Taichman N S, Iwase M, Lally E T, Shattil S J, Cunningham M E, Korchak H M
University of Pennsylvania, School of Dental Medicine, Department of Pathology, Philadelphia 19104.
J Immunol. 1991 Nov 15;147(10):3587-94.
Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.
伴放线放线杆菌产生一种细胞溶解肽白细胞毒素，可杀死包括人类中性粒细胞、单核细胞、淋巴细胞和HL - 60早幼粒细胞白血病细胞在内的易感靶细胞。细胞死亡是胶体渗透裂解的结果。在本研究中，通过流式细胞术和定量荧光光谱法研究了白细胞毒素诱导的早期膜通透性变化，研究对象为对白细胞毒素敏感和耐药的靶细胞。在5秒内，毒素敏感细胞的收缩期游离Ca2+呈现浓度依赖性持续增加，随后膜电位迅速逐渐下降。这些膜损伤的早期表现发生在细胞死亡前约10 - 15分钟，这可通过碘化丙啶染色细胞的流式细胞术分析反映出来。胞质Ca2+的升高几乎完全是由于细胞外Ca2+的内流。白细胞毒素对人中性粒细胞或HL - 60细胞Ca2+通透性作用的希尔图结果表明，两个或更多毒素分子参与了质膜中离子传导孔的组装。未观察到热灭活毒素引起的膜通透性或细胞活力变化。在适当条件下，毒素诱导的膜异常被白细胞毒素中和单克隆抗体或相对高浓度（大于或等于2.5 mM）的细胞外Ca2+抑制。白细胞毒素耐药靶细胞即使长时间暴露于高浓度白细胞毒素也未显示膜损伤迹象。这些细胞包括耐药的人K562红白血病细胞和小鼠SP2骨髓瘤细胞，先前已证明它们可吸附毒素，这表明它们具有一种保护机制，可阻止毒素插入或在脂质双层中组装。这些数据支持了伴放线放线杆菌白细胞毒素作为一种细胞特异性成孔蛋白，使易感靶细胞质膜通透性增加的概念。
