Susanne Radonjic-Hoesli1, Xiaoliang Wang2, Elisabeth de Graauw2, Christina Stoeckle2, Beata Styp-Rekowska3, Ruslan Hlushchuk3, Dagmar Simon4, Peter J Spaeth2, Shida Yousefi2, Hans-Uwe Simon5. 1. Institute of Pharmacology, University of Bern, Bern, Switzerland; Department of Dermatology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 2. Institute of Pharmacology, University of Bern, Bern, Switzerland. 3. Institute of Anatomy, University of Bern, Bern, Switzerland. 4. Department of Dermatology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland. 5. Institute of Pharmacology, University of Bern, Bern, Switzerland. Electronic address: hus@pki.unibe.ch.
Abstract
BACKGROUND: Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with 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. OBJECTIVE: We aimed to elucidate the molecular mechanism of eosinophil cytolysis. METHODS: Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. A morphologic 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 pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses. RESULTS: After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-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 means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions. CONCLUSION: We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy.
BACKGROUND: Eosinophils are a subset of granulocytes that can be involved in the pathogenesis of different diseases, including allergy. Their effector functions are closely linked to their cytotoxic granule proteins. Release takes place through several different mechanisms, one of which is cytolysis, which is associated with 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. OBJECTIVE: We aimed to elucidate the molecular mechanism of eosinophil cytolysis. METHODS: Isolated blood eosinophils were incubated on glass coverslips coated with intravenous immunoglobulin and inactive complement component 3b. A morphologic 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 pharmacologic inhibitors were performed to elucidate the sequence of events within the cascade. Tissue samples of patients with eosinophilic skin diseases or eosinophilic esophagitis were used for in vivo analyses. RESULTS: After eosinophil adhesion, we observed reactive oxygen species production, early degranulation, and granule fusion processes, leading to a distinct morphology exhibiting cytoplasmic vacuolization and, finally, cytolysis. Using a pharmacologic approach, we demonstrate the presence of a receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway in eosinophils, which, after its activation, leads to the production of high levels of reactive oxygen species in a p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase-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 means of pharmacologic inhibition of the mechanistic target of rapamycin counterregulates cell death. Moreover, MLKL phosphorylation, cytoplasmic vacuolization, and cytolysis were observed in eosinophils under in vivo inflammatory conditions. CONCLUSION: We report that adhesion-induced eosinophil cytolysis takes place through RIPK3-MLKL-dependent necroptosis, which can be counterregulated by autophagy.
Authors: Stephane Esnault; Alexander S Hebert; Nizar N Jarjour; Joshua J Coon; Deane F Mosher Journal: J Proteome Res Date: 2018-05-04 Impact factor: 4.466
Authors: Paneez Khoury; Praveen Akuthota; Steven J Ackerman; Joseph R Arron; Bruce S Bochner; Margaret H Collins; Jean-Emmanuel Kahn; Patricia C Fulkerson; Gerald J Gleich; Rashmi Gopal-Srivastava; Elizabeth A Jacobsen; Kristen M Leiferman; Levi-Schaffer Francesca; Sameer K Mathur; Michael Minnicozzi; Calman Prussin; Marc E Rothenberg; Florence Roufosse; Kathleen Sable; Dagmar Simon; Hans-Uwe Simon; Lisa A Spencer; Jonathan Steinfeld; Andrew J Wardlaw; Michael E Wechsler; Peter F Weller; Amy D Klion Journal: J Leukoc Biol Date: 2018-04-19 Impact factor: 4.962
Authors: Elizabeth A Kelly; Stephane Esnault; Lin Ying Liu; Michael D Evans; Mats W Johansson; Sameer Mathur; Deane F Mosher; Loren C Denlinger; Nizar N Jarjour Journal: Am J Respir Crit Care Med Date: 2017-12-01 Impact factor: 21.405
Authors: Stephane Esnault; Jonathan P Leet; Mats W Johansson; Karina T Barretto; Paul S Fichtinger; Frances J Fogerty; Ksenija Bernau; Sameer K Mathur; Deane F Mosher; Nathan Sandbo; Nizar N Jarjour Journal: Clin Exp Allergy Date: 2019-12-09 Impact factor: 5.018