Rationale Mast cells and eosinophils synthesize various mediators, including transforming growth factor b (TGF-b) and cysteinyl leukotrienes (cysLTs), that may promote subepithelial fibrosis in asthma. We sought to determine the roles of TGF-b and cysLTs in myofibroblast differentiation stimulated by coculture of fibroblasts with mast cells or eosinophils.
Methods IMR-90 human lung fibroblasts were serum starved and cocultured for 48 hours with either LAD-2 human mast cells or freshly isolated human blood eosinophils in the absence or presence of SB431542 (10 M) to block signaling through the TGF-b type I (ALK5) receptor; MK886 (1 M) to block leukotriene synthesis; MK571 (100 nM) to block the cysLT1 receptor; or Bay u9773 (3 M) to block the cysLT1 and cysLT2 receptors. a-Smooth muscle actin (a-SMA) expression was assessed as an index of myofibroblast differentiation.
Results Mast cells and eosinophils stimulated 2.50.2 (n = 5, p < .005)- and 1.80.2 (n = 5, p < .0001)-fold increases, respectively, in fibroblast a-SMA expression, determined by immunoblot analysis. In comparison, TGF-b (2.5 ng/mL) increased a-SMA expression by 5.50.5-fold (n = 6, p < .0001). SB431542 inhibited the increase in a-SMA expression stimulated by mast cells, eosinophils and TGF-b by 6,019%, 8,221% and 9,210%, respectively (all p < .0001). By contrast, none of the leukotriene pathway inhibitors significantly affected a-SMA expression. Immunofluorescence microscopy for a-SMA confirmed the immunoblot results. Conclusion: Our data indicate that mast cells and eosinophils stimulate differentiation of myofibroblasts by TGF-b signaling through the ALK5 receptor and that cysLTs are less important in this process. Inhibition of ALK5 receptor signaling may be a useful strategy to interrupt airway remodeling in asthma.
Funded by NIH R01HL072891, Crane Asthma Center of Northwestern University.
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