title: Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.
abstract: The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod-induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain-like protein. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase-8. Fingolimod-induced neutrophil death is independent of sphingosine-1-phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase-8 [Z-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone], receptor-interacting protein kinase 1 (necrostatin-1), receptor-interacting protein kinase 3 (necrosulfonamide), and heat shock protein 90 [geldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin]. Furthermore, NADPH oxidase 1 inhibition with diphenyleneiodonium chloride protects neutrophils against fingolimod-mediated cell death. Overall, these observations suggest that fingolimod acts through a mechanism involving the necrosome signaling complex and the oxidative stress machinery.
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skrzeczyńska-Moncznik
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Bzowska
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Anna
authors.initials: A
authors.lastname: Nogieć
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Sroka
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Mirosław
authors.initials: M
authors.lastname: Zarębski
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada.
authors.forename: Luc
authors.initials: L
authors.lastname: Vallières
authors.affiliation: Departments of *Immunology and Cell Biophysics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Kraków, Poland; and Department of Molecular Medicine, Laval University, Quebec, Canada krzysztof.guzik@uj.edu.pl.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Guzik
mesh_terms: D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D053938:DNA Fragmentation; D000068876:Fingolimod Hydrochloride / Q000494:pharmacology*; D005434:Flow Cytometry; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D018613:Microscopy, Confocal; D018832:Molecular Chaperones; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology
chemical_list: D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D018832:Molecular Chaperones; D000068876:Fingolimod Hydrochloride
keywords: MLKL; NADPH oxidase; apoptosis; necroptosis; phosphatidylserine