ANALYSIS OF THE NADPH OXIDASE COMPONENTS DURING DIFFERENTIATION OF HL-60 CELLS TO EOSINOPHILIC LINEAGE

HL-60 cells were induced to differentiate into eosinophil-like cells w ith sodium butyrate after passage under mild alkaline condition. The d ifferentiating cells gradually possessed the Luxol-fast-blue (LFB) sta ining-positive granules and the capacity to produce superoxide. The in crease in the amounts of cytochrome b-558 paralleled the superoxide an ion generating activity. Immunoblot analysis demonstrated that p47-pho x 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 i n 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 myris tate acetate (PMA) increased with increasing amounts of cytochrome b-5 58 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on t he 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.