published_or_final_version / Medicine / Master / Master of Philosophy

Lun Samantha Wei Man. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 181-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Asthma is a chronic inflammatory disease of the airways. Using a murine model which replicates many characteristic features of human asthma, this study evaluated the effects of treatment with anti-inflammatory drugs on the lesions of chronic asthma, and investigated potential underlying molecular mechanisms. Treatment with dexamethasone, a glucocorticoid, was compared with roflumilast, a novel phosphodiesterase-4 (PDE4) inhibitor. BALB/c mice sensitised to ovalbumin were challenged with a low mass concentration of aerosolised antigen for 30 min/day, 3 days/week for 6 weeks. In weeks 5 and 6, groups of animals were treated with either dexamethasone or roflumilast. Assessment included changes in acute-on-chronic inflammation, structural remodelling of the airways and airway hyper-responsiveness to a bronchoconstrictor stimulus. These were correlated with the expression of pro-inflammatory cytokines and growth factors. Compared to vehicle-treated control animals, dexamethasone- and roflumilast-treated mice exhibited reduced accumulation of intra-epithelial eosinophils and chronic inflammatory cells, including CD3+ T-lymphocytes in the airways. Similarly, both drugs inhibited subepithelial fibrosis and airway epithelial thickening, although only dexamethasone inhibited goblet cell hyperplasia/metaplasia. Airway hyper-reactivity was not diminished by either drug. Both treatments suppressed production of Th2 cytokines by ovalbumin-restimulated peribronchial lymph node cells. In selectively dissected airway tissue from vehicletreated animals, increased expression of mRNA for several pro-inflammatory cytokines (TNF-α, GM-CSF, IL-6) and cytokines characteristic of Th1 (IFN-γ), Th2 (IL-5, IL-13)and Th17 (IL-17A) cells was demonstrated using real-time PCR. Enhanced expression of growth factors (TGF-β1 and FGF-2) was also demonstrated in airway epithelium isolated by laser capture microdissection. Interestingly, whereas treatment with dexamethasone significantly inhibited expression of mRNA for all of the inflammationrelated cytokines examined, roflumilast inhibited only IL-17A, TNF-α, GM-CSF and IL-6. Both drugs inhibited mRNA expression of growth factors by epithelial cells. Because roflumilast was as effective as dexamethasone in suppressing inflammation and most changes of remodelling, the selective suppression of IL-17A, TNF-α, GM-CSF and IL-6 suggests that these mediators, or the cells that produce them, may have critical roles in pathogenesis. Furthermore, they may be particularly appropriate therapeutic targets in chronic asthma.

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.

Asthma is a chronic inflammatory disease of the airways. Using a murine model which replicates many characteristic features of human asthma, this study evaluated the effects of treatment with anti-inflammatory drugs on the lesions of chronic asthma, and investigated potential underlying molecular mechanisms. Treatment with dexamethasone, a glucocorticoid, was compared with roflumilast, a novel phosphodiesterase-4 (PDE4) inhibitor. BALB/c mice sensitised to ovalbumin were challenged with a low mass concentration of aerosolised antigen for 30 min/day, 3 days/week for 6 weeks. In weeks 5 and 6, groups of animals were treated with either dexamethasone or roflumilast. Assessment included changes in acute-on-chronic inflammation, structural remodelling of the airways and airway hyper-responsiveness to a bronchoconstrictor stimulus. These were correlated with the expression of pro-inflammatory cytokines and growth factors. Compared to vehicle-treated control animals, dexamethasone- and roflumilast-treated mice exhibited reduced accumulation of intra-epithelial eosinophils and chronic inflammatory cells, including CD3+ T-lymphocytes in the airways. Similarly, both drugs inhibited subepithelial fibrosis and airway epithelial thickening, although only dexamethasone inhibited goblet cell hyperplasia/metaplasia. Airway hyper-reactivity was not diminished by either drug. Both treatments suppressed production of Th2 cytokines by ovalbumin-restimulated peribronchial lymph node cells. In selectively dissected airway tissue from vehicletreated animals, increased expression of mRNA for several pro-inflammatory cytokines (TNF-α, GM-CSF, IL-6) and cytokines characteristic of Th1 (IFN-γ), Th2 (IL-5, IL-13)and Th17 (IL-17A) cells was demonstrated using real-time PCR. Enhanced expression of growth factors (TGF-β1 and FGF-2) was also demonstrated in airway epithelium isolated by laser capture microdissection. Interestingly, whereas treatment with dexamethasone significantly inhibited expression of mRNA for all of the inflammationrelated cytokines examined, roflumilast inhibited only IL-17A, TNF-α, GM-CSF and IL-6. Both drugs inhibited mRNA expression of growth factors by epithelial cells. Because roflumilast was as effective as dexamethasone in suppressing inflammation and most changes of remodelling, the selective suppression of IL-17A, TNF-α, GM-CSF and IL-6 suggests that these mediators, or the cells that produce them, may have critical roles in pathogenesis. Furthermore, they may be particularly appropriate therapeutic targets in chronic asthma.