title: Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.
abstract: Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.
authors.affiliation: Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain.
authors.forename: Juan I
authors.initials: JI
authors.lastname: Aguiló
authors.forename: Alberto
authors.initials: A
authors.lastname: Anel
authors.forename: Elena
authors.initials: E
authors.lastname: Catalán
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sebastián
authors.forename: Rebeca
authors.initials: R
authors.lastname: Acín-Pérez
authors.forename: Javier
authors.initials: J
authors.lastname: Naval
authors.forename: Reinhard
authors.initials: R
authors.lastname: Wallich
authors.forename: Markus M
authors.initials: MM
authors.lastname: Simon
authors.forename: Julián
authors.initials: J
authors.lastname: Pardo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D020169:Caspases / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000276:immunology; D053804:Granzymes / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000276:immunology / Q000378:metabolism; D019255:NADPH Oxidases / Q000276:immunology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D053804:Granzymes; D020169:Caspases