Moreover, Cell Cycle inhibitor differences in the nature of cell stimuli has
been proposed as the reason for NETs consisting of either nuclear DNA [3], mitochondrial DNA [6] or a combination of both [16]. While the majority of reports of NET release involve the eventual rupture of the neutrophil plasma membrane [3,4,15], early S. aureus-stimulated NET release (5–60 min) has been reported to occur via a process akin to exocytosis without plasma membrane rupture [16]. Furthermore, NETs comprising only mitochondrial DNA are reported to originate from cells remaining viable [6]. Here we demonstrate, for the first time, the requirement of hypochlorous acid (HOCl) for NET release and a potential regulatory role for endogenous taurine in this process. In our studies, for NET stimulation, PMA was employed to stimulate PKC in place of the endogenous activator, diacylglycerol. PMA, which is known to stimulate NADPH oxidase generation of ROS in neutrophils, has also been reported to elicit dramatic NET release [3]. Although less physiologically relevant, PMA provides direct intracellular stimulation, removing the complication of multiple simultaneous signalling pathways and responses that are likely to be evoked in neutrophils
stimulated with more physiologically relevant receptor-mediated stimuli such as un-opsonized (Toll-like receptor; TLR) or opsonized (Fcγ-receptor) bacteria. In addition, this form of stimulation is consistent with many previously reported studies. RPMI-1640 was obtained from Biosera (Ringmer, UK), Percoll from Volasertib cell line GE Healthcare (Little Chalfont, UK), SYTOX green nucleic acid stain was obtained from Invitrogen (Paisley, UK), 96-well plates were from Corning (Lowell, MA, USA), InnoZyme Myeloperoxidase Activity Kit was from Calbiochem (Nottingham, UK), micrococcal nuclease was from Worthington Biochemical Corporation (Lakewood, NJ, USA) and tryptone soy agar and broth were from Oxoid (ThermoFisher Scientific, Basingstoke, UK). All other chemicals were purchased from Sigma (Gillingham, UK). Unless specified otherwise, all ex-vivo experiments were conducted using human neutrophils from medically healthy volunteers and were isolated from venous blood by discontinuous
Rutecarpine Percoll density gradient followed by ammonium chloride lysis of red blood cells, as described previously [19]. Patients with chronic granulomatous disease (CGD) were recruited from the Department of Immunology, Birmingham Heartlands Hospital, following informed consent (West Midlands Research Ethics Committee number 10/H/1208/48). Neutrophils (1 × 105) in RPMI-1640 were seeded into bovine serum albumin (BSA)-coated [1% in phosphate-buffered saline (PBS)] 96-well plates and allowed to settle for 30 min at 37°C in the presence of the inhibitors or enzymes being tested. Cells were stimulated with 25 nM PMA or 0·75 mM HOCl and incubated for 3 h at 37°C [2]. NET-DNA was quantified using a modified version of a previously published method [20–22].