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Evaluation of the expression of NADPH oxidase components during maturation of HL‐60 cells to neutrophil lineage
Author(s) -
Hua Jian,
Hasebe Takeshi,
Someya Akimasa,
Nakamura Shinji,
Sugimoto Koichi,
Nagaoka Isao
Publication year - 2000
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1189/jlb.68.2.216
Subject(s) - promyelocyte , nadph oxidase , western blot , microbiology and biotechnology , biology , superoxide , staining , bone marrow , biochemistry , immunology , enzyme , gene , genetics
To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide‐producing activity using HL‐60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91 phox , p67 phox , and p47 phox were expressed after myelocyte stages, whereas p22 phox , p40 phox , and rac‐2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO‐induced HL‐60 cells indicated that gp91 phox , p67 phox , and p47 phox were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22 phox , p40 phox , and rac‐2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO‐induced HL‐60 cells were obtained using human bone‐marrow cells by immunocytochemical staining and NBT assay, except that p22 phox was detected by immunocytochemical staining after myelocyte stages in bone‐marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide‐producing activity is obtained after myelocyte stages during neutrophil maturation.