Prevention of hydrogen peroxide-induced apoptosis of human peripheral T cells by a lysosomotropic iron chelator, ammonium chloride

Human peripheral T cells are considered to be easily susceptible to oxidative stress because these cells lack peroxidase activity. Therefore, in a previous study, we investigated the site of ROS formation by utilizing MitoCapture, H 2 DCFDA (succinimidyl ester of dichlorodihydro-fluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization were observed in these cells. Based on the above-mentioned results, we concluded that an apoptotic cascade involving early lysosomal membrane destabilization exists in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis. Hydrogen peroxide-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes such as many kinds of cathepsins into the cell cytoplasm can lead to a cascade eventuating in cell death. To assess the importance of the intralysosomal pool of redox-active iron, we examined the effect of blockade of lysosomal digestion by exposing T cells to the lysosomotropic alkalinizing agent ammonium chloride (NH 4 Cl). Preincubation of human peripheral T cells with 10 mM NH 4 Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to hydrogen peroxide (H 2 O 2 ), and lysosomes and mitochondria showed almost normally preserved appearance. Therefore, we concluded here that lysosomal protease leakage caused by hydrogen peroxide in T cells was prevented by preincubation with ammonium chloride (NH 4 Cl).