Investigation of cytokine and midkine responses of human THP-1 leukemia cells induced by phorbol-12-Myristate-13-Acetate (PMA) at different concentrations and times

Abstract

Macrophages are accepted as cells that initially contact with the pathogens and initiate the innate immune response. They play effective roles in innate immune and inflammatory responses by intercellular relations and inflammatory mediator secretion. Human THP-1 leukemia cells are frequently used for the in vitro determination of the signal pathways, and the functions of macrophages. Phorbol-12-Myristate-13-Acetate (PMA) is commonly used to induce macrophage differentiation of monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. Midkine acts as a cytokine and growth factor which organizes proliferation, differentiation, survival, adhesion and migration of immune cells. The aim of this study was to observe the differences in the secretion of midkine, TNF-α, IL-10 and IFN-γ of macrophages differentiated from monocytes when stimulated with different doses of PMA for different durations. For this purpose, THP-1 monocytic cells were proliferated by PMA at 24, 48 and 72 hours by using the concentrations of 10 ng/ml, 20 ng/ml, 40 ng/ml and 60 ng/ml. Midkine, TNF-α, IL-10 and IFN-γ cytokine levels were determined by ELISA in the supernatants of the cells collected at the end of Incubation times. PMA stimuli initiated changes that were indicative of differentiation in the cell morphology. Differentiation of cells by PMA induced midkine, TNF-α, IL-10 and IFN-γ secretions in monocytic cells even at the lowest dosage (10 ng/ml). PMA caused cytotoxicity in the cells when the dosages were increased (> 20 ng/ml). THP-1 cells have a basal secretion of midkine and are increased by dosage dependent with PMA stimulation. Midkine secretion has shown changes dependent with dosage and time. A difference was also observed in the cytokine profile of PMA stimulated cells at different doses. The results indicated that the differentiation of THP-1 monocytes to macrophages requires optimization to ensure that this in vitro macrophage model more precisely reflects real in vivo physiologic conditions. As a conclusion the results have shown that a modified PMA differentiation protocol (20 ng/ml and 48 hours Incubation) might enhance macrophage differentiation of THP-1 cells without induced cell death (viability 92.2%) and cytokine secretion and midkine responses were the important discriminators of the level of macrophage differentiation.