Fig 1.
ACPA reactivity with PAD2- and PAD4-citrullinated fibrinogen.
(A-D) Microtiter plates were coated with native fibrinogen or fibrinogen citrullinated by either PAD2 or PAD4 (cit-Fib) at various concentrations. Pooled synovial fluid (SF) from 6 anti-CCP-positive RA patients was applied at different dilutions ranging from 1:50 to 1:800, and the binding of ACPAs was detected using HRP-anti-human IgG and OPD substrate. Shown are optical density (OD) values from citrullinated minus native fibrinogen. (E) The pooled SF was also tested in dilutions of 1:5 and 1:15 using fibrinogen citrullinated by 0.62 μg PAD per mg protein. Average and range of duplicate measurements are shown. (F) Western blotting showing the binding of ACPAs from pooled SF (diluted 1:100) to native fibrinogen and fibrinogen citrullinated by PAD2 or PAD4 (0.62 μg PAD per mg protein). Coomassie staining is shown as a loading control. The three bands show α-chain, β-chain and γ-chain of fibrinogen. (G) Anti-modified citrulline (AMC) western blot to detect citrullinated fibrinogen, citrullinated by 0.62 or 10 μg PAD per mg protein, respectively, (top) and brilliant blue stained gel to detect total protein (bottom). (H) The density of the visible citrullinated protein bands in the AMC blot was normalized to corresponding protein signal. Mean and SEM are shown for triplicate blots with *p<0.05, **p<0.01.
Fig 2.
Anti-citrullinated fibrinogen autoantibodies in SF and plasma from individual RA patients.
IgG autoantibodies against native and citrullinated fibrinogen were measured in (A, B) SF (diluted 1:50) and (C, D) plasma (diluted 1:50) from 32 anti-CCP-positive and 10 anti-CCP-negative RA patients using a direct ELISA based on microtiter plates coated with native fibrinogen or fibrinogen citrullinated by either PAD2 or PAD4 (cit-Fib) at 0.62 μg PAD per mg protein. Shown are (A, C) raw optical density (OD) values for all patients, and (B, D) net OD values all patients, adjusted for binding to native fibrinogen. Each data point represents duplicate measurements. ****p<0.0001.
Table 1.
Correlation between ACPA levels and disease parameters in 32 anti-CCP-positive RA patients.
Fig 3.
In vitro citrullination of ENO1, histone H3 and HSA by PAD2 and PAD4.
(A) ENO1, (B) histone H3 and (C) HSA were citrullinated with PAD2 and PAD4 (2 μg PAD per mg protein). Western blots (upper panels) show citrullination, as detected by the AMC kit, and gel electrophoresis followed by brilliant blue staining (lower panels) shows total protein. (D-F) The density of the visible bands corresponding to citrullinated protein in the AMC blot was normalized to the total protein signal. Mean and SEM are shown for triplicate blots, *p<0.05.
Fig 4.
Binding of antibodies from RA patients to citrullinated and native proteins.
Microtiter plates were coated with native protein or protein citrullinated by either PAD2 or PAD4 (2 μg PAD per mg protein), and SF samples from 15 anti-CCP-positive RA patients were applied at a 1:50 dilution. Binding of IgG antibodies to native or citrullinated (A) ENO1, (B) histone H3 and (C) HSA are shown as an average of duplicate optical density (OD) measurements, and horizontal bars represent median values. (D) ACPA binding to citrullinated ENO1 and (E) histone H3 was calculated for the 15 anti-CCP-positive RA patients and additional 9 anti-CCP-negative RA patients. Shown are averages and range of duplicate OD measurements, after subtraction of signals obtained for native protein.
Fig 5.
ACPA cross-reactivity assessed by competitive ELISA.
Microtiter plates were coated with fibrinogen citrullinated by either (A) PAD2 or (B) PAD4 (2 μg PAD per mg protein). A pool of SF from three RA patients, diluted 1:50, was pre-incubated with increasing concentrations of native fibrinogen, fibrinogen citrullinated by PAD2 (cit-Fib(PAD2)) or by PAD4 (cit-Fib(PAD4)), ENO1 citrullinated by PAD2 (cit-ENO1(PAD2)) or by PAD4 (cit-ENO1 (PAD4)), or histone H3 citrullinated with PAD2 (cit-H3(PAD2)) or PAD4 (cit-H3(PAD4)) for 30 minutes before addition to the plates. Binding signals obtained in the absence of added protein (n = 7) are shown as mean (solid line) and 95% CI (dotted line). The subsequent binding of IgG antibodies within the SF pool to (A) PAD2-citrullinated fibrinogen or (B) PAD4-citrullinated fibrinogen, as determined by ELISA, are shown as mean ± SEM of duplicate measurements.