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Keywords:

  • glucocorticoid receptor;
  • glucocorticoid therapy;
  • nasal inflammation;
  • nasal mucosa;
  • nasal polyps

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Poor response of nasal polyps to glucocorticoids (GCs) may be because of abnormal expression of GC receptors (GR) α and β or to downregulation of GRα. We aimed to evaluate the in vivo regulation of GR isoforms in GC-treated nasal polyps and to assess the relationship between clinical response to GCs and GR levels.

Methods:  Patients with nasal polyps were randomly (3:1) treated (n = 51) or not (n = 14) with oral prednisone and intranasal budesonide for 2 weeks, plus intranasal budesonide for 10 additional weeks. Nasal symptoms were evaluated. Biopsies were obtained before (w0) and after 2 (w2) and 12 (w12) weeks of treatment, and analysed for their inflammatory content and GR mRNA (102 cDNA copies/μg total RNA) and protein (% immunoreactive inflammatory cells) expression. Healthy nasal mucosa (n = 11) was also investigated. Data are presented as median and 25–75th percentile.

Results:  At w0, nasal polyps expressed less GRα mRNA (1343;683–2263; P < 0.05) and GR protein (41;29–54; P < 0.05) than nasal mucosa (2474;1346–2933; 60;51–72, respectively). GRβ immunoreactivity was higher in nasal polyps (11;4–19; P < 0.05) than in nasal mucosa (5;2–5). At w2, increased GRα mRNA (2010;1037–2732; P < 0.01) and GR protein (56;27–71; P = 0.056) were found compared with w0 (1177;759–2058; 37;29–55, respectively). At w12, GRα mRNA and GR protein were similar to w0. GRβ expression was unaltered by treatment. Neither GRα nor GRβ correlated with nasal symptoms. GR immunoreactivity negatively correlated with eosinophils (r = −0.478; P < 0.001).

Conclusions:  GRα is downregulated in nasal polyps and upregulated by GC treatment. Neither GRα nor GRβ appear to determine the sensitivity to GCs in nasal polyposis.

Glucocorticoids (GCs) exert their effects through the GC receptor (GR). At least two GR isoforms exist, GRα and GRβ, which are derived from alternative splicing of the GR primary mRNA. GRα binds GCs, translocates to the nucleus and mediates either transactivation or transrepression of target genes. The anti-inflammatory effects of GCs are mainly because of direct protein–protein interactions between GRα and transcription factors that activate the expression of pro-inflammatory genes, such as the activator protein-1 and nuclear factor kappa-B. GRβ is unable to bind GCs. When over-expressed with respect to GRα, GRβ inhibits GRα-mediated transactivation and transrepression in some cell types (1–3).

Functional responses of cells to both endogenous and exogenous GCs are likely to be affected by numerous factors, including the binding kinetics of the receptor to hormone, the degree of the receptor translocation to the nucleus and its interaction with co-factors from the transcriptional machinery (1–3). The expression levels of GRα and GRβ, as well as their regulation by supra-physiological doses of synthetic GCs, might also interfere with the response to exogenous GCs. In this respect, increased GRβ expression was reported in inflammatory cells from GC-insensitive patients suffering from asthma (4–6), nasal polyposis (7) and inflammatory bowel diseases (8, 9), although the clinical contribution of these findings to GC insensitivity is a matter of debate (1, 3, 10). Moreover, in vitro and in vivo studies demonstrate that GCs downregulate the expression of their own receptor in different tissues and cell types (1, 11–13), including nasal (14) and bronchial mucosa (15). This phenomenon is thought to be a feedback protector mechanism that would avoid the deleterious effects of prolonged exposure to hormone. Because of target tissue sensitivity to GCs directly correlates with receptor levels, hormone-induced downregulation of GRα might explain insensitivity to GC treatment.

Nasal polyposis is a chronic inflammatory disease of the sinus mucosa and the middle turbinate, often associated with asthma (16). Long-term therapy with potent intranasal GCs is widely advocated to control nasal polyp inflammation and growth, but the response to this treatment is often only partially successful and short courses of oral GCs have to be administered periodically in some patients to quickly improve nasal obstruction. Even with this regime, nasal polyps can progress in some patients and may require surgery (17). The underlying mechanisms leading to GC insensitivity in nasal polyps remain to be clarified, and the effect of GC treatment on GR expression is as yet unclear.

