Although originally cloned and characterized as a T-cell growth factor (30
), the precise role of IL-9 in inflammation and autoimmune diseases remained unclear. Although described as a pleiotropic cytokine, one of the most important functions for IL-9 appears to be a major role in mediating MC survival and function. IL-9R is strongly expressed on pluripotential precursors in bone marrow, committed immature, and mature MCs (30
) (). Furthermore, IL-9 has been shown to induce proliferation and survival of MCs (10
). Likewise, transgenic mouse studies have implicated a role for this cytokine in pulmonary mastocytosis (13
), and in an acute mouse model of asthma, IL-9 has been shown to regulate allergen-induced MC progenitor recruitment to the lung (21
). Notably, the airways of human subjects with chronic asthma exhibit features of ongoing mucosal MC activation, and in some studies this correlates with disease severity (31
). Furthermore, MC localization within ASM bundles is believed to contribute to the disordered airway physiology associated with this disease (33
). Thus, an impact on MC function may have therapeutic potential in all aspects of chronic asthma. In this study, we provide direct evidence for a pathogenic role of IL-9 in mechanisms that govern pulmonary MC numbers after allergen challenge and chronic remodeling of the airways that is associated with a decline in lung function.
We found that short-term IL-9 neutralization, during an acute allergen provocation, had no effect on resident lung MC numbers, albeit it did significantly reduce mucosal MC activation. Similarly, we observed no effect of IL-9 blockade on lung function during the acute phase, despite significant reductions in mMCP-1, IL-13 levels, and mucus production. However, the presence of functional and mature lung MCs in these mice would explain the lack of effect of short-term IL-9 neutralization on AHR and is consistent with the observation that IL-9 deficiency did not confer protection in a similar acute model (9
). In contrast, extended inhibition of IL-9 during prolonged allergen exposure markedly diminished mucosal MC activation and dramatically reduced the number of MCs in the lung. These data suggest that IL-9 regulates the recruitment of MC progenitors to the lung during chronic inflammation and support a role for IL-9 in MC hyperplasia (13
). Interestingly, we observed lower mMCP-1 levels in chronically, versus acutely, challenged mice even though MC numbers were significantly higher than animals undergoing acute exposure. The reason for this discrepancy is unclear given that mMCP-1 levels have previously been shown to correlate with MC numbers in tissue (34
). The acute and chronic models used in our study markedly differ in the allergen challenge regime and are thus difficult to compare. However, we have previously observed that consecutive, short-term MC activation (i.e., 3–4 d) results in a cumulative and significant enhancement of mMCP-1 levels in the absence of MC hyperplasia (data not shown). Importantly, the dramatic reduction in MC numbers correlated with a profound effect on the development of airway remodeling and subsequent changes in lung function after chronic allergen exposure. These changes included a reduction in mucus hyperplasia and subepithelial fibrosis as well as attenuation in airway smooth muscle mass and an overall improvement in lung function. Conclusively, these data predict that prolonged IL-9 inhibition is required to diminish MC activity and mediate significant beneficial effects in the lung.
The correlation between a reduction in MC numbers and decreased airway remodeling, after IL-9 inhibition, is consistent with reports that MC-deficient mice demonstrate significantly attenuated fibrosis and inflammation after silica (35
), ozone (36
), or bleomycin injury (37
). Additional evidence stems from reports indicating the abundance of MCs in diseases associated with fibrosis (38
). Of relevance was the recent report that MC-deficient mice have reduced structural changes after chronic allergen exposure (15
). We and others have previously identified a critical role for eosinophils in this process (22
). Moreover, we have observed that eosinophil-deficient and CCR3−/−
mice, the latter in which eosinophil trafficking is severely impaired to the lung, have significantly attenuated levels of IL-9 in the lung as well as reduced fibrosis after prolonged allergen exposure (A. A. Humbles and coworkers, unpublished observations). It is tempting to speculate that, in addition to lymphocytes, eosinophils are a major source of IL-9 in the lung and are indirectly contributing to fibrosis via an IL-9–MC dependent manner.
