AHR, a hallmark of asthma and cause of asthma morbidity, is induced by IL-4 and IL-13 in mice and associated with the same cytokines in humans. Because IL-4/IL-13R expression is ubiquitous, AHR could, in theory, result from IL-4/IL-13 effects on almost any cell type. Stimulation of lymphocytes, macrophages, mast cells, basophils, or dendritic cells, for example, might induce production of vasoactive mediators that promote AHR through their effects on epithelial cells, neurons, or smooth muscle cells or induce production of chemokines that attract inflammatory cells whose activities induce AHR. AHR-inducing effects of IL-4/IL-13 might also be direct. IL-4R stimulation of epithelial cells, which induces AHR in mice (Kuperman et al., 2002
), dramatically changes the expression of multiple epithelial cell genes and induces airway goblet cell hyperplasia and increased mucus production, which could contribute to AHR by narrowing airways. IL-4R stimulation of neurons might increase production and/or facilitate release of acetylcholine, which induces smooth muscle contraction (Goldhill et al., 1997
). IL-4R stimulation of smooth muscle cells might increase their contractility and/or stimulate them to proliferate or hypertrophy (Wills-Karp, 1997
; Laporte et al., 2001
; Shore and Moore, 2002
; Shore, 2004a
Although a previous study demonstrated that direct IL-13 activation of Stat6 in airway epithelial cells induces AHR in mice (Kuperman et al., 2002
) and AHR has been reported to depend on the products of epithelial cell–expressed genes that are induced by IL-4/IL-13 (Howarth et al., 1995
; Goto et al., 2000
; Poynter et al., 2002
; Zhu et al., 2004
; Kawada et al., 2007
), a study that demonstrated that AHR still develops in allergen-inoculated mice that have selectively deleted IL-4Rα from airway epithelium (Kuperman et al., 2005
) indicates that IL-4/IL-13 effects on other cell types must also be capable of inducing AHR. This justified consideration of smooth muscle, a cell type implicated in AHR by in vitro human and animal studies. These studies demonstrated that IL-13 increases the affinity of intestinal smooth muscle for acetylcholine (Kellner et al., 2007
), that IL-4 and IL-13 increase intestinal smooth muscle responses to nerve stimulation through a Stat6-dependent mechanism (Zhao et al., 2003
), that IL-13 acts in vitro to increase airway smooth muscle contractility and proliferation and stimulate smooth muscle production of the chemokine eotaxin (Shore, 2004b
), and that IL-13 induces calcium transients in cultured mouse airway smooth muscle cells and augments the calcium and contractile responses of these cells to leukotriene D4 (Eum et al., 2005
). However, until now, there has been no evidence that direct effects of IL-4/IL-13 on smooth muscle contribute to AHR development in vivo.
Our results indicate that the direct effects of these cytokines on smooth muscle contribute to AHR and are likely to be biologically important. In contrast to IL-4Rα–deficient mice, transgenic mice that express IL-4R only on smooth muscle cells and at approximately the same level as mice that have a single allele of the wild-type IL-4Rα gene developed AHR after i.t. inoculation with IL-4 or IL-13. This was observed despite the lack of development of airway eosinophilia or goblet cell hyperplasia in the smooth muscle–only mice. Consistent results were observed after i.t. inoculation of dust mite allergen instead of IL-4 or IL-13. However, allergen administration induced less AHR in smooth muscle–only mice than in wild-type mice, probably because endogenous IL-4 and IL-13 production was limited in the former strain by the absence of IL-4Rα on lymphoid cells. This conclusion is supported by the more impressive development of AHR in allergen-immunized smooth muscle–only mice that were inoculated with allergen-primed lymph node cells from wild-type mice before their own inoculation with allergen.
