{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T22:33:47Z","timestamp":1793140427096,"version":"4.5.0"},"reference-count":36,"publisher":"Springer Science and Business Media LLC","issue":"3","license":[{"start":{"date-parts":[[2014,6,13]],"date-time":"2014-06-13T00:00:00Z","timestamp":1402617600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2014,6,13]],"date-time":"2014-06-13T00:00:00Z","timestamp":1402617600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Arthritis Res Ther"],"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                        <jats:title>Introduction<\/jats:title>\n                        <jats:p>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.<\/jats:p>\n                     <\/jats:sec><jats:sec>\n                        <jats:title>Methods<\/jats:title>\n                        <jats:p>Neutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed <jats:italic>in vitro<\/jats:italic> 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.<\/jats:p>\n                     <\/jats:sec><jats:sec>\n                        <jats:title>Results<\/jats:title>\n                        <jats:p>Neutrophils from RA cases exhibited increased spontaneous NET formation <jats:italic>in vitro<\/jats:italic>, 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 &gt;97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases.<\/jats:p>\n                     <\/jats:sec><jats:sec>\n                        <jats:title>Conclusions<\/jats:title>\n                        <jats:p>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.<\/jats:p>\n                     <\/jats:sec>","DOI":"10.1186\/ar4579","type":"journal-article","created":{"date-parts":[[2014,6,13]],"date-time":"2014-06-13T09:11:27Z","timestamp":1402650687000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":322,"title":["Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility"],"prefix":"10.1186","volume":"16","author":[{"given":"Chanchal","family":"Sur Chowdhury","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stavros","family":"Giaglis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulrich A","family":"Walker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Buser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sinuhe","family":"Hahn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Hasler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2014,6,13]]},"reference":[{"key":"4223_CR1","doi-asserted-by":"publisher","first-page":"1532","DOI":"10.1126\/science.1092385","volume":"303","author":"V Brinkmann","year":"2004","unstructured":"Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science. 2004, 303: 1532-1535.","journal-title":"Science"},{"key":"4223_CR2","doi-asserted-by":"publisher","first-page":"231","DOI":"10.1083\/jcb.200606027","volume":"176","author":"TA Fuchs","year":"2007","unstructured":"Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A: Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 2007, 176: 231-241.","journal-title":"J Cell Biol"},{"key":"4223_CR3","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1038\/cdd.2011.1","volume":"18","author":"Q Remijsen","year":"2011","unstructured":"Remijsen Q, Kuijpers TW, Wirawan E, Lippens S, Vandenabeele P, Vanden Berghe T: Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ. 2011, 18: 581-588.","journal-title":"Cell Death Differ"},{"key":"4223_CR4","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1083\/jcb.200806072","volume":"184","author":"Y Wang","year":"2009","unstructured":"Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Coonrod SA: Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. 