This in vivo study was undertaken to answer the following questions: (1) are there differences in the expression of GR isoforms between healthy and inflamed nasal mucosa?; (2) does GC treatment downregulate the GR?; and (3) is the clinical response of nasal polyps to GCs related to the degree of expression of GR isoforms?

Subjects and methods

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Study subjects

A total of 65 patients (74% male, 51 ± 2 year) with diagnosis of severe nasal polyposis were selected; they comprised patients without asthma and patients with either aspirin-tolerant (ATA) or aspirin-intolerant asthma (AIA). The diagnosis of severe nasal polyposis was based on criteria described in the EP3OS document (17), i.e. nasal symptoms, nasal endoscopic examination and CT scan of paranasal sinus, as reported elsewhere (18). The diagnosis of asthma was established on the basis of the clinical history and the demonstration of a reversible bronchial obstruction, as previously reported (19). Diagnosis of aspirin intolerance was made on the basis of a clear-cut history of asthma attacks precipitated by nonsteroidal anti-inflammatory drugs (NSAID), and confirmed by aspirin nasal challenge in patients with an isolated episode of NSAID-induced asthma exacerbation, according to a method previously reported (20). All subjects agreed to participate in the study, which was approved by the Ethics Committee of our Hospital.

Study design

The study design used herein has been previously reported (18). After a 4-week washout period without any intranasal or oral steroids, patients were randomized (3:1) into two groups: (1) the GC-treated group included 51 patients who received oral prednisone (30 mg daily for 4 days followed by a tapering of 5 mg every 2 days) and intranasal budesonide (400 μg BID) for 2 weeks (w2), followed by intranasal budesonide (400 μg BID) alone for 10 additional weeks (w12); and (2) the nontreated group included 14 patients who did not receive any steroid treatment for 2 weeks following randomization (no treatment for 6 weeks in total). For ethical reasons, patients from the nontreated group were not kept for more than 6 weeks without any effective treatment. Nasal polyp biopsies were obtained at w0, w2 (treated and nontreated groups) and w12 (treated group).

We have previously reported the effects of GC therapy on nasal symptoms and other clinical data (18). Here, a total nasal symptoms score comprising nasal obstruction, loss of the sense of smell, rhinorrhea and sneezing was calculated and used to assess the relationship between clinical symptoms and GR expression levels.

Healthy nasal mucosa was obtained from 11 subjects undergoing nasal corrective surgery for turbinate hypertrophy or septal dismorphy (Table 1). None of them had a history of nasal or sinus disease, allergic rhinitis or upper respiratory tract infection, and they had not received GCs for any reason for at least 4 weeks prior to surgery.

Table 1.   Demographic data and clinical characteristics of the study population
 Nasal mucosaNasal polyps
Non-treated groupGC-treated group
Without asthmaWith asthma
ATAAIA
  1. ATA, aspirin-tolerant asthma; AIA, aspirin-intolerant asthma.

  2. Values of age are expressed as mean ± standard error of the mean.

Patients (n)1114181617
Gender (% female)36.414.316.72547
Age (y)38.4 ± 4.152.5 ± 4.853.8 ± 3.848.6 ± 4.749.1 ± 2.9
Skin prick test (% positivity)36.428.627.82529.4

Histology and immunohistochemistry

The inflammatory content of biopsies was characterized by histological and immunohistochemical analysis performed in 4 μm thick sections. Because of limitations in tissue availability, the histological and immunohistochemical analysis could only be carried out on 8 nasal mucosa and 29 nasal polyps. Mononuclear cells and neutrophils were counted from haematoxylin & eosin (H&E) stained sections, and eosinophils were analysed by both H&E staining and immunohistochemistry of BMK13. Anti-GRα/β antibody 57 (Affinity BioReagents, Golden, CO, USA) and anti-GRβ antibody BShGR (kindly provided by John A. Cidlowski) were used to characterize the GR (21, 22). Sections were counted blindly by light microscopy (×400 magnification). For the quantification of inflammatory cells, between 1.6 and 2 mm2 were counted for each section and cell counts were expressed as number of positive cells per square millimetre. For GR immunohistochemistry, the percentage of GR-positive cells was counted from a minimum of 400 inflammatory cells. The intensity of GR immunoreactivity in the epithelium was scored as follows: 0, negative result; 1, weak immunoreactivity; 2, moderate immunoreactivity; 3, strong immunoreactivity.

