Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage

Abstract

HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.