Illera V A, Perandones C E, Stunz L L, Mower D A, Ashman R F
VA Medical Center, Iowa City, IA.
J Immunol. 1993 Sep 15;151(6):2965-73.
Small dense splenic B lymphocytes from adult specific pathogen-free mice were shown to undergo apoptosis in vitro as indicated by internucleosomal DNA fragmentation, hypodiploid DNA content of isolated nuclei, and morphologic features by electron microscopy. Unstimulated cultures showed spontaneous apoptosis increasing gradually and monotonically from < 2 to 32% of B cells by 16 h. The rate of accumulation of apoptotic cells was reduced by the addition of IL-4 or PMA, but not by the inactive phorbol ester, 4 alpha PDD. In contrast, inhibitors of protein kinase C (H7 and staurosporine) increased the percentage of cells undergoing apoptosis to > 70% by 12 h; HA 1004, genistein, and herbimycin A all had no effect on apoptosis. Thus, protein kinase C activity regulates apoptosis, but there is no evidence that protein kinases A and G and tyrosine kinases are involved. Cycloheximide increased apoptosis, indicating that apoptosis may be restrained in B cells by the presence of one or more labile protective proteins. The percentage of apoptotic cells measured by flow cytometry and the percentage of fragmented DNA measured by the diphenylamine method were nearly equal, regardless of the method of apoptotic regulation. Together with the absence of nuclei with flow cytometric properties intermediate between normal and apoptotic, these results suggest that in individual B cells apoptosis progresses rapidly to completion. These data suggest a fundamental change in our concept of the life-style of the "resting" B cell: instead of a dormant cell remaining unchanged until it receives activation signals, the mature spleen B cell appears programmed to die by apoptosis unless rescued by specific agents, such protein kinase C activators or IL-4.
研究表明，来自成年无特定病原体小鼠的小而致密的脾脏B淋巴细胞在体外会发生凋亡，这可通过核小体间DNA片段化、分离细胞核的亚二倍体DNA含量以及电子显微镜下的形态学特征来表明。未受刺激的培养物显示，自发凋亡从<2%逐渐单调增加至16小时时的32%的B细胞。添加IL-4或PMA可降低凋亡细胞的积累速率，但无活性的佛波酯4αPDD则无此作用。相反，蛋白激酶C抑制剂（H7和星形孢菌素）在12小时时可使发生凋亡的细胞百分比增加至>70%；HA 1004、染料木黄酮和除莠霉素A对凋亡均无影响。因此，蛋白激酶C活性调节凋亡，但没有证据表明蛋白激酶A、G和酪氨酸激酶参与其中。放线菌酮增加凋亡，表明凋亡可能受到一种或多种不稳定保护蛋白的存在的抑制。无论凋亡调节方法如何，通过流式细胞术测量的凋亡细胞百分比和通过二苯胺法测量的DNA片段化百分比几乎相等。连同缺乏具有介于正常和凋亡之间流式细胞术特性的细胞核，这些结果表明在单个B细胞中凋亡迅速进展至完成。这些数据表明我们对“静止”B细胞生活方式的概念发生了根本性变化：成熟的脾脏B细胞并非是一个在接受激活信号之前保持不变的休眠细胞，而是似乎被编程为通过凋亡死亡，除非被特定因子拯救，如蛋白激酶C激活剂或IL-4。
