Effects of Anaplasma phagocytophila on NADPH Oxidase Components in Human Neutrophils and HL-60 Cells

The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O(2)(−)) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22(phox), but not gp91(phox), p47(phox), p67(phox), or P40(phox) reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47(phox), p67(phox), Rac2, and p22(phox) did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22(phox) levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47(phox) and p67(phox) or phosphorylation of p47(phox) upon stimulation by phorbol myristate acetate. The inhibitory signals for O(2)(−) generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22(phox) induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O(2)(−) generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage.

人粒细胞无形体病的病原体嗜吞噬细胞无形体（Anaplasma phagocytophila）仅在粒细胞的细胞质空泡中寄居并增殖。嗜吞噬细胞无形体可快速抑制人中性粒细胞在多种刺激下产生的超氧阴离子（superoxide anion, O₂⁻）。为明确其抑制机制，本研究探讨了嗜吞噬细胞无形体对NADPH氧化酶（NADPH oxidase）各组分的蛋白水平及其定位的影响。实验结果显示，嗜吞噬细胞无形体可降低人外周血中性粒细胞与HL-60细胞中p22(phox)的蛋白水平，但不影响各组分特异性抗体所识别的gp91(phox)、p47(phox)、p67(phox)及P40(phox)的表达量。双重免疫荧光标记实验表明，中性粒细胞与HL-60细胞内的p47(phox)、p67(phox)、Rac2及p22(phox)均未与嗜吞噬细胞无形体包涵体发生共定位，且p22(phox)的蛋白水平同样出现下调。经佛波醇肉豆蔻酸乙酸酯（phorbol myristate acetate）刺激后，嗜吞噬细胞无形体并未阻断胞浆p47(phox)与p67(phox)的膜转位过程，亦未抑制p47(phox)的磷酸化修饰。此外，超氧阴离子生成的抑制信号并不依赖于嗜吞噬细胞无形体内化入细胞所需的多种信号通路。上述结果提示，嗜吞噬细胞无形体结合中性粒细胞所诱导的p22(phox)快速改变，参与了其对超氧阴离子生成的抑制作用；而NADPH氧化酶组分与病原体包涵体未发生共定位，进一步保护了嗜吞噬细胞无形体免受氧化损伤。