Montelukast prevents microparticle-induced inflammatory and functional alterations in human bronchial smooth muscle cells

S Fogli1, F Stefanelli1, T Neri2, C Bardelli3, A Celi2, S Brunelleschi3, PL Paggiaro2, MC Breschi1. 1University of Pisa, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnologies, Italy, 2University of Pisa, Cardio-Thoracic and Vascular Department, Italy, 3University A. Avogadro, Department of Medical Sciences, Italy

Microparticles (MPs) are membrane fragments released from virtually any cell type upon activation o during apoptosis, which play a significant role in intercellular communication. Human airway smooth muscle has important synthetic functions in asthma and is a rich source of cytokines, chemokines, and inflammatory mediators, which contribute to the inflammatory process and airway remodelling. The present study was aimed at investigating the pro-inflammatory profile of monocyte/macrophage-derived MPs in bronchial smooth muscle cells (BSMC) of human origin and the effect of montelukast in this experimental setting. Human monocytes/macrophages (from which MPs were generated) were isolated from the buffy coats of blood donor by Ficoll gradient centrifugation followed by overnight culture of the mononuclear cell fraction. MPs generation was subsequently obtained from adherent cells stimulated with the calcium inophore, A23187, as previously described (Neri et al., 2011). Characterization tools used on this study included RT-PCR analysis of gene expression, immunoenzymatic determination of pro-inflammatory factors released into the cell supernatant, bioluminescent assay of intracellular cAMP levels and electromobility shift assay analysis of NF-κB nuclear translocation. Data were expressed as mean ± s.e.m. Statistical analysis was carried out by t test or one-way ANOVA followed by the Newman-Keuls test for multiple comparisons. Stimulation of human BSMC with MPs for 24 h significantly increased COX-2 mRNA levels (2.08±0.36-fold, as compared to control; n=5; p<0.05) without affecting COX-1 expression and was correlated with a considerably increase in PGE2 release (3.30±0.81-fold, as compared to control; n=5; p<0.05) in the culture medium. Following MPs stimulation, the ability of salbutamol to induce cAMP synthesis was reduced by 50% compared with unstimulated cells (108±19 and 218±25 nM, respectively; n=5; p<0.05) and treatment with indomethacin or the selective COX-2 inhibitor NS398 at 1 μM for 24 h restored salbutamol response in MP-desensitized BSMC. MPs also increased IL-8 mRNA expression and IL-8 release in culture medium (1048±175 and 350±66 pg/ml, MP-treated and control cells, respectively; n=5; p<0.05). Challenge with MPs markedly induced nuclear translocation of NF-κB and the selective NF-κB inhibitor Bay 11-7082 at 1 μM for 24 h decreased MP-induced IL-8 and PGE2 release in the supernatant, at levels comparable to those of unstimulated cells. Montelukast significantly reduced IL-8 release (-39.9±12.1%; n=5; p<0.05) as well as nuclear translocation of NF-κB from MP-stimulated cells, and completely restored salbutamol response in MP-desensitised BSMC. Our findings provide evidence that MPs play a role in the pathogenesis of airway inflammation by inducing COX-2 gene expression and β2-adrenoceptor desensitization and promoting cytokine release from human BSMC via activation of NF-κB. Finally, montelukast mitigates MP-induced IL-8 up-regulation and preserves the ability of salbutamol to induce cAMP synthesis in stimulated BSMC, suggesting that this drug may interfere with a potential novel mechanism involved in the progression of chronic respiratory diseases.

Neri T, Armani C, Pegoli A, et al. Eur Respir J. 2011; 37:1494-502