Characterization of expression and functional role of ß-adrenoceptors in human lung fibroblasts

Kurt Racké1, Fathi B. Lamyel1, Uwe R. Juergens2, Mareille Warnken1. 1Dept. Pharmacol., Univ. Bonn, Bonn, Germany, 2Univ. Hospital Bonn, Dept. Pulmonology, Bonn, Germany.

Fibrotic alterations are part of the airway remodelling processes observed in asthma and COPD. In addition to acute bronchodilatory effects, classical anti-obstructive drugs such as ß2-adrenoceptor (ß-AR) agonists may also modulate long term remodelling processes. Therefore the expression of ß-AR subtypes, their possible functional role and mechanisms controlling their expression in lung fibroblasts were studied.

The human lung fibroblast cell line MRC-5 and primary human lung fibroblasts (phLF) in culture were studied. In phLF and MRC-5 cells mRNA encoding ß2-AR, but not that encoding ß1- and ß3-AR was detected by RT-PCR. Expression of ß2-AR was confirmed at protein level by Western blot analysis. ß2-AR mRNA (determined by qPCR) was not altered after 24 h exposure to 100 nM isoprenaline or 10 nM formoterol, concentrations maximally effective in functional experiments, but reduced by about 25% after exposure to 100 nM formoterol, a supra-maximally effective concentration. 24 h exposure to dexamethasone (1 μM) or budesonide (100 nM) caused an up-regulation of ß2-AR mRNA by about 60%, whereas 24 h treatment with TGF-ß (5 ng/ml) caused a decrease by about 70%. Proliferation of MRC-5 cells as determined by 3H-thymidine incorporation was inhibited by isoprenaline (IC50 3 nM) and formoterol (IC50 57 pM), maximally by about 30-40%. The concentration response curve of formoterol was not affected by the ß1-AR selective antagonist GCP 20712 (3 μM), but shifted to the right by the ß2-AR selective antagonist ICI 118,551 (0.3 and 3 μM) (apparent pA2: 9.6). Furthermore, collagen synthesis as determined by 3H-proline incorporation was also inhibited by formoterol (IC50 91 pM) and isoprenaline (IC50 1 nM), maximally by about 30%. In phLF, isoprenaline and formoterol caused similar inhibition of 3H-thymidine and 3H-proline incorporation. Under basal culture conditions, MRC-5 show significant expression of α-smooth muscle actin, a marker of myo-fibroblast differentiation (determined by Western blot analysis). Basal α-smooth muscle actin expression was reduced following 24 h exposure to 1, 10 or 100 nM formoterol by about 50%.

In conclusion, human lung fibroblasts express ß2-AR which mediate inhibitory effects on different pro-fibrotic features. The pro-fibrotic cytokine TGF-ß caused a down-regulation whereas corticosteroids induced an up-regulation of ß2-AR mRNA expression.