibody, we found GR-{alpha} protein in all samples, but no GR-ß protein was detected in any PBMC sample, either before or after cytokine stimulation.

The molecular basis for glucocorticoid insensitivity induced by an increased expression of the GR-ß isoform has been demonstrated in several experimental studies (15, 16). However, the data are less convincing and more controversial with respect to the hypothesis that cellular levels of GR-ß isoforms, capable of interfering with GR-{alpha} function, can be achieved under conditions of tissue inflammation in patients with asthma or other inflammatory disease.

Indeed, marked differences have been reported by various groups concerning the expression of GR-ß isoforms in tissues and inflammatory cells. Differences in methodologic procedures are the most plausible explanation for these discrepancies. RT-PCR, Western blot, and immunohistochemistry techniques have been used to assess the level of expression of GR isoforms. Studies reporting increased expression of GR-ß in inflammatory processes relatively insensitive to glucocorticoids, such as asthma (5) and nasal polyps (7), have used immunohistochemistry. In contrast, those reporting little or no expression of GR-ß in similar patients with asthma (6), nasal polyps (8), and ulcerative colitis (9, 10) have measured GR-ß expression by RT-PCR and/or Western blot.

The specificity of antibodies raised against GR-ß has been demonstrated under experimental conditions with transfected cells (COS-7 and HeLa cells) (25, 26) expressing abundant GR-ß. However, it is less clear whether the specificity of the antibodies has always been tested in the detection of this GR isoform in inflammatory cells and in tissues (19).

The apparent discrepancies between RT-PCR/Western blot results and immunohistochemistry findings have still to be resolved. How can we explain the significant difference between such low levels of GR-ß mRNA and GR-ß protein analyzed through Western blot and the pronounced immunoreactive GR-ß reported in some studies of cytokine-stimulated PBMCs and of PBMCs from steroid-insensitive patients? One possible explanation is the existence of cross-reactivity between the antibody used in some studies and a GR-ß-like protein produced by cytokine-induced inflammation, a protein that is undetectable by more specific techniques, such as RT-PCR and Western blot.

Studies have demonstrated that regulation of the GR gene is complex. There are at least five different GR mRNAs with unique 5' untranslated regions within Exon 1 in addition to GR-{alpha} and GR-ß isoforms. Alternative translation initiation of the GR-{alpha} transcript results in a new GR-{alpha} protein, the B-isoform, in addition to the originally identified GR-{alpha} protein, now known as the A-isoform (12). A similar mechanism for alternative translation has been proposed for GR-ß, but a definitive demonstration is still lacking (12). It has been speculated that translation of the 7.0-kb message might give rise to additional GR-ß through an exon-skipping process. In addition, posttranslational modification of the GR opens up a wide range of possibilities for functionally different GR isoforms (12). It could then be speculated that transcriptional or posttranscriptional mechanisms might produce, under conditions of inflammation, a protein or proteins with significant homology for a GR-ß isoform, which cross-react with the polyclonal antibody used in immunohistochemistry studies.

In summary, the results of the present study suggest that the alternative splicing process leading to GR-ß expression is minimally activated in PBMCs of patients with asthma. In addition, induction of glucocorticoid insensitivity by in vitro cytokine stimulation of PBMCs is not accompanied by any significant change in the expression of GR-ß. Our findings do not support the hypothesis that increased GR-ß expression can contribute to cytokine-induced glucocorticoid insensitivity. Mechanisms other than an increase in expression of GR-ß should be involved in IL-1/IL-4-induced glucocorticoid insensitivity of PBMCs.


   FOOTNOTES
 
Supported by FUCAP 2002, FIS 99-0133, Generalitat de Catalunya 2001SGR-00384.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: A.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; L.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.R.-F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.X. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 16, 2003; accepted in final form May 30, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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