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

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

Not All Mouse Strains Respond Equal in a Model of Chemical-Induced Asthma

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The role of mast cells, IL-13 and TRP channels in a mouse model of chemical-induced airway hyperresponsiveness

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Is toluene diamine a sensitizer and is there cross-reactivity between toluene diamine and toluene diisocyanate?

Toluene diamine (TDA) is formed when toluene diisocyanate (TDI), a potent sensitizer, comes in contact with an aqueous environment. The sensitizing capacity of TDA and the cross-reactivity between TDI and TDA are unknown. TDA (5-25%) and TDI (0.3%), dissolved in acetone/olive oil (AOO) (4:1) were tested in the mouse local lymph node assay (LLNA). To determine the capacity of TDA to elicit an asthmatic response and to determine the cross-reaction with TDI, a locally developed experimental mouse model of chemical-induced asthma was used. On days 1 and 8, BALB/c mice received 20 µl of TDI (0.3%), TDA (20%), or AOO (4:1) on each ear. On day 15, they received an intranasal instillation of TDI (0.1%), TDA (0.5%) or AOO (3:2). The EC(3) of TDA in the LLNA is 19%. In the model of chemical-induced asthma, TDI induced a ventilatory response [increased Penh after challenge; increased airway hyperreactivity (AHR)], inflammatory changes (bronchoalveolar lavage neutrophils), and immunological changes (increased CD19(+) lymphocytes, IL-4 and total serum IgE), whereas TDA did not show any of these responses. Mice sensitized with TDI and challenged with TDA also did not show any airway or inflammatory response, although they had increased levels of total serum IgE. Mice sensitized with TDA and challenged with TDI did not show any response. According to the classification of sensitizers in the LLNA, TDA is a weak dermal sensitizer. In the experimental mouse model of chemical-induced asthma, TDA does not act as a respiratory sensitizer, at the concentration used. No cross-reactivity between TDI and TDA was found.status: publishe

Immunological determinants in a mouse model of chemical-induced asthma after multiple exposures

In a mouse model of chemical-induced asthma, we investigated the effects of multiple challenges, using toluene diisocyanate (TDI), a known cause of occupational asthma. On day 1 and 7, BALB/c mice received TDI or vehicle (acetone/olive oil). On days 10, 13 and 16 the mice received an intranasal instillation of TDI. Ventilatory function (Penh) was monitored by whole body plethysmography for 40 min after each challenge. Reactivity to methacholine was measured 22 h later. Pulmonary inflammation, TNF-a and MIP-2 levels were assessed 24 h after the last challenge by broncho-alveolar lavage (BAL). Other immunological parameters included total IgE, lymphocyte subpopulations in auricular and cervical lymph nodes, and IL-4, IFN-g and IL-13 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Early ventilatory function and airway reactivity increased in all groups that received a dermal application and one or multiple intranasal challenges of TDI. After multiple challenges, lung inflammation was characterized by neutrophils (~15%), and eosinophils (~4%), along with an increase in BAL MIP-2 and TNF-a levels. The auricular and cervical lymph node cells of all sensitized mice showed an increase in B-cells, Th-cells and an increased concentration of in vitro release of IL-4, IFN-g and IL-13 after stimulation with concanavalin A. Total serum IgE was elevated in dermally TDI-sensitized mice. This protocol including multiple challenges results in a model that resembles human asthma, indicating that responses found in the model using a single challenge could be a good first indication for the development of asthma.status: publishe

Validation of a mouse model of chemical-induced asthma using trimellitic anhydride, a respiratory sensitizer, and dinitrochlorobenzene, a dermal sensitizer

