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Ann Rheum Dis. Aug 2006; 65(8): 1013–1020.
Published online Jan 31, 2006. doi:  10.1136/ard.2005.044743
PMCID: PMC1798256

Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids

Abstract

Background

Anti‐citrullinated protein antibodies (ACPA) are specifically and frequently detected in sera of patients with rheumatoid arthritis (RA). Citrullinated fibrin or fibrinogen is a candidate autoantigen of such antibodies.

Objective

To investigate the presence of citrullinated fibrinogen (cFBG) in the plasma or synovial fluid of patients with RA and control patients, and to determine cFBG levels and their relationship with serum markers for RA if it is present.

Methods

A sandwich enzyme linked immunosorbent assay (ELISA) to measure cFBG was established using monoclonal antibodies cF16.1 and cF252.1, generated by immunising mice with R16Cit and R252Cit, the fibrinogen Aα chain derived sequences with citrulline at position 16 and 252, respectively, and the presence of cFBG was further investigated with immunoprecipitation‐western blotting.

Results

Positive signals were detected in 11/15 RA synovial fluids (RASFs), but not in osteoarthritis synovial fluids or RA plasma with sandwich ELISA for cFBG using cF16.1 and an anti‐modified citrulline (AMC) antibody. The presence of cFBG in RASFs was confirmed by immunoprecipitation‐western blotting. Furthermore, most RA sera strongly reacted against R16Cit. No relationship was seen between RASF cFBG levels and C reactive protein or anti‐cyclic citrullinated peptide antibody levels of the paired sera.

Conclusion

cFBG is detected as a soluble citrullinated autoantigen in RASFs and may therefore be a genuine candidate antigen for ACPA in patients with RA.

Keywords: anti‐citrulline‐containing peptide antibody, rheumatoid arthritis, synovial fluid

Anti‐citrullinated protein antibodies (ACPA), such as antifilaggrin antibodies and anti‐cyclic citrullinated peptide (CCP) antibodies, are useful serological markers for the diagnosis of rheumatoid arthritis (RA).1,2,3,4,5 Citrulline is formed by the post‐translational modification of peptidylarginine by peptidylarginine deiminase (PADI). Five isotypes have been identified6,7,8,9,10,11,12 and PADI1‐4, at least, require calcium ions. PADI2 and PADI4 are thought to be related to RA, both of which are found in the RA synovium.13,14,15,16 Our large scale, genome‐wide case‐control study using single nucleotide polymorphisms found that a PADI4 polymorphism is distinctly associated with RA and also with levels of antibodies to citrullinated filaggrin in the sera of Japanese patients with RA.15

Citrullinated forms of the α and β chains of fibrin have been identified in the RA synovium by Masson‐Bessiere et al,17 and these results were supported by subsequent reports.14,18,19 Fibrinogen, which constitutes a substrate for PADI2 and PADI4,16 is produced in the liver, and infiltrates synovial fluid (SF) through vessels. Then, under the activated coagulating pathway in the RA synovium,20,21,22,23,24,25 it polymerises into insoluble fibrin. The real target of the anti‐citrulline‐containing peptide antibody is still unknown, but because anti‐citrullinated fibrinogen (cFBG) antibody has been identified as a useful marker for RA with excellent sensitivity and specificity,26,27 we assume that citrullinated fibrin(ogen) is a genuine autoantigen for ACPA.

Though citrullinated fibrin is commonly observed, it is not yet known whether it is citrullinated only after fibrinogen polymerisation or whether fibrinogen is also citrullinated in blood or SF. Furthermore, if cFBG is present, its relevance to RA disease characteristics or activities is of exceptional interest. Thus, we were prompted to set up a sandwich enzyme linked immunosorbent assay (ELISA) to measure cFBG levels to investigate the presence of cFBG and its association with RA using monoclonal antibodies (mAbs) that recognise cFBG.

