Styrt B, Klempner M S
Infect Immun. 1985 Nov;50(2):415-9. doi: 10.1128/iai.50.2.415-419.1985.
Weak bases that alkalinize the pH within neutrophil lysosomes inhibit in vitro cell functions, including lysosomal enzyme release and superoxide production. To determine the relevance of this inhibition to microbicidal activity, the effect of lysosomotropic weak bases on interactions between human neutrophils and Staphylococcus aureus 502a was studied. After treatment with 1 mM chloroquine, neutrophils showed significantly impaired phagocytosis of 14C-labeled S. aureus. However, 50 mM ammonium chloride had no effect on phagocytosis, although we have previously shown that this concentration raises lysosomal pH and inhibits degranulation and superoxide production. This base was therefore used to study effects on intracellular microbicidal activity. Incubation of neutrophils with 50 mM ammonium chloride diminished killing of S. aureus (22.9 +/- 6.3% of bacteria surviving versus 8.2 +/- 1.3% in suspensions without ammonium chloride). At 1 mM, ammonium chloride had no significant effect. The inhibition of cellular function could be neither explained as a function of neutrophil death, as measured by trypan blue dye exclusion, nor attributed to direct promotion of bacterial growth (in the absence of neutrophils, colony counts were similar in the presence or absence of ammonium chloride) or enhanced resistance to neutrophil microbicidal mechanisms (bacteria treated with ammonium chloride and washed before neutrophil exposure showed no improvement in survival). Ammonium chloride at 50 mM also impaired neutrophil killing of S. aureus in an anaerobic chamber, but microbicidal activity against Escherichia coli S15 was not affected. These findings suggest that optimal neutrophil killing of staphylococci requires a highly acid intralysosomal compartment, but ingestion of bacteria does not. This may reflect primary failure of acidification of the phagocytic vacuole or differential pH requirements for fusion of the plasma membrane with itself and with lysosome membranes. The difference between effects on killing of S. aureus and E. coli is probably a result of the relative importance of the components of neutrophil microbicidal activity against the two organisms.
使中性粒细胞溶酶体内pH值碱化的弱碱会抑制体外细胞功能，包括溶酶体酶释放和超氧化物生成。为了确定这种抑制作用与杀菌活性的相关性，研究了溶酶体促渗弱碱对人中性粒细胞与金黄色葡萄球菌502a之间相互作用的影响。用1 mM氯喹处理后，中性粒细胞对14C标记的金黄色葡萄球菌的吞噬作用显著受损。然而，50 mM氯化铵对吞噬作用没有影响，尽管我们之前已经表明该浓度会提高溶酶体pH值并抑制脱颗粒和超氧化物生成。因此，使用该碱来研究其对细胞内杀菌活性的影响。将中性粒细胞与50 mM氯化铵一起孵育会减少对金黄色葡萄球菌的杀伤（存活细菌为22.9±6.3%，而在无氯化铵的悬液中为8.2±1.3%）。在1 mM时，氯化铵没有显著影响。细胞功能的抑制既不能解释为中性粒细胞死亡的作用（通过台盼蓝染料排除法测定），也不能归因于直接促进细菌生长（在无中性粒细胞的情况下，有无氯化铵时菌落计数相似）或增强对中性粒细胞杀菌机制的抗性（用氯化铵处理并在暴露于中性粒细胞之前洗涤的细菌在存活方面没有改善）。50 mM氯化铵在厌氧箱中也会损害中性粒细胞对金黄色葡萄球菌的杀伤，但对大肠杆菌S15的杀菌活性不受影响。这些发现表明，中性粒细胞对葡萄球菌的最佳杀伤需要高度酸性的溶酶体内隔室，但细菌的摄取则不需要。这可能反映了吞噬泡酸化的原发性失败或质膜自身融合以及与溶酶体膜融合的不同pH要求。对金黄色葡萄球菌和大肠杆菌杀伤作用的差异可能是中性粒细胞对这两种生物体杀菌活性成分相对重要性的结果。
