Glucocorticosteroid receptor characteristics of peripheral blood mononuclear cells in oral steroid dependent asthma : utilization of an in vitro model of steroid resistant asthma to investigate mechanisms of resistance and functional consequences of altered receptor affinity.

Abstract

Background: Although glucocorticoids are the most effective treatment for

asthma, some patients show a poor response. In such patients with steroid resistant asthma, this has been ascribed to altered glucocorticoid receptor (GR) ligand-binding affinity induced by IL-2 combined with IL-4 or IL-13

alone- all of which can also modulate glucocorticoid function in vitro.

Objective: We sought to assess the ligand-binding affinity in a distinct group

of oral steroid-dependent asthmatic subjects and examine the mechanisms by

which IL-2 and IL-4 (or IL-13) modify the ligand-binding affinity of the GR.

Methods: Using dexamethasone-binding assays, we examined PBMCs ex

vivo from healthy subjects, subjects with controlled asthma, and oral steroiddependent

subjects with severe asthma. In addition, IL-2 and IL-4 were used to alter GR affinity in vitro. We used mediators or inhibitors of signal

transduction to investigate the mechanisms of resistance. We also determined

cytokine production of PBMC's by means of ELISA.

Results: GR ligand-binding affinity was significantly reduced in the nucleus but not in the cytoplasm of oral steroid-dependent asthmatic subjects compared with that seen in steroid-sensitive and healthy subjects (dissociation

constant, 41.37 ± 17.83 vs. 25.36 ± 2.63 nmol/L vs. 9.40 ± 4.01 nmol/L,

respectively [p<.05 for both in comparison to normals] ).

This difference in ligand-binding affinity could be mimicked by IL-2 and

IL-4 co-treatment and was blocked by the p38 mitogen-activated protein

kinase (MAPK) inhibitor SB203580. PBMC's rendered resistant in vitro

demonstrated lower IL-10 and increased GM-CSF production following LPS

or PMA & PHA stimulation compared to cells with normal GR affinity.

Resistant cells also showed reduced dexamethasone repression of LPSstimulated

IL-10 release. These effects were also reversed by SB203580.

Inhibition of the ERK MAPK pathway by PD098059 (10 mol/L),

phosphoinositol 3 kinase by wortmannin (5 nmol/L) or treatment with IL-10

(10 ng/mL) failed to modulate the effect of IL-2 and IL-4 on receptor affinity.

Ro318220 (10 nmol/L), a specific protein kinase C inhibitor and theophylline,

similarly, had no effect on affinity.

Conclusion: GR ligand binding affinity is tiered; compared to normal

subjects; steroid responsive asthmatics have a mild reduction in ligand binding whereas oral steroid dependent asthmatics have greater reductions.

When mononuclear cells are rendered resistant in vitro, cytokine production

(low IL-10 and high GM-CSF) favours a pro-inflammatory state. Our data do

not support the ERK MAPK, phosphoinositol 3 kinase, protein kinase C

pathways in steroid resistance. Treatment with IL-10 and theophylline also

failed to modulate the effect of IL-2 and IL-4 on receptor affinity. However, P38 MAPK inhibitors may have potential in reversing glucocorticoid

insensitivity and re-establishing the beneficial effects of glucocorticoids in patients with severe asthma.