2009, 184: 205-213.","journal-title":"J Cell Biol"},{"key":"4223_CR5","doi-asserted-by":"publisher","first-page":"8674","DOI":"10.1073\/pnas.1301059110","volume":"110","author":"K Martinod","year":"2013","unstructured":"Martinod K, Demers M, Fuchs TA, Wong SL, Brill A, Gallant M, Hu J, Wang Y, Wagner DD: Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. Proc Natl Acad Sci U S A. 2013, 110: 8674-8679.","journal-title":"Proc Natl Acad Sci U S A"},{"key":"4223_CR6","volume-title":"PLoS One","author":"A Gupta","year":"2014","unstructured":"Gupta A, Giaglis S, Hasler P, Hahn S: Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. PLoS One. 2014, 12: doi:10.1371\/journal.pone.0097088"},{"key":"4223_CR7","doi-asserted-by":"publisher","first-page":"2689","DOI":"10.4049\/jimmunol.1201719","volume":"189","author":"MJ Kaplan","year":"2012","unstructured":"Kaplan MJ, Radic M: Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol. 2012, 189: 2689-2695.","journal-title":"J Immunol"},{"key":"4223_CR8","doi-asserted-by":"publisher","first-page":"1146","DOI":"10.1016\/j.humimm.2005.11.003","volume":"66","author":"AK Gupta","year":"2005","unstructured":"Gupta AK, Hasler P, Holzgreve W, Gebhardt S, Hahn S: Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia. Hum Immunol. 2005, 66: 1146-1154.","journal-title":"Hum Immunol"},{"key":"4223_CR9","doi-asserted-by":"publisher","first-page":"73ra19","DOI":"10.1126\/scitranslmed.3001180","volume":"3","author":"R Lande","year":"2011","unstructured":"Lande R, Ganguly D, Facchinetti V, Frasca L, Conrad C, Gregorio J, Meller S, Chamilos G, Sebasigari R, Riccieri V, Bassett R, Amuro H, Fukuhara S, Ito T, Liu YJ, Gilliet M: Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med. 2011, 3: 73ra19-","journal-title":"Sci Transl Med"},{"key":"4223_CR10","doi-asserted-by":"publisher","first-page":"982","DOI":"10.1002\/art.33432","volume":"64","author":"N Dwivedi","year":"2012","unstructured":"Dwivedi N, Upadhyay J, Neeli I, Khan S, Pattanaik D, Myers L, Kirou KA, Hellmich B, Knuckley B, Thompson PR, Crow MK, Mikuls TR, Csernok E, Radic M: Felty\u2019s syndrome autoantibodies bind to deiminated histones and neutrophil extracellular chromatin traps. Arthritis Rheum. 2012, 64: 982-992.","journal-title":"Arthritis Rheum"},{"key":"4223_CR11","doi-asserted-by":"publisher","first-page":"3150","DOI":"10.4049\/jimmunol.1103414","volume":"188","author":"K Tillack","year":"2012","unstructured":"Tillack K, Breiden P, Martin R, Sospedra M: T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses. J Immunol. 2012, 188: 3150-3159.","journal-title":"J Immunol"},{"key":"4223_CR12","doi-asserted-by":"publisher","first-page":"73ra20","DOI":"10.1126\/scitranslmed.3001201","volume":"3","author":"GS Garcia-Romo","year":"2011","unstructured":"Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, Punaro M, Baisch J, Guiducci C, Coffman RL, Barrat FJ, Banchereau J, Pascual V: Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011, 3: 73ra20-","journal-title":"Sci Transl Med"},{"key":"4223_CR13","doi-asserted-by":"publisher","first-page":"178ra140","DOI":"10.1126\/scitranslmed.3005580","volume":"5","author":"R Khandpur","year":"2013","unstructured":"Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, Yalavarthi S, Knight JS, Friday S, Li S, Patel RM, Subramanian V, Thompson P, Chen P, Fox DA, Pennathur S, Kaplan MJ: NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med. 2013, 5: 178ra140-","journal-title":"Sci Transl Med"},{"issue":"7","key":"4223_CR14","doi-asserted-by":"publisher","first-page":"1414","DOI":"10.1136\/annrheumdis-2012-202765","volume":"73","author":"F Pratesi","year":"2014","unstructured":"Pratesi F, Dioni I, Tommasi C, Alcaro MC, Paolini I, Barbetti F, Boscaro F, Panza F, Puxeddu I, Rovero P, Migliorini P: Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps. Ann Rheum Dis. 2014, 73 (7): 1414-1422. doi:10.1136\/annrheumdis-2012-202765","journal-title":"Ann Rheum Dis"},{"key":"4223_CR15","doi-asserted-by":"publisher","first-page":"1609","DOI":"10.1373\/clinchem.2006.084509","volume":"53","author":"XY Zhong","year":"2007","unstructured":"Zhong XY, von M\u00fchlenen I, Li Y, Kang A, Gupta AK, Tyndall A, Holzgreve W, Hahn S, Hasler P: Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis. Clin Chem. 2007, 53: 1609-1614.","journal-title":"Clin