IS THERE A ROLE FOR GLUCOCORTICOID RECEPTOR BETA IN GLUCOCORTICOID-DEPENDENT ASTHMATICS?

To the Editor:

In our study, we questioned the role of GR as a dominant negative receptor in GC-dependent asthma (1). In the accompanying editorial, Leung and Chrousos expressed criticisms regarding design and methods used in our study (2). We provide here a response to their comments that were based on the first manuscript submitted and not on the published paper. Thus, we will only address issues that were not sufficiently clarified in the published paper.

The authors of the editorial claimed that we used an anti-GR antiserum. As indicated in our paper, we did not use an anti-GR antiserum, but an anti-GR/ antiserum that recognizes GR and GR equally well (3). GR and GR were distinguished by their molecular weight, and this antiserum detected only GR in peripheral blood mononuclear cells (PBMC) from all individuals. Data obtained with the anti-GR antiserum only confirmed the absence of detectable amount of GR in these PBMC, GR-transfected A549 cells being an appropriate positive control. The second technical point raised relates to the protein samples we analysed. These did contain the nuclear fraction because the procedure we used lyses both the cells and their nuclei, as checked by microscopy. In contrast to the statements of Leung and Chrousos, there is no indication in the literature that molybdate is required to reveal the presence of GR (3). The third technical point concerned the analysis of GR/ expression by RT-PCR. We have used exactly the same primers as Oakley and colleagues. These authors and we have checked that amplification efficiencies for the GR and GR fragments with these pairs of primers were equivalent by using as templates plasmids containing GR or GR cDNA (Reference 6 and our unpublished observation). Computer analysis of the 3' divergent region in GR and GR mRNAs indicates that both sequences contain 35% of G+C and can not form stable secondary structures at 37° C, the temperature at which reverse transcription was performed. Therefore, the undetectable level of GR message after 30 cycles of PCR is not due to an inefficient reverse transcription or an inefficient amplification of GR message. Moreover, our data are in agreement with those of Oakley and colleagues who needed to increase the number of PCR cycles above 30 to amplify GR message from human tissues.

Finally, we have examined GR/ expression in PBMC because the authors of the editorial have found that, in GC-resistant asthma, the increase in GR expression observed in airway tissues was reflected in PBMC (7). We agree with Leung and Chrousos that GR may be predominant in a particular cell type or subset that was not specifically examined in our study. Alternatively, the differences between the results reported in our study and those of the authors of the editorial may be due to the heterogeneity of GC-dependent and GC-resistant asthmatics.

Hôpital Arnaud de Villeneuve, Montpellier, France

1. Gagliardo R, Chanez P, Vignola AM, Bousquet J, Vachier I, Godard P, Bonsignore G, Demoly P, Mathieu M. Glucocorticoid receptor  and  in glucocorticoid dependent asthma. Am J Respir Crit Care Med 2000; 162: 7-13 [Abstract/Free Full Text].

2. Leung DYM, Chrousos GP. Is there a role for glucocorticoid receptor beta in glucocorticoid-dependent asthmatics? Am J Respir Crit Care Med 2000; 162: 1-3 [Free Full Text].

3. Oakley R, Webster J, Sar M, Parker CJ, Cidlowski J. Expression and subcellular distribution of the beta-isoform of the human glucocorticoid receptor. Endocrinology 1997; 138: 5028-5038 [Abstract/Free Full Text].

4. de Castro M, Elliot S, Kino T, Bamberger C, Karl M, Webster E, Chrousos G. The non-ligand binding beta-isoform of the human glucocorticoid receptor (hGR beta): tissue levels, mechanism of action, and potential physiologic role. Mol Med 1996; 2: 597-607 [Medline].

5. Hect K, Carlstedt-Duke J, Stierna P, Gustafsson J-Å, Brönnegård M, Wikström A-C. Evidence that the beta-isoform of the human glucocorticoid receptor does not act as a physiologically significant repressor. J Biol Chem 1997; 272: 26659-26664 [Abstract/Free Full Text].

6. Oakley R, Sar M, Cidlowski J. The human glucocorticoid receptor beta isoform: expression, biochemical properties, and putative function. J Biol Chem 1996; 271: 9550-9559 [Abstract/Free Full Text].

7. Leung D, Hamid Q, Vottero A, Szefler S, Surs W, Minshall E, Chrousos G, Klemm D. Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J Exp Med 1997; 186: 1567-1574 [Abstract/Free Full Text].

Dr. Donald Leung was given an opportunity to respond to this letter, but declined to do so.