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. 2016 Jun;75(6):1099-107.
doi: 10.1136/annrheumdis-2014-206785. Epub 2015 Jul 9.

Identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis

Maria Juarez  1 , Holger Bang  2 , Friederike Hammar  2 , Ulf Reimer  3 , Bernard Dyke  1 , Ilfita Sahbudin  1 , Christopher D Buckley  1 , Benjamin Fisher  1 , Andrew Filer  1 , Karim Raza  4
Affiliations

Identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis

Maria Juarez et al. Ann Rheum Dis. 2016 Jun.

Abstract

Objective: To investigate serum antibody reactivity against a panel of post-translationally modified vimentin peptides (PTMPs) in patients with early inflammatory arthritis.

Methods: A panel of PTMPs was developed. Microtitre plates were coated with peptides derived from vimentin that were identical in length and composition except at one amino acid that was changed to introduce one of three post-translational modifications (PTMs)-either a citrullinated, carbamylated or acetylated residue. Sera of 268 treatment-naive patients with early inflammatory arthritis and symptoms ≤3 months' duration were tested. Patients were assigned to one of three outcome categories at 18-month follow-up (rheumatoid arthritis (RA), persistent non-RA arthritis and resolving arthritis).

Results: Antibodies against citrullinated, carbamylated and acetylated vimentin peptides were detected in the sera of patients with early inflammatory arthritis. The proportion of patients seropositive for all antibody types was significantly higher in the RA group than in the other groups. Anti cyclic citrullinated peptide (CCP)-positive patients with RA had higher numbers of peptides recognised and higher levels of antibodies against those peptides, representing a distinct profile compared with the other groups.

Conclusions: We show for the first time that antibodies against acetylated vimentin are present in the sera of patients with early RA and confirm and extend previous observations regarding anticitrullinated and anticarbamylated antibodies.

Keywords: Autoantibodies; Early Rheumatoid Arthritis; Rheumatoid Arthritis.

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Figures

Figure 1
Figure 1
Frequency of seropositivity for IgG and IgA antibodies against different post-translationally modified peptides in rheumatoid arthritis (RA) (dotted bars), persistent non-RA (black bars) and resolving (grey bars) groups. (A) IgG antibody panel; (B) IgA antibody panel. *p<0.05, ***p≤0.001.
Figure 2
Figure 2
Competition studies. (A) Preincubation of post-translationally modified peptides with sera containing anticitrullinated, anticarbamylated and antiacetylated antibodies specifically blocks full-length modified vimentin protein binding. Representative western blot image shown. In each western blot, the following antigens were applied to the SDS-PAGE separation: lane 1, human recombinant vimentin; lane 2, citrullinated human recombinant vimentin; lane 3, carbamylated human recombinant vimentin; lane 4, acetylated vimentin-albumin conjugate. (A) Immunoblot with serum from a patients with rheumatoid arthritis (RA) cross-reacting with three different modified peptides. (B–D) Same experimental conditions as in (A), but the serum was preincubated with 10 µg/mL of citrullinated vimentin (B), carbamylated vimentin (C) or acetylated isoform (D) for 2 h prior to the immunoblotting experiment. Immunoassay competition experiments: (B) preincubation of patient sera with unmodified vimentin peptide did not inhibit IgG binding for any of the antibodies tested. Control denotes non-preincubated sera. (C) Preincubation of sera with corresponding modified peptides inhibited IgG binding in a dose-dependent manner. (D) Cross-reactivity between antibodies against different modifications. Binding of IgG antibodies to anti-CitVimentin peptides is inhibited by preincubation with citrullinated vimentin predominantly with low degree of inhibition by preincubation with carbamylated or acetylated vimentin. The same pattern is observed for anti-AcVimentin antibodies. Binding of anti-CarVimentin IgG antibodies is predominantly inhibited by preincubation with carbamylated vimentin but inhibition is also observed when sera is preincubated with citrullinated vimentin indicating a degree of cross-reactivity. Bars represent mean, error bars SD. CitVim, citrullinated vimentin peptide; CarVim, carbamylated vimentin peptide; AcVim, acetylated vimentin peptide.
Figure 3
Figure 3
Frequency of antibody positivity to different post-translationally modified peptides in each outcome group. Anti cyclic citrullinated peptide (CCP)-positive rheumatoid arthritis (RA) (checkered bar), anti-CCP-negative RA (vertical lines bar), persistent non-RA arthritis (black bar) and resolving arthritis (grey bar). (A) IgG reactivities: statistically significant differences in the percentages of patients with positive antibodies were observed between patients with anti-CCP-positive RA and the remaining groups for all peptides. (B) IgA reactivities: statistically significant differences in the frequency of anti-CitVimentin IgA antibody reactivities seen between patients with anti-CCP-positive RA and the remaining groups but not for the remaining post-translationally modified peptides. ***p≤0.001. (C) Distribution of the number of peptides recognised according to outcome group. The highest proportion of patients with anti-CCP-positive RA recognised three peptides while the highest proportions of patients in the remaining outcome groups did not recognise any peptides.
Figure 4
Figure 4
Graphical representation of IgG antibody reactivity results. Patients with anti cyclic citrullinated peptide (CCP)-positive rheumatoid arthritis (RA) showed a distinct antibody profile that differed from that of the remaining outcome groups. This patient group had reactivities against a higher number of peptides and the antibody levels against those peptides were also higher. Shaded squares represent positive tests: light grey, low-level positivity (OD >98th percentile to 1.0); dark grey, medium positivity (OD >1.0–1.9); black, high positivity (OD >1.9). Each row represents an individual patient, each column represents a peptide. Control, native unmodified vimentin peptide; CitVim, citrullinated vimentin peptide; CarVim, carbamylated vimentin peptide; AcVim, acetylated vimentin peptide.
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