Chem"},{"key":"4223_CR16","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1196\/annals.1368.015","volume":"1075","author":"A Gupta","year":"2006","unstructured":"Gupta A, Hasler P, Gebhardt S, Holzgreve W, Hahn S: Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia: a link with elevated levels of cell-free DNA?. Ann N Y Acad Sci. 2006, 1075: 118-122.","journal-title":"Ann N Y Acad Sci"},{"key":"4223_CR17","doi-asserted-by":"publisher","first-page":"13076","DOI":"10.1073\/pnas.1200419109","volume":"109","author":"M Demers","year":"2012","unstructured":"Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT, Wagner DD: Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci U S A. 2012, 109: 13076-13081.","journal-title":"Proc Natl Acad Sci U S A"},{"key":"4223_CR18","doi-asserted-by":"publisher","first-page":"3210","DOI":"10.1111\/trf.12203","volume":"53","author":"TA Fuchs","year":"2013","unstructured":"Fuchs TA, Alvarez JJ, Martinod K, Bhandari AA, Kaufman RM, Wagner DD: Neutrophils release extracellular DNA traps during storage of red blood cell units. Transfusion. 2013, 53: 3210-3216.","journal-title":"Transfusion"},{"key":"4223_CR19","doi-asserted-by":"publisher","first-page":"1777","DOI":"10.1161\/ATVBAHA.111.242859","volume":"32","author":"TA Fuchs","year":"2012","unstructured":"Fuchs TA, Brill A, Wagner DD: Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2012, 32: 1777-1783.","journal-title":"Arterioscler Thromb Vasc Biol"},{"key":"4223_CR20","doi-asserted-by":"publisher","first-page":"369","DOI":"10.1189\/jlb.0711387","volume":"91","author":"H Parker","year":"2012","unstructured":"Parker H, Albrett AM, Kettle AJ, Winterbourn CC: Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide. J Leukoc Biol. 2012, 91: 369-376.","journal-title":"J Leukoc Biol"},{"key":"4223_CR21","doi-asserted-by":"publisher","first-page":"623","DOI":"10.1038\/nm.1959","volume":"15","author":"K Kessenbrock","year":"2009","unstructured":"Kessenbrock K, Krumbholz M, Schonermarck U, Back W, Gross WL, Werb Z, Grone HJ, Brinkmann V, Jenne DE: Netting neutrophils in autoimmune small-vessel vasculitis. Nat Med. 2009, 15: 623-625.","journal-title":"Nat Med"},{"key":"4223_CR22","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1021\/tx00026a012","volume":"5","author":"CP LeBel","year":"1992","unstructured":"LeBel CP, Ischiropoulos H, Bondy SC: Evaluation of the probe 2\u2032,7\u2032-dichlorofluorescein as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol. 1992, 5: 227-231.","journal-title":"Chem Res Toxicol"},{"key":"4223_CR23","doi-asserted-by":"publisher","first-page":"3193","DOI":"10.1016\/j.febslet.2010.06.006","volume":"584","author":"AK Gupta","year":"2010","unstructured":"Gupta AK, Joshi MB, Philippova M, Erne P, Hasler P, Hahn S, Resink TJ: Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death. FEBS Lett. 2010, 584: 3193-3197.","journal-title":"FEBS Lett"},{"key":"4223_CR24","first-page":"413","volume":"3","author":"V Brinkmann","year":"2012","unstructured":"Brinkmann V, Goosmann C, Kuhn LI, Zychlinsky A: Automatic quantification of in vitro NET formation. Front Immunol. 2012, 3: 413-","journal-title":"Front Immunol"},{"key":"4223_CR25","doi-asserted-by":"publisher","first-page":"1445","DOI":"10.1038\/nprot.2007.202","volume":"2","author":"D Shechter","year":"2007","unstructured":"Shechter D, Dormann HL, Allis CD, Hake SB: Extraction, purification and analysis of histones. Nat Protoc. 2007, 2: 1445-1457.","journal-title":"Nat Protoc"},{"key":"4223_CR26","doi-asserted-by":"publisher","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","volume":"55","author":"SA Bustin","year":"2009","unstructured":"Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009, 55: 611-622.","journal-title":"Clin Chem"},{"key":"4223_CR27","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","volume":"25","author":"KJ Livak","year":"2001","unstructured":"Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408.","journal-title":"Methods"},{"key":"4223_CR28","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1083\/jcb.201006052","volume":"191","author":"V