Eosinophils and activation markers after allergen challenge – a pilot study for three‐dimensional analysis in the bronchial mucosa
Authors: Frauke Prenzler, Thomas Tschernig, Katherina Sewald, Tibor Z Veres, Susanne Rittinghausen, Norbert Krug, Jens M. Hohlfeld, Armin Braun
Journal: Allergy (2021)

TO THE EDITOR, The experimental procedure of segmental allergen challenge (SAC) in mild asthmatic subjects is an extremely valuable study tool to investigate mechanisms of bronchial asthma in patients in general and in particular for the role of eosinophils. In this procedure, BAL and bronchial mucosa can be analysed simultaneously after the induction of allergic inflammation. Older studies yielded data on different time points after the challenge with increasing numbers of eosinophils in the BAL.1 There are no data published on increased numbers of eosinophils within the bronchial mucosa 24 hours after SAC. Here, eosinophils were studied in mucosa and airway lumen of mild asthmatics undergoing segmental allergen challenge as described before.2, 3 Eosinophils and their release products were investigated in thick sections of the bronchial biopsies using advanced three-dimensional analysis. Subject data, methods and detailed results are given in the supplement. All subjects showed a clear eosinophilic response in the airway lumen and mucosa 24 hours after the challenge (Figure 1; FigureS1). The data on neutrophils were inconclusive (Figure 1; Figure S2). There was an increase of eosinophils in the mucosa and in the BAL. In the BAL, the concentrations of IL-5 and ECP were elevated (supplement). In the mucosa, ECP-stained volumes were found elevated as well as signs of eosinophil activation and degranulation (Figure 1B,C). In higher resolution, the ECP expression was associated with either cellular structures or small granules or dispersed over a large area beneath the epithelium (Figure 2A-E). In the presented study not cell associated, free ECP-positive granules were seen at baseline but only to a small extent. However, after allergen challenge the ECP-expressing volumes were massively increased (Figure 1B,C). These results might suggest that in human tissue eosinophils degranulate after a single allergen challenge and release their inflammatory mediators into the surrounding tissue. This is in contrast to animal models of asthma where eosinophilic degranulation after allergen challenge does not occur extensively.4, 5 In the present study, subjects with a strong IL-5 reaction in BAL showed a strong eosinophilic response in the lumen. However, BAL IL-5 levels did not correlate with eosinophil numbers in the mucosa or volume of ECP-positive surfaces in the mucosa (Figure S3). It is a long known fact that IL-5 levels in the BAL correlate highly with absolute numbers of eosinophils in the same compartment. In the present study, the allergic reaction is comparable to those of other studies. The missing correlation between IL-5 in the BAL and numbers of tissue eosinophils showed that the relationship between both compartments is not as simple as assumed. Here, the data for the volume of ECP-positive surfaces may give an important hint. Interestingly, the volume of ECP-positive surfaces was inversely correlated with TNF-α and IL-8. TNF-α enhances migration of eosinophils from mucosa to lumen shown in an in vitro cell culture model.6 Therefore, one possible explanation is that increased TNF-α in the BAL leads to a migration of activated eosinophils from the mucosa into the airways. In conclusion, the findings in subjects with mild asthma are in alignment with other published results and suggest that 1) human tissue eosinophils release their granules in non-provoked state, and 2) toxic content of these cells is significantly released into the surrounding tissue after a single allergen challenge, whereas the distribution and the degree of activation and degranulation of eosinophils differ widely between subjects. Many eosinophils in the airways indicate many eosinophils in the mucosa. Three-dimensional analysis in thick tissue sections using confocal microscopy is a valuable tool in the investigation of bronchial biopsies from patients suffering from bronchial asthma. We would like to thank Isabelle Bleeker for processing the biopsy samples of the classical immunohistology. None of the authors has any financial interest. AB, JH and NK planned and conducted the study. FP, AB and TT wrote the manuscript, all other authors read, corrected and approved the manuscript. TZV, KS, SR and FP established and performed the morphology and made the evaluation of tissue data. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.