 in Airway Cells
of Glucocorticoid-insensitive Asthma
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ABSTRACT |
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INTRODUCTION
METHODS
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Glucocorticoid (GC)-insensitive asthma is a challenging clinical problem that can be associated with
life-threatening disease progression. The molecular basis of GC insensitivity is unknown. Alternative
splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCR
, which does not
bind GC but antagonizes the transactivating activity of the classic GCR. Thus increased expression of
GCR could account for glucocorticoid insensitivity. Bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMC) were examined for GCR immunoreactivity using a GCR-specific antibody by immunohistochemical staining. Cell localization of GCR expression was performed
using a double immunostaining technique. Patients with GC-insensitive asthma expressed a significantly higher number of GCR-immunoreactive cells in their BAL and peripheral blood than GC-sensitive asthmatics or normal control subjects. Furthermore, GCR expression in GC-insensitive asthma
was particularly high in airway T cells, which are thought to play a major role in the pathogenesis of
asthma. We also examined the expression of GCR in specimens from the airways of patients with
chronic bronchitis. In chronic bronchitis, few cells were GCR-positive and their numbers did not differ significantly from normal control subjects. We conclude that GC-insensitive asthma is associated with increased expression of GCR in airway T cells.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Recent guidelines for the treatment of chronic asthma have focused on early intervention with anti-inflammatory therapy, particularly inhaled glucocorticoids (GC) (1). However, a subset of asthmatics fails to demonstrate a satisfactory response even to systemic GC therapy and have been termed "glucocorticoid-insensitive" (2). It is important to recognize these patients early because failure to respond to steroids often leads to prolonged courses of high-dose GC therapy and serious adverse effects despite persistent airway compromise. Peripheral blood mononuclear cells (PBMC) from patients with GC-insensitive asthma have a reversible defect in GC receptor (GCR) ligand and DNA binding affinity, which can be sustained in vitro by the addition of interleukin (IL)-2 and IL-4 but not the individual cytokines (3). Bronchoscopy studies indicate that airway T cells of GC-insensitive, as compared with GC-sensitive, asthmatics have significantly higher levels of IL-2 and IL-4 gene expression (6). These data support the concept that GC-insensitive asthma results from immune activation, which leads to reduced GCR binding affinity.
Recently, it has been found that alternative splicing of the
GCR pre-messenger RNA (pre-mRNA) gives rise to an additional homologous mRNA and protein isoform, termed GCR,
which is distinct from the ligand-activated classic GCR, GCR
.
Both mRNAs contain the first eight exons of the GCR gene
(7). The remainder is derived by alternative splicing of the last
exon of the GCR gene, corresponding to either exon 9 or 9.
The two protein isoforms have the same first 727 NH2-terminal amino acids. GCR differs from GCR only in its carboxy
terminus with replacement of the last 50 amino acids of GCR
with a unique 15 amino acid sequence. These differences render GCR unable to bind GC hormones, reduce its binding affinity for DNA recognition sites and its ability to transactivate
GC-sensitive genes (5, 8). In the current study, we demonstrate for the first time that airway cells from patients with
GC-insensitive asthma express significantly higher levels of
GCR than patients with GC-sensitive asthma, chronic bronchitis, or normal subjects. Colocalization studies reveal that
GCR expression in GC-insensitive asthma is particularly high
in airway T cells, which likely play a major role in the pathogenesis of asthma (3, 5).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Patient Selection
The protocol was approved by the institutional review board and informed consent was obtained from all patients before their entry into
this study. Patients with a diagnosis of asthma, based on the American
Thoracic Society criteria (11), were selected for evaluation. They
were included if they had a morning prebronchodilator FEV1 < 70%
of predicted values and a 
15% increase in FEV1 after two inhalations of albuterol (90 µg per actuation) and excluded if they had evidence for other types of lung disease. If there was any doubt about the
diagnosis, a complete set of pulmonary function tests with lung volume and diffusion capacity/total lung capacity ratio, methacholine
challenge study as well as a lung computed tomographic (CT) scan or
chest radiograph was obtained. None of the asthmatics enrolled had
a history of smoking. Patients were classified as GC-sensitive or GC-
insensitive based on their prebronchodilator morning FEV1 response
to a 1-wk course of 40 mg/d oral prednisone (3). Asthmatic patients
were defined as GC-insensitive if they failed to improve their morning
prebronchodilator FEV1 by 15% and GC-sensitive if they had an
increase in baseline FEV1 of 30% or greater (Table 1). Normals had
no evidence of respiratory disease. As a disease control group, lung
biopsies were also obtained from seven patients with chronic bronchitis and airway obstruction (FEV1 < 80% predicted). They were all
males, smokers, had no history of asthma, negative skin prick tests to aeroallergens and were not on inhaled GCs. These latter specimens were compared with lung biopsies from seven normal control subjects.
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Cell Preparation
All patients entering into this study agreed to undergo fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) performed according to American Thoracic Society guidelines (12). BAL cells suspended at a concentration of 1 × 10 5 cells per ml in phosphate-buffered saline (PBS) were cytospun at 800 rpm onto poly-L-lysine-coated slides (6). Peripheral blood was also collected in heparinized syringes and PBMC isolated by Ficoll-Paque (Pharmacia, Piscataway, NJ) gradient centrifugation (3) were cytospun onto slides as described for BAL cells.
Immunohistochemistry Staining
GCR immunoreactivity was detected in acetone:methanol-fixed
cytospins of PBMC by the use of a GCR-specific polyclonal rabbit antibody raised against human GCR. This antibody has previously been shown to be specific for GCR, and has no cross-reactivity against GCR (5, 9). It was raised in New Zealand white rabbits immunized with a 15-mer synthetic peptide (NVMWLKPESSHTLI) derived from the carboxyl terminal amino acid sequences of the corresponding residues 728-742 of the human GCR protein as previously described (9). The antibody was affinity purified on a column prepared by coupling of the synthetic human GCR-specific oligopeptide to UltraLink (Pierce, Rockford, IL) as specified by the manufacturer. The specificity of the anti-human GCR antisera was demonstrated by its reactivity (using Western blot and immunohistochemical analysis) to GCR protein expressed in COS-7 cells or HeLa cells following transfection with the pRShGR expression vector, but not GCR
protein (following expression in COS-7 cells or HeLa cells with the
pRShGR expression vector). Furthermore, nonimmunized rabbit
serum did not react with the GCR protein. Specificity of the immunoreaction for GCR was demonstrated by the lack of reactivity of
nonimmunized rabbit serum to the cytospins and the observation that
anti-GCR staining of BAL cells or PBMC from GC-insensitive asthmatics was blocked by purified GCR immunizing peptide. The reaction was visualized using an avidin-alkaline phosphatase complex and
fast red technique (13). For negative control preparations, the primary antibody was replaced by either nonspecific rabbit immunoglobulin or Tris-buffered saline. The percentage of cells positive for GCR
in each preparation was enumerated by a blinded assessor counting a
minimum of 1,000 total cells. The expression of GCR in specimens
from chronic bronchitis patients was expressed as number of cells/mm2.
Cell localization of GCR expression was performed using double
immunostaining with the GCR-specific polyclonal rabbit antibody raised against human GCR (9) and monoclonal antibodies to CD3
(T cells) and CD68 (macrophages) as previously described (14).
Statistical Analysis
Data were analyzed using the JMP software program (SAS Institute, Cary, NC). Group means were compared by one-way analysis of variance, with individual linear contrasts to compare pairs of means if the overall test was significant at the 0.05 level. Paired comparisons were performed using paired t tests.
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RESULTS |
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INTRODUCTION
METHODS
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DISCUSSION
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BAL cell and PBMC cytospins from seven GC-insensitive
asthmatics, eight GC-sensitive asthmatics, and eight normal
subjects were stained by immunohistochemistry for the expression of GCR. Positive staining for GCR was observed
on all samples stained with anti-GCR (Figure 1, upper left,
upper right, lower left), but not the immunoglobulin isotype
control (Figure 1, lower right). This staining was blocked in
the presence of purified GCR immunizing peptide indicating that the staining was specific for GCR (data not shown).
BAL preparations from GC-insensitive asthmatics were associated with a significantly higher percentage of GCR+ cells (p < 0.0001) than GC-sensitive asthmatics or normal control
subjects (Figure 2). PBMC from GC-insensitive asthmatics also
had a significantly higher (p < 0.0001) expression of GCR
than PBMC from GC-sensitive asthmatics or normal control
subjects. Interestingly, BAL cells from GC-insensitive and
GC-sensitive asthmatics had a significantly higher percentage
of GCR+ cells when compared with their respective PBMC
(p < 0.01). In contrast, the expression of GCR was similar in
the BAL and PBMC of normal subjects.
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To ensure that the high expression of GCR was not related to nonspecific inflammation and airway obstruction, the
GCR immunoreactivity was assessed in lung biopsies of
seven patients with airway obstruction due to chronic bronchitis compared with seven normal subjects. There were very few
cells expressing GCR immunoreactivity in the airways of patients with chronic bronchitis. Indeed, the number of these
cells did not differ from normal control subjects (6.8 ± 2.9 versus 5.5 ± 1.2, respectively).
To determine whether the increased GCR expression in
airway cells of patients with GC-insensitive asthma was derived
from T cells or macrophages, we carried out double immunostaining on BAL from four GC-insensitive and four GC-sensitive asthmatics. BAL cells from GC-insensitive asthmatics expressed GCR immunoreactivity in 95 to 100% of CD3+ T
cells (Table 2). This was significantly greater (p < 0.0001) than
the GCR staining found in GC-sensitive asthmatics where
only 16 to 28% of T cells expressed GCR. In contrast, 15 to
30% of macrophages expressed GCR in both groups.
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DISCUSSION |
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INTRODUCTION
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DISCUSSION
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Previous in vitro studies have demonstrated that overexpression of GCR is associated with decreased GC-induced gene
transactivation and decreased GC insensitivity (5, 8). Recently,
we found that patients with GC-insensitive asthma had significantly higher numbers of GCR-immunoreactive cells in their
peripheral blood than GC-sensitive asthmatics or normal control subjects (5). Our current observation that significantly
higher numbers of GCR-reactive cells are found in the BAL
of GC-insensitive asthmatics than peripheral blood suggests
that the inflammatory milieu of their airways is contributing to
GC insensitivity.
The exact mechanism for increased GCR expression in
GC-insensitive airways requires further study. However, we
have previously found that IL-2 and IL-4 gene expression is
abnormally high in GC-insensitive asthma (6), and that the incubation of these two cytokines with PBMC increases GCR
expression (5). These observations suggest that increased IL-2
and IL-4 expression in the airways could cause the elevated
GCR-reactive airway cells in GC-insensitive asthma. Previous studies have demonstrated that increased expression of
GCR protein after transfection of the pRShGR plasmid
into various cell lines induces GC insensitivity (5, 8). The degree of positivity for GCR in the current study should result
in a functional GC-insensitive state because PBMC with approximately 20% reactivity with GCR following incubation with the combination of IL-2/IL-4 have been associated with
functional insensitivity to GCs (5, 15).
Although several cell types are likely to be involved in GC-insensitive asthma, it is thought that T cells are one of the key
cell types (2). In this regard, activation of T cells from GC-
insensitive asthmatics is not inhibited by GCs, and these patients have increased expression of T-cell activation antigens despite treatment with GCs (16, 17). Our current observation that T cells from the airways of GC-insensitive, as compared
with GC-sensitive, asthmatics have proportionately higher expression of GCR than macrophages is consistent with an important role for T cells in the pathogenesis of GC-insensitive
asthma (3, 6). In GC-insensitive, but not GC-sensitive, asthma
the expression of T-cell-derived cytokines such as IL-4, IL-5,
and IL-13 remains elevated even after treatment with systemic
GCs (6). Inability to inhibit these cytokines could lead to prolonged inflammatory events associated with severe asthma.
Thus, selective expression of GCR in T cells could have
downstream effects leading to GC insensitivity of a number of
cell types involved in chronic asthma and thus contribute to
significant morbidity.
In summary, our data indicate that increased T-cell expression of GCR may be an important marker in characterizing
and understanding GC-insensitive asthma. This observation
may also have important implications for the pathogenesis of
other allergic inflammatory or autoimmune conditions as this
may represent a common mechanism for GC insensitivity. The
current study provides a biologic marker for early identification of patients with GC insensitivity and a new therapeutic
target for patients who do not respond to steroids.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Donald Y. M. Leung, M.D., Ph.D., National Jewish Medical and Research Center, 1400 Jackson Street, Room K926, Denver, CO 80206. E-mail: leungd{at}njc.org
(Received in original form April 28, 1998 and in revised form September 30, 1998).
Acknowledgments: The authors thank David Iklé for assistance in our statistical analyses, Leigh Hume, Carolyn Swartz, and Caroline Bronchick in the General Clinical Research Center for their nursing assistance during the bronchoscopies, and Maureen Plourd-Sandoval for assistance in preparing this manuscript.
Supported in part by Public Health Services Research Grants HL36577, AR41256, HL 37260, and General Clinical Research Center Grant 5 MO1 RR00051 from the Division of Research Resources, the University of Colorado Cancer Center, an American Lung Association Asthma Research Center Grant, MRC Canada and Inspiraplex.
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References |
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METHODS
RESULTS
DISCUSSION
REFERENCES
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 P. J. Hauk, E. Goleva, I. Strickland, A. Vottero, G. P. Chrousos, K. O. Kisich, and D. Y. M. Leung Increased Glucocorticoid Receptor {beta} Expression Converts Mouse Hybridoma Cells to a Corticosteroid-Insensitive Phenotype Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 361 - 367. [Abstract] [Full Text] [PDF]
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A. ten BRINKE, A. H. ZWINDERMAN, P. J. STERK, K. F. RABE, and E. H. BEL Factors Associated with Persistent Airflow Limitation in Severe Asthma Am. J. Respir. Crit. Care Med., September 1, 2001; 164(5): 744 - 748. [Abstract] [Full Text] [PDF]
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K G Tantisira and S T Weiss Complex interactions in complex traits: obesity and asthma Thorax, September 1, 2001; 56(90002): ii64 - 74. [Full Text] [PDF]
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 J. C. Webster, R. H. Oakley, C. M. Jewell, and J. A. Cidlowski 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 PNAS, May 24, 2001; (2001) 121455098. [Abstract] [Full Text] [PDF]
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C. B. Whorwood, S. J. Donovan, P. J. Wood, and D. I. W. Phillips Regulation of Glucocorticoid Receptor {{alpha}} and {beta} Isoforms and Type I 11{beta}-Hydroxysteroid Dehydrogenase Expression in Human Skeletal Muscle Cells: A Key Role in the Pathogenesis of Insulin Resistance? J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2296 - 2308. [Abstract] [Full Text]
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M. KRAFT, Q. HAMID, G. P. CHROUSOS, R. J. MARTIN, and D. Y. M. LEUNG Decreased Steroid Responsiveness at Night in Nocturnal Asthma . Is the Macrophage Responsible? Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1219 - 1225. [Abstract] [Full Text]
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L. Pujols, J. Mullol, M. Pérez, J. Roca-Ferrer, M. Juan, A. Xaubet, J. A. Cidlowski, and C. Picado Expression of the Human Glucocorticoid Receptor {alpha} and {beta} Isoforms in Human Respiratory Epithelial Cells and Their Regulation by Dexamethasone Am. J. Respir. Cell Mol. Biol., January 1, 2001; 24(1): 49 - 57. [Abstract] [Full Text]
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S. Wenzel Proceedings of the ATS Workshop on Refractory Asthma . Current Understanding, Recommendations, and Unanswered Questions Am. J. Respir. Crit. Care Med., December 1, 2000; 162(6): 2341 - 2351. [Full Text]
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D. Y. M. LEUNG and G. P. CHROUSOS Is There a Role for Glucocorticoid Receptor Beta in Glucocorticoid-dependent Asthmatics? Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 1 - 3. [Full Text]
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R. GAGLIARDO, P. CHANEZ, A. M. VIGNOLA, J. BOUSQUET, I. VACHIER, P. GODARD, G. BONSIGNORE, P. DEMOLY, and M. MATHIEU Glucocorticoid Receptor alpha and beta in Glucocorticoid Dependent Asthma Am. J. Respir. Crit. Care Med., July 1, 2000; 162(1): 7 - 13. [Abstract] [Full Text]
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J. C. Webster, R. H. Oakley, C. M. Jewell, and J. A. Cidlowski 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 PNAS, June 5, 2001; 98(12): 6865 - 6870. [Abstract] [Full Text] [PDF]
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