Hua J, Hasebe T, Someya A, Nakamura S, Sugimoto K, Nagaoka I
Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan.
Inflamm Res. 2001 Mar;50(3):156-67. doi: 10.1007/s000110050740.
Superoxide-generating NADPH oxidase consists of the membrane-bound cytochrome b558 (gp91phox and p22Phox) and the cytosolic components (p67phox, p47phox, p40phox and rac). In this study, we evaluated the superoxide-generating activity and the expression of NADPH oxidase components during eosinophilic maturation using HL-60 clone 15 cell line.
HL-60 clone 15 cells were matured to eosinophils by incubation with 0.5 mM butyrate for 7 days, and NADPH oxidase components were detected by Northern blot, Western blot analyses and immunocytochemical staining. Moreover, superoxide-generating activity was examined by nitro blue tetrazolium (NBT) assay.
Northern blot and Western blot analyses revealed that mRNAs and proteins for gp91phox, p67phox and p47phox were expressed after eosinophilic myelocyte stages, whereas mRNAs and proteins for p40phox and rac-2 were expressed from the promyelocyte stage. Interestingly, p22phox mRNA was expressed from the promyelocyte stage, but its protein was expressed after eosinophilic myelocyte stages. Consistent with the results of Western blotting, immunocytochemical staining of butyrate-induced HL-60 clone 15 cells indicated that gp91phox, p22phox, p67phox and p47phox were detected after eosinophilic myelocyte stages (eosinophilic myelocytes, eosinophilic metamyelocytes, eosinophilic band cells and eosinophilic-segmented cells), whereas p40phox and rac-2 were expressed from the promyelocyte stage. Moreover, almost the same results as those with butyrate-treated HL-60 clone 15 cells were obtained using human bone marrow cells by immunocytochemical staining. Furthermore, nitro blue tetrazolium (NBT) assay indicated that superoxide could be produced after eosinophilic myelocyte stages but not produced before the promyelocyte stage.
Together these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during eosinophilic maturation.
产生超氧化物的NADPH氧化酶由膜结合细胞色素b558（gp91phox和p22Phox）和胞质成分（p67phox、p47phox、p40phox和rac）组成。在本研究中，我们使用HL-60克隆15细胞系评估了嗜酸性粒细胞成熟过程中NADPH氧化酶成分的超氧化物生成活性和表达情况。
将HL-60克隆15细胞与0.5 mM丁酸盐孵育7天使其成熟为嗜酸性粒细胞，通过Northern印迹、Western印迹分析和免疫细胞化学染色检测NADPH氧化酶成分。此外，通过硝基蓝四唑（NBT）试验检测超氧化物生成活性。
Northern印迹和Western印迹分析显示，gp91phox、p67phox和p47phox的mRNA和蛋白质在嗜酸性晚幼粒细胞阶段后表达，而p40phox和rac-2的mRNA和蛋白质在早幼粒细胞阶段即开始表达。有趣的是，p22phox mRNA在早幼粒细胞阶段表达，但其蛋白质在嗜酸性晚幼粒细胞阶段后表达。与Western印迹结果一致，丁酸盐诱导的HL-60克隆15细胞的免疫细胞化学染色表明，gp91phox、p22phox、p67phox和p47phox在嗜酸性晚幼粒细胞阶段（嗜酸性晚幼粒细胞、嗜酸性中幼粒细胞、嗜酸性杆状核细胞和嗜酸性分叶核细胞）后被检测到，而p40phox和rac-2从早幼粒细胞阶段开始表达。此外，通过免疫细胞化学染色，使用人骨髓细胞获得了与丁酸盐处理的HL-60克隆15细胞几乎相同的结果。此外，硝基蓝四唑（NBT）试验表明，超氧化物可在嗜酸性晚幼粒细胞阶段后产生，但在早幼粒细胞阶段之前不产生。
这些观察结果共同表明，NADPH氧化酶的所有成分均有表达，且在嗜酸性粒细胞成熟过程中，超氧化物生成活性在晚幼粒细胞阶段后获得。
