Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation

Immunol Cell Biol. 2010 Jul;88(5):545-54. doi: 10.1038/icb.2010.5. Epub 2010 Feb 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / immunology
  • Caspases / immunology*
  • Caspases / metabolism
  • Cell Line
  • Enzyme Activation / immunology
  • Granzymes / immunology*
  • Granzymes / metabolism
  • Humans
  • Immunoblotting
  • Mice
  • Mice, Inbred C57BL
  • Mitochondria / immunology
  • Mitochondria / metabolism
  • NADPH Oxidases / immunology
  • NADPH Oxidases / metabolism
  • Reactive Oxygen Species / immunology*
  • Reactive Oxygen Species / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / immunology*
  • T-Lymphocytes, Cytotoxic / immunology*
  • T-Lymphocytes, Cytotoxic / metabolism


  • Reactive Oxygen Species
  • NADPH Oxidases
  • Granzymes
  • Caspases