Papayannopoulos","year":"2010","unstructured":"Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A: Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol. 2010, 191: 677-691.","journal-title":"J Cell Biol"},{"key":"4223_CR29","doi-asserted-by":"publisher","first-page":"1853","DOI":"10.1084\/jem.20100239","volume":"207","author":"P Li","year":"2010","unstructured":"Li P, Li M, Lindberg MR, Kennett MJ, Xiong N, Wang Y: PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med. 2010, 207: 1853-1862.","journal-title":"J Exp Med"},{"key":"4223_CR30","doi-asserted-by":"publisher","first-page":"11727","DOI":"10.1021\/bi061180d","volume":"45","author":"Y Luo","year":"2006","unstructured":"Luo Y, Arita K, Bhatia M, Knuckley B, Lee YH, Stallcup MR, Sato M, Thompson PR: Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization. Biochemistry. 2006, 45: 11727-11736.","journal-title":"Biochemistry"},{"key":"4223_CR31","doi-asserted-by":"publisher","first-page":"2205","DOI":"10.1056\/NEJMra1004965","volume":"365","author":"IB McInnes","year":"2011","unstructured":"McInnes IB, Schett G: The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011, 365: 2205-2219.","journal-title":"N Engl J Med"},{"key":"4223_CR32","doi-asserted-by":"publisher","first-page":"801","DOI":"10.1016\/j.autrev.2012.02.009","volume":"11","author":"I Auger","year":"2012","unstructured":"Auger I, Charpin C, Balandraud N, Martin M, Roudier J: Autoantibodies to PAD4 and BRAF in rheumatoid arthritis. Autoimmun Rev. 2012, 11: 801-803.","journal-title":"Autoimmun Rev"},{"key":"4223_CR33","doi-asserted-by":"publisher","first-page":"186ra165","DOI":"10.1126\/scitranslmed.3005370","volume":"5","author":"E Darrah","year":"2013","unstructured":"Darrah E, Giles JT, Ols ML, Bull HG, Andrade F, Rosen A: Erosive rheumatoid arthritis is associated with antibodies that activate PAD4 by increasing calcium sensitivity. Sci Transl Med. 2013, 5: 186ra165-","journal-title":"Sci Transl Med"},{"key":"4223_CR34","doi-asserted-by":"publisher","first-page":"516","DOI":"10.1016\/j.copbio.2011.12.024","volume":"23","author":"FC Simmel","year":"2012","unstructured":"Simmel FC: DNA-based assembly lines and nanofactories. Curr Opin Biotechnol. 2012, 23: 516-521.","journal-title":"Curr Opin Biotechnol"},{"key":"4223_CR35","doi-asserted-by":"publisher","first-page":"483","DOI":"10.1136\/annrheumdis-2013-203844","volume":"73","author":"N Dwivedi","year":"2014","unstructured":"Dwivedi N, Radic M: Citrullination of autoantigens implicates NETosis in the induction of autoimmunity. Ann Rheum Dis. 2014, 73: 483-491.","journal-title":"Ann Rheum Dis"},{"key":"4223_CR36","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1186\/ar3740","volume":"14","author":"BA Fisher","year":"2012","unstructured":"Fisher BA, Venables PJ: Inhibiting citrullination in rheumatoid arthritis: taking fuel from the fire. Arthritis Res Ther. 2012, 14: 108-","journal-title":"Arthritis Res Ther"}],"container-title":["Arthritis Research &amp; Therapy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/ar4579.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/ar4579\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/ar4579.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,10,14]],"date-time":"2024-10-14T16:17:57Z","timestamp":1728922677000},"score":1,"resource":{"primary":{"URL":"https:\/\/arthritis-research.biomedcentral.com\/articles\/10.1186\/ar4579"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,6,13]]},"references-count":36,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2014,6]]}},"alternative-id":["4223"],"URL":"https:\/\/doi.org\/10.1186\/ar4579","relation":{},"ISSN":["1478-6354"],"issn-type":[{"value":"1478-6354","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,6,13]]},"assertion":[{"value":"12 July 2013","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"21 May 2014","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 June 2014","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"R122"}}