Adhesion-induced eosinophil cytolysis requires the RIPK3-MLKL signaling pathway which is counter-regulated by autophagy

Publication date: Available online 12 April 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): Susanne Radonjic-Hoesli, Xiaoliang Wang, Elisabeth de Graauw, Christina Stoeckle, Beata Styp-Rekowska, Ruslan Hlushchuk, Dagmar Simon, Peter J. Spaeth, Shida Yousefi, Hans-Uwe Simon
BackgroundEosinophils are a subset of granulocytes which can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. The release takes place by several different mechanisms, one of which is cytolysis, which is associated with the release of intact granules, so-called clusters of free eosinophil granules. The mechanism underlying this activation-induced form of cell death in eosinophils has remained unclear.ObjectiveWe aimed to elucidate the molecular mechanism of eosinophil cytolysis.MethodsIsolated blood eosinophils were incubated on glass cover slips coated with intravenous immunoglobulin (IVIG) and inactive complement component 3b (iC3b). A morphological characterization of the distinct stages of the proposed cascade was addressed by means of time-lapse automated fluorescence microscopy, electron microscopy, and immunohistochemistry. Experiments with pharmacological inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients suffering from eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses.ResultsFollowing eosinophil adhesion, we observed reactive oxygen species (ROS) production, early degranulation, and granule fusion processes leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, to cytolysis. Using a pharmacological approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3) – mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, following its activation, leads to the production of high levels of reactive oxygen species (ROS) in a p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K) - dependent manner. All these steps are required for cytoplasmic vacuolization and subsequent cytolysis to occur. Interestingly, triggering cytolysis is associated with an induction of autophagy in eosinophils and additional stimulation of autophagy by pharmacological inhibition of the mechanistic target of rapamycin (mTOR) counter-regulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions.ConclusionWe report that adhesion-induced eosinophil cytolysis takes place by a RIPK3-MLKL-dependent necroptosis which can be counter-regulated by autophagy.The receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase-like (MLKL) signaling pathway mediates eosinophil cytolysis, a phenomenon often observed in eosinophilic tissues of patients suffering from diseases with eosinophil-mediated immunopathology.

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