Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta.

In many chronic inflammatory disorders, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (R) pre-messenger RNA generates a second GCR, termed GCR-beta, which does not bind GCs but antagonizes the transactivating activity of the classic GCR, termed GCR-alpha. In the current study, we demonstrate that GC-insensitive asthma is associated with a significantly higher number of GCR-beta-immunoreactive cells in peripheral blood than GC-sensitive asthmatics or normal controls. Furthermore, we show that patients with GC-insensitive asthma have cytokine-induced abnormalities in the DNA binding capability of the GCR. These abnormalities can be reproduced by transfection of cell lines with the GCR-beta gene resulting in significant reduction of their GCR-alpha DNA binding capacity. We conclude that increased expression of GCR-beta is cytokine inducible and may account for GC insensitivity in this common inflammatory condition.

Allergen exposure decreases glucocorticoid receptor binding affinity and steroid responsiveness in atopic asthmatics.

Allergen exposure can confound the management of asthma. To understand the potential mechanisms by which allergens increase the steroid requirements in atopic asthmatics, we examined the effects of allergens on glucocorticoid receptor (GCR) binding affinity and glucocorticoid (GC) responsiveness of peripheral blood mononuclear cells (PBMC) from atopic asthmatics. A significant reduction (p < 0.001) in the GCR binding affinity (Kd) was observed in ragweed-allergic asthmatics during ragweed pollen season compared with PBMC obtained before and after ragweed season. In vitro effects of allergen on PBMC GCR Kd were also examined by incubating PBMC from atopic asthmatics with allergen (ragweed and cat) versus Candida albicans. GCR binding affinity was significantly reduced after incubation with ragweed (p < 0.001) or cat allergen (p < 0.001) compared with baseline or C. albicans stimulation. This effect was limited to atopic asthmatics in that in vitro cat allergen incubation for 48 h failed to significantly alter GCR binding affinity in nonasthmatic, atopic individuals. These allergen-induced reductions in GCR binding affinity also rendered the PBMC less sensitive to the inhibitory effects of hydrocortisone and dexamethasone on allergen-induced proliferation (p < 0.01). To test the hypothesis that allergen-induced alterations in GCR binding affinity were cytokine-induced, we examined the effects of interleukin-2 (IL-2)and IL-4 neutralization using anticytokine antibodies. Addition of both anti-lL-2 and anti-lL-4 antibodies resulted in a significant (p < 0.001) inhibition of allergen-induced alterations in GCR binding affinity. Furthermore incubation with cat allergen induced significantly higher concentrations of IL-2 (p = 0.03) and IL-4 (p = 0.02) by PBMC from atopic as compared with nonatopic subjects. Our current observations suggest that allergen exposure may contribute to poor asthma control by reducing GCR binding affinity in mononuclear cells. This appears to be mediated through IL-2 and IL-4. These findings may have important implications for novel approaches to the treatment of poorly controlled asthma.

Effects of glucocorticoids on lymphocyte activation in patients with steroid-sensitive and steroid-resistant asthma.

BACKGROUND: Glucocorticoids are important medications used to control the airway inflammation associated with asthma. Synthetic glucocorticoids vary in their binding affinity for the glucocorticoid receptor (GCR). METHODS: We compared hydrocortisone, beclomethasone dipropionate, triamcinolone acetonide, flunisolide, and budesonide with regard to their capacity to inhibit phytohemagglutinin-induced peripheral blood mononuclear cell proliferation from six patients with steroid-sensitive asthma and seven patients with steroid-resistant asthma. Peripheral blood mononuclear cell GCR binding affinities for dexamethasone and budesonide were also determined for both patient groups by using a radioligand binding assay and Scatchard analysis. RESULTS: Dose-dependent inhibition was demonstrated for all glucocorticoids in both patient groups, with the steroid-resistant group requiring approximately 2 log-fold more glucocorticoids for an equivalent degree of inhibition. The mean concentrations necessary to cause 50% inhibition of lymphocyte proliferation (IC50s) for the steroid-sensitive group ranged from 2 x 10(-10) mol/L for budesonide to 7 x 10(-8) mol/L for hydrocortisone, whereas the mean IC50s for the steroid-resistant group ranged from approximately 2 x 10(-8) mol/L for budesonide to greater than 10(-6) mol/L for hydrocortisone. In addition, a significant correlation was noted between the degree of inhibition of lymphocyte proliferation (IC50) and the binding affinity of dexamethasone to the GCR. Patients with steroid-resistant asthma have been shown to have a reduced GCR binding affinity. The GCR binding affinity for budesonide was significantly higher in both groups (i.e., lower dissociation constant) than that obtained for dexamethasone. CONCLUSION: These data suggest that glucocorticoids such as budesonide, by virtue of their high GCR binding affinities and greater ability to suppress lymphocyte proliferation, may therefore be beneficial in the management of difficult-to-control asthma.

A novel action of IL-13: induction of diminished monocyte glucocorticoid receptor-binding affinity.

