Primary airway fibroblasts in the understanding of asthma and its severity

Asthma is characterised by both chronic inflammation and a remodelling of the airways. Airway remodelling in asthma includes increase myofibroblast numbers and increased extra cellular matrix (ECM) deposition. Hypothesis. It is hypothesised that fibroblasts in the severe asthmatic airway are contributing to asthma severity by possessing an increased ability to proliferate, synthesise ECM proteins and propagate the inflammatory process, and that the airway environment in asthma may contributes to this behaviour. Aims. The aims of this thesis were to use broncho-alveolar lavage (BAL) and primary airway fibroblasts to construct a simple in vitro model of the in vivo environment of the airways, and by using this model we are able to compare and quantify the mitogenic ability of asthmatic fibroblasts to that of healthy fibroblasts, as well as comparing their ability to induce mRNA synthesis, which would represent an ability to synthesise ECM and pro-inflammatory proteins, in response to the local environment. Methods. Fibroblasts from 6 healthy, 6 mild asthmatic and 6 severe asthmatic patients were grown from biopsies and challenged with BAL from 6 healthy, 6 mild asthmatic or 6 moderate/severe asthmatic volunteers. The [3H] thymidine incorporation assay and TaqMan real time RT -PCR were used to assess their mitogenic potential and ability to synthesise collagen III mRNA. Interleukin-8 protein levels were also measured in the supematents of these BAL challenged fibroblasts using ELISA. The phosphorylation status of a variety of MAPKs within healthy, mild asthmatic and severe asthmatic fibroblasts was determined after challenge with moderate/severe asthmatic BAL using the R & D systems MAPK phosphorylation assay kit. Results. BAL stimulated eH] thymidine incorporation in fibroblasts grown from biopsies from healthy and mild asthmatics but not in those from severe asthmatics (p<O.OOOl), indicative of an altered fibroblast mitogenic potential in severe asthma. BAL from those with moderate/severe asthma , however, induced significantly more collagen III mRNA expression by the fibroblasts cultured from severe asthmatics than in fibroblasts cultured from the airways of either healthy or mild asthmatics subjects (p<0.05) at 1 hour. IL-8 protein generated by severe asthmatic fibroblasts was significantly decreased compared to that from healthy and mild asthmatic fibroblasts after a 1 and 4 hour challenge with healthy and moderate/severe asthmatic BAL (p<0.05). There was also Aktl and Akt2 phosphorylation in mild and severe fibroblasts after a 30 minute challenge with moderate/severe asthmatic BAL, which was not present in healthy fibroblasts. Conclusion. Fibroblasts from severe asthma thus have an altered phenotype favouring a synthetic rather influencing Akt phosphorylation may be implicated in this process. These findings have relevance to structural airway changes in asthma and processes underlying disease severity. than proliferative phenotype. Signalling pathways