We have shown that specific reduction of eosinophil numbers in the bronchial mucosa in mild asthmatic subjects using an anti–IL-5 monoclonal antibody (mepolizumab) is associated with a significant decrease in the expression of the ECM proteins tenascin and lumican in the reticular basement membrane of the airway mucosa. Furthermore, these changes were associated with significant reductions in TGF-β1 expression by airway eosinophils. These data support a novel role for the eosinophil and suggest a link between eosinophilic infiltration and airway remodeling in asthma.
We confirmed previous reports that the thickness of tenascin and lumican within the RBM was significantly greater in asthmatic individuals than in normal subjects (6
). With tenascin, mepolizumab reduced RBM expression to values observed in nonasthmatic, nonatopic individuals. We also showed that tenascin thickness, density, and total expression correlated with bronchial mucosal eosinophil numbers. Our findings are consistent with those previously reported in animal studies. In an IL-5 gene knockout mouse model of airways remodeling, abrogation of eosinophil infiltration following repeated allergen challenge was associated with an absence of structural changes, including fibronectin production and mucus gland hyperplasia observed in the WT controls (35
). In a separate animal model of atopic asthma, prophylactic treatment of ovalbumin-sensitized BALB/c mice with anti–IL-5 significantly reduced the subepithelial fibrosis produced by repeated allergen challenge (36
). However, these authors did not report the effects of anti–IL-5 treatment on established fibrosis.
Compared with lumican or procollagen III, there was a particularly close correlation between baseline eosinophils and tenascin expression and a more marked decrease in tenascin expression in response to anti–IL-5 therapy. With procollagen III, mepolizumab did not significantly decrease the expression of this ECM protein, although the changes observed were significantly different than with placebo. This suggests a particularly strong association between eosinophils and tenascin deposition, as previously shown in allergen-induced tenascin formation in atopic skin (24
). It is possible that longer periods of treatment may be required to reverse procollagen and lumican expression to the same degree as that observed with tenascin. Evidence from studies investigating the effect of inhaled corticosteroids on RBM thickness indicates that although short-term treatment is associated with a significant decrease in tenascin thickness in the RBM (5
), longer-term studies are required to show a reduction in collagen III thickness (37
). In the placebo group, there were nonsignificant increases, over the study period, in eosinophils, tenascin, and procollagen, raising a further possibility that these changes reflect the natural history of untreated mild atopic asthma and that anti–IL-5 may have prevented such an increase.
The value for tenascin expression before placebo was lower than before mepolizumab, albeit not significantly lower. Because the treatment groups were otherwise well matched both in terms of clinical and histopathological features and were selected randomly, this difference is most likely the consequence of the relatively small numbers and the large standard deviation of the baseline values for tenascin.
TGF-β1 is a potent differentiation factor for the formation of myofibroblasts in vivo and in vitro (39
) and has been shown to upregulate the expression by fibroblasts of an array of ECM proteins, including tenascin (41
). Consistent with previous studies (42
), we have shown that most of the TGF-β1 mRNA+
cells in the submucosa in these mild atopic asthmatics were eosinophils. Selective depletion of eosinophils with anti–IL-5 significantly reduced the number of TGF-β1 mRNA+
eosinophils in the bronchial mucosa and decreased TGF-β1 concentrations in BAL fluid. In addition, the proportion of cells expressing TGF-β1 mRNA decreased significantly in the mepolizumab-treated group. This suggests that anti–IL-5 therapy not only decreased the number of eosinophils producing TGF-β1, but also reduced the degree of activation of these cells for TGF-β1 expression. The fact that the majority of TGF-β1mRNA+
cells were eosinophils, and that levels of TGF-β1 in BALF were also reduced supports our hypothesis that eosinophils were an important source of TGF-β1 in the bronchial mucosa of asthmatics.
We did not quantify bronchial mucosal myofibroblasts in the present study. The myofibroblast marker α-smooth muscle actin is also expressed by a large number of other cells in the bronchial mucosa, and the ultrastructural identification of myofibroblasts using electron microscopy as used by Gizycki and co-workers (44
) was beyond the scope of this study. Our previous in vitro work, however, has demonstrated that coculture of eosinophils and fibroblasts is associated with myofibroblast differentiation and the generation of tenascin transcripts and protein, an effect that is inhibited by antibodies directed against TGF-β (24
). We suggest, therefore, that eosinophil-derived TGF−β1 may influence the expression of tenascin and other ECM proteins via the activation of myofibroblasts.
It is also possible that the observed effect of anti–IL-5 on matrix protein expression was mediated by the effect of the drug on IL-5 itself, rather than through the reduction in eosinophil numbers. Although IL-5 has been shown to influence airway smooth muscle contraction (45
), it is not known to directly release growth factors from smooth muscle or other cells appropriate to the remodeling changes studied. In addition, the consistent correlation between eosinophil numbers and tenascin thickness before and after mepolizumab therapy makes a direct action of IL-5 unlikely — at least for tenascin. It is equally unlikely that the changes in ECM protein expression could be explained by the action of anti–IL-5 on other cell types in the bronchial mucosa. There were no significant changes in the numbers of other cells in response to anti–IL-5. Although there was a trend towards a reduction in basophil numbers, this did not correlate with changes in ECM protein expression.
The relevance of our findings in terms of asthma pathophysiology is unclear because the effect of airways remodeling on lung function in asthma is uncertain. Deposition of ECM proteins in the airway wall may have a mechanical effect, amplifying the degree of narrowing associated with a given degree of smooth muscle contraction (46
). A number of studies have reported a correlation between the thickness of the RBM and airways hyperreactivity (AHR), FEV1
, and other markers of asthma severity (6
). We found no significant correlation between baseline PC20
, and the thickness, density, and expression of tenascin, lumican, or procollagen III. Our subjects were mild asthmatics, and the study was not powered to detect significant correlations in clinical variables such as FEV1
or AHR. Furthermore, although we observed a significant reduction in the expression of tenascin, lumican, and procollagen III in the RBM, we did not measure overall RBM thickness in these biopsies, and it is possible that eosinophil depletion was not associated with so marked a reduction in the expression of other matrix proteins such as fibronectin and types I and V collagen.
Although anti–IL-5 administration significantly reduced the numbers of tissue eosinophils in the bronchial submucosa, it did not deplete them to the same extent as that reported previously in sputum and blood (28
). GM-CSF, IL-3, and eotaxin are also involved in eosinophil homeostasis, and there is evidence that inhibition of these, in conjunction with IL-5 blockade, may be associated with more complete eosinophil depletion from the bronchial mucosa (48
). Equally, studies showing the most marked effects of corticosteroids on RBM thickness have tended to be of longer duration, and so sustained eosinophil depletion may be required to achieve a maximal effect (50
In summary, we have shown that the selective reduction of eosinophils in mild asthmatics by anti–IL-5 treatment is associated with a significant decrease in expression of ECM proteins in the airway RBM. Furthermore, this reduction was accompanied by a decrease in TGF-β1 expression by airway eosinophils. These data provide strong evidence of a causal association between eosinophils and airway remodeling in mild atopic asthma.