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Open Access Highly Accessed Research article

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility

Chanchal Sur Chowdhury1, Stavros Giaglis12, Ulrich A Walker3, Andreas Buser4, Sinuhe Hahn1* and Paul Hasler2*

Author Affiliations

1 Department of Biomedicine, University Hospital Basel, Hebelstrasse 20, 4032 Basel, Switzerland

2 Department of Rheumatology, Kantonsspital Aarau, Tellstrasse, 5001 Aarau, Switzerland

3 Department of Rheumatology, University Hospital Basel, Basel, Switzerland

4 Division of Haematology, Department of Internal Medicine, University Hospital Basel, Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland

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Arthritis Research & Therapy 2014, 16:R122  doi:10.1186/ar4579

Published: 13 June 2014

Abstract

Introduction

Neutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness.

Methods

Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated.

Results

Neutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases.

Conclusions

Signaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.