E-cadherin is a marker protein of epithelium and is indispensable for the maintenance of the epithelial phenotype [24, 25]. Suppression of E-cadherin expression leads to the disassembly of cell–cell adhesion and subsequent loss of epithelial polarity, and initiates the differentiation of epithelial cells into fibroblast-type mesenchymal cells [25–27]. Vimentin is a protein expressed in mesenchymal cells, but it can also be expressed in epithelial cells where EMT occurs. Epithelial cells undergoing EMT are also characteristic of irregularly spindle-shaped appearance and powerful migration capacity [28]. In the present study, we found that mast cells alone did not affect significantly E-cadherin and vimentin expression in the co-cultured 16-HBE cells, nor did affect the epithelial morphology and migration ability, suggesting that mast cells alone have not the potential to induce EMT in 16- HBE cells. Unlike mast cells, IL-1β significantly down-regulated E-cadherin expression while significantly upregulated vimentin expression in 16-HBE cells. Moreover, the IL-1β-treated 16-HBE cells exhibited the morphological features of mesenchymal cells and an increasing mobility. The results indicate that IL-1β is able to induce 16-HBE cells to undergo EMT.
TGF-β1is the most potent and most well described inducer of EMT identified so far [28]. In the normal airways, a low of level of TGF-β1 can be secreted by airway epithelial cells and other structural cells, and is an essential growth factor for the maintaining of epithelial integrity [10]. However, repeated aggression of inflammation or exogenous irritants (allergens, infections and cigarette smoke) leads to the release of large amounts of TGF-β1 from the damaged epithelial cells in asthmatic airways [10, 29]. Increased levels of TGF-β1 have been reported in bronchoalveolar lavage fluid and bronchial biopsies of asthmatic patients [30, 31]. A lot of studies have shown that TGF-β1 could directly induce EMT in human bronchial epithelial cells [11–14]. Yasukawa et al. reported that eosinophils induce EMT in airway epithelial cells via increasing TGF-β1 production [32]. In our experiment, we observed that epithelial cells cultured alone produced only a small amount of TGF-β1, however, mRNA expression and protein production of TGF-β1 were significantly increased in IL-1β-treated 16-HBE cells compared with 16-HBE cells alone. In addition, in the presence of anti-TGF-β1 mAb, IL-1β-induced suppression of E-cadherin expression and enhancement of vimentin expression in 16-HBE cells were abrogated completely. These results indicate that the IL-1β could stimulate 16-HBE cells to produce TGF-β1 which mediated the conversion of 16-HBE cells to mesenchymal cells.
Previous reports have demonstrated that human mast cell line LAD2 and the cultured mast cells from the progenitors in human cord blood or peripheral blood can constitutively express mRNA for TGF-β1 and produce bioactive TGF-β1 [33, 34]. In our study, the peripheral blood-derived mast cells could releasea small amount of TGF-β1, but IL-1β stimulation significantly enhanced expression of both TGF-β1 mRNA and protein in the cultured mast cells. The results provide further evidence that mast cells are also a potential source of TGF-β1 and inflammatory stimulation is able to activate mast cells to release more TGF-β1. It is likely that the amount of TGF-β1 produced by 16-HBE cells and/or mast cells in a quiescent state is too small to activate TGF-β signaling pathway to affect the expression of E-cadherin and vimentin in 16-HBE cells, therefore, mast cells failed to induce EMT in the co-cultured 16-HBE cells.
Our study found that mast cells and 16-HBE cells in the co-culture were incapable of interacting with each other in the expression of TGF-β1, however, when IL-1β was added to the co-culture, TGF-β1 expression was significantly higher in the co-cultured mast cells than in IL-1β-stimulated mast cells, and higher in the co-cultured 16-HBE cells than in IL-1β-stimulated 16-HBE cells. The result shows that addition of IL-1β could significantly enhance mRNA expression and protein production of TGF-β1 in both 16-HBE cells and mast cells. Since activated epithelial cells or mast cells also have potential to produce a variety of biologically active mediators such as IL-4, TNF-α and IL-1β, which in turn further promote TGF-β1 expression in mast cells and epithelial cells [10, 16, 35], therefore, the enhancing TGF-β1 expression in the co-cultured cells with IL-1β may result from the stimulation of active mediators other than TGF-β1 secreted by 16-HBE cells and/or mast cells in an autocrine/paracrine fashion.
In our experiment, mast cells alone had no effect on E-cadherin or vimentin expression in the co-cultured 16-HBE cells, however, when IL-1β was added to the co-cultures, E-cadherin expression was significantly decreased while vimentin expression was significantly increased in 16-HBE cells. The result indicates that EMT could be induced in the 16-HBE cells co-cultured with mast cells and IL-1β. When compared with IL-1β-induced EMT of 16-HBE cells, EMT in co-cultured 16-HBE cells with mast cells was significantly enhanced by the addition of IL-1β, and could not be abrogated significantly by TGF-β1 neutralizing antibody, suggesting that the EMT was independent on TGF-β1. Given that IL-1β itself induced EMT of 16-HBE through a TGF-β1-dependent mechanism, mast cells may play a vital role in promoting the transformation of 16-HBE cells to mesenchymal cells in the present of IL-1β. As mentioned above, in addition to TGF-β1, activated mast cells can also release multiple other profibrotic factors such as epidermal growth factor, connective tissue growth factor, fibroblast growth factor-2, IL-6, IL-1β and TNF-α, which have been reported to participate in epithelial EMT [7, 13, 14, 16, 29]. Thus, it is likely that mast cells induced the TGF-β1-independent EMT in the co-cultured 16-HBE cells by releasing some unknown EMT inducers in the presence of IL-1β.
In conclusion, we for the first time demonstrated that IL-1β alone induced a TGF-β1-dependent EMT in 16-HBE cells, but human peripheral blood-derived mast cells alone failed to induce EMT of 16-HBE cells. Mast cells combined with IL-1β induced EMT in 16-HBE cells through a TGF-β1-independent mechanism. Our results suggest the possibility that mast cells contribute to EMT in human bronchial epithelial cells in the inflammatory airway tissues of asthmatics.