Polymyxin-Induced Cell Death of Human Macrophage-Like THP-1 and Neutrophil-Like HL-60 Cells Associated with the Activation of Apoptotic Pathways.

ABSTRACT

Innate immunity is crucial for the host to defend against infections, and understanding the effect of polymyxins on innate immunity is important for optimizing their clinical use. In this study, we investigated the potential toxicity of polymyxins on human macrophage-like THP-1 and neutrophil-like HL-60 cells. Differentiated THP-1 human macrophages (THP-1-dMs) and HL-60 human neutrophils (HL-60-dNs) were employed. Flow cytometry was used to measure the concentration-dependent effects (100 to 2,500 µM for THP-1-dMs and 5 to 2,500 µM for HL-60-dNs) and time-dependent effects (1,000 µM for THP-1-dMs and 300 µM for HL-60-dNs) of polymyxin B over 24 h. Effects of polymyxin B on mitochondrial activity, activation of caspase-3, caspase-8, and caspase-9, and Fas ligand (FasL) expression in both cell lines were examined using fluorescence imaging, colorimetric, and fluorometric assays. In both cell lines, polymyxin B induced concentration- and time-dependent loss of viability at 24 h with 50% effective concentration (EC50) values of 751.8 µM (95% confidence interval [CI], 692.1 to 816.6 µM; Hill slope, 3.09 to 5.64) for THP-1-dM cells and 175.4 µM (95% CI, 154.8 to 198.7 µM; Hill slope, 1.42 to 2.21) for HL-60-dN cells. A concentration-dependent loss of mitochondrial membrane potential and generation of mitochondrial superoxide was also observed. Polymyxin B-induced apoptosis was associated with concentration-dependent activation of all three tested caspases. The death receptor apoptotic pathway activation was demonstrated by a concentration-dependent increase of FasL expression. For the first time, our results reveal that polymyxin B induced concentration- and time-dependent cell death in human macrophage-like THP-1 and neutrophil-like HL-60 cells associated with mitochondrial and death receptor apoptotic pathways.