Mast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation. Human lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells. The migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration. Mast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases.

Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.

Background: Protease activated receptor 2 (PAR2) is highly expressed on various cells. Although the well documented effects of PAR2 in innate immune responses to inflammation, the knowledge of PAR2 in the pathogenesis of chronic lung diseases is limited. The objective was to investigate the role of PAR2 and its effect on fibroblasts regarding changes in phenotype profile and mediator release in fibroblast monoculture and in co-culture with mast cells. Methods: The experiments were performed in 2D in vitro cultures on cell culture plastic and in a 3D ex vivo model where the cells were seeded on decellularized human lung scaffolds in order to mimic a more physiological milieu for the cells to interact. Human lung fibroblasts (HFL-1) were co-cultured with LAD2 mast cells with and without the PAR2 antagonist, P2pal-18s. The fibroblast morphology, distribution of a-smooth muscle actin protein and mediator profile were analysed. Results: The PAR-2 antagonist induced an altered fibroblast phenotype to a more elongated shape and increased aSMA expression. The PAR2 antagonist significantly induced a different mediator profile, where Il-6 (p=0.003), VEGF (p=0.004) and HGF synthesis (p=0.001) increased in fibroblasts co-cultured with mast cells. The mediators were differently synthesised when the cells were co-cultured in human lung scaffolds compared to 2D culture on plastic. Conclusions: PAR2 antagonism promote important effects on human lung fibroblast function with alterations in mediator release and morphological changes. These data indicate an important role of PAR2 in acute as well as chronic inflammatory diseases in which fibroblasts and mast cells are involved.