An assay to evaluate the long‐term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNFα up‐regulates 5‐HT_2A‐mediated contraction

Abstract

1 Asthma research is arguably limited by an absence of appropriate animal models to study the pharmacology of in¯ammatory mediators that a ect airway hyperresponsiveness and remodelling. Here we assessed an assay based on mouse tracheal segments cultured for 1 ± 32 days, and investigated contractile responses mediated by muscarinic and 5-hydroxytryptamine (5-HT) receptors following long-term exposure to tumour necrosis factor-alpha (TNFa). 2 Following culture, in the absence of TNFa, maximum contractile responses to KCl and carbachol were similar, with an increase in response up to day two and a decrease to a stable level after 8 days. Maximal relaxations to isoprenaline were not a ected by the culture procedure. The potency of KCl and isoprenaline increased throughout the study. DNA microarray data revealed that global gene expression changes were greater when tissues were introduced to culture than when they were maintained in culture. The morphology of smooth muscle cells was maintained throughout the culture period. 3 5-HT induced a weak contraction in both fresh and cultured (up to 8 days) segments. Culture with TNFa produced a time-and concentration-dependent increase in the maximal contraction to 5-HT, evidently mediated by 5-HT 2A receptors, whereas, the potency for carbachol was reduced. 4 In conclusion, the phenotype of airway smooth muscle remained largely intact during the culture period, even though minor changes were obtained during the ®rst days of culture. The timedependent e ect of TNFa indicates the importance of studying the long-term e ect of cytokines on the smooth muscle cells in relation to airway hyperresponsiveness and remodelling.

Toll‐like receptor stimulation induces airway hyper‐responsiveness to bradykinin, an effect mediated by JNK and NF‐κB signaling pathways

“…Individual tracheae were used in whole for molecular studies or divided into segments of three cartilage rings for functional studies [16]. Briefly, segments were cultured for 1 or 4 days in 300 or 1,000 ?…”

“…Tracheal segments were mounted on two L-shaped metal prongs, with a resting tension of 0.8 mN. The organ bath, containing Krebs-Henseleit buffer at 37°C, was continuously equilibrated with 5% CO 2 and 95% O 2 [16]. Indomethacin (3 ?…”

“…Recently, we have developed an organ culture assay for evaluation of the effects of inflammatory mediators on isolated mouse airways [16]. In the cultured tissue, contact between indigenous structures such as extracellular matrix, epithelium, smooth muscle and nerve cells is maintained, reducing phenotypic alteration of the cells [17] while avoiding the complexity of in vivo models [18].…”

“…In the presented study the effects of TLR2, TLR3 and TLR4 stimulation were examined using the mouse organ culture model [16], focusing on the role of bradykinin in airway hyper-reactivity. The expression of TLR was evaluated by reverse transcription (RT)-PCR and immunohistochemistry.…”

Inflammation-induced airway smooth muscle responsiveness is strain dependent in mice

“…In mice, studies have shown that long-term inflammatory stimulation with TNFα, IL-1β and IL-4, alters the responses to contractile agonists such as 5-hydroxytryptamine and bradykinin [9][10][11][12][13]. Moreover, smooth muscle cells can be activated directly by stimuli of microbial origin through Toll-Like receptors (TLRs) [14,15].…”