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. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/2527 |
| Date | January 2007 |
| Creators | Irusen, Elvis Malcolm. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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