Muscarinic Stimulation Of Expression And Release Of Interleukin-8 (IL-8) In Human Lung Fibroblasts

Margarita Fuhrmann1, Mareille Warnken1, Michael Paul Pieper2, Kurt Racké1.1Inst. Pharmacol & Toxicol, Univ. Bonn, Bonn, Germany, 2Pulmonary Research, Boehringer Ingelheim, Biberach, Germany.

IL-8 is a pro-inflammatory cytokine of particular importance for the neutrophil infiltration associated with chronic obstructive airway diseases. Human lung fibroblasts are increasingly recognized as a cellular source of pro-inflammatory cytokines. Since human lung fibroblasts have been shown to be controlled by muscarinic mechanisms (for review see Racké et al., 2008), and the long acting-muscarinic antagonist tiotropium has been shown to exert, in addition to its broncho-dilatatory action, various anti-inflammatory effects (for review see Bateman et al., 2009). The pre-sent study explored in human lung fibroblasts a possible muscarinic modulation of IL-8 expression and release.

MRC-5 human lung fibroblasts were cultured for 24 h in absence or presence of test sub-stances, followed by determination of IL-8 mRNA by qPCR and of IL-8 accumulation in the culture medium by ELISA.

Under control conditions the IL-8 mRNA levels, expressed as ΔCt over GAPDH, amounted to 10.9±2.6 (mean±s.e.mean, number of experiments (n) = 9). The muscarinic agonist oxotremorine (10 µM) induced an increase in IL-8 mRNA by 282±91% (n = 7, p<0.01 vs controls), an effect pre-vented by 10 nM tiotropium, which alone had no effect (difference vs controls 2±9%). Tumor necro-sis factor-α (TNF-α, 10 pg/ml) induced an increase in IL-8 mRNA by 386±69% (n = 7, p<0.01 vs controls), and the effects of TNF-α and oxotremorine were additive. Thus, in presence of both stimu-lators, IL-8 mRNA levels were increased by 784±124% (n = 6, p<0.05 vs TNF-α alone). Observations on IL-8 release paralleled those on mRNA expression. Basal IL-8 accumulation in the culture medi-um amounted to 150±42 pg/ml and oxotremorine (10 µM) induced an increase by 302±95% (n = 7, p<0.01 vs controls). Like the effects on mRNA, this effect was prevented by 10 nM tiotropium, which alone had no effect (difference vs controls -7±4%). TNF-α (10 pg/ml) caused an increase in IL-8 accumulation by 291±70% (n = 7, p<0.01 vs controls) and the effects of TNF-α and oxotremorine were again at least additive, resulting in an increase by 1275±325% by TNF-α in combination with oxotremorine (n = 7, p<0.01 vs TNF-α alone).

Conclusions: IL-8 expression and release in human lung fibroblasts is markedly stimulated by muscarinic receptor activation and the effects of muscarinic receptor agonists were additive to those of TNF-α. Inhibition of the cholinergic drive of IL-8 expression and release in human lung fibroblasts may contribute to anti-inflammatory effects, and by this to long-term beneficial effects of long-acting muscarinic antagonists such as tiotropium in the treatment of COPD.

Racké K, Haag S, Bahulayan A, Warnken M (2008) Naunyn-Schmiedeberg´s Arch Pharmacol 378:193-201.

Bateman ED, Rennard S, Barnes P et al. (2009) Pulm Pharmacol Ther 22:533-542.