title: A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.
abstract: A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: t.kraaij@lumc.nl.
authors.forename: Tineke
authors.initials: T
authors.lastname: Kraaij
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: C.F.Tengstroem@lumc.nl.
authors.forename: Fredrik C
authors.initials: FC
authors.lastname: Tengström
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: s.w.a.kamerling@lumc.nl.
authors.forename: Sylvia W A
authors.initials: SW
authors.lastname: Kamerling
authors.affiliation: Department of Medicine, Renal and Vascular Inflammation Section, Imperial College London, United Kingdom. Electronic address: c.pusey@imperial.ac.uk.
authors.forename: Charles D
authors.initials: CD
authors.lastname: Pusey
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: H.U.Scherer@lumc.nl.
authors.forename: H Ulrich
authors.initials: HU
authors.lastname: Scherer
authors.affiliation: Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: R.E.M.Toes@lumc.nl.
authors.forename: Rene E M
authors.initials: RE
authors.lastname: Toes
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: a.j.rabelink@lumc.nl.
authors.forename: Ton J
authors.initials: TJ
authors.lastname: Rabelink
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: C.van_Kooten@lumc.nl.
authors.forename: Cees
authors.initials: C
authors.lastname: van Kooten
authors.affiliation: Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. Electronic address: y.k.o.teng@lumc.nl.
authors.forename: Y K Onno
authors.initials: YK
authors.lastname: Teng
mesh_terms: D000936:Antigen-Antibody Complex / Q000276:immunology*; D001327:Autoimmune Diseases; D004247:DNA / Q000276:immunology*; D065206:Extracellular Traps / Q000276:immunology*; D006801:Humans; D009504:Neutrophils / Q000276:immunology*; D017382:Reactive Oxygen Species
chemical_list: D000936:Antigen-Antibody Complex; D017382:Reactive Oxygen Species; D004247:DNA
keywords: Autoimmune disease; Neutrophil extracellular traps; Neutrophils