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

Abstract

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.