To generate mAbs, we prepared two peptides, R16Cit and R252Cit as immunogens, respectively corresponding to positions 11–21, 247–257 of the secreted Aα chain with the citrulline substituted from arginine at position 16 and 252, both of which are recognised by PADI4.16 Thrombin recognises 16Arg for the cleavage of fibrinopeptide A (FPA), and thus holds a key to fibrinogen polymerisation into fibrin. We also investigated RA sera reactivity against these peptides to determine whether these positions are citrullinated or not, based on the study by Schellekens et al,3 in which amino acid sequences adjoining citrulline were shown to be crucial in the determination of antigenicity.

Materials and methods

Citrullination of human fibrinogen

Human fibrinogen (100 μl; 5 mg/ml; American Diagnostics, Pendleton, IN, USA) was citrullinated with 30 μl of recombinant human PADI4 (prepared as previously described,16 1.3 U/mg fibrinogen, for the standard of sandwich ELISA) or 10 μl of rabbit PADI2 (Sigma, St Louis, MO, USA, 5 U/mg fibrinogen, for the selection of mAbs) in a 1 ml reaction buffer (100 mM Tris, 93 mM NaCl, 20 mM CaCl2, 5 mM dithiothreitol, pH 7.4). The incubation was performed for 2 hours at 37°C and, finally, 200 μl of 0.2 M EDTA was added to stop the reaction. For the control, 0.2 M EDTA was added to native fibrinogen (nFBG) and other reaction mix components before the incubation.

Generation of murine monoclonal antibodies against human citrullinated fibrinogen

BALB/c mice were immunised with synthetic peptides R16Cit and R252Cit (fig 1A1A)) conjugated with keyhole limpet haemocyanin. After immunisation, the spleen cells of the mice were fused with myeloma cells and the hybridoma cell lines were cloned with a limiting dilution method. The heavy chain isotypes of mAbs were identified with a mouse mAb isotyping kit (Amersham, Town, UK), according to the manufacturer's protocol. Selected clones were cultured in RPMI 1640 with 10% fetal bovine serum, and antibodies from culture media were purified by a HiTrap IgM purification column (Amersham) after pretreatment with a HiTrap protein G.

figure ar44743.f1
Figure 1 Generation and selection of mAbs against cFBG. (A) The sequences of position 1–49 and 241–284 of the fibrinogen Aα chain are shown, and the sequences of R16R and R252R are underlined. The mRNA encoded fibrinogen ...

The clones obtained more or less recognised the non‐citrulline control R16R or R252R (fig 1A1A),), as well as R16Cit and R252Cit. A further selection of clones that preferentially recognise the cFBG Aα chain was performed by western blotting. cFBG and nFBG were subjected to 10% SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad, Hercules, CA, USA). After blocking by 5% skimmed milk in Tris buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated for 2 hours at room temperature with mAbs. After washes with TBST, 1:2000 goat antimouse immunoglobulin, horseradish peroxidase (HRP) conjugate (Sigma) was added and they were incubated for 2 hours. After washes, the membranes were visualised with electrochemiluminescence western blotting detection reagents (Amersham).

Plasma, serum, and synovial fluid samples

Twenty seven plasma and 36 serum samples were collected from patients who fulfilled the American College of Rheumatology criteria for RA, and normal control sera were collected from eight healthy subjects. Fifteen RA synovial fluids (RASFs) and five osteoarthritis synovial fluids (OASFs) were collected in EDTA‐containing or heparinised tubes at the time of therapeutic arthrocentesis, centrifuged at 450 g for 5 minutes to remove any debris, and the supernatants were collected. Informed consent was obtained for all the samples.

Sandwich ELISA for cFBG using the AMC antibody

cF16.1 and cF252.1, the selected mAbs (described in detail in “Results”), were coated on plate wells (Nunc Maxisorp, Roskilde, Denmark) at 10 μg/ml in 50 mM carbonate buffer, pH 9.5. After an incubation at 4°C overnight the wells were washed with phosphate buffered saline with 0.05% Tween 20 (PBST), then blocked with 3% bovine serum albumin (BSA; Sigma)/PBST for 1 hour. Then, the standard cFBG, control nFBG, plasma, and SF samples diluted in 1% BSA/HEPES buffered saline: 100 mM HEPES, 100 mM NaCl, pH 7.2 with 0.1% Tween 20(HBST) were applied to the wells. After incubation at 4°C overnight, the wells were washed, incubated with 1% glutaraldehyde/PBS for 1 hour, then with 0.2 M Tris‐Cl, pH 7.8 for 30 minutes. After washes, reagent A and B (modified citrulline detection kit; Upstate, Chicago, IL, USA) were applied and the wells were incubated overnight at 37°C.

