The influence of agents differentiating HL‐60 cells toward granulocyte‐like cells on their ability to release neutrophil extracellular traps

Abstract Studies on neutrophil extracellular traps ( NET s) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL‐60) cells toward granulocyte‐like cells able to release NET s. HL ‐60 cells were differentiated with all‐trans retinoic acid ( ATRA ), dimethyl sulfoxide ( DMSO ) or dimethylformamide ( DMF ) and stimulated with phorbol 12‐myristate 13‐acetate ( PMA ) or calcium ionophore A23187 ( CI ). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NET s release were measured fluorometrically and analyzed microscopically. LC 3‐ II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL ‐60 cells toward granulocyte‐like cells. ATRA ‐ dHL ‐60 cells released NET s only upon PMA stimulation, DMSO ‐ dHL ‐60 cells only post CI stimulation, while DMF ‐ dHL ‐60 cells formed NET s in response to both stimuli. Oxidative burst was induced in ATRA ‐, DMSO ‐ and DMF ‐ dHL ‐60 cells post PMA stimulation and only in DMF ‐ dHL ‐60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO ‐ and DMF ‐, but not in ATRA ‐ dHL ‐60 cells. The calcium influx was diminished in ATRA ‐ dHL ‐60 cells. Significant increase in autophagosomes formation was observed only in PMA ‐stimulated DMF ‐ dHL ‐60 cells. Phagocytic index was higher in ATRA ‐ dHL ‐60 cells than in control, DMSO ‐ and DMF ‐ dHL ‐60 cells. We conclude that ATRA , DMSO and DMF differentiate HL ‐60 in different mechanisms. DMF is the best stimulus for HL ‐60 cell differentiation for NETs studies.