Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct 30;5(209):209ra150.
doi: 10.1126/scitranslmed.3006869.

Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis

Violeta Romero  1 , Justyna Fert-Bober, Peter A Nigrovic, Erika Darrah, Uzma J Haque, David M Lee, Jennifer van Eyk, Antony Rosen, Felipe Andrade
Affiliations

Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis

Violeta Romero et al. Sci Transl Med. .

Abstract

Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. RA SF cells show hypercitrullination and extrinsic apoptotic cell death
(A) Protein citrullination in SF cells from 12 RA patients was analyzed by immunoblotting electrophoresed cell lysate using AMC and γ actin (loading control) antibodies. For RA-SF2, data from three samples collected on different dates is shown. Ionomycin was used as a positive control for citrullination. Intervening lanes containing irrelevant data between lanes 2 and 3 were spliced out. PMN, polymorphonuclear cells.(B and E) SF cell lysate was immunoblotted using PR3, CD14, caspase-3 (B), and BID (E) antibodies. Neutrophilsdying by anti-Fas (B and E) or spontaneous apoptosis (E) were positive controls for caspase-3activation, and granzyme B/perforin (GzmB/Pfn) and TNFα/cycloheximide (CHX) (E) were positive controls for BID activation. Filled arrows denote intact proteins, and the unfilled arrows mark active products. pBID, phosphorylated BID; tBID, truncated BID. (C, D, and F) Expression of PR3 and CD14 and cleavage of caspase-3and BID were quantified by densitometry and normalized to actin. Linear correlations were analyzed using Pearson’s correlation coefficient using a two-tailed α of 0.05. GraphPad Prism 5.0 was used to perform analysis and generate figures. (G) Citrullination, expression of PR3 and CD14, and caspase-3 and BID cleavage were quantified by densitometry and normalized to actin. The normalized values were distributed from 0 (low) to 2 (high) and subjected to unsupervised hierarchical clustering using the Cluster and TreeView software programs (71).
Fig. 2
Fig. 2. The granzyme B/perforin pathway efficiently induces hypercitrullination in neutrophils compared to other death and activation stimuli
(A and B) Apoptosis was induced in purified neutrophils by overnight incubation (spontaneous apoptosis), ultraviolet radiation (UVR), granzyme B/perforin (GzmB/Pfn), anti-Fas, and TNFα/CHX. NETosis was induced using phorbol 12-myristate 13-acetate (PMA) or LPS, and autophagy/necroptosis was induced by incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and anti-CD44. Cell stimulation with TNFα was also studied. Neutrophils incubated for 0 or 4 hours were used as negative controls. (C) Neutrophils were incubated for 4 hours at 37°C in the absence (Control-4 hr) or presence of granzyme B/perforin, interleukin 6 (IL6), IL8 endothelial-derived (IL8e), IL8 monocyte-derived (IL8m), G-CSF, GM-CSF, fMLP, or ligands for Toll-like receptor 2 (TLR2) [heat-killed Listeria monocytogenes (HKLM)], TLR5 (flagellin), TLR7/8 (CL075), and TLR9 (CpG-C). Neutrophils without incubation (Control-0′) were also included as a negative control. In (A), samples were analyzed by immunoblotting using anti–caspase-3 antibodies. In (B) and (C), general protein citrullination was visualized by AMC immunoblotting (upper panel) and histone H3 citrullination (Cit-H3) using antibodies against citrullinated histone H3 (citrulline 2 + 8 + 17) (middle panel). The piece of the membrane-containing histone H3 was stripped and reprobed using anti–histone H3 antibodies as loading control (lower panel). The experiments were performed on at least three separate occasions with similar results.
Fig. 3
Fig. 3. Purified perforin induces hypercitrullination in target cells
(A and B) Purified neutrophils from 2 different healthy donors (A) or 293T cells expressing PAD2, PAD3 or PAD4 (B), were incubated in the absence or presence of sublytic amounts of perforin for 4 hr at 37°C. After terminating the reactions, samples were analyzed by electrophoresis and proteins visualized by immunoblotting using antibodies against AMC (A and B), PAD2, PAD3 or PAD4 (B), and β-actin as a loading control (A and B). The experiments were performed on at least 4 separate occasions, with similar results.
Fig. 4
Fig. 4. Cytotoxic cells induce perforin-dependent and apoptosis independent hypercitrullination of human primary neutrophils
Purified neutrophils sensitized with anti-CD16 were preincubated in the absence or presence of CMA (lane 5), z-DEVD-FMK (lane 6) or z-VAD-FMK (lane 7), followed by incubation in the absence or presence of LAK cells (killer:target ratio 5:1). Killing assays were also performed in the presence of 8mM EGTA (lane 4). After terminating the reactions, the samples were electrophoresed and immunoblotted using anti-gelsolin, anti-granzyme B (GzmB) (LAK loading control), anti-PR3 (neutrophil loading control), or AMC antibodies. Filled and unfilled arrows denote intact gelsolin and its apoptotic fragments, respectively. The experiments were performed on at least four separate occasions with similar results.
Fig. 5
Fig. 5. MAC is a potent inducer of hypercitrullination in neutrophils
(A and B) TNBS-treated neutrophils sensitized with anti-DNP antibodies were incubated in the absence or presence of increasing amounts of human serum (A) or in the absence (−) or presence (+) of complement C7-deficient serum without or with increasing amounts of purified human C7 (B). After terminating the reactions, the samples were electrophoresed and immunoblotted using anti-gelsolin, anti-C9, anti-PR3 (as loading control), or AMC antibodies. The filled arrow denotes intact gelsolin. The experiments were performed on at least three separate occasions with similar results.(C) RA SF cells were electrophoresed and immunoblotted using anti-C9 or anti-γ-actin (loading control) antibodies. Control (lane 1) and complement treated sensitized neutrophils (lane 2) were used as negative and positive controls for C9 deposition, respectively. (D) C9 levels in C were quantified by densitometry and normalized to actin. The data was then subjected to unsupervised hierarchical clustering (using the Cluster and TreeView software programs) (71) together with the data of citrullination, PR3, CD14, caspase-3 and BID cleavage from Fig. 1G.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays. 2003;25:1106–1118. - PubMed
    1. Wegner N, Lundberg K, Kinloch A, Fisher B, Malmstrom V, Feldmann M, Venables PJ. Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunol. Rev. 2010;233:34–54. - PubMed
    1. Mortier A, Gouwy M, Van DJ, Proost P. Effect of posttranslational processing on the in vitro and in vivo activity of chemokines. Exp. Cell Res. 2011;317:642–654. - PubMed
    1. 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. - PMC - PubMed
    1. Masson-Bessiere C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T, Serre G. The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the alpha- and beta-chains of fibrin. J. Immunol. 2001;166:4177–4184. - PubMed

Publication types