We have recently demonstrated that the combination of IL-2 and IL-4 blunts T cell responses to glucocorticoids in steroid resistant (SR) asthma by reducing glucocorticoid receptor (GCR)-binding affinity. Since immune activation appears to be involved in the acquisition of steroid resistance, we sought to identify whether other cytokines could also induce diminished GCR-binding affinity. In the current report, utilizing a [3H]dexamethasone radioligand-binding assay and Scatchard analysis, we found that IL-13, a cytokine with similar actions as IL-4, could induce diminished GCR binding-affinity (GCR Kd = 34.4 +/- 2.3 nM with IL-13 vs Kd = 8.8 +/- 0.7 nM for unstimulated control cells; p < 0.001) in PBMC from normal subjects. In contrast, PBMC incubated with IL-1, IL-3, IL-5, IL-7, IL-8, IL-12, or granulocyte-macrophage-CSF had no effect on GCR-binding affinity; and no additive effect to the decreased GCR-binding affinity was noted when IL-13 was cocultured with IL-2 or IL-4. The cell target of IL-13-induced GCR effects was studied and found to reside in the non-T cell population; specifically, the monocyte fraction. To determine the functional significance of the decreased GCR-binding affinity, monocytes were pretreated with and without IL-1 3 prior to stimulation with LPS and hydrocortisone. IL-13 pretreatment of monocytes significantly diminished (p = 0.005) the suppressive effects of hydrocortisone on LPS-induced IL-6 production. IL-13, by virtue of its ability to induce diminished GCR-binding affinity, may contribute to impaired GC responsiveness during inflammatory illnesses.

Reduced glucocorticoid binding affinity in asthma is related to ongoing allergic inflammation.

Recent studies indicate that chronic asthma is associated with a spectrum of glucocorticoid receptor (GCR) binding abnormalities that are cytokine-inducible. These GCR abnormalities may contribute to poor asthma control and failure to respond to glucocorticoid (GC) therapy. The purpose of this study was to determine whether GCR defects are associated with poorly controlled asthma, and whether diminished GCR binding is reversible following a course of GC therapy. We enrolled 12 patients with poorly controlled asthma characterized by nocturnal awakening with cough or wheezing, AM FEV1 < 70%, or FEV1 variability of > 25% requiring a short course of high dose GC therapy. GCR binding affinity was measured in peripheral blood mononuclear cells using a radioligand binding assay before and after the GC course. Spirometry, serum cortisol, eosinophil cationic protein (ECP), and soluble IL-2 receptor (sIL-2R) levels were also performed before and after the GC course. At baseline, all subjects had airflow obstruction that significantly improved (median FEV1 increased from 65.0% to 89.5% of predicted, median FEV1/FVC ratio increased from 0.60 to 0.72) with therapy. A diminished GCR binding affinity at baseline was noted with an elevated median dissociation constant (Kd) of 29.0 nM (interquartile range at the 25th and 75th percentile [IQ] of 22.3 and 44.5 nM) compared with normal controls (Kd 8.0 nM [IQ 7.0, 9.2]). Following the GC course, a significant decrease in the Kd was seen. Serum ECP and sIL-2R levels at baseline were elevated, with serum ECP demonstrating a significant reduction following the GC course.(ABSTRACT TRUNCATED AT 250 WORDS)

Coexistence of glucocorticoid receptor and pharmacokinetic abnormalities: factors that contribute to a poor response to treatment with glucocorticoids in children with asthma.

Rapid glucocorticoid clearance and abnormal glucocorticoid receptor binding have been described as factors that contribute to an inadequate response to treatment with glucocorticoids in patients with asthma. We report the coexistence of these abnormalities in children with severe asthma who respond poorly to systemic glucocorticoid therapy.

Steroid-resistant asthma. Cellular mechanisms contributing to inadequate response to glucocorticoid therapy.

The current study examined whether alterations in glucocorticoid receptor (GR) binding contribute to poor response to glucocorticoid therapy in asthma. 29 asthma patients with forced expiratory volume in 1 s (FEV1) < 70% predicted were studied. Patients were classified as steroid sensitive (SS) if their morning FEV1 increased > 30% after a 1-wk course of oral prednisone 20 mg twice daily and steroid resistant (SR) if they failed to increase > 15%. PBMC obtained from these two groups, 17 SR and 12 SS, as well as 12 normal controls were analyzed. SR patients had two distinguishable GR binding abnormalities: 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity, as compared with SS patients (P = 0.0001) and normal controls (P = 0.0001). This defect was localized to T cells and reverted to normal after 48 h in culture media. However, incubation with a combination of IL-2 and IL-4 sustained this abnormality. The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells. Furthermore, GR number failed to normalize after incubation in media alone or IL-2 and IL-4. Therefore, SR asthma may be due to more than one abnormality, the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number. These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma.

Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids.

