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. 2015 Feb 4:10:283-91.
doi: 10.2147/COPD.S72403. eCollection 2015.

Reversal of corticosteroid insensitivity by p38 MAPK inhibition in peripheral blood mononuclear cells from COPD

Nadia Khorasani  1 , Josephine Baker  1 , Malcolm Johnson  2 , Kian Fan Chung  1 , Pankaj K Bhavsar  1
Affiliations

Reversal of corticosteroid insensitivity by p38 MAPK inhibition in peripheral blood mononuclear cells from COPD

Nadia Khorasani et al. Int J Chron Obstruct Pulmon Dis. .

Abstract

Background: Corticosteroids (CS) have limited efficacy in the treatment of chronic obstructive pulmonary disease (COPD). p38 mitogen-activated protein kinase (MAPK) activation is increased in lung macrophages of COPD. We investigated whether p38 MAPK inhibition can modulate CS insensitivity of peripheral blood mononuclear cells (PBMCs) from patients with COPD.

Methods: PBMCs from patients with COPD (n=8) or healthy smokers (n=8) were exposed to lipopolysaccharide (LPS) with a selective p38 MAPK inhibitor (GW856553; 10(-10)-10(-6) M), with dexamethasone (10(-10)-10(-6) M), or with both. Phosphorylated glucocorticoid receptor (GR) was measured by Western blot.

Results: Baseline (P<0.01) and LPS-induced (P<0.05) CXCL8 release was greater in PBMCs from COPD compared to healthy smokers. Inhibition of LPS-induced CXCL8 release by dexamethasone (10(-6) M) was reduced, and baseline and LPS-induced p38 MAPK activation increased in PBMCs of COPD. GW856553 (10(-9) and 10(-10) M) synergistically increased the inhibitory effect of dexamethasone (10(-8) and 10(-6) M) on LPS-induced CXCL8 release in COPD. Similar results were obtained for IL-6 release. GW856553 inhibited dexamethasone- and LPS-activated phosphorylation of serine 211 on GR. CS insensitivity in COPD PBMCs is reversed by inhibition of p38 MAPK activity, partly by preventing phosphorylation of GR at serine 211.

Conclusion: p38 MAPK inhibition may be beneficial in COPD by restoring CS sensitivity.

Keywords: glucocorticoid receptor; p38 mitogen-activated protein kinase.

