单核-巨噬细胞和中性粒细胞抗问号钩端螺旋体感染能力及吞噬溶酶体形成差异性研究

Differences in engulfing ability and phagolysosome formation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection

目的了解单核-巨噬细胞和中性粒细胞抗问号钩端螺旋体(简称钩体)感染能力及吞噬溶酶体形成是否存在差异。方法人THP-1单核细胞和HL-60细胞分别用佛波酯(PMA)和全反式维甲酸(ATRA)预处理,使其分化为单核-巨噬细胞和中性粒细胞。采用激光共聚焦显微镜法检测THP-1-PMA单核-巨噬细胞和HL-60-ATRA中性粒细胞吞噬问号钩体能力。采用透射电镜法检测胞内钩体形态。采用激光共聚焦显微镜法和荧光分光光度法检测胞内问号钩体活力和死亡率。采用激光共聚焦显微镜法检测不同吞噬细胞内吞噬溶酶体形成率。结果THP-1-PMA单核-巨噬细胞和HL-60-ATRA中性粒细胞均能吞噬问号钩体,但前者吞噬问号钩体能力显著强于后者(P<0.05)。胞内问号钩体都以吞噬泡的形式存在于上述两种吞噬细胞内。THP-1-PMA单核-巨噬细胞杀灭胞内问号钩体能力显著强于HL-60-ATRA中性粒细胞(P<0.05)。THP-1-PMA单核-巨噬细胞内问号钩体与溶酶体共定位率显著高于相应的HL-60-ATRA中性粒细胞(P<0.05)。结论单核-巨噬细胞而非中性粒细胞作为主要的吞噬细胞在吞噬、杀灭入侵问号钩体过程中起着重要作用。中性粒细胞内较低的吞噬溶酶体形成率可能是导致其杀灭问号钩体能力较低的原因。

Objective To understand the differences in engulfing ability and phagolysosome formation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection.Methods Human THP-1 monocytes and HL-60 cells were pretreated with PMA(phorbol-12-myristate-13-acetate)and ATRA(all-trans retinoic acid)to differentiate them into mononuclear-macrophages and neutrophils,respectively.The phagocytosis of Leptospira interrogans in THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils was detected by confocal microscopy.The morphology of intracellular Leptospira was determined by transmission electron microscopy.The viability of phagocytized Leptospira and the percentages of dead Leptospira were analyzed by confocal microscopy and spectrofluorimetry,respectively.Confocal microscopy was used to measure the formation of phagolysosomes in different phagocytes.Results Both THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils could phagocytize Leptospira interrogans,but the phagocytic ability of the former was notably stronger than that of the latter(P<0.05).Intracellular Leptospira were surrounded by phagocytic vesicles in both types of phagocytes.THP-1-PMA mononuclear-macrophages were better than HL-60-ATRA neutrophils in killing intracellular Leptospira(P<0.05).More phagolysosomes were formed in THP-1-PMA mononuclear-macrophages than in HL-60-ATRA neutrophils(P<0.05).Conclusions Human mononuclear-macrophages but not neutrophils act as major phagocytes that play an important role in phagocytizing and killing Leptospira during infection.Less fusion of the phagosomes with lysosomes may be responsible for the lower Leptospira-killing ability of neutrophils.