Brown Norway rat asthma model of diphenylmethane-4,4′-diisocyanate (MDI): Impact of vehicle for topical induction

Abstract

The development of this Brown Norway (BN) rat asthma model was focused on the duplication of at least some hallmarks of human diisocyanate asthma using the skin as the initial priming route of exposure. Equal total doses of polymeric diphenylmethane-diisocyanate (MDI) were applied to similar surface areas either dissolved in di-n-octyl sebacic acid ester (20%) (SEBA), in acetone:olive oil (20%) (AOO) or undiluted. The elicitation of respiratory allergy utilized four repeated nose-only inhalation challenges of 30 min with 39 mg/m³ MDI–aerosol approximately every 2 weeks. Emphasis was directed towards the analysis of respiratory responses delayed in onset. Endpoints suggestive of an allergic inflammatory response were examined by bronchoalveolar lavage (BAL) 1 day after the last inhalation challenge and comprised protein, LDH, cytodifferentiation of BAL cells, MCP-1, and some Th1 and Th2 cytokines. MCP-1 and cytokines were comparatively determined in three compartments: BAL fluid, BAL cells, and lung-associated lymph nodes (LALN). In all groups sensitized topically to MDI typical delayed-onset respiratory responses occurred. The lung and LALN weights, BAL-protein and -LDH were significantly increased as compared to the naïve control group challenged identically. There was compelling evidence of a neutrophilic rather than an eosinophilic inflammatory response. The patterns of interleukin (IL) IL-1α, TNF-α, IFN-γ, GM-CSF, and IL-4 differed appreciably from one compartment to another and were essentially maximal in BAL cells. In contrast, MCP-1 was increased to the same extent in all compartments measured. Collectively, changes were slightly, although consistently more pronounced when using SEBA as vehicle when compared with the vehicle AOO or undiluted MDI. Notable was a discordance of cytokine profiles and respiratory responses. In conclusion, the priming potency of topically administered MDI and subsequent asthma-like responses following repeated inhalation exposures appear to be dependent on multiple factors, one of them appears to be associated with the type of matrix used to dissolve MDI. This animal model provides a versatile and robust experimental tool to evaluate and assess at least some features of MDI-related asthma.

Introduction

When designing an in vivo model of respiratory allergy, it is important to clearly decide which aspects of this complex disease is the focus. In addition to expedience and practicability, the species and strain selection, the sensitization and challenge protocol as well as the endpoint(s) chosen are paramount for the outcome of test. The relative abundance of biomarkers to probe responsiveness may change from one compartment to another and, for example, it may be different in serum, lung parenchyma and draining lymph nodes or bronchoalveolar lavage fluid (BALF) (Schuster et al., 2000, Pauluhn, 2006). However, how these tissue microenvironments shape the function of professional antigen-presenting cells remains elusive. In terms of endpoints, those that integrate independently a series of complex events might be most practical to probe for positive responses in animal models in order to detect and categorize lung-sensitizing agents. Once identified, dose (concentration × time)–response analyses may facilitate risk assessment. Suffice it to say, particularly for dose–response analyses, the inhalation route is considered to be superior to expedient ‘shortcut’ bolus dosing procedures as they may not adequately dose the bronchial airways, the site asthma prevails. Limitations of most of the currently applied bioassays are that they model the acute rather than the chronic manifestation of asthma. Apart from the difficulties in transposing rodent to human data, it has to be borne in mind that each animal model is a trait associated with asthma, rather than for modeling the entire asthma phenotype (Kips et al., 2003, Kumar and Foster, 2002, Redlich et al., 2002).

Allergic asthma is a pulmonary disease characterized by episodes of acute airway inflammation superimposed on a background of chronic inflammation and structural changes collectively referred to as airway wall remodeling and airway hyper-responsiveness to a variety of stimuli (Kumar, 2001, Maddox and Schwartz, 2002). It was sought to develop a Brown Norway (BN) rat model of chronic asthma in which topically sensitized animals were repeatedly challenged by inhalation to a carefully controlled, minimally irritant concentration × time relationship of MDI–aerosol, to minimize the likelihood of eliciting marked parenchymal inflammation. Currently, most rodent models are more correctly designated models of allergic bronchopulmonary inflammation rather than of a bronchial airway-specific inflammation. However, when using this repeated challenge protocol, BN rats exhibited many of the lesions that typify chronic human asthma (Pauluhn et al., 2005, Pauluhn, 2006). The focus of this animal model was to elicit an acute-on-chronic allergic inflammation of the airways and to consider the compartmentalization of pulmonary responses (Schuster et al., 2000).