Reverse transcription and real-time PCR

The extraction of total RNA from the specimens and the reverse transcription step were performed as previously reported (21). Quantification of GRα and GRβ transcripts was achieved by extrapolation to a plasmid double-stranded DNA external standard curve added in each PCR run (19).

Statistical data analysis

Data are expressed as median and 25th to 75th percentile. Nonparametric statistical analysis was performed by using the Friedman test and Wilcoxon rank test for within-group comparisons, and the Kruskal–Wallis test and Mann–Whitney U test for between-group comparisons. Spearman rank correlation was used when analysing relationships between data. Statistical significance was set at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects

Table 1 summarizes the demographic data and clinical characteristics of the subjects.

Healthy vs inflamed nasal mucosa

At baseline (w0), nasal polyp biopsies (n = 29) contained a higher number of eosinophils (215;96–409; P < 0.001), neutrophils (8;2–28; P < 0.05) and total inflammatory cells (1128;801–2107; P < 0.05) than healthy nasal mucosa (0;0–1, 2;1–5 and 721;604–920, n = 8, respectively).

GRα mRNA was detected in all tissues and its expression (×102 cDNA copies/μg total RNA) was lower in nasal polyps (1343;683–2263; n = 64; P < 0.05) than in nasal mucosa (2474;1346–2933; n = 11) (Fig. 1A). GRβ mRNA was expressed (×102 cDNA copies/μg total RNA) at very low levels and did not significantly differ between nasal mucosa (3.42;2.72–9.13; n = 11) and nasal polyps (2.53;0.56–3.31; n = 15; ns) (Fig. 1B).

image

Figure 1.  Basal expression of GR isoforms. (A) GRα mRNA in nasal mucosa (n = 11) and nasal polyps (n = 64). (B) GRβ mRNA in nasal mucosa (n = 11) and nasal polyps (n = 15). (C) GR and (D) GRβ proteins in nasal mucosa (n = 7) and nasal polyps (n = 29). Box plots show the 25th, 50th (median) and 75th percentile values. Whiskers show the 10th and 90th percentiles. *P < 0.05 compared with nasal mucosa by Mann–Whitney U test.

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Glucocorticoid receptor protein was localized in numerous cell types, including inflammatory cells, epithelial cells, fibroblasts, glands and endothelial cells. Eosinophils mostly stained negative for the GR (Fig. 2). Nasal polyps had a lower percentage of GR-positive inflammatory cells (41;29–54; n = 29; P < 0.05) (Fig. 1C) and less intensity of GR immunoreactivity in the epithelium (2;1–2; n = 29; P < 0.05) than nasal mucosa (cells: 60;51–72; n = 7; epithelium: 3;2–3; n = 7). A negative correlation was found between the percentage of GR-positive cells and eosinophil numbers (r = −0.475; n = 36; P < 0.01).

image

Figure 2.  Representative image of GR immunohistochemistry in (A) nasal mucosa and (B) nasal polyp tissue at ×200 magnification. C and D correspond to ×400 magnification images of A and B, respectively. Note the negative staining in eosinophils (arrows).

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Immunoreactivity for GRβ was mostly detected in mononuclear inflammatory cells and, with low intensity, in epithelial cells (Fig. 3). Eosinophils were GRβ-negative. GRβ immunostaining was localized in both the cell nucleus and cytoplasm. Nasal polyps had a higher percentage of GRβ-positive inflammatory cells (11;4–19; n = 29; P < 0.05) than nasal mucosa (5;2–5; n = 7) (Fig. 1D). No significant difference in GRβ immunoreactivity of the epithelium was found between nasal mucosa (0;0–1; n = 7) and nasal polyps (1;0–1; n = 28; ns).

image

Figure 3.  Representative image of GRβ immunohistochemistry in (A) nasal mucosa and (B) nasal polyp tissue and (C) an adjacent section of B stained with GRβ antibody preabsorbed with the peptide antigen at ×200 magnification. (D) ×400 magnification image of B.

