title: Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.
abstract: BACKGROUND
  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.
authors.forename: Chanchal
authors.initials: C
authors.lastname: Sur Chowdhury
authors.forename: Stavros
authors.initials: S
authors.lastname: Giaglis
authors.forename: Ulrich A
authors.initials: UA
authors.lastname: Walker
authors.forename: Andreas
authors.initials: A
authors.lastname: Buser
authors.forename: Sinuhe
authors.initials: S
authors.lastname: Hahn
authors.forename: Paul
authors.initials: P
authors.lastname: Hasler
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood / Q000175:diagnosis / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D004247:DNA / Q000276:immunology / Q000378:metabolism; D065206:Extracellular Traps / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006657:Histones / Q000276:immunology / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007150:Immunohistochemistry; D019272:Leukocyte Elastase / Q000276:immunology / Q000378:metabolism; D008297:Male; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D009707:Nucleosomes / Q000276:immunology / Q000378:metabolism; D010456:Peptides, Cyclic / Q000276:immunology; D009195:Peroxidase / Q000276:immunology / Q000378:metabolism; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D013582:Synovial Fluid / Q000276:immunology
chemical_list: D001323:Autoantibodies; D006657:Histones; D009707:Nucleosomes; D010456:Peptides, Cyclic; D017382:Reactive Oxygen Species; C487763:cyclic citrullinated peptide; D002956:Citrulline; D004247:DNA; D009195:Peroxidase; D006867:Hydrolases; D019272:Leukocyte Elastase; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases