Is There a Role for Glucocorticoid Receptor Beta in Glucocorticoid-dependent Asthmatics?

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Glucocorticoid (GC)-dependent and GC-resistant asthma are both challenging clinical problems that are costly to the health care system. Suboptimal responses to steroids often lead to prolonged courses of high-dose GC therapy accompanied by serious adverse effects despite persistent airway compromise. An understanding of the mechanisms that lead to these two conditions is important for the development of new therapeutic approaches. It is also not known whether GC-dependent and GC-insensitive asthma are part of the same disease spectrum with GC-resistant asthma simply being a more severe form of GC-dependent asthma owing to a higher level of immune activation accompanied by structural changes, or alternatively, they may be distinct entities. Because T cells from GC-resistant asthmatics generally have a shift to the right in their dose-response to corticosteroids, i.e., an insensitivity rather than an absolute resistance, it is reasonable to consider the possibility that GC-dependent asthma and GC-insensitive asthma are part of the same pathogenic process.

Peripheral blood mononuclear cells (PBMC) from most patients with GC-resistant asthma have a reversible defect in their T-cell GC receptor (GCR) ligand and DNA binding affinity which can be sustained in vitro by the addition of interleukin-2 (IL-2) and IL-4 but not the individual cytokines (1, 2). Bronchoscopy studies indicate that airway T cells of GC-resistant, as compared with GC-sensitive, asthmatics have significantly higher levels of IL-2 and IL-4 gene expression (3). Involvement of non-T cells requires other cytokines, e.g., IL-13 for monocytes (4) or IL-8 for neutrophils (5), in the induction of GC insensitivity. These data support the concept that GC-resistant asthma results from immune activation which leads to reduced GCR binding affinity.

Alternative splicing involving exon 9 of the GCR gene gives rise to two homologous messenger ribonucleic acids (mRNAs) and protein isoforms, termed GCR and GCR (6). Both mRNAs contain the first eight exons of the GCR gene. GCR is the classic ligand binding protein for corticosteroids, which mediates its metabolic effects primarily by interaction of the GCR with GC response elements (GREs) in the promoters of GC responsive genes. Its anti-inflammatory actions are thought to be exerted via protein-protein interactions with activating transcription factors, such as activator protein-1 (AP-1) and nuclear factor kappa B (NF-B), blocking their ability to induce transcription of proinflammatory cytokine genes. 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. Several groups have shown that these differences render GCR unable to bind GC hormones, inhibit its ability to transactivate GC-sensitive genes, and make it a dominant negative inhibitor of GCR on activating GRE-containing enhancers or transrepressing NF-B (7). Of note, GCR does not inhibit GR-mediated transrepression of AP-1-responsive promoters (10). Hecht and coworkers (11) have challenged the concept of GCR as a dominant negative inhibitor of GR activity because they could find no evidence for a specific dominant negative effect of GCR on transactivation induced by GCR in COS 7 cell lines. However, these discrepant results could be explained by the use of different vectors, cell/tissue specificity, or inadequate GCR expression connected to the transient transfection systems used. The generation of transgenic animals and stable cell lines expressing varying levels of GCR isoforms are needed to resolve these issues. Indeed, Oakley and coworkers have shown that formation of transcriptional impaired GCR-GCR heterodimers may be an important component of the mechanism responsible for the dominant negative activity of GCR (9).

In our experience, overexpression of GCR induces GC insensitivity, reproducing the ligand and DNA binding abnormality found in PBMC from GC-resistant asthma (Reference 1 and D. Y. M. Leung, unpublished observations). Using immunohistochemistry, we have reported that airway cells and PBMC from patients with GC-resistant asthma express significantly higher levels of GCR than patients with GC-sensitive asthma or normal subjects (12). GCR expression was significantly higher in airway T cells than peripheral blood T cells. In patients with GC-resistant asthma, approximately 20% of PBMC and nearly 100% of airway T cells expressed GCR at high levels. In contrast, GC-sensitive asthmatics only expressed GCR in 5 to 10% of PBMC and normal control subjects had GCR in approximately 5% of PBMC. The synthesis of GCR was inducible with the combination of IL-2 and IL-4. Animal models of systemic GC resistance such as New World monkeys have approximately 10-fold higher GCR than GCR levels (6). Interestingly, mice, known to be extremely steroid-sensitive animals, do not appear to have GCR.

In the current issue, Gagliardo and coworkers (13) report that they were unable to find increased GCR in patients with GC-dependent asthma using Western blot or polymerase chain reaction (PCR) analyses of total PBMC. These data would suggest that GCR does not have a role in GC-dependent asthma. Therefore, this study raises the interesting possibility that GC-dependent versus GC-resistant asthma may have different mechanisms. While that might be true, enthusiasm for this conclusion is dampened by several methodologic problems and design flaws in this study which precludes interpretation of their data regarding analysis of GCR in GC-dependent asthma.

In particular, their Western analysis data comparing GCR and GCR levels do not include any quantitation standard (13). Without this, it is difficult to determine the detection limits of the anti-GCR antiserum compared with the anti-GCR antiserum. It cannot be concluded that GCR levels exceed those of GCR unless the sensitivity of the two antisera for their respective targets is quantitated. The observation that a broad and heavy band can be detected from the GCR-transfected A549 cells is not convincing evidence that GCR is not present in the patient samples, because the A549 cells would be expected to produce very high levels of protein from the plasmid. Indeed, their inability to find any GCR protein in patient or control PBMC in the cytoplasmic fraction of a mixed cell population suggests a technical problem with GCR degradation as it is at odds with data from multiple groups that have found GCR protein in normal control subjects or asthmatics (1, 6, 12, and J. A. Cidlowski, personal communication). Because GCR does not bind ligand, it is extremely sensitive to proteolysis, especially when no sodium molybdate is present in the preparation buffer as was the case in this study. Furthermore, these investigators examined the cytosolic rather than the nuclear fraction where GR exerts its dominant negative effects on GR (6, 8).

