Anti-inflammatory effects of levalbuterol-induced 11β-hydroxysteroid dehydrogenase type 1 activity in airway epithelial cells

Abstract

Airway epithelial NF-κB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting β2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-κB activity. Since β2-agonists can induce expression of 11β-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-κB activation induced by the β-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R + S)- (racemic) albuterol, augmented mRNA expression of 11β-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-κB (NF-κB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-κB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luc activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-, but not with (S)- or racemic albuterol, augmented 11-keto corticosteroid (cortisone) induced luc activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA), indicating that there was a conversion of inactive 11-keto to active 11-hydroxy corticosteroids. LPS- and TNFα-induced NF-κB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine production to an extent similar to that of dexamethasone. These results demonstrate that levalbuterol augments expression of 11β-HSD1 in airway epithelial cells, reducing LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production through the conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form in this cell type.

Keywords: 11beta-hydroxysteroid dehydrogenase; albuterol; anti-inflammatory; epithelium; glucocorticoid.

Figure 1

Exposure to (R)-albuterol, but not (S)-albuterol or racemic (R + S)-albuterol, induces mRNA expression of 11β-HSD1, but not 11β-HSD2, in airway epithelial cells. MTCC were exposed to 10⁻⁶M (R)-albuterol (R), (S)-albuterol (S), racemic (R + S)-albuterol, or 10 ng/ml TNFα. Twenty-four hours later, RNA was isolated, cDNA was generated, and 11β-HSD1 (Hsd11b1), 11β-HSD2 (Hsd11b2), and GAPDH (Gapdh) gene expression were analyzed by quantitative PCR. Relative expression of 11β-HSD1 (A) and 11β-HSD2 (B) were calculated. Data were pooled from three separate experiments. n = 6–9/group; *p ≤ 0.05 and **p ≤ 0.01 compared to vehicle.

Figure 2

Pre-exposure to (R)-albuterol reduces LPS- and TNFα-induced NF-κB activity in airway epithelial cells. NF-κB-luciferase MTCC were exposed to vehicle (veh.) or 10⁻⁶M (R)-albuterol (R), (S)-albuterol (S), or (R + S)-albuterol for 24 h. All cells except control were then exposed to 100 ng/ml LPS (A) or 10 ng/ml TNFα (B). Sixteen hours later, cell lysates were prepared, luciferase activity was measured, and protein concentration was determined. n = 5–6/group and the experiment was repeated twice; **p ≤ 0.01 compared to control, ***p ≤ 0.001 compared to control, ^#p ≤ 0.05 compared to vehicle, ^##p ≤ 0.01 compared to vehicle.

Figure 3

Pre-exposure of airway epithelial cells to (R)-albuterol for 24 h, followed by cortisone exposure for 16 h, augments GRE-luciferase activity, which requires the activity of 11β-HSD. GRE-luciferase MTCC were exposed to 10⁻⁸M dexamethasone or 10⁻⁶M (R)-albuterol. Twenty-four hours later, 10⁻⁶M cortisone alone or with 10⁻⁶M glycyrrhetinic acid was added for 16 h and luciferase activity and total protein were measured. n = 5–6 samples/group and the experiment was repeated twice; ***p ≤ 0.001 compared to untreated, *p ≤ 0.05 compared to untreated, ^#p ≤ 0.05 compared to (R)-albuterol + cortisone.

Figure 4

Pre-exposure of MTCC to (R)-albuterol and cortisone for 24 h, followed by exposure to LPS or TNFα for 16 h, diminishes NF-κB-luciferase activity, which requires the activity of 11β-HSD. NF-κB-luciferase MTCC were exposed for 24 h to 10⁻⁶M cortisone alone or to 10⁻⁶M (R)-albuterol with or without 10⁻⁶M cortisone and 10⁻⁶M glycyrrhetinic acid. Twenty-four hours later, 100 ng/ml LPS (A) or 10 ng/ml TNFα (B) were added to the cell culture medium. Cells were then lysed 16 h later using reporter lysis buffer. Luciferase activity and total protein were then measured. n = 4–6 samples/group and the experiment was repeated twice; ***p ≤ 0.001 compared to untreated, ^###p ≤ 0.001 compared to LPS (A) or TNFα (B), ^#p ≤ 0.05 compared to LPS (A) or TNFα(B).

Figure 5

Pre-exposure of MTCC to (R)-albuterol and cortisone diminishes LPS- and TNFα-induced pro-inflammatory cytokine production. MTCC were exposed to 10⁻⁶M (R)-albuterol, with or without 10⁻⁶M cortisone or 10⁻⁶M glycyrrhetinic acid, or to 10⁻⁸M dexamethasone. Twenty-four hours later, 100 ng/ml LPS (A) or 10 ng/ml TNFα (B) were added to the cell culture medium. Cell-free conditioned media were then collected 16 h later and pro-inflammatory cytokine levels were measured. n = 3 samples/group and the experiment was repeated twice; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to LPS (A) or TNFα (B).

Figure 6

Proposed mechanism of action. By signaling through the β2-adrenergic receptor, (R)-albuterol transcriptionally upregulates the mRNA expression and oxidoreductase activity of 11β-HSD1 in airway epithelial cells, thereby potentiating the anti-inflammatory effects of endogenous glucocorticoids to inhibit activity of the pro-inflammatory transcription factor NF-κB.