title: Inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen.
abstract: BACKGROUND
  Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Citrullinated fibrinogen is bound by ACPA but it is less well understood how extracellular proteins are citrullinated. The cells that produce fibrinogen, hepatocytes, do not express peptidyl arginine deiminase (PAD) enzymes nor do PAD enzymes include N-terminal signal peptides to direct them into the secretory pathway. We hypothesized that dying neutrophils release PAD in the extracellular space, and that this could cause citrullination of target extracellular antigens relevant to RA such as fibrinogen.
  
  
  METHODS
  HL60 cells were differentiated into neutrophil-like cells by treatment with all-trans retinoic acid (ATRA). Differentiation was confirmed by CD11b staining, PAD4, PAD2 and myeloperoxidase expression, cell division, and nuclear morphology. Death was induced with various stimuli, including freeze-thaw to induce necrosis, Ionomycin and PMA to induce NETosis, and UV-B to induce apoptosis. Death markers were assessed by immunohistochemistry and flow cytometry. To quantify extracellular citrullination, dying ATRA-differentiated HL60 cells were cultured with fibrinogen for 24 hours and supernatants were probed for fibrinogen citrullination, PAD2 and PAD4 by western blot.
  
  
  RESULTS
  While both NETotic and necrotic ATRA differentiated HL60 cells citrullinated fibrinogen, apoptotic cells did not citrullinate fibrinogen, even when allowed to undergo secondary necrosis. Incubation of necrotic neutrophil lysates with fibrinogen also causes fibrinogen citrullination. PAD2 and PAD4 were detected by western blot of supernatants of ATRA-differentiated HL60 cells undergoing necrotic and NETotic death, but not apoptotic or secondarily necrotic cell death.
  
  
  CONCLUSIONS
  We implicate granulocytes undergoing inflammatory cell death as a mechanism for altering extracellular self-proteins that may be targets of autoimmunity linked to inflammatory diseases such as rheumatoid arthritis.
authors.affiliation: Laboratory of Neuro-Oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. blachen@rockefeller.edu.|Howard Hughes Medical Institute, New York, New York, 10065, USA. blachen@rockefeller.edu.
authors.forename: Nathalie E
authors.initials: NE
authors.lastname: Blachère
authors.affiliation: Laboratory of Neuro-Oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. sparveen@rockefeller.edu.
authors.forename: Salina
authors.initials: S
authors.lastname: Parveen
authors.affiliation: Laboratory of Neuro-Oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. fakj@rockefeller.edu.
authors.forename: John
authors.initials: J
authors.lastname: Fak
authors.affiliation: Laboratory of Neuro-Oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. frankm@rockefeller.edu.|New York Genome Center, 101 Avenue of the Americas, New York, NY, 10013, USA. frankm@rockefeller.edu.
authors.forename: Mayu O
authors.initials: MO
authors.lastname: Frank
authors.affiliation: Laboratory of Neuro-Oncology, The Rockefeller University, 1230 York Avenue, New York, NY, 10065, USA. dorange@rockefeller.edu.|Division of Rheumatology, Hospital for Special Surgery, New York, NY, 10021, USA. dorange@rockefeller.edu.|New York Genome Center, 101 Avenue of the Americas, New York, NY, 10013, USA. dorange@rockefeller.edu.
authors.forename: Dana E
authors.initials: DE
authors.lastname: Orange
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D001323:Autoantibodies / Q000378:metabolism*; D002956:Citrulline / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D018836:Inflammation Mediators; D002956:Citrulline; D005340:Fibrinogen