The mechanisms contributing to persistent T cell activation and poor response to glucocorticoids in chronic inflammatory illnesses such as steroid resistant (SR) asthma are poorly defined. We examined the possibility that certain cytokines, specifically IL-2 and IL-4, could affect T cell response to glucocorticoids. A [3H]dexamethasone radioligand-binding assay was used to measure the number of glucocorticoid receptors (GR) and dissociation constant (Kd) in PBMC from normal donors and patients with SR asthma, cultured in the absence and presence of these cytokines. PBMC from normal donors incubated for 48 h in the presence of combination IL-2 + IL-4 had nuclear GR with significantly reduced binding affinity (GR Kd = 36.1 +/- 1.63 nM, mean +/- SEM; p = 0.0001) as compared with PBMC incubated with medium alone (GR Kd = 6.74 +/- 0.46 nM). The cytosolic GR Kd remained unchanged. However, when PBMC were incubated with IL-2 alone or IL-4 alone, no change in GR-binding affinity was observed. Furthermore, when T cells and non-T cells were individually stimulated with combination IL-2 + IL-4, a significant reduction in GR-binding affinity was observed only in the T cell population (p = 0.0001). The IL-2 + IL-4-induced alteration in PBMC GR Kd was associated with an increase in GR number (8348 +/- 964 vs 1710 +/- 228 sites/cell; p = 0.0003). More importantly, the alteration in PBMC GR-binding affinity with IL-2 + IL-4 was associated with a functional change in T cell response to methylprednisolone MPN, i.e., a reduced inhibitory effect of MPN on PMA/ionomycin-induced T cell proliferation. These effects of IL-2 + IL-4 on PBMC GR affinity and response to MPN were blocked by co-incubation with IFN-gamma. Freshly isolated PBMC from four patients with SR asthma had a significantly reduced GR-binding affinity (Kd = 40.0 +/- 2.68 nM; p = 0.0001) when compared with seven normal subjects (7.15 +/- 0.41 nM). The altered PBMC GR binding from patients with SR asthma reversed to normal when incubated with medium alone, but was sustained with IL-2 + IL-4. These observations suggest that with persistent inflammation certain cytokines may contribute to an impaired response to glucocorticoids. Furthermore, the effects of IL-2 and IL-4 were blocked by IFN-gamma.

Steroid-resistant asthma: immunologic and pharmacologic features.

Glucocorticoids play an important role in asthma therapy; however, a subset of patients are poorly responsive. We evaluated immunologic and pharmacologic features of 17 patients with steroid-resistant (SR) asthma (six male and 11 female patients) between the ages of 16 and 69 years (mean age, 29 years). SR asthma was defined as failure to improve morning prebronchodilator FEV1 greater than 60% predicted after a 2-week course of oral prednisone (mean dose, 45 mg/day). These patients were compared to 24 steroid-sensitive (SS) patients, aged 5 to 70 years (mean age, 17 years; 17 male and seven female patients), and 47 healthy control subjects, aged 20 to 40 years. Mean prednisone dose in SS patients was 25 mg/day. Steroid pharmacokinetics were evaluated in six patients with SR asthma. All studies were within normal limits. Peripheral blood mononuclear cells (PBMCs) from all subjects were stimulated with 10 micrograms/ml of phytohemagglutinin and incubated for 72 hours with 10(-5) to 10(-9) mol/L of methylprednisolone (Mpn). The Mpn dose-response curve for PBMCs from patients with SR asthma demonstrated a significant (p less than 0.05) increase in DNA synthesis, that is, more T cell proliferation than PBMCs stimulated with phytohemagglutinin in the presence of Mpn, as compared to SS patients and normal subjects. This augmentation of DNA synthesis was reversible with 10 micrograms/ml of troleandomycin in vitro. We conclude that PBMCs from patients with SR asthma demonstrate altered response to Mpn in the presence of a T cell mitogen. This abnormality in cellular response may contribute to persistent airway inflammation in patients with SR asthma despite glucocorticoid therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma histamine, epinephrine, cortisol, and leukocyte beta-adrenergic receptors in nocturnal asthma.

Plasma histamine, cortisol, epinephrine, cyclic adenosine monophosphate (cAMP), and leukocyte beta-adrenergic receptors were measured in asthmatic patients with (n = 7) and without (n = 10) nocturnal asthma at 4 PM and 4 AM and compared with those of normal subjects (n = 10). A twofold higher plasma histamine concentration was observed at 4 AM compared with 4 PM in all groups, with no change in plasma cortisol, epinephrine, and cAMP concentrations. At 4 AM compared with 4 PM, only patients with nocturnal asthma had a significant 33% decrease (p less than 0.05) in mononuclear and polymorphonuclear leukocyte beta-adrenergic receptor density, with no difference in binding affinity in all three groups. Polymorphonuclear leukocytes from patients with nocturnal asthma had significantly impaired response to isoproterenol at 4 AM (17% +/- 7.3% SEM increase in cAMP; p less than 0.05) compared with those of patients without nocturnal asthma (69.4% +/- 13.7%) and normal (80.2% +/- 21.3%) subjects. A significant change in beta-adrenergic receptor density and function occurs at night in patients with nocturnal asthma.