Abstract
BackgroundNeutrophil 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.MethodsHL60 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.ResultsWhile 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0890-0) contains supplementary material, which is available to authorized users.
Highlights
Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA)
These data confirmed that all-trans retinoic acid (ATRA) treatment differentiated HL60 cells (ATRA/ HL60) into a neutrophil-like cell, with increased expression of PAD2 and PAD4 and cell surface expression of CD11b
Perhaps the more surprising finding is that apoptotic and even secondarily necrotic cells did not citrullinate extracellular fibrinogen in culture. This result indicates that death by apoptosis deactivates the peptidyl arginine deiminase (PAD) enzymes before they are released into the extracellular space and is consistent with prior work demonstrating that PAD4mediated histone deimination is induced by inflammatory stimuli in neutrophils but not apoptosis [16]
Summary
Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Autoantibodies to citrullinated fibrinogen are 98 % specific for RA [3] and proteomic studies of synovial tissue and fluid have identified citrullinated fibrinogen as one of the major sources of citrullinated antigen in the joints in RA [4,5,6] It is not known how extracellular proteins such as fibrinogen are citrullinated because the cells that produce fibrinogen, hepatocytes, do not express any of the peptidylarginine deiminase (PAD) family of enzymes nor do any of the PAD enzymes include Nterminal signal peptides that would direct them into the secretory pathway [7]. A better understanding of the mechanisms by which fibrinogen can become citrullinated in the context of inflammation would facilitate dissection of the immune pathways that lead to autoreactivity to citrullinated extracellular antigens
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