Background/Purpose:

Protein citrullination, the post-translational conversion of arginine to citrulline, mediated by peptidylarginine deiminase (PAD) enzymes, is considered a likely mechanism for the stimulation of anti-citrullinated protein antibodies (ACPA) in patients with rheumatoid arthritis (RA).  Hypercitrullination, the citrullination of multiple intracellular proteins, was recently demonstrated in synovial fluid cells from RA patients but was not found in other physiologic processes involving citrullination, such as NETosis (Romero et al., Sci Transl Med, 2013). This unique form of citrullination is proposed to occur via immune-mediated, pore-forming membranolytic pathways causing neutrophil cell death; one mediated by cytotoxic cells through the perforin-granzyme pathway, and the other mediated by complement activation and formation of the membrane attack complex (MAC). Since the evidence to support this hypothesis was derived from simulated in vitro systems, we explored the naturally occurring humoral mechanisms required for induction of complement-mediated neutrophil lysis.  

Methods:

Neutrophils (PMNs) isolated from synovial fluid (SF) or peripheral blood (PB) of RA patients as well as in healthy controls (HC) were exposed to the inflammatory milieu found in RA joints. PMNs isolated from SF (n=3) and PB (n=25)  of ACPA-positive RA patients as well as PB of HC (n=10) were screened for anti-neutrophil binding IgG, C3bi and C5b-9 complement component deposition, as well as PMN viability using annexin V and propidium iodide staining by flow cytometry analysis.  In addition, PMNs isolated from HC were incubated with serum or SF from RA patients or normal human serum from the same healthy controls. 

Results:

In the periphery, in the majority of patient samples, RA PMNs had highly increased mean fluorescence intensity (MFI) ratio for C3bi cell-surface deposition as compared to healthy controls (p<0.05), but no differences were seen in MFI of C5b-9 deposition. However, in SF samples, PMNs had elevated MFI ratio of C5b-9 as compared to PB PMNs from HC (p<0.05) and paired RA patients (P< 0.0001). Complement deposition on SF PMNs was also associated with decreased cell surface expression of the complement regulatory molecule CD46, as well as increased cell death, with more than 50% of the SF PMNs undergoing secondary necrosis, consistent with the notion of complement-mediated killing of SF PMNs.  Moreover, incubation of HC PMNs with 15% RA SF, but not 15% serum from paired RA patients or HC induced C3bi and C5b-9 deposition, reduction in CD46 expression, and PMN lysis via secondary necrosis.

Conclusion:

RA PMNs, in particular SF PMNs, demonstrate increased complement deposition associated with PMN cell death both in vivo and in vitro. Elucidating the cause of complement deposition and the role of complement regulatory proteins in promoting PMN lysis may help to uncover a mechanism for hypercitrullination, ACPA formation and RA disease propagation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/complement-mediated-neutrophil-lysis-a-mechanism-promoting-hypercitrullination-in-rheumatoid-arthritis/