Assessment of eosinophil degranulation in allergic diseases and experimental models

Sammanfattning: In allergic disorders, the eosinophilic granulocytes migrate to affected tissues and release granule proteins with cytotoxic, immunoregulatory, and remodeling-promoting properties. Given that degranulation is reflected by a loss in eosinophil granule density of living cells, or by cell membrane rupture and release of intact granules, these morphological changes may represent a biomarker of the allergic disease. In this thesis, eosinophil structural changes were quantified by transmission electron microscopy. The main objective was to reveal the degranulation status of blood eosinophils during allergic disease, and to determine the eosinophil morphology in common experimental cell- and animal models used for studying eosinophil activation and its pathogenic consequences. First, the eosinophil ultrastructure in common mouse models of allergic airway inflammation was determined and the relevance of these models to human disease was assessed. Both in vivo and in vitro studies revealed that eosinophil degranulation occurred in human but not in mouse eosinophils. Thus, eosinophil-driven pathologic events, reflecting human allergic disease, should not unconditionally be expected in current mouse models. Furthermore, the granule structure of isolated human blood eosinophils was studied and compared with the baseline morphology in blood to examine whether the transformation from an intact eosinophil to a degranulating phenotype can be accurately studied in vitro. The standard procedures of erythrocyte lysis were shown to induce artefactual eosinophil degranulation that also increased the susceptibility of cells to further treatment. Hence, caution should be taken when assessing data and concepts generated in the many previous studies on isolated human blood eosinophils. However, by a novel protocol described herein, eosinophils with minimal granule abnormalities can now be recovered and used for studies on the events regulating the early degranulation. Finally, using a novel approach to assess the ultrastructure of blood eosinophils the degranulation status of circulating eosinophils in a variety of allergic diseases was determined. The results showed that in symptomatic allergic disease, eosinophils retain their granule contents until they have reached their target organ. Hence, eosinophil degranulation in the circulation cannot be used as a biomarker of allergic disease. This finding also suggests that eosinophils in different body compartments have different effector functions and may have distinct susceptibility to therapeutic interventions. Altogether, this thesis underlines the general importance of validating samples and applied methodology when exploring eosinophil degranulation in vitro or in allergic disease.