title: Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.
abstract: A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.
authors.affiliation: Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091, Warsaw, Poland. Electronic address: magdalena.ostafin@wum.edu.pl.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ostafin
authors.affiliation: Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091, Warsaw, Poland; Postgraduate School of Molecular Medicine, 02-091, Warsaw, Poland.
authors.forename: Michal Przemyslaw
authors.initials: MP
authors.lastname: Pruchniak
authors.affiliation: Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091, Warsaw, Poland.
authors.forename: Olga
authors.initials: O
authors.lastname: Ciepiela
authors.affiliation: Faculty of Medicine, Technion-Israel Institute of Technology, Technion City, Haifa, 32000, Israel.
authors.forename: Abraham Zeev
authors.initials: AZ
authors.lastname: Reznick
authors.affiliation: Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091, Warsaw, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Demkow
mesh_terms: D065206:Extracellular Traps / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D009861:Onium Compounds; D017382:Reactive Oxygen Species; C007517:diphenyleneiodonium
keywords: Diphenyleneiodonium chloride; Free radicals; NADPH oxidase; Neutrophil extracellular traps; Reactive oxygen species