Chronic intranasal administration of mould spores or extracts to unsensitized mice leads to lung allergic inflammation, hyper-reactivity and remodelling

Abstract

Allergic asthma is a serious multifaceted disease characterized by eosinophil-rich airway inflammation, airway hyperreactivity and airway wall modifications known as remodelling. We previously demonstrated that the spores of two allergenic moulds, Alternaria alternata and Cladosporium herbarum, were potent inducers of immunoglobulin E (IgE) production. Moreover, mice sensitized by two intraperitoneal injections before intranasal challenge with A. alternata or C. herbarum spores developed an allergic lung inflammation and hyperreactivity. Here we report on the effect of chronic intranasal administration of C. herbarum spores or A. alternata extracts to unsensitized BALB/c mice. Our results demonstrate that this chronic treatment led to an increase of total serum IgE and the appearance of specific IgE and IgG1. Total cell number in bronchoalveolar lavage fluid from treated mice was highly increased compared to phosphate-buffered-saline-treated mice because of the accumulation of macrophages, neutrophils, lymphocytes and eosinophils. Airway hyperreactivity appeared after 3 weeks (extract) and 7 weeks (spores) and was maintained during the whole treatment. Increased interleukin-13 mRNA expression in the lungs and T helper type 2 cytokines (interleukin-4, -5, -6 and -13) and transforming growth factor-beta secretion in bronchoalveolar lavage fluid were also observed. Lung hydroxyproline and fibronectin contents indicated increased fibrosis in mice treated with mould allergen. These observations were confirmed by histological analysis demonstrating airway wall remodelling and strong mucus production. These observations show that this model, using chronic intranasal administration of relevant particulate allergens, is an interesting tool for the study of mechanisms leading to allergic pulmonary diseases and lung remodelling.

Figure 3

Chronic i.n. instillations of Alternaria alternata extract do not influence the T-cell response induced by immunization with a model antigen. BALB/c mice were treated chronically twice a week i.n. with PBS or with 5 μg A. alternata extract during the entire experiment. Three weeks after the beginning of instillations, mice were immunized i.p. twice with 50 μg KLH emulsified in CFA or in alum. Mice were boosted 2 weeks later with 50 μg KLH emulsified in IFA or in alum, respectively. One week after the KLH boost, spleen cells were restimulated in vitro for 3 days with medium only or with medium supplemented with KLH or PWM. The concentrations of IFN-γ, IL-4 and IL-13 in supernatants were measured by ELISA.

Figure 1

Polyclonal IgE synthesis induced by chronic i.n. instillations of Alternaria alternata extract and Cladosporium herbarum spores. BALB/c mice were chronically administered i.n. with PBS, with 5 μg A. alternata extract (Alt Ext) or with 2 × 10⁵ spores of C. herbarum (Clado Sp) for 10 weeks. Twenty-four hours after the last instillation mice were bled and total IgE levels were determined. Significant differences are shown between values from extract or spores treated mice versus PBS-treated mice; **P < 0·05, ***P < 0·01.

Figure 2

Specific IgE and IgG1 antibody synthesis induced by chronic i.n. instillations of Alternaria alternata extract and Cladosporium herbarum spores. BALB/c mice were given PBS, 5 μg A. alternata extract or 2 × 10⁵ spores of C. herbarum chronically i.n. for 10 weeks. Twenty-four hours after the last instillation mice were bled and specific IgE and IgG1 were determined. Data represent the mean ± SEM of the A₄₅₀ values of five to seven mice per group.

Figure 4

Inflammatory cell recruitment in the lungs of Alternaria alternata extract and Cladosporium herbarum spore-treated mice. BALB/c mice were chronically treated i.n. with PBS, with 5 μg A. alternata extract (Alt Ext) or 2 × 10⁵ spores of C. herbarum (Clado Sp) for 10 weeks. (a) BALF was collected 24 hr after the last instillation and the total cell number was determined. (b) Differential cell count of the inflammatory subpopulation in BALF 24 hr after the last instillation. Data represent the mean ± SD of five or six individual mice per group.

Figure 5

Mice chronically treated with Alternaria alternata extract or Cladosporium herbarum spores develop a sustained AHR to inhaled methacholine. BALB/c mice were administered PBS, 5 μg A. alternata extract (Alt Ext) or 2 × 10⁵ spores of C. herbarum (Clado Sp) chronically i.n. for 10 weeks. P_enh were measured by whole body plethysmography after 3, 5, 7, 9 and 11 weeks. Results are expressed as means ± SEM of five to seven mice per group.

Figure 6

(a) Chronic instillations of Alternaria alternata extracts induce the production of IL-13 mRNA in the lungs. BALB/c mice were left untreated (NT), or were chronically treated i.n. with PBS or with 5 μg A. alternata extract (Alt Ext) for 10 weeks. At the indicated time-points (24 hr after the last instillations) total RNA was extracted from the lungs and transcriptional levels of IL-13 were analysed by quantitative RT-PCR. Data represent the mean ± SD of triplicate assays. (b) Chronic instillations of A. alternata extracts or C. herbarum spores induce an accumulation of IL-13 into the BALF. BALB/c mice were treated chronically i.n. with instillations of PBS, 5 μg Alternaria alternata extract (Alt Ext) or 2 × 10⁵ spores of Cladosporium herbarum (Clad Sp) for 10 weeks. Twenty-four hours after the last instillation BALF were collected and IL-13 concentrations in BALF were analysed by ELISA. Significant differences between values from extract-treated or spore-treated mice versus PBS-treated mice are shown; ***P < 0·01. There was no significant difference between extract-treated versus spore-treated mice.

Figure 7

Quantification of IL-4, IL-5, IL-6, IL-10 and TGF-β concentrations in the BALF of mice chronically treated with instillations of Alternaria alternata extract. BALB/c mice were treated chronically i.n. with PBS or with 5 μg A. alternata extract (Alt Ext) during 10 weeks. BALF were collected 24 hr after the last instillation and IL-4, IL-5, IL-6, IL-10 and TGF-β concentrations were determined by ELISA. Significant differences between values from extract-treated mice versus PBS-treated mice for IL-4, IL-5, IL-6 and TGF-β are shown; **P < 0·05, ***P < 0·01. Non-significant (NS) for IL-10.

Figure 8

Levels of hydroxyproline in lung homogenates, soluble collagen in supernatents of lung homogenates and fibronectin in BALF of BALB/c mice were administered instillations of PBS or Alternaria alternata extracts (Alt Ext) for 10 weeks. **P < 0·05.

Figure 9

Histopathological changes in the lungs of mice chronically instilled with Alternaria alternata and Cladosporium herbarum. BALB/c mice were given i.n. instillations PBS (left column), A. alternata extracts (middle column) or C. herbarum (right colum) for 10 weeks. The first two rows show lung sections stained with Masson trichrome (a–f). The third row shows lung sections stained with periodic acid Schiff (g–i) and the last row shows lung sections stained with haematoxylin–eosin–safran (j–l).