Doi T, Motoyama N, Tokunaga A, Watanabe T
Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
Int Immunol. 1999 Jun;11(6):933-41. doi: 10.1093/intimm/11.6.933.
B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades.
B细胞抗原受体（BCR）介导的细胞死亡被认为是骨髓中B细胞分化过程中清除抗自身特异性免疫库的一种机制。凋亡性细胞死亡的某些方面需要线粒体改变和半胱天冬酶的激活，但线粒体和半胱天冬酶如何促成BCR介导的细胞死亡尚不清楚。在本研究中，我们使用小鼠WEHI-231 B细胞系来证明线粒体改变和半胱天冬酶的激活确实参与了BCR介导的细胞死亡。半胱天冬酶的肽抑制剂N-苄氧羰基-Val-Ala-Asp-氟甲基酮（z-VAD-fmk）可阻断聚（ADP-核糖）聚合酶的切割以及核凋亡的各种表现，如核碎裂、亚二倍体和DNA片段化，表明来自BCR的信号诱导了半胱天冬酶的激活。此外，z-VAD-fmk延迟了凋亡相关的细胞还原-氧化电位变化，这是通过超氧阴离子的过度产生以及外质膜中磷脂酰丝氨酸残基的暴露来确定的。相比之下，尽管z-VAD-fmk延缓了细胞溶解，但即使在它完全阻断核凋亡的相同条件下，它也无法阻止质膜的破坏。z-VAD-fmk也不能阻断线粒体膜电位的丧失。邦克雷酸是线粒体通透性转换孔的特异性抑制剂，它不仅抑制线粒体膜电位，还抑制质膜通透性的变化。Bcl-xL的过表达可预防由BCR信号转导引发线粒体功能障碍、核凋亡和膜通透性细胞死亡。这些观察结果表明，来自BCR的死亡信号可能首先导致线粒体改变，随后激活坏死和凋亡级联反应。
