Salbutamol increases MKP-1 expression and inhibits the production of inflammatory mediators TNF and NO in activated macrophages.

T Keränen, T Hömmö, M Hämäläinen, E Moilanen, R Korhonen. The Immunopharmacology Research Group, University of Tampere School of Medicine and Tampere University Hospital, Tampere, Finland

Introduction: Mitogen-activated protein kinase phosphatase-1 (MKP-1) is an endogenous anti-inflammatory mechanism known to dephosphorylate (i.e. inactivate) p38 MAP kinase, inhibit cytokine production and suppress the inflammatory response. Salbutamol is a β2-receptor agonist and it is used as a bronchodilator in obstructive lung diseases. In addition, salbutamol regulates cellular responses by a cAMP-dependent manner. Becauce MKP-1 promotor contains a cAMP response element, we hypothesized that salbutamol may regulate MKP-1 expression.

Methods: The effects of salbutamol were investigated on MKP-1 expression, on p38 MAP kinase phosphorylation and on the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO) in primary mouse peritoneal macrophages (PM) and in J774 macrophage cell line. MKP-1 expression was determined by quantitative RT-PCR and Western blot, and p38 MAP kinase phosphorylation by Western blot following 1h incubation. TNF and NO accumulated in the culture medium in 24h incubation were measured by ELISA and Griess reaction, respectively. One-way ANOVA with Bonferroni’s or Dunnett’s post-test was used in the statistical analysis

Results: Salbutamol (1-1000 nM) alone and in combination with LPS (10 ng/mL) enhanced MKP-1 expression in J774 macrophages in a dose-dependent manner (2.5- fold increase by 100 nM salbutamol vs. control, n=3; and 30% increase by LPS + 100 nM salbutamol vs. LPS, n=3) at 1h. cAMP analog 8-Br-cAMP (100 µM) also enhanced LPS-induced MKP-1 expression (by 20%, p<0.01, n=3) at 1h. Salbutamol (100 nM) inhibited LPS-induced TNF production from 7709 ± 563 pg/mL to 840 ± 12 pg/mL (p<0.01, n=6) and NO production from 41 ± 1.5 µM to 21 ± 1 µM (p<0.01, n=6) during 24h incubation. Salbutamol (100 nM) also reduced LPS-induced phosphorylation of p38 MAP kinase at 1 h (75 ± 10% inhibition, p<0.01, n=4). p38 MAP kinase inhibitors BIRB 796 (1 µM) and SB202190 (1 µM) inhibited TNF and NO production in J774 macrophages and TNF production in PMs by 37-62%.

Conclusions: Salbutamol increased expression of MKP-1 and concomitantly inhibited activation of p38 MAP kinase and limited production of TNF and NO in mouse J774 macrophages and PMs. The results suggest that the anti-inflammatory effects of salbutamol on TNF and NO production are mediated by increased MKP-1 expression which limits p38 MAP kinase pathway and, subsequently, inflammatory gene expression.