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Figures

Figure 1
Figure 1
Baseline and LPS-induced CXCL8 release from PBMCs of patients with COPD and healthy smokers. Notes: (A) Comparison of baseline (NS) and LPS-induced CXCL8 release in PBMCs from healthy smokers (n=10) or COPD patients (n=11). Cells were stimulated with LPS (10 ng/mL) for 24 hours. CXCL8 release was determined by enzyme-linked immunosorbent assay. Horizontal bar represents median. *P<0.05, **P<0.01, ***P<0.001. (B) Spearman’s rank correlation between baseline CXCL8 release and FEV1. (C) Spearman’s rank correlation between LPS-induced CXCL8 release and FEV1. Abbreviations: COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 second; LPS, lipopolysaccharide; NS, not stimulated; PBMCs, peripheral blood mononuclear cells.
Figure 2
Figure 2
Relative corticosteroid insensitivity in PBMCs of COPD patients. Notes: PBMCs from healthy smokers (▼, n=8) and COPD patients (•, n=11) were pretreated with (A) dexamethasone or (B) GW856553 for 1 hour and then stimulated with LPS (10 ng/mL) for 24 hours. CXCL8 release was determined by enzyme-linked immunosorbent assay. (A) Inhibition of LPS-induced CXCL8 release from PBMCs of healthy smokers and COPD patients by dexamethasone. (B) Inhibition of LPS-induced CXCL8 release from PBMCs of healthy smokers and COPD patients by the p38α MAPK inhibitor, GW85655. **P<0.01 compared to healthy smokers. Abbreviations: COPD, chronic obstructive pulmonary disease; Dex, dexamethasone; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells.
Figure 3
Figure 3
Comparison of baseline and induced p38 MAPK activation in PBMCs of COPD patients and healthy smokers. Notes: Phosphorylated and total p38 MAPK expression were determined in whole-cell protein extracts by Western blotting and were normalized to β-actin. The ratio of phospho-p38 MAPK to total p38 MAPK expression, determined by Western blotting and subsequent densitometric analysis, was taken as a measure of p38 MAPK activation. (A and B) Baseline phosphorylated and total p38 MAPK expression in PBMCs of healthy smokers and COPD patients. (C) LPS-induced p38 MAPK activation in PBMCs of healthy smokers and COPD patients. PBMCs were stimulated with LPS (10 ng/mL) for 30 minutes. (B and C) Horizontal bar represents median. *P<0.05. Abbreviations: C, COPD; COPD, chronic obstructive pulmonary disease; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells; S, healthy smoker.
Figure 4
Figure 4
Effect of p38 MAPK inhibition on dexamethasone-mediated suppression of LPS-induced CXCL8 release from PBMCs of COPD patients. Notes: Cells were pretreated with dexamethasone or GW856553 alone or in combination for 1 hour and then stimulated with LPS (10 ng/mL) for 24 hours. (A) Inhibition of LPS-induced CXCL8 release from PBMCs of COPD patients by the p38 MAPK inhibitor, GW856553 (n=8), in the presence of dexamethasone, and (B) by dexamethasone in the presence of GW856553. Data shown as mean ± standard error. *P<0.05, **P<0.01, ***P<0.001 compared to dexamethasone, alone. Abbreviations: COPD, chronic obstructive pulmonary disease; Dex, dexamethasone; GW, GW856553; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells.
Figure 5
Figure 5
Effect of p38 MAPK inhibition on dexamethasone-mediated suppression of LPS-induced IL-6 release from PBMCs of COPD patients. Notes: Cells were pretreated with dexamethasone or GW856553 alone or in combination for 1 hour and then stimulated with LPS (10 ng/mL) for 24 hours. (A) Inhibition of LPS-induced IL-6 release from PBMCs of COPD patients by the p38 MAPK inhibitor, GW856553 (n=8), in the presence of dexamethasone, and (B) by dexamethasone in the presence of GW856553. Data shown as mean ± standard error. *P<0.05, **P<0.01, ***P<0.001 compared to dexamethasone alone. Abbreviations: COPD, chronic obstructive pulmonary disease; Dex, dexa-methasone; GW, GW856553; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells.
Figure 6
Figure 6
Reversal of corticosteroid insensitivity in PBMCs of COPD patients. Notes: PBMCs of patients with COPD (•, n=8) or healthy smokers (▼, n=8) were pretreated with dexamethasone (10−6 M) alone or in combination with GW856553 for 1 hour and then stimulated with LPS (10 ng/mL) for 24 hours. CXCL8 was determined by enzyme-linked immunosorbent assay. Data shown as mean ± standard error. Abbreviations: COPD, chronic obstructive pulmonary disease; Dex, dexamethasone; LPS, lipopolysaccharide; PBMCs, peripheral blood mononuclear cells.
Figure 7
Figure 7
Effect of GW865553 on dexamethasone-induced phosphorylation of Ser211 on the glucocorticoid receptor in PBMCs of COPD patients. Notes: Cells were pretreated with dexamethasone (10−6 M) alone or in combination with GW856553 (10−6 M) for 1 hour and then stimulated with LPS (10 ng/mL) for 30 minutes. Phosphorylated Ser211 in whole-cell protein extracts (n=5) was determined by Western blotting (A), followed by densitometric analysis (B) and was normalized to β-actin. (B) Data shown as mean ± standard error. #P<0.05 compared to NS. *P<0.05. Abbreviations: COPD, chronic obstructive pulmonary disease; Dex, dexamethasone; GR, glucocorticoid receptor; GW, GW856553; LPS, lipopolysaccharide; NS, unstimulated control; PBMCs, peripheral blood mononuclear cells; Ser211, serine 211.
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