Stearylamine Causes the Formation of Neutrophil Extracellular Traps Independently of Reactive Oxygen Species

Abstract

Neutrophils can form extracellular traps that consist of chromatin and granule proteins and can, acting as a net, trap bacteria. The trap formation process called NETosis that has been thoroughly characterized in cells exposed to phorbol ester (PMA) takes 2–3 h and depends on reactive oxygen species produced by NADPH oxidase (this is called classical NETosis). The aim of the present work was to study the distinctive features of the NETosis process evoked by stearylamine (SA) dissolved in DMSO or incorporated into phosphatidylcholine (PC) liposomes and to compare it to NETosis evoked by PMA. Human neutrophils were incubated with 0.2 mg/mL SA (at a 2% DMSO content in the medium) or with cationic PC liposomes that contained SA (PC–SA liposomes; PC and SA concentrations 1.8 and 0.2 mg/mL, respectively). Confocal fluorescence microscopy of fixed neutrophil preparations showed that SA (dissolved in DMSO or incorporated into liposomes) caused the formation of neutrophil extracellular traps. NETosis kinetics were analyzed in the real-time mode in live cells exposed to fluorescently labeled PC–SA liposomes. PC–SA liposomes added to the neutrophils were shown to adsorb to isolated patches of the plasma membrane at first and to occupy the entire membrane surface as the incubation time was prolonged. This was accompanied by chromatin decondensation and fusion of the nuclear material and the cytoplasm, that is, the stages observed for phorbol ester-induced NETosis. However, SA-evoked NETosis was much faster (30–90 min) than PMA-evoked NETosis. It is necessary to emphasize that SA did not induce an oxidative burst (in contrast to PMA), as revealed by analysis of luminol-dependent chemiluminescence. Trap formation by neutrophils exposed to PC–SA liposomes was not affected by apocynin and DPI (NADPH oxidase inhibitors) or catalase, and this also shows that SA stimulates ROS-independent formation of neutrophil extracellular traps.