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Treatment of nasal polyps with glucocorticoids

Clinical response.  At w0, there were no significant differences in the nasal symptoms score between GC-treated and nontreated groups (Table 2). When analysing both groups together, a higher score was found in asthmatic patients (9;6–11; n = 41; P < 0.05), particularly in AIA patients (9.5;7.3–11; n = 20; P < 0.05), compared with nonasthmatics (8;6–9; n = 24).

Table 2.   Effect of glucocorticoid treatment on the nasal symptoms score in the different subtypes of nasal polyps
 nw0w2w12
  1. Results expressed as median and 25–75th percentile.

  2. w0, week 0; w2, week 2; w12, week 12; n, number of cases.

  3. **P < 0.01 and ***P < 0.001, symptoms improving compared to w0 (Wilcoxon test); †P < 0.05 and ††P < 0.01, symptoms worsening compared to w2 (Wilcoxon test).

Non-treated group148.5 (6–10.2)9 (7–11.2)
GC-treated group519 (6–10)4 (2–5)***5 (3–6)***,†
 No asthma188 (5.7–9)4.5 (3.7–6)**5 (4.5–6)**
 Asthma339 (6.5–11)3 (1.2–4.7)***5 (2–6)***,
 Aspirin-tolerant asthma168.5 (6–11.5)4 (1–4)**3.5 (2–5.7)**
 Aspirin-intolerant asthma179 (7.5–11)3 (2–5)***5 (3–7)**,

At w2, no change in the nasal symptoms score was found in the nontreated group compared with w0. In contrast, GC-treated patients showed a marked reduction in nasal symptoms in all groups. At w12, nasal symptoms decreased compared with w0 but increased compared with w2 (Table 2). The decrease in nasal symptoms as a result of GC treatment was found in both nonasthmatic and asthmatic patients. In the asthmatics, particularly in the AIA patients, however, nasal symptoms worsened at w12 compared with w2 (Table 2).

Histological findings.  With the exception of neutrophils, at w0 no significant differences in the number of inflammatory cells were found between GC-treated and nontreated groups (Table 3). When analysing both groups together, an increased number of eosinophils – as measured by BMK13 immunostaining – was found in asthmatic patients (173;91–260; n = 39; P < 0.05), particularly in AIA patients (193;123–286; n = 18; P < 0.05), compared with nonasthmatics (86;41–249; n = 19).

Table 3.   Effect of glucocorticoid treatment on the inflammatory cell counts of nasal polyp biopsies
 Non-treated groupGC-treated group (all patients)
w0w2w0w2w12
  1. Results expressed as median and 25–75th percentile of cells/mm2.

  2. w0, week 0; w2, week 2; w12, week 12; n, number of cases; H&E, haematoxylin & eosin. Total inflammatory cells correspond to the sum of mononuclear cells, eosinophils, and neutrophils.

  3. *P < 0.05 and **P < 0.001, compared to w0 from GC-treated group (Wilcoxon test); ††P < 0.01 compared to w0 from non-treated group (Mann–Whitney U test).

n77222018
Mononuclear cells (H&E)928 (484–1893)1277 (711–1482)892 (581–1351)1040 (582–1785)1061 (540–1503)
Eosinophils (H&E)169 (86–215)227 (117–1039)243 (100–480)20 (5–39)**22 (3–85)*
Neutrophils (H&E)34 (13–69)42 (12–195)3 (1–14)††20 (1–43)*6 (1–26)
Total inflammatory cells (H&E)1127 (721–2531)1641 (1373–2181)1207 (832–2101)1168 (634–1807)1294 (579–2191)

At w2, no change in the number of inflammatory cells was found in the nontreated group compared with w0. In contrast, GC-treated patients showed a marked downregulation in the number of eosinophils, which remained low at w12, and an increase in the number of neutrophils compared with w0 (Table 3). Further analysis of eosinophils by BMK13 immunostaining revealed that the reduction in eosinophils at w2 was found in both nonasthmatic and asthmatic patients (Fig. 4). However, at w12, eosinophils remained low in nonasthmatics but increased significantly in asthmatics compared with w2. This increase was found in both ATA and AIA patients (not shown).

image

Figure 4.  Eosinophil counts in nonasthmatic (n = 12) and asthmatic nasal polyps (n = 24). Empty boxes, light grey boxes and dark grey boxes correspond to weeks 0, 2 and 12 of GC treatment, respectively. See‘Methods’ for GC treatment regimen. Box plots show the 25th, 50th (median) and 75th percentile values. Whiskers show the 10th and 90th percentiles. *P < 0.05 and **P < 0.01 compared with week 0 by Wilcoxon test. †P < 0.05 compared with week 2 by Wilcoxon test.

