Prolonged allergen challenge in mice leads to persistent airway remodelling

Abstract

Background: Inflammatory infiltrates, airway hyper-responsiveness, goblet cell hyperplasia and subepithelial thickening are characteristic of chronic asthma. Current animal models of allergen-induced airway inflammation generally concentrate on the acute inflammation following allergen exposure and fail to mimic all of these features.

Objective: The aim of this study was to use a murine model of prolonged allergen-induced airway inflammation in order to characterize the cells and molecules involved in the ensuing airway remodelling. Moreover, we investigated whether remodelling persists in the absence of continued allergen challenge.

Methods: Acute pulmonary eosinophilia and airways hyper-reactivity were induced after six serial allergen challenges in sensitized mice (acute phase). Mice were subsequently challenged three times a week with ovalbumin (OVA) (chronic phase) up to day 55. To investigate the persistence of pathology, one group of mice were left for another 4 weeks without further allergen challenge (day 80).

Results: The extended OVA challenge protocol caused significant airway remodelling, which was absent in the acute phase. Specifically, remodelling was characterized by deposition of collagen as well as airway smooth muscle and goblet cell hyperplasia. Importantly, these airway changes, together with tissue eosinophilia were sustained in the absence of further allergen challenge. Examination of cytokines revealed a dramatic up-regulation of IL-4 and tumour growth factor-beta1 during the chronic phase. Interestingly, while IL-4 levels were significantly increased during the chronic phase, levels of IL-13 fell. Levels of the Th1-associated cytokine IFN-gamma also increased during the chronic phase.

Conclusion: In conclusion, we have demonstrated that prolonged allergen challenge results in persistent airway wall remodelling.

Fig. 1

Effect of prolonged ovalbumin (OVA) challenge on lung tissue pathology. Representative photomicrographs of (a) haematoxylin/eosin (H&E)- (arrows depict eosinophils), (b) periodic acid-Schiff (PAS)- and (c) Martius scarlet blue (MSB)-stained lung sections from alum control mice, OVA challenged mice at day 24 (acute phase) and day 55 (chronic phase), and mice that were no longer exposed to OVA from day 55 and killed at day 80 are shown (original magnification × 40). Data is representative of n = 9–17/group/time point.

Fig. 2

Effect of prolonged ovalbumin (OVA) challenge on matrix deposition. (a) Image analysis of Martius scarlet blue-stained lung sections from mice treated with alum or OVA during acute and prolonged allergen challenge. Random measurements from the basement membrane into the submucosa (10 measurements of 20 μm in length) were taken and the mean density calculated from four bronchioles per mouse, n = 4 mice per group. (b) Levels of total collagen were measured in lung homogenate samples taken throughout the model. Data is expressed as mean ± SEM, n = 4/group; *P<0.05 in comparison with alum control mice.

Fig. 3

Effect of prolonged ovalbumin (OVA) challenge on airway smooth muscle cell proliferation. (a) Representative photomicrographs of paraffin-embedded lung sections from day 24 acute OVA challenged mice and mice from day 55 during prolonged allergen challenge, stained with antibodies against proliferating cell nuclear antigen (PCNA) (original magnification × 40. PCNA-positive cells stain brown). Data is representative of n = 4/ group/time point. Ig control sections were negative. (b) Total and proliferating airway smooth muscle cell counts from lung tissue sections taken from alumand OVA-treated mice throughout the model. Data is expressed as mean ± SEM, n = 4/group; *P<0.05 in comparison with alum control mice.

Fig. 4

Effect of prolonged ovalbumin (OVA) challenge on cytokine levels. IL-4 (a, b), IL-5 (c, d), IL-13 (e, f) and IFN-γ (g, h) levels were measured in bronchoalveolar lavage (left panel) and lung tissue homogenates (right panel) by ELISA in samples from alum controls, acute and chronic phase killed 24 h after the final OVA challenge and mice from day 80. Data are expressed as mean ± SEM; n = 4–17/group and *P<0.05 in comparison with alum control mice. ND, non-detectable.

Fig. 5

Effect of prolonged ovalbumin (OVA) challenge on levels of TGF-β. (a) Representative photomicrographs of paraffin-embedded lung sections from alum controls (i), OVA challenged mice at day 24 (ii), day 55 (iii) and day 80 (iv), stained with antibody against TGF-β1. Data is representative of n = 4/group/ time point. Ig controls were negative (original magnification × 40). (b) Levels of active TGF-β1 were measured in lung homogenates by ELISA taken from acute and chronic OVA challenged mice. Data is expressed as mean ± SEM, n = 5–10/group; *P<0.05 in comparison with alum control mice. (c) Levels of eotaxin were measured in lung tissue homogenates by ELISA from mice during prolonged allergen challenge. Data is expressed as mean ± SEM, n = 4/group; *P<0.05 in comparison with alum control mice.

Fig. 6

Effect of prolonged allergen challenge on airway hyper-responsiveness. Airway hyper-responsiveness was measured 24 h after each final OVA challenge on days 24 (acute phase), 35 and 55, and in the absence of further allergen exposure on day 80 using a Buxco system where mice were exposed to increasing concentrations of methacholine (3–100 mg/mL). Values are expressed as mean ± SEM, n = 8–12/group/time point. *P<0.05 in comparison with alum control mice.