Of significance was our finding that anti–IL-9 pretreatment also protected mice from chronic HDM-induced remodeling. HDM is a clinically relevant aeroallergen and is, in contrast to OVA, a complex antigen with proteolytic activity. These allergens are believed to elicit very different immune responses, which is supported by recent data identifying opposing roles for TGFβ blockade in both of these models (2
). Remarkably, we found a protective and similar phenotype with anti–IL-9 therapy in both OVA- and HDM-driven responses, suggesting that an IL-9–MC axis is central to allergen-induced chronic inflammation.
Importantly, attenuated airway remodeling by anti–IL-9 antibody was also associated with a decrease in lung expression of the growth factors TGFβ, VEGF, and FGF-2. TGFβ has been reported to play a critical role in airway remodeling and is a key player in the tissue repair response (2
). Similarly, overexpression of VEGF in mouse lung provokes not only a vascular phenotype but also marked subepithelial fibrosis, mucus hyperplasia, and AHR (41
), and FGF-2 is up-regulated in the airways of subjects with asthma undergoing segmental challenge (42
) as well as in a nonhuman primate model of asthma wherein expression was associated with remodeling (43
). Moreover, MC activation/proteases have been linked to the release of many of these growth factors, including FGF-2 release after allergen challenge (44
). Our analysis of VEGF and FGF-2 expression in lung tissue from subjects with mild asthma clearly implicates MC as a potential and relevant source of these growth factors in vivo
. Moreover, we found increased numbers of VEGF-expressing MCs in subjects with asthma compared with control subjects. These data are consistent with a recent study demonstrating that tryptase+
) are increased in asthma and correlate with the number of VEGF+
cells within the vascular area of the bronchial mucosa (48
). Thus, the combined reduction of TGFβ, VEGF, and FGF-2 during chronic inflammation provides a possible mechanism whereby IL-9 blockade could prevent fibrosis and potentially alleviate asthma symptoms.
Airway remodeling is believed to be a determinant of AHR and associated with the accelerated loss of lung function that is observed over time in chronic asthma. Structural changes, including subepithelial fibrosis, are believed to contribute to the thickened airway walls and progression of airway dysfunction in subjects with asthma by potentiating airway narrowing (49
). Furthermore, a recent study in subjects with asthma has shown that the persistence of AHR is associated with airway remodeling and not dependent on sustained inflammatory cell recruitment (50
). Notably, this is consistent with the observation that AHR persists in more severe disease despite prolonged treatment with inhaled corticosteroids (51
). Interestingly, we found that the effects of anti–IL-9 therapy on changes in lung physiology were very specific. The observed increases in total lung and smaller airways elastance were not affected by IL-9 blockade. Changes in these specific airway parameters are believed to be associated with inflammation (28
) and therefore concur with anti–IL-9 treatment having no effect on leukocyte accumulation in the lung. In contrast, increases in total airway resistance (R) and resistance of the smaller airways (G) were completely attenuated by IL-9 blockade, implying that airway remodeling significantly contributes to changes in the resistance of the peripheral airways. Similar findings describing a correlation between subepithelial fibrosis and progression of AHR have also been reported in mice after chronic HDM exposure (52
). Notably, and in agreement with our observations, a recent study in allergic cynomolgus monkeys suggests that treatments aimed at improving smaller airways dysfunction might be more effective in asthma (53
In conclusion, our studies demonstrate a critical role for IL-9 in regulating MC numbers in the airways after allergen challenge. Prolonged IL-9 neutralization results in a profound inhibition of MC numbers, airway remodeling, and AHR after chronic allergen exposure. Furthermore, we correlate a reduction in MCs with decreased expression of TGFβ, VEGF, and FGF-2 in the lung. Our data suggest a novel IL-9–MC axis that regulates airway fibrosis and is further supported by studies in human asthmatic airways, demonstrating that lung MCs express the IL-9 receptor and can exhibit colocalization with VEGF and FGF-2 expression. These data suggest that IL-9 may play a critical role in allergic inflammation as well as other respiratory fibrotic-related diseases, such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease. Furthermore, these studies highlight the MC-enhancing activity of IL-9, which is unique among the Th2 cytokines, and predict that targeting IL-9 could provide a novel approach for the treatment of asthma.