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A positive correlation was found between nasal symptoms and eosinophil counts (H&E: r = 0.724; n = 74; P < 0.0001; BMK13+: r = 0.410; n = 128; P < 0.0001).

GRα and GRβ mRNA.  At w0, there were no significant differences in GRα mRNA expression between GC-treated and nontreated groups (Fig. 5), nor between nonasthmatic and asthmatic patients (not shown).

image

Figure 5.  Regulation of GRα mRNA in nontreated patients (n = 14) and GC-treated patients (n = 50). Empty boxes, light grey boxes and dark grey boxes correspond to weeks 0, 2 and 12 of treatment, respectively. See‘Methods’ for treatment regimes. Box plots show the 25th, 50th (median) and 75th percentile values. Whiskers show the 10th and 90th percentiles. **P < 0.01 compared with week 0 by Wilcoxon test.

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There were no significant changes in GRα mRNA between w0 and w2 in the nontreated group, while GRα mRNA was found increased in GC-treated patients at w2 (2010;1037–2732; P < 0.01) compared with w0 (1177;759–2058). At w12, GRα mRNA expression was similar to w0 (Fig. 5). The same pattern of regulation, i.e., increase of GRα mRNA at w2 and return to basal levels at w12, was observed in both nonasthmatic and asthmatic patients (not shown).

No correlation was found between GRα mRNA levels and nasal symptoms (ns), while a weak but significant negative correlation was found between GRα mRNA and eosinophil counts (H&E: r = −0.270; n = 73; P < 0.05; BMK13+: r = −0.246; n = 125; P < 0.01). In addition, no significant correlation was found between baseline GRα mRNA levels and changes in either nasal symptoms or eosinophils after the 2-week treatment with oral and intranasal GCs (not shown).

GRβ mRNA expression was analysed in 15 GC-treated patients. No significant change in GRβ mRNA was found at w2 (2.5;2–4.4; ns) and w12 (3.2;1.9–4; ns) compared with w0 (2.5;0.6–3.3).

GR immunohistochemistry.  At w0, no differences in GR immunoreactivity were found between GC-treated and nontreated group, nor between nonasthmatic and asthmatic patients (not shown).

There were no significant changes in the number of GR-positive inflammatory cells between w0 and w2 in nontreated patients, while a near significant increase in the number of GR-positive inflammatory cells was found in GC-treated patients (w0: 37;29–55; w2: 56;27–71; n = 22; P = 0.056). GR immunoreactivity at w12 (37;18–72) was similar to w0. A different response to GC treatment was found between nonasthmatic and asthmatic patients. Thus, in nonasthmatics, no significant change in GR immunoreactivity was found at w2 and w12 compared with w0, whereas in asthmatics, the number of GR-positive cells increased significantly at w2 and returned to basal levels at w12 (Fig. 6A). GR immunoreactivity of the epithelium was not significantly modified by GC treatment in any group of patients (not shown).

image

Figure 6.  Percentage of GR-positive (A) or GRβ-positive (B) inflammatory cells in GC-treated patients who either had no asthma (n = 9) or asthma (n = 13). Empty boxes, light grey boxes and dark grey boxes correspond to weeks 0, 2 and 12 of treatment, respectively. See‘Methods’ for treatment regimes. Box plots show the 25th, 50th (median) and 75th percentile values. Whiskers show the 10th and 90th percentiles. *P < 0.05 compared with week 0 and week 12 (A) or week 0 and week 2 (B), by Wilcoxon test.

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No significant correlation was found between the number of GR-positive cells and nasal symptoms (ns), while a negative correlation was found between GR immunoreactivity and eosinophil counts (H&E: r = −0.362; n = 78; P = 0.001; BMK13+: r = −0.478; n = 50; P < 0.001). In addition, no significant correlation was found between baseline GR immunoreactivity and changes in either nasal symptoms or eosinophils after the 2-week treatment with oral and intranasal GCs (not shown).