After washes, the wells were blocked with 5% BSA/PBST for 30 minutes, then incubated with 1:2500 AMC antibody (Upstate) in 2.5% BSA/PBST for 3 hours at 37°C, successively with goat F(ab′)2 antirabbit immunoglobulin HRP conjugate, 1:50 000 (Biosource, Camarillo, CA, USA) for 2 hours at 37°C. After washes, 3,3′,5,5′‐tetramethylbenzidine substrate (KPL, Gaithersburg, MD, USA) was applied and the colour development was stopped with 0.5 M H2SO4. Then, the absorbance at a wavelength of 450 nm (A450) was read.

For all the ELISAs in our study, the samples were tested in duplicate and the A450 of blank wells was subtracted from that of the other wells.

Fibrinogen is very abundant in plasma (1.5–3 g/l) and is normally expected to be present in native forms. Thus, we investigated whether the mAbs could capture cFBG from nFBG‐rich samples such as plasma. Normal plasma (1:50), which contains 100–1000‐fold as much nFBG as the cFBG used for the standard curve, was added to the diluent and the assays were performed as above. For the comparison, goat F(ab′)2 antihuman fibrinogen (Cappel, Aurora, OH, USA) was used as the capture antibody.

Sandwich ELISA for cFBG using an antihuman fibrinogen antibody for the detection and quantification of fibrinogen in plasma and SFs

To detect cFBG, plate wells were coated with cF16.1 and cF252.1, respectively, incubated at 4°C overnight, and blocked with 3% BSA/PBST for 1 hour. Then cFBG, nFBG, plasma, and SFs in 1% BSA/HBST were applied, and the wells were incubated for 3 hours at 37°C. After washing, the wells were incubated with 1:250 antihuman fibrinogen antibody, HRP conjugate (FG‐EIA‐D; Affinity Biologicals, Ontario, Canada) for 2 hours at room temperature. After washes and colour development, A450 was read.

For the quantification of fibrinogen the procedures were the same, except that goat F(ab′)2 antihuman fibrinogen (Cappel) was coated, and normal referential plasma was used as the standard.

Immunoprecipitation and western blotting

cF16.1 (5 μg) was added to 400 μl of 1:20 diluted RASFs and OASFs and incubated for 2 hours at 4°C. Then, Dynabeads coupled with rat antimouse IgM (Dynal, Oslo, Norway) were added to the samples and they were incubated for a further 2 hours at 4°C. Next, the beads were collected and washed as instructed by the manufacturer. Laemmli sample buffer was added to the beads and they were heated for 5 minutes at 95°C. Then, the supernatants were collected after centrifugation and subjected to SDS‐PAGE and western blotting. SF samples diluted 1:15 were prepared for comparison.

For the detection of citrullination, blotted PVDF membranes were disposed of as instructed by the manufacturer (modified citrulline detection kit; Upstate). After chemical modification, the membrane was washed with distilled water and blocked with 5% skimmed milk/TBST for 30 minutes, incubated with 1:1000 AMC antibody in 2.5% BSA/TBST for 1 hour, then successively with 1:5000 antirabbit immunoglobulin antibody (Upstate) for 1 hour after washes with TBST. The membrane was visualised with Supersignal WestDura substrate (Pierce, Rockford, IL, USA). To detect fibrinogen, membranes were blocked with 5% skimmed milk/TBST for 30 minutes, incubated with 1:1500 rabbit antihuman fibrinogen (Hyphen BioMed, Andresy, France) for 1 hour, then with 1:30 000 antirabbit immunoglobulin HRP conjugate (Biosource) for 1 hour. The membranes were visualised with electrochemiluminescence‐plus.