BACKGROUND: Occupational asthma can be caused by chemicals. Previously, we established a murine model of immunologically mediated chemical-induced asthma using toluene diisocyanate. OBJECTIVE: We sought to verify this model using trimellitic anhydride (TMA), a respiratory sensitizer, and 1-chloro-2,4-dinitrobenzene (DNCB), a dermal sensitizer. METHODS: BALB/c mice received dermal applications (vehicle or chemical) on days 1 and 7. On day 10, they received an intranasal instillation (vehicle or chemical). Whole-body plethysmography (enhanced pause) was used to monitor changes in ventilatory function and methacholine reactivity. Pulmonary inflammation was assessed by using bronchoalveolar lavage (cells, TNF-alpha levels, and macrophage inflammatory protein 2 levels). Immunologic parameters included total serum IgE levels, lymphocyte distribution in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells incubated with or without concanavalin A. RESULTS: Mice dermally treated and intranasally challenged with TMA experienced markedly increased enhanced pause immediately after intranasal challenge and increased methacholine reactivity (24 hours later). Mice similarly treated with DNCB did not show any ventilatory changes. Neutrophil influx and increased macrophage inflammatory protein 2 and TNF-alpha levels were found in bronchoalveolar lavage fluid in both TMA- and DNCB-treated mice. The proportion of CD19+ B cells was increased in auricular and cervical lymph nodes of TMA-treated mice. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular and cervical lymph node cells of TMA- or DNCB-treated mice; however, the relative proportions of IL-4 and IFN-gamma levels differed between TMA- and DNCB-treated mice. Serum total IgE levels were increased in TMA-treated mice only. CONCLUSION: Both compounds induce a mixed T(H)1-T(H)2 response, but only TMA induced ventilatory changes. CLINICAL IMPLICATIONS: In the workplace avoiding skin contact with chemical sensitizers might be advised to prevent chemical-induced asthma.status: publishe

Secreted frizzled related proteins inhibit fibrosis in vitro but appear redundant in vivo

The pathogenesis of pulmonary fibrosis remains poorly understood. The Wnt signaling pathway regulates fibrogenesis in different organs. Here, we studied the role of two extracellular Wnt antagonists, secreted frizzled-related protein-1 (SFRP1) and frizzled-related protein (FRZB) on lung fibrosis in vitro and in vivo. For this purpose, we used an alveolar epithelial cell line and a lung fibroblast cell line, and the bleomycin-induced lung fibrosis model, respectively.status: publishe

Oropharyngeal aspiration: An alternative route for challenging in a mouse model of chemical-induced asthma

Background: To assess the importance of the route of challenge in an existing mouse model of chemicalinduced asthma, we replaced intranasal instillation by oropharyngeal aspiration. To our knowledge, oropharyngeal aspiration as a challenge route has not yet been investigated in a mouse model of chemicalinduced asthma. Methods:Ondays 1 and 8, micewere dermally sensitized with toluene diisocyanate (TDI) (0.3%) [or vehicle (acetone/olive oil)] and on day 15 they received a single challenge, via oropharyngeal aspiration, with TDI (0.01%) or vehicle. One day after challenge, airway reactivity to methacholine was measured by a forced oscillation technique (FlexiVent) and total and differential cell counts, as well as levels of KC, IL-5, IL-17 and TNF-a, were assessed in the bronchoalveolar lavage (BAL) fluid. Lymphocytes from the auricular and mediastinal lymph nodeswere cultured to determine the concanavaline A-induced secretion of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN-g. Total serum IgE was measured. Results: In TDI-sensitized mice, a significant increase in airway reactivitywas found after a single oropharyngeal challenge with TDI. BAL neutrophils and eosinophils were increased 7- and 5-fold, respectively. An upregulation of Th1 (IFN-g), Th2 (IL-4, IL-10, IL-13) and Th17 (IL-17) cytokines was found in the auricular lymph nodes, in the mediastinal lymph nodes only IL-4 was upregulated. The total serum IgE level in TDI-sensitized mice was significantly increased when compared to control mice. Conclusion: We conclude that challenging mice via oropharyngeal aspiration mimics the characteristics of human asthma well, without the possible drawbacks of other techniques.status: publishe

Methylene diphenyl diisocyanate in a mouse model of chemical-induced asthma

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