Wang J F, Jerrells T R, Spitzer J J
Department of Physiology, Louisiana State University Medical Center, New Orleans 70112-1393, USA.
Free Radic Biol Med. 1996;20(4):533-42. doi: 10.1016/0891-5849(95)02085-3.
Thymocyte apoptosis is one of the best characterized experimental models of apoptosis that can be induced by a variety of stimuli such as glucocorticoids, ionizing radiation, antibodies, and toxins. Recently, it has been suggested that oxidative stress is a common mediator of apoptosis. However, little is known about the production and possible function of reactive oxygen intermediates (ROI) in thymocytes. We used a highly sensitive flow cytometric assay with the hydrogen peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), to measure intracellular ROI production in rat thymocytes, to study its primary sources, and to compare ROI levels in normal and apoptotic thymocytes. Apoptosis was induced by incubating the cells in the presence or absence of dexamethasone (Dex) at 37 degrees C in vitro. Normal thymocytes spontaneously produced significant amounts of ROI. Catalase or superoxide dismutase did not affect this intracellular fluorescence, presumably due to their failure to penetrate into the cells. However, N-acetyl-L-cysteine significantly attenuated the fluorescence in a dose-dependent manner. Significant inhibition of the intracellular fluorescence was also observed by addition of N-nitro-L-arginine methyl ester (L-NAME), that could not be reversed by L-arginine. The addition of N-nitro-D-arginine methyl ester (D-NAME) also caused considerable inhibition. This indicates that the inhibition by L-NAME or D-NAME is due to a direct scavenging effect, and nitric oxide production is not likely to be involved. In contrast to neutrophils and macrophages whose superoxide anions are released from membrane-bound NADPH oxidase, the production of ROI in thymocytes is likely to originate mainly from mitochondria, as indicated by the inhibitory effect of the addition of rotenone or antimycin A. The addition of lymphocyte simulators phytohemagglutinin (PHA), concanavalin A (Con A), or phorbol 12-myristate 13-acetate (PMA) enhanced intracellular fluorescence of thymocytes. This increase was abrogated by addition of rotenone or antimycin A. The ROI production was decreased with time after incubation of the thymocytes for 1, 3, and 6 h in vitro. The appearance of apoptosis of thymocytes in vitro, as indicated by DNA content of cells by flow cytometry and DNA ladder formation in agarose gel electrophoresis, was delayed, as compared to the time course of the decreased ROI production. The addition of Dex to the culture medium accelerated both of these processes. The results suggest that a decreased spontaneous production of ROI in thymocytes precedes the spontaneous in vitro apoptosis and Dex exaggerates these changes.
胸腺细胞凋亡是特征最为明确的凋亡实验模型之一，可由多种刺激诱导，如糖皮质激素、电离辐射、抗体和毒素等。最近，有人提出氧化应激是凋亡的常见介质。然而，关于胸腺细胞中活性氧中间体（ROI）的产生及其可能的功能却知之甚少。我们使用对过氧化氢敏感的染料2',7'-二氯荧光素二乙酸酯（DCFH-DA）进行高灵敏度流式细胞术检测，以测量大鼠胸腺细胞内ROI的产生，研究其主要来源，并比较正常和凋亡胸腺细胞中的ROI水平。通过在37℃体外有或无地塞米松（Dex）存在的情况下孵育细胞来诱导凋亡。正常胸腺细胞会自发产生大量ROI。过氧化氢酶或超氧化物歧化酶对这种细胞内荧光没有影响，推测是由于它们无法穿透进入细胞。然而，N-乙酰-L-半胱氨酸以剂量依赖的方式显著减弱了荧光。添加N-硝基-L-精氨酸甲酯（L-NAME）也观察到细胞内荧光的显著抑制，且L-精氨酸无法逆转这种抑制。添加N-硝基-D-精氨酸甲酯（D-NAME）也导致了相当程度的抑制。这表明L-NAME或D-NAME的抑制是由于直接清除作用，不太可能涉及一氧化氮的产生。与中性粒细胞和巨噬细胞不同，它们的超氧阴离子是从膜结合的NADPH氧化酶释放的，胸腺细胞中ROI的产生可能主要源于线粒体，这一点从添加鱼藤酮或抗霉素A的抑制作用可以看出。添加淋巴细胞刺激剂植物血凝素（PHA）、刀豆球蛋白A（Con A）或佛波酯12-肉豆蔻酸13-乙酸酯（PMA）可增强胸腺细胞的细胞内荧光。添加鱼藤酮或抗霉素A可消除这种增加。在体外孵育胸腺细胞1、3和6小时后，ROI的产生随时间减少。与ROI产生减少的时间进程相比，通过流式细胞术检测细胞DNA含量以及琼脂糖凝胶电泳中DNA梯带形成所表明的体外胸腺细胞凋亡的出现有所延迟。向培养基中添加Dex加速了这两个过程。结果表明，胸腺细胞中ROI自发产生的减少先于体外自发凋亡，而Dex会加剧这些变化。
