Background. Inhaled corticosteroids represent the most common treatment for asthma. Although most asthmatic patients respond well, a significant proportion of severe asthmatic patients either require high doses or even fail to respond to oral or inhaled corticosteroids. We have previously reported that glucocorticoid receptor-beta is associated with corticosteroid resistance. We have also shown that in severe asthmatic patients, Th-17 cytokines increase steroid insensitivity in airway epithelial cells via a mechanism involving GR-beta upregulation.
Objective. To investigate whether IL-17A and F cytokines enhance steroid unresponsiveness in PBMCs isolated from normal and severe asthmatic subjects via the upregulation of GR-beta isoform.
Methods. PBMCs were isolated from the blood of healthy and severe asthmatics subjects and cultured for 48 hours in the presence or absence of IL-17A, IL-17F, IL-17A+IL-17F, or IL-2+IL-4 cytokines. Inhibition of proliferation by Dexamethasone (IC50) was then assessed on PHA-treated cells using 3H-thymidine pulse labeling. Expression of GR-alpha, GR-beta, GILZ and IL-6 was determined using Real Time RT-PCR and/or Western blotting.
Results. Treatment of PBMCs with IL-17A+F cytokines significantly decreased the level of expression of GR-alpha m-RNA while the of expression of GR-beta m-RNA was significantly upregulated. Moreover, while treating PBMCs with IL-2+IL-4 had a more remarkable decrease in GR-alpha expression, this cytokine combination had no effect on GR-beta receptors. Both cytokine combinations significantly decreased the inhibitory effect of Dexamethasone on PBMC proliferation (IL-17A+F, IC50=190 nM Dex; IL-2+4, IC50=1060 nM Dex); no significant differences were observed between the PBMCs from normal subjects and severe asthmatics. Treatment with IL-2+4, but not IL-17A+F, inhibited the dexamethasone-induced expression of the glucocorticoid-inducible leucine zipper gene (GILZ) in PBMCs from both normal (60%) and asthmatics (45-50%).
Conclusion. IL-17A, IL-17F, IL-2 and IL-4, which are known to be upregulated in the blood and lung tissue of asthmatics, contribute to steroid insensitivity of severe asthmatic patients by modulating the expression of GR-alpha and GR-beta receptors on peripheral blood PBMCs. Furthermore, the increased GR-beta/GR-alpha ratios by both IL-17A+F and IL-2+4 cytokines correlates with the decreased inhibitory effect of Dexamethasone on PHA-induced PBMC proliferation.