Since the first description of NETs , studies have attempted to elucidate the molecular signalling pathways regulating their release. While there are likely to be a multitude of
converging factors regulating this process, research has focused upon the pathway involving nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of ROS. The importance of the NADPH oxidase to NET release was first demonstrated by studies employing the oxidase inhibitor, Selleck DMXAA diphenylene iodonium (DPI) which, when added extracellularly and prior to stimulation of ‘NETosis’, reduced NET release . The NADPH oxidase generates superoxide which either dismutates spontaneously to hydrogen peroxide (H2O2) or is reduced more efficiently by the enzyme family of superoxide dismutases  (SOD; Fig. 1). The generation of H2O2 was shown to be sufficient to elicit NET release and the requirement for this signalling molecule was confirmed subsequently by studies utilizing catalase to remove H2O2 (by reduction to H2O and O2) and which was found to inhibit NET PD98059 research buy release. In contrast, the catalase inhibitor 3-aminotriazole (3-AT) increased NET release by elevating levels of available H2O2. Most recently, an inhibitor of myeloperoxidase (MPO) (aminobenzoic acid hydrazide,
4-ABAH) has been reported to reduce NET release, Lck indicating the potential requirement for this enzyme in the process [12,13]. Independently, NADPH oxidase generation of ROS has been found to be required specifically for the chromatin decondensation step that is a necessary prerequisite for NET formation . The decondensation of neutrophil nuclear chromatin prior to NET extrusion into the extracellular space has also been demonstrated to require citrullination
of histones by the enzyme peptidylarginine deiminase-4 , and also neutrophil elastase . NET biology is a relatively new area of study and with the literature growing rapidly there are various reports of apparently conflicting data concerning the mechanisms of NET release. This may be due in part to the inherent challenges associated with quantifying NET release, such that descriptive analyses form a substantial component of the reported evidence base. For example, NET release has been reported to be both NAPDH oxidase-independent  and NADPH oxidase-dependent [3,6,17]. The reason for the apparently discordant data may, in part, relate to different stimuli being employed; for example, although phorbol myristate acetate (PMA) and Helicobacter pylori elicit NADPH oxidase-dependent NET release, this activation occurs via different pathways, either protein kinase C (PKC)-dependent or -independent, respectively .