Measurement of reactivity against R16Cit and R252Cit by sera

The peptides R16R, R16Cit, R252R, R252Cit conjugated with BSA were coated on wells at 10 μg/ml and incubated at 4°C overnight. The wells were washed and blocked with 3% BSA/PBST and incubated with 1:200 diluted sera in 1% BSA/PBST at 4°C overnight. Then, the wells were washed and incubated with 1:50 000 goat F(ab′)2 antihuman IgG, HRP conjugate (Biosource) at 37°C for 2 hours. Colour development was performed and A450 of the reaction against R16Cit minus that against R16R (ΔR16Cit−R16R), and A450 of the reaction against R252Cit minus that against R252R (ΔR252Cit−R252R) were assessed.

Measurement of autoantibodies to CCP

CCP antibody levels were measured with a DIASTAT anti‐CCP ELISA kit (Axis‐Shield, Cambridgeshire, UK).

Statistical analyses

The non‐parametric data obtained for the sera reactivity against peptides and the correlations between CCP antibody or C reactive protein and A450 levels measured with cF16.1‐AMC ELISA were analysed with Wilcoxon's rank sum test and Spearman's rank correlation test, respectively, for significance (p<0.05).

Results

Preparation and characterisation of murine anti‐cFBG mAbs

As described in “Materials and methods”, the two clones cF16.1 and cF252.1 were selected by western blotting (fig 1B–D). These clones, both IgM, preferentially recognised the cFBG Aα chain.

Establishment of sandwich ELISA standard curves for citrullinated fibrinogen

The standard curves were established with cF16.1 and cF252.1 as capture antibodies and AMC and antihuman fibrinogen as detection antibodies. Though not definitely specific to fibrinogen, the ELISA with AMC was specific to citrulline up to 10 μg/ml and the standard curves were almost unaffected by the addition of 1:50 normal plasma (figs 2A and BB),), which is obvious when compared with the case in which the anti‐fibrinogen antibody was used for capturing (fig 2C2C),), whereas the ELISA with antihuman fibrinogen was preferential for cFBG, but cross reacted with nFBG at a higher concentration (figs 3A and BB)

figure ar44743.f2
Figure 2 The standard curves of sandwich ELISA for cFBG using the AMC antibody for detection. (A) cF16.1, (B) cF252.1, (C) goat F(ab′)2 antihuman fibrinogen antibodies were used as capture antibodies. cFBG + plasma means cFBG was ...
figure ar44743.f3
Figure 3 Standard curves of sandwich ELISA for cFBG using the antihuman fibrinogen antibody for detection. (A) cF16.1, (B) cF252.1 were used as the capture antibodies. In (A) and (B) cFBG was preferentially recognised, but nFBG was also detected ...

All the samples were first tested with AMC for citrulline specificity and negligible interference by nFBG, which is abundantly present in plasma and SF, and the positive samples with AMC were further tested with anti‐fibrinogen.

Sandwich ELISA using cF16.1 or cF252.1 and the AMC antibody

The sensitivity of the ELISA was determined to be 16 ng/ml and 30 ng/ml for cF16.1 and cF252.1, respectively, and because all the samples were tested at a dilution of 1:25, the cut off lines for positivity were set at 400 and 750 ng/ml, respectively.

With cF16.1, positive signals were detected in 11/15 RASFs, while the signals of OASFs and plasma samples were below the level of detection (fig 44).

figure ar44743.f4
Figure 4 The levels of cFBG measured by cF16.1‐AMC sandwich ELISA. All the samples were tested at 1:25 and the cut off line for sensitivity was 400 ng/ml. Signals for cFBG were detected in 11/15 RASFs, while all of the RA plasma ...

Assuming that the degree of citrullination was the same between samples and the standard cFBG citrullinated in vitro with PADI4, the levels of cFBG were 500 ng/ml to 15.2 μg/ml for positive RASFs.

With cF252.1 as the capture antibody, only two RASFs were positive for cFBG. Both were also positive with cF16.1, although the concentrations were 1.2 μg/ml and 1.7 μg/ml, much lower than with cF16.1 (8.7 and 15.2 μg/ml, respectively).