Our results also suggest that smooth muscle IL-4R signaling increases AHR by increasing the expression of several genes that likely act in parallel to promote smooth muscle differentiation, proliferation, migration, and contractility and to promote the accumulation of extracellular matrix, which enhances smooth muscle contractility (). Although the extent to which these changes in gene expression affect smooth muscle protein expression and the exact mechanisms by which changes in the expression of these genes promote AHR remain unknown, our observations provide useful leads for further investigation as well as potential targets for asthma therapy. It is also likely that IL-4R signaling increases or decreases the expression of additional genes in smooth muscle but that these changes are not seen in a whole lung analysis because they are obscured by IL-4R–independent expression of the same genes in other cell types.
Although selective smooth muscle IL-4R expression is sufficient for AHR induction by allergen or cytokine inhalation, it is not required. This is consistent with the previous demonstration of AHR in transgenic mice that overexpress IL-13 in their lungs and express Stat6 only in airway epithelial cells (Kuperman et al., 2002
). Most likely, AHR can be induced in mice either by airway narrowing caused by epithelial cell enlargement and mucus production, despite normal smooth muscle contractility, or by increased smooth muscle contractility, despite normal airway diameter. Alternatively, it is possible that IL-4/IL-13 effects on airway epithelium indirectly increase smooth muscle contractility.
The significance of AHR in rodents and the best way to determine it has been debated (Finkelman, 2008
). There are considerable differences between murine and human lung structure, and it is impossible to characterize maximal voluntary inhalations and exhalations in rodents (these are used to measure airway responsiveness in people). Noninvasive techniques performed with awake mice, such as barometric plethysmography, avoid changes in airway physiology that are induced by anesthesia and intubation but are indirect and can be influenced by upper airway inflammation and factors other than airway diameter and distensibility. Invasive techniques allow more direct measurement of airway characteristics, but the results they provide may be distorted by instrumentation and the use of anesthetics and paralytics. We approached the issues raised by these considerations by using three different techniques to measure responsiveness to cholinergic stimulation. Despite their differences, each technique demonstrated that exogenous and endogenously produced IL-4/IL-13 induce AHR in mice that express IL-4Rα only on smooth muscle cells but not in fully IL-4Rα–deficient mice.
This still leaves open the issue of whether our results with mouse models are applicable to human asthma. Although it is impossible to resolve this issue without performing human studies that are unethical and probably technically impossible, similar in vitro observations made with IL-4/IL-13–stimulated rodent and human smooth muscle cells (Shore, 2004a
) make it seem likely that the in vivo mouse observations will also extend to humans. Indeed, the much greater airway diameter in humans than in mice and the development of considerable smooth muscle hyperplasia by human asthmatics (Ebina et al., 1993
) but not by mice in our study make it likely that effects of IL-4/IL-13 on smooth muscle contribute more to AHR in human asthma than in our mouse models. In this regard, our model in which AHR depends directly on IL-4/IL-13 effects on smooth muscle should be useful for evaluating potential therapeutics for human asthma.
Two other issues raised by our observations remain unresolved and are attractive subjects for future study. First, why do IL-4 and IL-13 differ in their relative effects on airway responsiveness in IL-4Rα+/−
mice? One possible explanation is that IL-4 but not IL-13 stimulates NK and T cells to produce IFN-γ and IL-10 (Finkelman et al., 2005
; Morris et al., 2006
; unpublished data), cytokines which can suppress airway responsiveness. Failure of IL-4 to induce these cytokines in IL-4Rα−/−
mice might account for the greater IL-4 stimulation of AHR in IL-4Rα−/−
mice than IL-4Rα+/−
or wild-type mice. The failure of IL-13 to induce IFN-γ or IL-10 expression would account for the greater effect of IL-13 on AHR in wild-type mice, in which it directly affects epithelial and inflammatory cells, than on IL-4Rα−/−
mice, in which it only directly affects smooth muscle.
Second, do IL-4/IL-13 effects on smooth muscle and epithelial cells totally account for AHR induction by these cytokines, or might effects of these cytokines on other non–bone marrow–derived cell types (neurons, fibroblasts, and vascular endothelium) also contribute? Mice that express a floxed IL-4Rα gene and both smooth muscle– and epithelial cell–specific Cre are currently being bred to address this question.