ng-bo Li

Inhibition of Histone Deacetylase 2 Expression by Elevated Glucocorticoid Receptor β in Steroid-resistant Asthma

RATIONALE Cross-talk between glucocorticoid receptors and histone deacetylases (HDACs) under steroid-insensitive conditions has not been explored. OBJECTIVES To evaluate expression and interaction of HDACs with glucocorticoid receptor isoforms in bronchoalveolar lavage and peripheral blood mononuclear cells from steroid-resistant versus steroid-sensitive patients with asthma. METHODS Expression of HDACs 1 through 11 was measured by real-time polymerase chain reaction in primary cells and in the DO11.10 cell line, designed to overexpress glucocorticoid receptor β. Glucocorticoid receptor β expression was inhibited in bronchoalveolar lavage cells by small interfering RNA. Human HDAC2 promoter fragments were cloned into a luciferase reporter vector, and transiently transfected with glucocorticoid receptor α- and β-encoding plasmids into the cells. Luciferase activity was then assayed in response to glucocorticoids. MEASUREMENTS AND MAIN RESULTS Levels of HDAC2 mRNA, but not other histone deacetylases, were significantly decreased in bronchoalveolar lavage cells but not in peripheral blood mononuclear cells from steroid-resistant patients with asthma. Overexpression of glucocorticoid receptor β in DO11.10 cells selectively reduced HDAC2 mRNA and protein levels. Silencing of glucocorticoid receptor β in bronchoalveolar lavage cells from patients with asthma significantly increased HDAC2 mRNA. Luciferase activity assays with HDAC2 promoter reporter constructs identified two glucocorticoid-inducible regions in the HDAC2 promoter. Promoter activity was increased more than fourfold in dexamethasone-treated cells cotransfected with glucocorticoid receptor α. Cotransfection of glucocorticoid receptor β abolished this effect in a dose-dependent manner. CONCLUSIONS Glucocorticoid receptor β controls expression of histone deacetylase 2 by inhibiting glucocorticoid response elements in its promoter. This highlights a novel mechanism by which glucocorticoid receptor β promotes steroid insensitivity (Li et al.: J Allergy Clin Immunol 2009;123:S146; and Li et al.: J Allergy Clin Immunol 2010;125:AB104).

Divergent expression and function of glucocorticoid receptor β in human monocytes and T cells

Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune‐mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12‐myristate 13‐acetate/ionomycin‐induced tumor necrosis factor α and interleukin‐6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid‐responsive gene, mitogen‐activated protein kinase phosphatase‐1 (MKP‐1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor β (GCRβ) is a splicing variant of the classic GCR, GCRα, which functions as a dominant‐negative inhibitor of GCRα in humans, not mice (as mice do not express GCRβ mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRβ than T cells. Furthermore, GCRβ was found in the cytoplasm and nucleus of monocytes, and GCRβ was localized to the nucleus of T cells. This raised the possibility that GCRβ in the cytoplasm could affect GCRα cellular shuttling in response to DEX. Indeed, we found that DEX‐induced nuclear translocation of GCRα was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRβ in human monocytes resulted in enhanced steroid‐induced GCRα transactivation and transrepression. Our data suggest that GCRβ contributes to variation in the GC responses of monocytes versus T cells.

IFN-γ Reverses IL-2– and IL-4–Mediated T-Cell Steroid Resistance

Corticosteroids are the most common therapeutic approach for control of tissue inflammation. Combination IL-2/IL-4 is known to induce T-cell steroid resistance. This can be reversed with IFN-gamma; however, the mechanism by which this occurs is unknown. In the current study, we found that treatment of peripheral blood mononuclear cells with combination IL-2/IL-4 for 48 hours, but not with IL-2 or IL-4 alone, abrogated dexamethasone (DEX)-induced glucocorticoid receptor (GCR)-alpha nuclear translocation in both CD4(+) and CD8(+) T cells. The presence of IL-4 significantly down-regulated IFN-gamma production by IL-2-stimulated cells. Importantly, addition of IFN-gamma to the IL-2/IL-4 combination restored GCRalpha nuclear translocation in response to DEX. Furthermore, DEX-induced mitogen-activated protein kinase (MAPK) phosphatase-1 induction, used as a readout for corticosteroid-induced transactivation, was significantly greater (P < 0.05) in media and IL-2/IL-4/IFN-gamma-treated conditions compared with IL-2/IL-4-treated cells. The combination of IL-2/IL-4 induced p38 MAPK activation in CD3(+) cells (30.5 +/- 5.7% cells expressed phospho-p38 MAPK versus no phospho-p38 MAPK expression after media treatment). The presence of the p38 MAPK inhibitor, SB203580, or IFN-gamma inhibited p38 MAPK phosphorylation and enhanced GCRalpha nuclear translocation in response to DEX. These data indicate that combination IL-2/IL-4 inhibits GCRalpha nuclear translocation in human T cells, and this effect is reversed by IFN-gamma via inhibition of p38 MAPK activation.

Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics.

Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics’ peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance.

CD56+ Cells Induce Steroid Resistance in B Cells Exposed to IL-15

Interleukin-2 can induce steroid resistance in T cells. IL-15 shares biological activities with IL-2, as both cytokines use IL-2Rγ for signal transduction. We therefore sought to determine whether IL-15 contributes to induction of PBMC corticosteroid resistance. Surprisingly, we found that incubation of unfractionated PBMC with IL-15 for 48 h resulted in the inhibition of glucocorticoid receptor (GCR) nuclear translocation in response to dexamethasone (DEX) treatment in CD19-positive B cells significantly greater than CD19-negative non-B cells (p < 0.01). However, pure B cells incubated with IL-15 responded normally with nuclear translocation of GCR in response to steroids, but failed to translocate GCR when they were grown in the presence of CD19− cells. Coculture of B cells with CD3+ (T cells), CD14+ (monocytes), or CD56+ (NK and NKT cells) in the presence of IL-15 revealed that only CD56+ cells contributed to the steroid insensitivity of B cells. IL-15 stimulation significantly increased production of IL-4 by CD56+ cells (p < 0.02). Treatment of purified B cells with combination IL-15/IL-4 resulted in abrogation of glucocorticoid receptor nuclear translocation and the inability of DEX to suppress cytokine production by B cells. In the presence of IL-4-neutralizing Ab, when B cells were cocultured with CD56+ cells and IL-15, the B cells were found to be steroid sensitive, i.e., DEX induced GCR nuclear translocation. This study demonstrates that B cells develop steroid resistance in the presence of CD56+ cells after IL-15 stimulation. Furthermore, IL-15 and IL-4 have the capacity to induce B cell insensitivity to steroids.