Suppressive activity of tiotropium bromide on matrix metalloproteinase production from lung fibroblasts in vitro

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling with an accumulation of inflammatory cells. There is also increasing evidence that metalloproteinases (MMPs) may contribute to the pathogenesis of COPD, but the influence of agents that used for the treatment of COPD is not well understood.

Objective: We evaluated whether tiotropium bromide hydrate (TBH), a M3 muscarinic receptor antagonist, could inhibit MMP production from lung fibroblasts (LFs) in response to tumor necrosis factor (TNF)-alpha stimulation.

Methods: LFs were established from normal lung tissues taken from patients with lung tumors. LFs (5 x 10(5) cells/ml) were stimulated with TNF-alpha in the presence of various concentrations of TBH. After 24 h, culture supernatants were obtained and assayed for the levels of MMPs and tissue inhibitor of metalloproteinases (TIMPs) by ELISA. The influence of TBH on mRNA expression of MMPs and TIMPs in 4h-cultured cells was also examined by real-time RT-PCR. Furthermore, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) in LFs treated with TBH for 4h was examined by ELISA.

Results: TBH at more than 15 pg/ml inhibited the production of MMP-2 from LFs after TNF-alpha stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected by TBH through the suppression of both mRNA expression and transcription factor, NF-kappaB, activation in LFs induced by TNF-alpha stimulation.

Conclusion: These results suggest that the attenuating effect of TBH on MMP-2 production from LFs induced by inflammatory stimulation may be additional beneficial therapeutic effects not directly relating to its bronchodilatory effects.

Figure 1

Influence of tiotropium on metalloproteinase (MMP)-2 production from lung fibroblasts induced by tumor necrosis factor (TNF)-α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 24 h. MMP-2 levels in culture supernatants were examined by ELISA. Data are expressed as the mean ng/ml + SE of five different subjects. Notes: *significant (P < 0.05) as compared with TNF alone.

Figure 2

Influence of tiotropium on the production of tissue inhibitor of metalloproteinase (TIMP)-1 (a) and TIMP-2 (b) from lung fibroblasts in response to tumor necrosis factor (TNF)-α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 24 h. TIMP-1 and TIMP-2 levels in culture supernatants were examined by ELISA. Data are expressed as the mean ng/ml + SE of five different subjects.

Figure 3

Influence of tiotropuim on matrix metalloproteinase (MMP)-2 mRNA expression in lung fibroblasts after tumor necrosis factor (TNF)-α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 4h. mRNA expression was examined by real-time RT-PCR. Data are expressed as the mean ratio + SE of five different subjects. Notes: *significant (P < 0.05) as compared with TNF alone.

Figure 4

Influence of tiotropium on mRNA expression for tissue inhibitor of metalloproteinases (TIMP)-1 (a) and TIMP-2 (b) in lung fibroblasts in response to tumor necrosis factor (TNF)-α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 4 h. mRNA expression was examined by real-time RT-PCR. Data are expressed as the mean ratio + SE of five different subjects.

Figure 5

Influence of tiotropium on nuclear factor (NF)-κB activation in lung fibroblasts induced by tumor necrosis factor (TNF)-α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 4 h. NF-κB, p-50 and p-65, activities were assayed by ELISA. Data are expressed as the mean optical density (OD) at 450 nm + SE of five different subjects. Notes: *significant (P < 0.05) as compared with TNF alone.

Figure 6

Influence of tiotropium on activator protein (AP)-1 activation in lung fibroblasts induced by tumor necrosis factor (TNF)– α stimulation. Cells (5 × 10⁵ cells/ml) were stimulated with 25.0 ng/ml TNF-α in the presence of various concentrations of tiotropium for 4 h. AP-1, Fra 2 and Jun B, activities were assayed by ELISA. Data are expressed as the mean optical density (OD) at 450 nm + SE of five different subjects.