TGF-β has been previously shown to exert pleiotropic, context-specific effects on allergic asthma, mostly through effects on T cells, fibroblasts, and potentially smooth muscle cells (14
). Our findings suggest novel roles for TGF-β as a potent switch regulating mast cell protease expression and as an inhibitor of mast cell accumulation within the airway epithelium. These effects appear to play an important role in regulating contractile responses of airway smooth muscle and in vivo AHR after chronic allergen challenge. Previous studies using mast cell–deficient mice have shown that mast cells contribute to AHR development when mice are sensitized and challenged with an adjuvant-free regimen (5
), as we used here. Our results suggest that these effects are probably caused by TGF-β–dependent differentiation of intraepithelial mast cells expressing mMCP-1, but not those expressing mMCP-4. Our results also suggest that mast cell–dependent induction of AHR might be prevented by inhibition of the αvβ6 integrin within the airway epithelium.
Although previous work suggested that expression of mMCP-1 and mMCP-2 in intestinal intraepithelial mast cells depends on TGF-β activation through the αvβ6 integrin and that TGF-β is required for differentiation of mMCP-1– and mMCP-2–expressing mast cells in vitro (23
), to our knowledge, our finding that mMCP-4 and mMCP-6 expression was inhibited by TGF-β is novel. We were also surprised to find significant accumulation of mMCP-4– and mMCP-6–containing mast cells within the epithelium in the absence of αvβ6-mediated TGF-β activation. Previous studies of intraepithelial mast cells in the intestinal epithelium of β6 KO mice identified mast cells by mMCP-1 immunostaining (29
), and might thus have missed these intraepithelial mast cells that do not express mMCP-1. In the present study, we did not determine the mechanisms that lead to accumulation of these mast cells in the absence of local TGF-β activation. TGF-β has been reported to promote mast cell proliferation and migration (30
). Mitogen-activated protein kinase activity and integrins have previously been shown to be involved in TGF-β–regulated mast cell migration (31
). However, TGF-β1 has also been reported to act as a negative regulator of mast cell function (34
), and it is well accepted that mast cell proliferation and migration can be regulated by different mechanisms in different locations (35
). Mast cells are also known to accumulate at sites of inflammation (36
), so it is possible that the increase in baseline intraepithelial mast cells in β6 KO mice is caused by their low-level baseline inflammation, as we have previously described (37
It is noteworthy that the significant differences in intraepithelial mast cell numbers and mast cell protease expression we describe here were not at all apparent from microarray or qRT-PCR analysis of whole lung samples. These effects could only be identified by sampling the airway epithelial microenvironment. The simple airway brush we have developed should thus be a useful tool for future investigations of cellular and gene expression changes that are restricted to the epithelium.
The protective effects of mMCP-4 on airway smooth muscle contractility were somewhat surprising, given the extensive literature describing the contributions of mast cells to the pathophysiology of allergic asthma and AHR (1
). However, our findings are consistent with a previous report of enhanced AHR after allergen challenge in mice lacking mMCP-4 (40
). Furthermore, mMCP-4 is the closest functional homolog of human mast cell chymase (27
), and epithelial chymase expression has been suggested to be protective against asthma severity in human subjects with asthma (41
). Given the increased number of intraepithelial mast cells we observed in β6 KO mice, it seems likely that the increased levels of Mcpt4
we observed were caused by the combined effects of increased mast cell accumulation and increased Mcpt4
expression in these cells.
Interestingly, mMCP-1 and mMCP-4 not only exert opposite effects on airway contractility, they do so by acting on distinct cell types. Our observation that the contractility-enhancing effect of mMCP-1 was completely abrogated by removal of airway epithelium suggests that mMCP-1 is released within the epithelium and acts directly on a substrate within the epithelial compartment to enhance airway smooth muscle contraction and airway narrowing. In contrast, the inhibitory effects of mMCP-4 on IL-13–enhanced airway contractility were completely preserved in tracheal rings denuded of epithelium, which suggests that mMCP-4 is acting on substrates outside of the epithelial compartment. One attractive hypothesis is that mMCP-4 acts directly on substrates on airway smooth muscle cells. However, given the cellular complexity of tracheal rings, the evidence presented here cannot exclude effects on substrates on other cells or extracellular compartments in the airway wall.
One obvious issue not addressed by the current work is the precise nature of the proteolytic substrates for mMCP-1 and mMCP-4 that regulate the opposing effects of these proteases on airway smooth muscle contractility. Although not much is known about substrates for mMCP-1, several substrates have been described for mMCP-4, including angiotensin I, pro-gelatinase, hepatocyte growth factor, thrombin, and fibronectin (27
). However, the in vivo relevance of these substrates to airway responsiveness is unclear. Identification of the substrates responsible for the effects described herein could lead to new strategies for protection against AHR and human asthma. Although there is no precise human ortholog for mMCP-1, once the proteolytic substrate and downstream molecular cascade responsible for enhancement of airway smooth muscle contractility is identified, the potential relevance of this pathway to human asthma could be more readily evaluated.