Our findings reveal an important and previously unsuspected role for IFN-γ–dependent activation of mast cells in the development of the inflammation and tissue pathology associated with this mouse model of chronic allergic inflammation of the airways. As summarized in Supplemental Table 1, mast cell expression of IFN-γR is required for optimal development of many features of this model, including AHR to methacholine, inflammation of the airways (with infiltrates containing many neutrophils and eosinophils), airway remodeling (i.e., increases in both numbers of epithelial goblet cells and lung collagen), and markedly increased lung expression of several cytokines, chemokines, and markers of an alternatively activated macrophage response. It bears emphasis that in KitW-sh/W-sh mice engrafted with Ifngr1–/– mast cells, mast cells are the only cells that genetically lack IFN-γR. The poor development of many important features of this model in such mice therefore demonstrates that these features require functions of IFN-γ–responsive mast cells that cannot be provided adequately by any of the other IFN-γ–responsive cell types present in this setting.
Mast cell expression of IFN-γR is required for optimal expression of most of the effects of mast cells in this model that also require mast cell expression of FcεRIγ (Supplemental Table 1), and this model is associated with significant, but mast cell–independent, elevations of levels of IFN-γ in the affected lungs (Figure A). In vitro, costimulation of mouse mast cells with IFN-γ significantly enhanced their ability to release histamine, IL-6, and IL-13 in response to activation with IgE and specific antigen (Figure ), and IFN-γ dose-dependently enhanced the IgE+Ag-dependent release of IL-6 and IL-13 by mast cells in concentrations as low as 0.1 ng/ml (Supplemental Figure 2). In vivo, the significant increases in lung levels of IL-6, IL-13, and IL-33 in this model occurred by wholly (for IL-6 and IL-13) or largely (for IL-33) mast cell–dependent mechanisms (Figure , A–C). Moreover, such mast cell–dependent elevations in lung levels of these cytokines required that mast cells express both FcεRIγ and IFN-γR1 (Figure , A–C). Taken together, these findings are consistent with the hypothesis that elevations of lung levels of IFN-γ are mechanistically upstream of mast cells in this model and that costimulation of mast cells with IFN-γ (via IFN-γR) and via FcεRIγ results in increased production of IL-6, IL-13, and IL-33, by mast cells (especially for IL-6 and IL-13) and/or other cells that produce these cytokines in a mast cell–dependent manner.
Most of the other features of the model that we analyzed — including striking mast cell–dependent increases in airway responses to antigen or airway hyperreactivity to methacholine (Figure ), tissue neutrophils and eosinophils (Figure , K and L, respectively) and BAL fluid leukocytes (Figure ), and lung levels of a panel of chemokines (Supplemental Figure 6) — were affected equally (and, in general, markedly diminished) by a lack of either mast cell IFN-γR or mast cell FcεRIγ (Supplemental Table 1). These results indicate that signaling via both IFN-γR and FcεRIγ is required for mast cells to perform the functions important for the development of these mast cell–dependent features of the model. However, some of the in vivo effects of mast cell activation via IFN-γR or FcεRIγ are distinct. The increase in lung mast cells in this model is critically dependent on mast cell FcεRIγ, as revealed both in this (Figure M) and in our prior study (29
), but this expansion of lung mast cells occurs independently of mast cell IFN-γR (Figure M). Because mast cells that lack FcεRIγ exhibit ablation of signaling through FcεRIγ and, as a consequence of that, are significantly impaired in their ability to expand in numbers in the lungs in this model of chronic allergic inflammation, changes in the pathology that are affected by the lack of mast cell expression of FcεRIγ might reflect the lack of FcεRIγ-dependent signaling in those mast cells that are present in such animals, the reduced numbers of lung mast cells in these mice, or both.
Regarding features of lung remodeling, mast cells lacking either IFN-γR or FcεRIγ were equally and markedly impaired in their ability to increase levels of lung collagen (Figure F) and TIMP-1 protein (Figure H). However, the marked mast cell–dependent increase in lung levels of integrin α7 protein in this model was completely ablated if mast cells lacked IFN-γR but only modestly, albeit significantly, reduced if mast cells lacked FcεRIγ (Figure I). Similarly, the marked and apparently entirely mast cell–dependent increase in lung levels of MARCO protein in this model required that mast cells express IFN-γR but not FcεRIγ (Figure D). By contrast, two other markers of an alternatively activated macrophage response, substantially increased lung levels of arginase-1 protein and Saa3 mRNA, were also induced in a largely or entirely mast cell–dependent manner, but in this case the effect was either ablated (in the case of arginase-1 protein; Figure E) or markedly reduced (in the case of Saa3 mRNA; Figure F) if mast cells lacked either IFN-γR or FcεRIγ. Finally, our data indicate that one important feature of the model, the development of large numbers of intraepithelial goblet cells, is entirely mast cell dependent but that this function can be largely sustained by mast cells lacking either IFN-γR or FcεRIγ (Figure G).