The investigators also tried to quantitate mRNA encoding GCR and GCR via reverse transcriptase/polymerase chain reaction (RT-PCR). Oligo dT was used as a primer to initiate reverse transcription of GCR, GCR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR was then performed on the mixtures using primers specific for GCR, GCR, and GAPDH. The investigators attempted to normalize the PCR products of the GCR to the product of the GAPDH. This technique makes several assumptions. First, that the efficiency of reverse transcription was the same for GCR and GCR mRNA. Because the sequences of the two mRNAs differs primarily at the 3' end across which reverse transcriptase must read, this assumption is not valid. Second, the two presumed products of reverse transcription were amplified with heterologous primer sets, which were assumed to amplify equivalently. This assumption is also not valid without evidence. Quantitative RT-PCR (QRT-PCR) usually requires use of homologous RNA standards, which are likely to have the same or very similar reverse transcription and PCR efficiencies as the target mRNA. Homologous RNA standards are generally created from the native complementary DNA (cDNA) via in vitro transcription. Techniques for QRT-PCR have been published (14, 15), and could be applied to quantitation of these two mRNAs to strengthen the authors' claims of different amounts present in the patient samples.

Their claims would also have been stronger had they shown normal GCR binding affinities in their GC-dependent asthmatics as patients with GC-resistant asthma have defects in their GC receptor ligand and DNA binding affinity (1, 2). Indeed, it is of concern that the PBMC from their GC-dependent patients did respond to corticosteroids in vitro but had poor clinical response to GCs in vivo. Of note, PBMC from GC-resistant patients fail to respond to GCs in vitro and in vivo. This raises questions about compliance to therapy. Unfortunately, they did not monitor cortisol levels to be certain their GC-dependent asthmatics were adherent to therapy. In a study by Spahn and coworkers (16) of GC-dependent asthmatics, nearly 50% of patients were nonadherent to therapy with oral corticosteroids. The possibility that adherence to therapy could have been a problem is suggested by their observation that GC-dependent asthma was not associated with a decrease in GCR levels. Based on their current observation and those of others (17) that GC therapy reduces GCR levels, one would have expected that if their GC-dependent patients were taking their corticosteroids regularly they should have had decreased GCR expression.

Another concern with the study of Gagliardo and coworkers (13) relates to inadequate patient characterization and age/ treatment matching to disease controls. Their GC-dependent asthmatics were significantly older than their control groups. Their GC-dependent asthmatics also had asthma for twice as long as their asthma control groups. Of note, five of their 14 GC-dependent asthmatics had less than 15% improvement after -agonist treatment, suggesting they may have a structural basis for irreversible airway change accounting for poor response to GCs. There was also considerable variation in the oral GC dose patients were thought to be on at the time of study. GCs are known to alter GCR binding characteristics in GC-dependent patients (16). Because there was inconsistent use of the same patients for each laboratory study, this raises the question: could the differences in cytokine production they observed be age- or treatment- related rather than an intrinsic difference in the form of asthma?

The major limitation of this study, however, was that they restricted their analyses only to total PBMC. Indeed, neutrophils are known to be steroid-resistant and we have recently found that they constitutively have high level expression of GCR  particularly after IL-8 stimulation (5). Thus, the failure of patients to resolve their airway inflammation may be due to persistent neutrophilic inflammation rather than GC insensitivity of their PBMC. Furthermore, asthma is a disease of the airways rather than the peripheral blood. Our previous studies demonstrate that GCR expression is significantly higher in the airway T cells than in the peripheral blood. In the peripheral blood only a small subset of cells express GCR at high levels (12). Therefore, Western blot or PCR analyses total PBMC may miss a critical T-cell subset involved in the disease as the majority of PBMC express GCR only at low levels.

In future studies, these potential confounding variables should be taken into account when characterizing and studying patients with either GC-dependent or GC-resistant asthma. Aside from abnormalities in GCR expression, it will also be important to look at other factors that contribute to suboptimal response to corticosteroid therapy. These include abnormalities in corticosteroid pharmacokinetics (e.g., reduced absorption or increased metabolism and rapid clearance), transcription factor protein interactions with the GCR leading to decreased cellular activation as well as GCR modification, e.g. phosphorylation, which may modulate its function. None of these factors are mutually exclusive. Indeed pharmacokinetic abnormalities have been described in patients with GCR binding abnormalities (18). Increased GCR expression has also been described in GC-resistant asthmatics with increased AP-1 transcriptional activity (17, 19). Thus, it is important to keep an open mind to the possible heterogeneity and complexity of mechanisms contributing to GC dependence and resistance and how they might affect the natural history of chronic asthma.

DONALD Y. M. LEUNG

Division of Pediatric Allergy-Immunology

National Jewish Medical and Research Center

Department of Pediatrics

University of Colorado Health Sciences Center

Denver, Colorado

GEORGE P. CHROUSOS

National Institutes of Child Health and

  Human Development

National Institutes of Health

Bethesda, Maryland

	Footnotes

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. Email: leungd{at}njc.org

	References

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