GRβ immunohistochemistry.  At w0, we found no differences in GRβ immunoreactivity between GC-treated and nontreated groups, nor between nonasthmatic and asthmatic patients (not shown).

There were no significant changes in the number of GRβ-positive inflammatory cells between w0 and w2, either in the nontreated or the GC treated (w0: 13;6–20; w2: 11;6–25; n = 22; ns) group. As with total GR, a different response to GC treatment was found between asthmatic and nonasthmatic patients. Thus, in nonasthmatics, no significant change in GRβ immunoreactivity was found at w2 and w12 compared with w0, whereas in asthmatics a significant decrease in the number of GRβ-positive cells was found at w12 when compared with both w0 and w2 (Fig. 6B). GRβ immunoreactivity of the epithelium was not significantly modified by GC treatment in any group of patients (not shown).

No significant correlation was found between GRβ immunoreactivity and either nasal symptoms or inflammatory cell counts. In addition, no significant correlation was found between baseline GRβ immunoreactivity and changes in either nasal symptoms or eosinophils after the 2-week treatment with oral and intranasal GCs (not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this in vivo study we show that: (1) GRα is downregulated in nasal polyps; (2) short-term potent oral GC and intranasal budesonide upregulate GRα; and (3) the clinical response to GC treatment is not related to the level of expression of GR isoforms.

GRα is downregulated in nasal polyps

We found decreased GRα mRNA and protein expression in nasal polyps when compared with nasal mucosa. These findings could be ascribed to the differential cellular composition between both tissues. As shown, nasal polyps contain more inflammatory cells – especially eosinophils – than nasal mucosa, and most of the eosinophils stained negative for the GR. Accordingly, a negative correlation was found between the percentage of GR-positive inflammatory cells and eosinophil numbers. Alternatively, or added to this, our findings showing decreased GRα mRNA and protein in nasal polyps might indicate that the local inflammatory milieu of nasal polyps downregulates GRα. Because of the cellular complexity of whole tissues, it is very difficult to discern which of the two explanations prevails. In contrast to our results, Choi and co-workers detected higher GRα mRNA expression in nasal polyps than in nasal mucosa (23). Similar GRα mRNA levels have been reported in epithelial cells isolated from both nasal mucosa and nasal polyps (24), and in cells and tissues from both GC-insensitive and GC-sensitive asthmatic patients (1, 6, 19, 25–27).

In agreement with previous data (1, 3, 6, 13, 19, 21, 22, 24, 28), the expression of GRβ mRNA was very low in all nasal samples. We detected very low immunoreactivity for GRβ in nasal mucosa and a higher number of GRβ-positive inflammatory cells in nasal polyps. In line with our results, Hamilos and co-workers (7) reported an increased percentage of inflammatory cells expressing GRβ in nasal polyps (40%) compared with nasal mucosa (16%). We localized GRβ in mononuclear inflammatory cells and the nasal epithelia. Epithelial cells in liver, thymus and lung (29), and the bronchial epithelium (27) also express GRβ protein. In common with other reports (6, 28), we detected GRβ in both the nucleus and cytoplasm of cells.

The increased GRβ immunoreactivity in inflammatory cells from nasal polyps might result from the effect of local inflammatory cytokines present in this tissue. In line with this, numerous in vitro studies report upregulation of GRβ in a variety of cells after incubation with pro-inflammatory stimuli (reviewed in 1, 3, 10).

Whether the increased expression of GRβ that we and others report in nasal polyps (7), – a finding also reported in bronchial asthma (4, 5, 27) and ulcerative colitis (9) – is sufficient to account for GC insensitivity, is a matter of debate (1, 3, 10). GRβ represses the functional activity of GRα only when the former is over-expressed relative to GRα (6, 30, 31). Since this has not been reported in vivo in patients suffering from GC insensitive/resistant diseases, the clinical relevance of mild increases in GRβ expression remains to be clarified.