For patients with paired sera available (n = 8), no clear correlation was observed between RASF cFBG levels and serum C reactive protein (fig 5A, SSpearman'spearman's coefficient (rs) = 0.57, p = 0.13) or anti‐CCP antibody levels (fig 5B5B,, rs = −0.26, p = 0.48).

figure ar44743.f5
Figure 5 The correlations between RASF cFBG levels measured by cF16.1‐AMC ELISA and C reactive protein (CRP) (A) or anti‐CCP antibody levels (B) of the paired sera. No significant correlation was observed between them. (rs = 0.57 ...

Sandwich ELISA using cF16.1 and cF252.1 and an antihuman fibrinogen antibody for the detection

Samples were serially diluted and positive signals of plasma samples, proved to be citrulline negative in the ELISA with AMC, were observed up to 1:200 owing to the cross reactivity against nFBG. They disappeared at 1:400 completely, at which level nFBG interference in SFs was also negligible because the fibrinogen concentration was below 1000 mg/l for all the SFs (data not shown), and was lower than plasma levels. Eight samples selected from SF and plasma were diluted 1:400 and subjected to the assay. cFBG positive RASFs with AMC were also positive in this assay (table 11).). This further demonstrated the presence of cFBG.

Table thumbnail
Table 1 A450 levels of 1:400 diluted plasma and synovial fluid samples in cF16.1‐anti fibrinogen sandwich ELISA

Immunoprecipitation and western blot

To further confirm the citrullination of fibrinogen in RASFs, RASF3, 4 (cFBG concentrations measured by cF16.1‐AMC ELISA were 8.7, 15.2 μg/ml, respectively) were immunoprecipitated with cF16.1, and the precipitates were subjected to western blotting by anti‐fibrinogen (fig 6A6A)) and AMC (fig 6B6B).

figure ar44743.f6
Figure 6 A demonstration of the presence of cFBG in RA synovial fluids using the immunoprecipitation (IP)‐western blotting (WB) method. Samples diluted 1:15 and immunoprecipitates using cF16.1 were obtained for cFBG positive RASFs (RASF3, ...

As shown in fig 6A6A,, three bands at the levels of the fibrinogen Aα, Bβ, γ chains were immunoprecipitated (lanes 3, 4) from numerous fibrinogen related bands observed in the same SFs (lanes 5, 6). For the same precipitates Aα and Bβ were positive for AMC, with Aα more citrullinated than Bβ (fig 6B6B).). Thus, the presence of cFBG in RASFs was demonstrated.

ELISA for measurement of the reactivity of RA sera against R16Cit and R252Cit

The mAbs were expected to recognise the immunised peptides R16Cit and R252Cit, but it is difficult to tell whether they exclusively recognised the epitopes. Amino acids adjoining citrulline are critical in the determination of antigenicity, and if RA sera react against the peptides differently or disproportionately, a difference in the degree of citrullination at positions 16 and 252 is likely.

The median (2SD) of ΔR16Cit−R16R was 0.301 (1.55) (range −0.609 to 2.263) and that of ΔR252Cit−R252R was 0.262 (1.202) (range −0.490 to 1.811) for 36 patients with RA, and 0.010 (0.019) (range −0.013 to 0.032) and 0.008 (0.021) (range −0.024 to 0.027), respectively, for eight normal controls. Although the patterns of reactivity differed among samples (fig 77),), R16Cit was significantly more strongly antigenic to RA sera (p<0.05), at least in comparison with R252Cit.

figure ar44743.f7
Figure 7 The A450 levels of RA sera reactivity against R16Cit and R252Cit. RA sera in general reacted against R16Cit significantly more strongly (p<0.05), some even exclusively, but the patterns showed a wide variety depending on the patient. ...

