Persistence of respiratory and inflammatory responses after dermal sensitization to persulfate salts in a mouse model of non-atopic asthma

Persulfate salts are highly reactive low molecular weight (LMW) chemical compounds which are present in considerable proportions (10–20 %) in the bleaching powders used by hairdressers during hair-bleaching procedures [1]. Exposure to these salts has been identified as the main cause of immunological sensitization and subsequent allergic diseases such as contact dermatitis and bronchial asthma, and it has been associated with a high risk of occupational asthma (OA) in hairdressers [2‐4].

Nevertheless, the mechanisms by which persulfate salts induce sensitization and OA are not well established [5]. An immunologic mechanism has been postulated; various authors have suggested an IgE-driven mechanism, based on skin prick test positivity to persulfate salts and the finding of high levels of total serum IgE in hairdressers with OA [4, 6]. However, other data seem to suggest that persulfate salts act through an immunological mechanism without driving an IgE response [7]. Therefore, studies of OA using suitable animal models may be able to shed light on the processes involved in the onset and persistence of bronchial hyperresponsiveness and airway inflammation and remodeling.

In a previous study using local lymph node assays [8], our research group identified ammonium persulfate (AP) as a moderate dermal sensitizer. In later work we developed and validated a mouse model of chemical-induced asthma using AP. In this model, mice were dermally sensitized with AP and then underwent a single airway challenge with AP, which triggered the responses typical of human OA [8, 9].

It has been reported that asthma symptoms and non-specific airway hyperresponsiveness persist even after cessation of exposure. The reason for this is not clear. In the present study, we examined how long the asthmatic response to AP persists after dermal sensitization. The aim of the study was to compare the airway responses, lung inflammation, and immune responses induced by a single intranasal AP challenge administered at variable intervals (between 1 and 90 days) after dermal sensitization to AP.

We investigated the time course of immunologic and respiratory responses after dermal sensitization in a validated mouse model of OA due to persulfate salts [9]. We were able to induce both respiratory responsiveness to methacholine and pulmonary inflammation in AP-sensitized mice with a single intranasal challenge with AP up to 40 days after initial AP sensitization. Even 60 days after initial AP sensitization, a single challenge could still induce respiratory responsiveness (without neutrophilic inflammation), while 90 days afterwards, a single challenge with AP no longer induced these asthma-like symptoms. In terms of the immune response, there was evidence of systemic sensitization (with an increase in IgE) at early stages, while high IgG levels appeared later.

Exposure to persulfate salts is associated with a high risk of developing OA, although the mechanisms by which these substances induce sensitization and OA are not well understood [2, 4]. It has been suggested that persulfate-induced OA is mediated by an immunological mechanism [2, 4, 12]. Positive skin prick tests to persulfates have been reported, suggesting an IgE-mediated mechanism [4, 12‐14]. An increase in total IgE levels has been described in studies with human patients and with mouse models [4, 9]. In this connection, in a mouse model of chemical-induced asthma previously developed by our group using AP [9], we showed that AP, after two dermal applications and only one airway challenge, can induce features of human occupational asthma in mice, including respiratory responsiveness to methacholine, neutrophil inflammation in BAL, T- and B cell proliferation and a Th2 cytokine profile in the auricular lymph nodes (the site of sensitization), and also increased total serum IgE levels.

Recent animal and human data collectively support a central role for skin barrier function and skin exposure in the development of Th2-like sensitization and the subsequent development of asthma. Mouse models have also shown that chemicals can induce mixed Th1/Th2 responses [15, 16]. The skin may play a role both as an important route of exposure and as an immunological organ that can contribute to pulmonary immune diseases [17]. The epidermis contains keratinocytes and Langerhans cells, a major dendritic cell in the skin which can acquire antigen, migrate to draining lymph notes, and initiate immune responses [18]. The activation of dendritic cells with subsequent T-lymphocyte transformation in the lymph nodes draining the skin produces activated effector T-lymphocytes or memory cells in the systemic circulation [19]. Once in the lung, effector T-cells will produce cytokines and chemokines or undertake cytotoxic functions. Repeated lung exposure to the irritant by inhalation doses may synergistically amplify this allergic inflammation and asthma. These cells are able to express high-affinity receptors for IgE and, upon re-exposure, binding of the allergen to IgE orchestrates the immune system to initiate a more aggressive and rapid memory response [20].

In this study, we found a trend towards an increase in total serum IgE levels already at 15 days after initial dermal application of AP. IgE levels remained high until 22 days. A previous study by our group [21] described the course of bronchial hyperresponsiveness and immunologic test results in patients with OA due to persulfate salts, and found that total IgE levels remained increased even in patients who ceased exposure. On the other hand, levels of total serum IgG1 tended to increase from day 60, when total serum IgE levels had returned to baseline values. This is compatible with a Th2 immunological response, despite the unexpected increases in levels of total serum IgG2a (characteristic of a Th1 stimulation in mice) from day 60. These results are consistent with other studies carried out by our group with the same animal model and other mouse models of asthma using LMW agents [9, 11, 20‐23], which suggested a mixed Th2-Th1-type immune response in sensitized mice. It has also been proposed that an increase in IgG levels may have a protective effect in this animal model [24], as the percentage of neutrophils decreased at the same time point that IgG started to increase.

Nevertheless, respiratory responsiveness to methacholine still persisted at 45 days despite the increased levels of IgG. Thus, inflammation and respiratory responsiveness were not associated after AP exposure. These results suggest that the presence of abnormal airway smooth muscle function is determinant for respiratory responsiveness to LMW agents in this OA model, while the presence of mucosal airway inflammation may aggravate the situation but is not the cause. Swedin et al. [25] also reported dissociation between airway inflammation and airway hyperresponsiveness (AHR) in an ovalbumin allergic mouse model, suggesting that inflammatory cells in BAL do not change in parallel with AHR. Regarding LMW agents, Vanoirbeek et al. [22] observed the same pattern of dissociation in a mouse model of OA due to isocyanate. These results are consistent with previous observations in subjects with asthma in whom BAL inflammation was not a predictive surrogate marker of AHR [26], suggesting that other factors such as airway wall remodelling, the activation state of inflammatory cells, T-cell activation or autonomic dysfunction may play a more important role in the development of AHR. Recently, in a mouse model of severe asthma, Raundhal et al. [27] demonstrated a role for IFN-ϒ in the induction of AHR, whereas IL-17 promotes neutrophilic airway inflammation. These observations suggest that IFN-ϒ is the predominant cytokine associated with AHR in severe asthma and that airway inflammation and AHR may not always be linked.

The progressive reduction in responsiveness over time also occurs in patients with OA who cease exposure, although the results of the present study only partially reflect this situation. It is well established that patients with OA generally become less responsive to the sensitizer after complete exposure removal [4]. Nevertheless, there is insufficient scientific evidence to assert that cessation of exposure improves asthma symptoms, and many patients do not become completely unresponsive to the sensitizer [28]. In our study, asthma symptoms and functional airway abnormalities had disappeared after 90 days. However, our results did not prove that these mice would not become responsive if repeated exposures to the causal agent were given.

In conclusion, we show that both AHR and airway inflammation responses decrease with increasing time between sensitization and challenge. These findings suggest that dermal contact with a chemical can cause long-term sensitization and may lead to asthmatic symptoms. Moreover, many days after sensitization, exposure to the causal agent may produce various responses of which AHR is the most persistent; for its part, the inflammation response may be decreased. In any case, the mechanisms underlying the process remain undefined and more studies in this direction are needed.