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FRI0059 Complement-Mediated Neutrophil Lysis - A Mechanism Promoting Hypercitrullination in Rheumatoid Arthritis?
  1. T. Gazitt,
  2. C. Lood,
  3. J. Ledbetter,
  4. K.B. Elkon
  1. Rheumatology, University of Washington, Seattle, United States

Abstract

Background Protein citrullination, the post-translational conversion of arginine to citrulline, is thought to participate in the development of anti-citrullinated protein antibodies (ACPA) in patients with rheumatoid arthritis (RA). Recently, synovial fluid cells from RA patients were found to have citrullination of several intracellular proteins, a process named hypercitrullination. This unique form of citrullination did not occur in other types of cell death such as apoptosis or NETosis, but was proposed to be induced only through pore-forming membranolytic pathways, including complement-mediated cell lysis through the membrane attack complex.

Objectives To explore the naturally occurring humoral mechanisms required for induction of complement-mediated neutrophil lysis in the RA joint.

Methods Neutrophils (PMNs) isolated from synovial fluid (SF) (n=4) or peripheral blood (PB) (n=25) of ACPA-positive RA patients as well as PB of healthy controls (HC) (n=12) were analyzed for anti-PMN binding IgG, C3bi and C5b-9 complement 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 4 matched RA serum and SF samples as well as 7 additional unmatched RA SF samples, or normal human serum (NHS); complement deposition, complement regulatory protein CD46 expression, and PMN viability were then measured. Finally, the kinetics by which HC PMNs underwent cell death upon stimulation with complement-fixing antibodies (Abs) and NHS were analyzed using the IncuCyte ZOOM® live cell imaging system.

Results In the periphery, RA PMNs had highly increased mean fluorescence intensity (MFI) ratio for C3bi cell-surface deposition as compared to HC (p=0.0183), but no differences were seen for C5b-9 deposition. However, in SF, RA PMNs had elevated MFI ratio for both C5b-9 deposition (p=0.0121) as well as anti-PMN binding IgG deposition (p=0.0011) as compared to PB PMNs from HC, or paired PB PMNs from RA patients (p=0.0177, p=0.0113, respectively). Consistent with the selective increase in complement C5b-9 deposition in SF, we observed a decrease in the expression of CD46 in SF, but not PB PMNs of RA patients (p=0.0270), suggesting a potential mechanism through which SF PMNs are sensitized to complement-mediated lysis. Further, PMNs isolated from SF were, to a higher extent than PB PMNs, undergoing secondary necrosis, supporting the notion of complement-mediated lysis of SF PMNs in vivo. Finally, adding RA SF, but not RA serum, to HC PMNs induced markedly elevated C3bi and C5b-9 deposition, reduction in CD46 levels and PMN cell death, clearly demonstrating the PMN cytotoxic properties of RA SF in 10/11 samples, as well as suggesting an important role of complement activation in this process. IgG- and complement-mediated lysis of PMNs was also verified in vitro by live cell imaging.

Conclusions RA 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 CD46 in promoting PMN lysis may help to uncover a mechanism for hypercitrullination, ACPA formation and RA disease propagation.

  1. Holers, V.M. Annu. Rev. Immunol (2014) 32:433–59.

  2. Romero, V. et al. Sci Transl Med (2013) 5 (209):1–10.

Disclosure of Interest None declared

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