Discussion

Fibrin has been shown to be citrullinated in synovial tissues of inflamed joints.14,17,18,19 The presence of a soluble citrullinated antigen, cFBG, was newly demonstrated in our study. cFBG was not detected in RA plasma, whereas it was detected in 11/15 RASFs, and this showed that fibrinogen is citrullinated, not in blood, but exclusively in joints before its polymerisation, suggesting leakage of PADI enzymes in SFs and highly activated citrullination.

cF252.1 did not work effectively in capturing cFBG. This may be due to the lower sensitivity compared with cF16.1 or the interference by citrullinated fibrin degradation products, in which R252Cit is included, but position 252 may be less citrullinated than position 16. In contrast, cF16.1 captured cFBG from RASFs successfully, and RA sera strongly reacted against R16Cit, which implies the citrullination of position 16, the thrombin cleavage site, in cFBG. Dysfibrinogenaemia is often associated with the mutation of position 16 and impaired FPA release.28,29,30,31,32 The replacement of basic arginine with neutral citrulline at this position may hamper the binding of thrombin. Thus protected from polymerisation and made stable as a soluble antigen, cFBG may become antigenic as a modified form of the self antigen, fibrinogen. cFBG was detected only in RASFs and not in OASFs in our study, while the citrullination of synovial fibrin, which shares almost the same structure as fibrinogen, was shown to be a common phenomenon in any synovitis, including OA.18,19 We recently found that PADI4, expressed in neutrophils, is associated with RA in Japanese subjects,15 and is immunohistochemically colocalised with apoptotic cells.14 Therefore, the discrepancy may be explained by the difference in the degree of neutrophil infiltration between RASF and OASF. Whereas almost no neutrophils are present in OASFs, they massively infiltrate RASFs and at the time of their apoptosis PADI4 is assumed to leak out abundantly and recognise and citrullinate fibrinogen at an early stage before its polymerisation. On the other hand, fibrin deposits are very stable and may be citrullinated with as minute an amount of PADI as is detectable in OA synovium. A larger number and a wider variety of SFs should be tested to examine whether cFBG is specifically detected in RA. If cFBG is specifically observed in RA, it may be a real target for ACPA.

Aα chains were more visible than Bβ in the immunoprecipitate of RASFs. This is consistent with our previous report using liquid chromatography with tanden mass spectrometry,16 in which more peptidylarginines proved to be citrullinated in Aα than in Bβ. The γ chain was shown to be less efficiently citrullinated than Aα or Bβ16,17 and unrecognised by sera positive for ACPA,17,19 and we assume that it was undetectable owing to this even lower citrullination.

For the association with RA disease characteristics, cFBG may work not only as a pathogenic antigen but also as a disease exacerbating factor by forming immunocomplexes in RASFs with anti‐cFBG antibodies that are frequently detected in patients with RA.26,27,33

In conclusion, we detected, for the first time, cFBG in RASFs as a soluble citrullinated autoantigen and confirmed that fibrinogen citrullination takes place in joints. The thrombin cleavage site is thought to be citrullinated and this makes cFBG antigenic to patients with RA. Also, cFBG may be a genuine antigen for soluble immunocomplexes. Further studies are required to evaluate the precise role of cFBG and its association with PADI4.

Acknowledgements

This work was supported by a grant from the Japanese Millennium Project. We thank Dr Akihiro Yamaguchi and Dr Yuko Okazaki (Department of Allergy and Rheumatology, The University of Tokyo Hospital) for their technical advice and assistance.

Abbreviations

ACPA - anti‐citrullinated protein antibodies

AMC - anti‐modified citrulline

BSA - bovine serum albumin

cFBG - citrullinated fibrinogen

CCP - cyclic citrullinated peptide

ELISA - enzyme linked immunosorbent assay

FPA - fibrinopeptide A

HBST - HEPES buffered saline with 0.1% Tween 20

HRP - horseradish peroxidase

IP - immunoprecipitation

mAb - monoclonal antibody

nFBG - native fibrinogen

OA - osteoarthritis

PADI - peptidylarginine deiminase

PBST - phosphate buffered saline with 0.05% Tween 20

PVDF - polyvinylidene difluoride

RA - rheumatoid arthritis

SDS‐PAGE - sodium dodecyl sulphate‐polyacrylamide gel electrophoresis

SF - synovial fluid

TBST - Tris buffered saline with 0.1% Tween 20

Footnotes

Competing interests: None.

Ethical approval: This study was approved by the University of Tokyo ethical committee and the ethical committee of The Institute of Physical and Chemical Research, Japan.

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