Evaluation of the expression of NADPH oxidase components during maturationof HL-60 clone 15 cells to eosinophilic lineage

Objective: Superoxide-generating NADPH oxidase consists of the membrane-bou nd cytochrome b(558) (gp91(phox) and p22(phox)) and the cytosolic component s (p67(phox), g47(phox), p40(phox) and rac). In this study, we evaluated th e superoxide-generating activity and the expression of NADPH oxidase compon ents during eosinophilic maturation using HL-60 clone 15 cell line. Materials and Methods: HL-60 clone 15 cells were matured to eosinophils by incubation with 0.5 mM butyrate for 7 days, and NADPH oxidase components we re detected by Northern blot? Western blot analyses and immunocytochemical staining. Moreover, superoxide-generating activity was examined by nitro bl ue tetrazolium (NBT) assay. Results: Northern blot and Western blot analyses revealed that mRNAs and pr oteins for gp91(phox), p67(phox) and p47(phox) were expressed after eosinop hilic myelocyte stages, whereas mRNAs and proteins for p40(phox) and rac-2 were expressed from the promyelocyte stage. Interestingly, p22(phox) mRNA w as expressed from the promyelocyte stage, but its protein was expressed aft er eosinophilic myelocyte stages. Consistent with the results of Western bl otting, immunocytochemical staining of butyrate-induced HL-60 clone 15 cell s indicated that gp91(phox), p22(phox), p67(phox) and p47(phox) were detect ed after eosinophilic myelocyte stages (eosinophilic myelocytes, eosinophil ic metamyelocytes, eosinophilic band cells and eosinophilic-segmented cells ), whereas p40(phox) and rac-2 were expressed from the promyelocyte stage. Moreover, almost the same results as those with butyrate-treated HL-60 clon e 15 cells were obtained using human bone marrow cells by immunocytochemica l staining. Furthermore, nitro blue tetrazolium (NBT) assay indicated that superoxide could be produced after eosinophilic myelocyte stages but not pr oduced before the promyelocyte stage. Conclusions: Together these observations indicate that all the components f or NADPH oxidase are expressed, and the superoxide-producing activity is ob tained after myelocyte stages during eosinophilic maturation.