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OP0221 Live cell imaging of human neutrophil responses to MSU crystals
  1. S. Chatfield1,2,
  2. B. McKenzie3,
  3. I. Wicks1,2
  1. 1Inflammation Division, Walter and Eliza Hall Institute
  2. 2Rheumatology, Royal Melbourne Hospital
  3. 3CSL Ltd. Bio21 Institute, Parkville, Australia

Abstract

Background Neutrophil Extracellular Traps (NETs) are a recently described antibacterial mechanism (1). Neutrophils stimulated with live bacteria and other stimuli including phorbol myristate acetate (PMA) release nuclear DNA coated with neutrophil granule proteins including myeloperoxidase (MPO) and neutrophil elastase (NE). Gout is an inflammatory disease triggered by monosodium Urate (MSU) crystals (2) and is characterised by neutrophil infiltration and activation. The role of NET formation in the pathogenesis of gout is unclear.

Objectives To assess the kinetics of NET formation in human neutrophils following exposure to MSU crystals in vitro.

Methods Fixed confocal microscopy and live cell imaging were used to characterise extracellular DNA release in response to synthetic MSU crystals (0.05–0.2 mg/ml) and the classical NET stimulus, PMA (27 nM). A computerised quantification system was used to compare the kinetics of NET release with different stimuli and to assess the effect of potential inhibitors.

Results MSU crystals caused DNA release from healthy control human neutrophils. Extracellular DNA structures were coated with MPO and NE, consistent with NETs. MSU crystals caused more rapid DNA release than PMA (Fig. 1, Mean ± SEM, n=3, in duplicate). DNA release following MSU crystal stimulation was evident as early as 20 minutes after stimulation, compared to one hour with PMA. DPI, an inhibitor of NADPH oxidase completely suppressed PMA-induced NET release but had minimal effect on MSU-induced DNA release. Addition of autologous serum significantly abrogated PMA-induced NET release but only delayed MSU-induced DNA release.

Conclusions MSU crystals cause DNA release consistent with NETosis. MSU crystal-induced NET release was not dependent on reactive oxygen species and occurred more rapidly than PMA NET release. This suggests different biochemical pathways are involved, further investigation of which may provide insight into the pathogenesis of gout.

  1. Brinkmann et al. Science. 2004 Mar 5;303(5663):1532-5.

  2. Faires JS, McCarty JD. The Lancet. 1962 Oct 6;280(7258):682-685

Disclosure of Interest None Declared

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