Yang Ya-Wun, Wu Ching-An, Morrow W J W
School of Pharmacy, College of Medicine, National Taiwan University, Room 1214, 1, Jen-Ai Road, Section 1, Taipei 100, Taiwan.
Vaccine. 2004 Mar 29;22(11-12):1524-36. doi: 10.1016/j.vaccine.2003.08.048.
Many vaccine adjuvants contain surface-active agents, but the immunological roles played by these components have been essentially ignored. The objective of this study was to examine possible apoptotic and necrotic effects of the surface-active agents, Pluronic L121 and Tween 80, which are components of L121-adjuvant (a formulation we synthesized with the aim of representing several commercially produced adjuvants), on EL4 lymphoma cells. Cell viability and cytolytic effects were analyzed using the MTT and LDH release assays, and the distribution of cells in different stages of the cell cycle after treatment with these agents was analyzed by propidium iodide (PI) staining and flow cytometry. L121-adjuvant was shown to induce cell cycle arrest and inhibit cell proliferation. Treatment of EL4 cells with surface-active agents resulted in a concentration-dependent increase in the apoptotic/necrotic cell populations. Fluorescence microscopy using Hoechst 33342 staining demonstrated chromosome condensation and DNA fragmentation in cells treated with surfactants or adjuvant. The apoptotic and necrotic effects of vaccine adjuvant containing surface-active agents were confirmed by Annexin V/propidium iodide staining and flow cytometric analysis. Pretreatment of EL4 cells with zVAD-fmk, a broad range caspase inhibitor, partially prevented apoptosis induced by Pluronic L121, but did not prevent the cell death induced by Tween 80 or L121-adjuvant. These findings suggested that Tween 80 and L121-adjuvant induced apoptosis in EL4 cells via a "non-classical" caspase-independent pathway. Results presented in this study suggest mechanisms of elicitation of CD8(+), class I-restricted CTL response by soluble antigens mediated by the vaccine adjuvant containing surface-active agents.
许多疫苗佐剂都含有表面活性剂，但这些成分所发挥的免疫作用基本上被忽视了。本研究的目的是检测表面活性剂普朗尼克L121和吐温80对EL4淋巴瘤细胞可能产生的凋亡和坏死作用，这两种表面活性剂是L121佐剂（我们合成的一种制剂，旨在代表几种商业生产的佐剂）的成分。使用MTT和LDH释放试验分析细胞活力和细胞溶解作用，并用碘化丙啶（PI）染色和流式细胞术分析用这些试剂处理后细胞在细胞周期不同阶段的分布。结果表明，L121佐剂可诱导细胞周期停滞并抑制细胞增殖。用表面活性剂处理EL4细胞导致凋亡/坏死细胞群体呈浓度依赖性增加。使用Hoechst 33342染色的荧光显微镜观察显示，用表面活性剂或佐剂处理的细胞中出现染色体浓缩和DNA片段化。通过膜联蛋白V/碘化丙啶染色和流式细胞术分析证实了含表面活性剂的疫苗佐剂的凋亡和坏死作用。用泛半胱天冬酶抑制剂zVAD-fmk预处理EL4细胞可部分阻止普朗尼克L121诱导的凋亡，但不能阻止吐温80或L121佐剂诱导的细胞死亡。这些发现表明，吐温80和L121佐剂通过“非经典”的不依赖半胱天冬酶的途径诱导EL4细胞凋亡。本研究结果提示了含表面活性剂的疫苗佐剂介导可溶性抗原引发CD8(+)、I类限制性CTL反应的机制。