Short-term oral GCs and intranasal budesonide upregulate GRα

Treatment of nasal polyps with oral and intranasal GCs for 2 weeks resulted in a significant increase in GRα mRNA expression and a mild increase in the percentage of GR-positive inflammatory cells, which was significant in the asthmatic population. In these patients, the number of GR-positive cells went back to basal levels after 10 weeks of intranasal GC treatment. In parallel, the 2-week oral and intranasal GC treatment provoked a marked reduction in inflammatory cell counts, particularly eosinophils, in all subgroups of patients. These results concur with previous studies showing a decrease in eosinophil numbers after treatment of nasal polyps with intranasal GCs (32, 33). We found an increased presence of neutrophils after GC treatment, which is probably related to the ability of GCs to reduce apoptosis in these cells (34, 35). However, after the 10-week intranasal GC treatment, eosinophils remained low in the nonasthmatic group but increased significantly in the asthmatic group. A negative correlation was found between eosinophil counts and the number of GR immunoreactive inflammatory cells. The reason for this association may be that eosinophils – abundant in nontreated nasal polyps – mostly stained negative for the GR. Thus, the decrease in eosinophils induced by GC treatment indirectly results in an increase in the number of GR immunoreactive cells. In line with this, the asthmatic population, which was more insensitive to eosinophil apoptosis induced by intranasal GC treatment, showed a return of GR cell immunoreactivity to basal levels after intranasal GCs.

Our results showing no downregulation of the GR, either in inflammatory cells or nasal epithelia, after in vivo treatment of nasal polyps with oral and intranasal GCs, contrast with data showing downregulation of the GR after in vitro treatment of different airway cell types with GCs (1, 12, 13). In keeping with our results, Henriksson and co-workers (36) reported absence of GR mRNA downregulation after in vivo treatment of nasal polyps with intranasal GCs. In contrast, in vivo treatment of healthy nasal mucosa (14) with intranasal GCs resulted in a transient downregulation of GRα mRNA. As explained before, the absence of GC-induced GR downregulation in nasal polyps might be ascribed, at least in part, to the effects of GCs on the inflammatory infiltrate. However, the lack of GC-induced GR downregulation in the epithelia of nasal polyps, as opposed to GRα downregulation in nasal mucosa epithelial cells after in vitro treatment with dexamethasone (13), suggests that homologous downregulation of the GR is uncommon in the nasal polyp epithelia.

With regard to GRβ, neither oral nor intranasal GCs significantly altered the low levels of GRβ mRNA and protein.

The clinical response to GC treatment is not related to the level of expression of GR isoforms

We found that the 2-week treatment with oral and intranasal GCs resulted in a marked decrease in nasal symptoms in all patients, which relapsed after 10 weeks of intranasal GCs. This loss of effect after intranasal GC therapy was more prominent in the AIA patients. A positive correlation was found between nasal symptoms and eosinophil counts, which suggest that the improvement in clinical symptoms is at least in part linked to resolution of the eosinophilic inflammation.

However, we did not find any correlation between either GRα or GRβ expression and nasal symptoms. In contrast, Hamilos and co-workers (7) found an association between GRβ expression and insensitivity to GCs in nasal polyposis. More specifically, the authors (7) reported an inverse correlation between baseline inflammatory cell GRβ expression and the reduction after GC treatment of eosinophils, T lymphocytes, VCAM-1 expression and IL-4 mRNA-positive cells. However, we did not find any correlation between baseline expression of either GRβ or GRα and the decrease in eosinophils after the 2-week treatment with oral and intranasal GCs. Thus, our results do not support any role for GRα or GRβ as markers of GC insensitivity in nasal polyposis. However, it cannot be ruled out that GR function – for instance its nuclear translocation, or its interaction with both the transactivating and transrepressing machinery – is altered in this or other inflammatory diseases (1, 2).

In summary, we have analysed the expression of GRα and GRβ in the nasal respiratory mucosa before and after in vivo treatment with oral and intranasal GCs. We have found that nasal polyps express less GRα than healthy nasal mucosa, and that short oral and intranasal GC therapy upregulates, rather than downregulates, GRα expression in nasal polyps. These effects might be partly explained by changes in intensity of the eosinophilic inflammation. The absence of GRα downregulation by GC treatment in nasal polyposis might ultimately contribute to increase the anti-inflammatory activity of GCs in this disease. Finally, neither GRα nor GRβ appear to determine the sensitivity to GCs in nasal polyposis.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors thank Dr Josep Ramírez, Department of Pathology, Hospital Clínic, who provided assistance in the identification of cell types in the tissue biopsies. This study was supported in part by grants from FIS (050069 and 050108), SEPAR-FEPAR and CIBERes (Isciii CB06/06/0010).

References

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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