Ogawa Yasuhiro, Kobayashi Toshihiro, Kariya Shinji, Nishioka Akihito, Nakayama Koichi, Seguchi Harumichi, Yoshida Shoji
Department of Radiology, Medical School, Kochi University, Kochi-Prefecture 783-8505, Japan.
Int J Mol Med. 2004 Dec;14(6):1007-13.
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 Mito-Capture, H(2)DCFDA (succinimidyl ester of dichlorodihydrofluorescein 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).
人类外周血T细胞被认为极易受到氧化应激的影响，因为这些细胞缺乏过氧化物酶活性。因此，在之前的一项研究中，我们利用线粒体捕获试剂、二氯二氢荧光素二乙酸琥珀酰亚胺酯（H(2)DCFDA）、4',6-二脒基-2-苯基吲哚（DAPI）和溶酶体传感器，研究了活性氧（ROS）的形成位点。我们的结果表明，在用0.1 mM过氧化氢进行氧化应激处理的T细胞中，ROS的形成明显呈弥散分布。此外，在这些细胞中观察到溶酶体肿胀和变形，这可能表明溶酶体膜不稳定。基于上述结果，我们得出结论，在过氧化氢诱导的人类外周血T细胞凋亡中，存在一个涉及早期溶酶体膜不稳定的凋亡级联反应。因此，溶酶体中形成的羟基自由基导致溶酶体蛋白酶泄漏（可能导致线粒体膜功能障碍），被认为在过氧化氢诱导的T细胞凋亡中起重要作用。过氧化氢介导的溶酶体膜不稳定，伴随着多种组织蛋白酶等水解酶释放到细胞质中，可导致一系列最终导致细胞死亡的事件。为了评估溶酶体内氧化还原活性铁池的重要性，我们通过将T细胞暴露于溶酶体促渗碱化剂氯化铵（NH(4)Cl）来检测溶酶体消化受阻的影响。将人类外周血T细胞与10 mM NH(4)Cl预孵育4小时，可显著降低随后暴露于过氧化氢（H(2)O(2)）所导致的凋亡死亡，并且溶酶体和线粒体的外观几乎保持正常。因此，我们在此得出结论，氯化铵（NH(4)Cl）预孵育可防止过氧化氢导致的T细胞溶酶体蛋白酶泄漏。
