A Mouse Model of Chemical-Induced Asthma: Optimization - Validation - Mechanisms

Abstract

Asthma and allergy are a worldwide health problem and especially in industrialized countries their incidence has increased substantially. Allergic asthma is a chronic airway disease characterized by a reversible allergen-specific airway obstruction, airway inflammation and non-specific airway hyper-reactivity. In adults, 9-15% of asthma cases can be attributed to occupational exposures and this makes occupational asthma the most frequent work-related lung disease. Occupational asthma can be caused by high molecular weight (pollen, flour, enzymes, ) as well as low molecular weight (chemicals, metals, resins, ) agents. Diisocyanates are chemical agents, used in isolation material, varnishes, spray paints, rubbers, etc., that frequently cause occupational asthma. The research unit of Lung Toxicology has succeeded in developing a mouse model that reflects several characteristics of chemical-induced asthma. Two dermal sensitizations followed by a single airway challenge with toluene diisocyanate resultedin a specific early airway response, followed by an airway inflammationwith an influx of neutrophils and a non-specific airway hyper-reactivity. Furthermore, we found an increase in T-helper, T-cytotoxicand B-lymphocytes in the draining lymph nodes as well as an increased in vitro production of the cytokines IL-4, IFN-γ and an increase in total serum IgE. In this doctoral thesis we focused on: a/ Optimization of the mouse model b/ Validation of the mouse model c/ Unravel the mechanisms of chemical-induced asthma To optimize the mouse model, we attempted to obtain a chronic asthma modelby exposing the mice to repeated airway challenges. However,the multiple challenges did not result in a more chronic phenotype but induced instead a state of tolerance. Multiple intranasal instillations with toluene diisocyanate led to a full waning of the airway response and airway inflammation as well as a disappearance of immunologic parameters. Nevertheless, the humoral response was still increased after multiple challenges.Next, a further optimization was obtained by changing the challenge technique from intranasal instillation to oropharyngeal aspiration. Using the latter technique, wewere able to achieve a better exposure of the lower airways while bypassing the nose. Oropharyngeal aspiration appeared to be a goodalternative for intratracheal instillation without the drawbacks of thelatter. The use of this technique yielded, besides neutrophils, also an influx of eosinophils in the bronchoalveolar lavage.In alast step of the optimization we tested besides BALB/c mice, six other mouse strains in our mouse model. We concluded that BALB/c mice reproduce the characteristics of chemical-induced asthma most closely. Large differences could be found in bronchial reactivity, immunologic and humoral responses between the different mouse strains. Furthermore, no clear distinction can be made between more T-helper 1 or T-helper 2 prone mouse strains. The mouse model was validated by testing the potential dermal sensitization capacity and subsequent response to an airway challenge of toluene diamine and ammonium persulfate. We found that toluene diamine, produced when toluene diisocyanate reacts with water, is a weak dermal sensitizer and weak asthmogen and that there is no cross-reactivity between toluene diamine and toluene diisocyanate in our asthma model. Ammonium persulfate, a known cause of occupational asthma among hairdressers, appeared to be a moderate dermal sensitizer and to produce a robust response in our asthma model.These validation studies proved that our mouse model can be used to test less potent and even weak airway sensitizers. The mechanisms of chemical-induced asthma are still unclear. In an attempt to understand the mechanisms of this disease we developed an adoptive transfer protocol focusing on cellular responsesand used this to investigate the antibody-independent role of B-lymphocytes in chemical-induced asthma.The transfer of 0.5 x 106 lymphocytes obtained from auricular lymph nodes of toluene diisocyanate sensitizedmice was sufficient to produce an asthma-like response in naïve mice after an airway challenge with toluene diisocyanate.Adoptive transfer experiments with B-lymphocytes into wild type, B knock-out and SCID miceresulted in airway hyper-reactivity and airway inflammation upon challenge with toluene diisocyanate. These data suggest an important antibody-independent role for B-lymphocytes in chemical-induced asthma. The optimization and validation of our mouse model of chemical-induced asthma has led to a better model that, in the future, could be used to identify potential asthmogens and to understand the mechanisms of action. A possibly important role for B-lymphocytes can bring a new insight into the mechanisms of non-atopic asthma.status: publishe