The majority of current rodent models of asthma include a priming phase, when animals are sensitized by topical administration of the inciting agent. Sometimes this administration is together with a Th2-skewing adjuvant. The exact roles played by Th1 and Th2 cytokines during the initial phase of sensitization in the development of eosinophils-mediated and neutrophil-mediated asthma remain unclear (Fischer et al., 2007, Johnson et al., 2004). Although not typically considered to be a Th2 cytokine, TNF-α is prominent in asthmatic airways, and genotypes that correlate with increased TNF-α secretion are associated with an increased risk of asthma (Moffatt and Cookson, 1997), suggesting that TNF-α plays an important role in the development of asthma and allergic airways inflammation. Because of their unique profile of cytokine production, it was proposed to distinguish ‘inflammatory Th2’ cells (IL-4, IL-5, IL-13, IL-10) from ‘conventional Th2’ cells (IL-4, IL-5, IL-13, TNF-α) (Liu et al., 2007). However, it should be recognized that bronchial asthma is not the only isocyanate-induced disorder. Nonobstructive bronchitis, rhinitis, cutaneous hypersensitivity, and hypersensitivity pneumonitis represent further diagnoses that may be associated with antibody responses (Baur, 2007).

Diisocyanates are widely used in industry and are known to be involved as causative agents for occupational asthma (Chan-Yeung and Malo, 1994). The pathogenesis associated with low-molecular-weight diisocyanates is not well understood and the current literature indicates that pathomechanisms involved in isocyanate-induced disorders are complex and heterogeneous. This includes the role of the dermal route of induction for priming and the inhalation route to elicit an asthma-like disease. More recently, concerns have been raised that skin exposure to isocyanates may prime exposed individuals to the subsequent development of asthma as a result of inhalation exposures (Bello et al., 2007). So far the sequence and frequency of exposure as well as the additional determining factors leading to disease remain poorly defined (Basketter et al., 2006, Boukhman and Maibach, 2001, Pauluhn, 2006). Among the issues contributing to controversy is the lack of knowledge whether the total dose, the concentration of the hapten (allergen), adjuvants (or irritation), absorption enhancers (vehicles) or skin injury, the exposed skin surface area, the frequency or repeated contact with the skin, and subsequent single or recurrent high-level inhalation excursions either alone or in combinations might be the critical determinant(s) leading to disease in susceptible individuals.

It has been recognized for many years that the vehicle matrix in which a chemical allergen (hapten) is encountered on the skin may have profound effects on the response elicited and the extent to which skin sensitization will be acquired (Kligman, 1966, Basketter et al., 2001). Although not all chemical allergens are affected similarly, for certain substances a greater than 10-fold vehicle dependent change in potency is observed (Basketter et al., 2001). In the guinea pig, it has been shown that the vehicle can substantially affect the rate of sensitization, although this could not be related to the apparent bioavailability (Andersen et al., 1985). However, the view that the vehicle matrix is likely to have an important impact on the sensitizing effect of a chemical is also based on the underlying knowledge of skin penetration.

Especially for potentially irritant chemicals, such as MDI, skin damage-associated factors may be released by stressed tissue or necrotic cells which then modulate the functional maturation of antigen presenting cells (Langerhans’ cells) (Lambrecht and van den Toorn, 2007). Thus, selection of the vehicle solvent for delivery of MDI to the skin and the dose metric (total dose or skin surface area concentration) are important determinants of the outcome and deserve better appreciation during study design and execution. Chemical reactivity of MDI with skin proteins, water, sweat components, and self polymerization reactions, as well as limited understanding of the kinetics of these processes on the skin and the role of solvent on skin penetration and bioavailability of MDI, further emphasize the need to pay closer attention to these factors. For these reasons, quantitative assessment of the vehicle effect and dose metric are time-consuming and technically challenging.

The objective of this study was to study the modifying impact on MDI-induced respiratory allergy (asthma) of three modes of topical skin exposure while maintaining an equivalent total dose and surface area. For this purpose groups of rats were topically sensitized to MDI dissolved in the two different vehicles, namely di-n-octyl sebacic acid ester (20%) (SEBA) and acetone:olive oil (20%) (AOO). Similarly dosed rats using undiluted MDI served as reference. The modifying role of these priming procedures on pro-inflammatory cytokines/chemokines and the recruitment of inflammatory cells in various lung compartments in repeatedly MDI–aerosol challenged BN rats were evaluated. SEBA was considered to be a nonreactive, nonvolatile lipophilic vehicle, whilst AOO (4:1) has been suggested as the vehicle of choice for other topical sensitization bioassays (Basketter and Kimber, 1996). Neither vehicle has any relevance to occupational exposure but may alter the stability, duration of topical exposure and thus the potential for skin penetration and bioavailability of this reactive chemical. Accordingly, the modifying properties caused by different formulation matrices were the focus of study.