Our results support the conclusion that optimal expression of many critical mast cell functions in this model requires costimulation of the cells via both IFN-γ/IFN-γR and FcεRIγ, whereas other mast cell functions can be influenced by each receptor independently of the other. We do not know why costimulation of mast cells with IFN-γ is important for the development of so many, but not all, of the features of this model of chronic allergic inflammation. Nor can our mast cell engraftment approach permit one to determine whether particular “mast cell–dependent” features of the model reflect direct actions of mast cells (such as effects of mast cell–derived mediators on leukocytes or structural cells) or indirect effects (such as those mediated by other cells of innate or acquired immunity that are recruited to, and/or influenced in, the lungs in a mast cell–dependent manner). However, our study clearly establishes that mast cell responsiveness to IFN-γ, a cytokine classically regarded as a marker of a Th1 response, is as critical for the development of many features of the inflammation, tissue remodeling, and airway functional changes observed in this model of chronic allergic inflammation of the airways as is mast cell expression of FcεRIγ. Moreover, our in vitro findings show that even low concentrations of IFN-γ can enhance IgE+Ag-dependent release of cytokines by mast cells (Figure and Supplemental Figure 2), suggesting that this mechanism may influence IgE-dependent mast cell functions in many settings, not just those associated with marked elevations of IFN-γ.
Others have shown that various populations of rodent and human mast cells can express IFN-γR or respond to IFN-γ in vitro (31
), but the importance of mast cell expression of IFN-γR in vivo was not clear. The unexpected identification of an IFN-γ/mast cell axis in this mouse model of airway allergic inflammation challenges the notion that the mast cell’s function in this and perhaps other models of chronic allergic inflammation is entirely or predominantly under the control of the Th2 cell response. Moreover, treatment with a single injection of a neutralizing antibody to IFN-γ, administered to mice 3 hours after the last OVA challenge, reduced the magnitude of both the Penh response and the increases in BAL fluid leukocytes induced by antigen challenge (Supplemental Figure 1), findings indicating that IFN-γ represents a therapeutic target in this mouse model.
Are these findings relevant to human asthma? One always must be cautious in attempting to extrapolate findings from any animal model of a disease to its human counterpart, and the relevance of our observations in mice to asthma in humans remains to be determined. For example, while it is well established that the phenotypic features of mast cells can vary based on species, anatomical site, and exposure to cytokines and other factors in the cells’ microenvironments (57
), little is known about the regulation of the surface expression of IFN-γR by mast cells, whether in the airways or in any other site. Moreover, the relationship of our mouse model of chronic allergic inflammation of the airways to particular clinical subtypes of human asthma is not clear. However, as a first step toward addressing the relationship of our mouse model to human asthma, we used gene set enrichment analysis (GSEA) (59
) to compare the set of genes that Laprise et al. (60
) reported were upregulated in the bronchial biopsy specimens from 4 allergic asthmatic subjects with mild asthma (compared with those from 4 control subjects without asthma) and those genes that were upregulated in our mouse model in OVA-sensitized and -challenged (vs. PBS-treated control) mice. We detected significant (i.e., P
< 0.05) similarity between genes upregulated in the human asthma specimens compared with those upregulated in our model in WT mice (P
~ 0.021) or WT mast cell–engrafted KitW-sh/W-sh
~ 0.020), but not in identically sensitized and challenged mast cell–deficient KitW-sh/W-sh
mice or in KitW-sh/W-sh
mice engrafted with Ifngr1–/–
mast cells (Supplemental Figures 7–12 and Supplemental Tables 2 and 3). While these findings are intriguing, it will be of interest to repeat such GSEA studies using data sets derived from additional subsets of asthma subjects, including those with increased numbers of airway neutrophils and/or increased levels of IFN-γ in the lung, when data of this type become available. Such work, together with other studies in human subjects, will be important in the attempt to decide whether IFN-γ merits investigation as a therapeutic target for reducing the pathology characteristic of IgE-associated allergic inflammation in asthma and perhaps other settings.