Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Serum and synovial fluid were collected from patients fulfilling the American College of Rheumatology criteria for RA [27] and European Spondyloarthropathy Study Group criteria for SpA [28] (for patient information see Table 1). Sera from healthy donors were used as controls. Informed consent was obtained from patients and healthy controls and the study was approved by the local ethics committee. Detailed information on the identity of the samples used in each experiment is provided in Table 1. RF titers were determined with the Waaler Rose test and ACPA titers were measured with anti-CCP-EliA (Phadia, Freiburg, Germany).

IC were further purified by affinity immunoprecipitation with Immobilized Protein G (Pierce, Rockford, IL, USA). 400 μL beads were washed twice with 500 μL phosphate buffered saline (PBS). A total of 50 μL serum or SF was mixed with 450 μL PBS and added to the beads. Sample and beads were placed on a rocker for four hours at 4°C. The beads with the bound IC were washed five times in 500 μL PBS. The pellet of protein G beads was resuspended in reducing Laemmli sample buffer for five minutes at 95°C. After centrifugation (5 minutes, 460 g) the supernatant was stored at -20°C.

Protein concentrations were determined by Coomassie (Bradford) Protein Assay (Pierce, Rockford, IL, USA) and 2 D Quant (GE Healthcare, Uppsala, Sweden).

Protein samples were dissolved in Laemmli buffer (50 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, bromophenol blue) with 5% β-mercapto-ethanol and incubated at 95°C for five minutes. The samples were loaded on a 10% TrisHCl polyacrylamidegel (Biorad, Hercules, CA, USA) and electrophoresis was performed by applying 150 V for 30 minutes, followed by 200 V for one hour.

For 2D-PAGE, protein samples were first precipitated overnight in acetone at -20°C. After centrifugation at 18,000 g for 10 minutes the samples were air dried. A total of 100 μg was resuspended in 200 μL rehydration buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 0.2% carrier ampholytes, 100 mM DTT, bromophenol blue). The sample was introduced passively in an IPG strip (11 cm, pH 4 to 7) (Biorad) as previously described [29]. Iso-electric focussing was performed in a Protean IEF Chamber (Biorad) according to the following program: 100 V, 30 minutes, linear voltage slope - 250 V, 30 minutes, linear - 500 V, one hour, linear - 1,000 V, one hour, linear - 8,000 V, four hours, rapid - 8,000 V, 35,000 V hours, rapid - 500 V, 20 h, rapid. Subsequently the strips were equilibrated in equilibration buffer (50 mM TrisHCl, pH 8.8, 6 M Urea, 20% glycerol, 2% SDS) containing 1.5% DTT for 15 minutes, followed by 4% IAA in equilibration buffer for 15 minutes.

Gel electrophoresis was carried out on a 10% TrisHCl PAGE using 150 V for 30 minutes, followed by 200 V for one hour.

After a 15-minute equilibration of the gels and the nitrocellulose membranes (Biorad) in CAPS (pH 11), electrophoretic transfer of proteins was performed by tank blotting in a Trans Blot Cell (Biorad), with CAPS (pH = 11), at 50 V for three hours. Successful transfer of proteins was checked by means of Ponceau S staining.

The presence of citrullinated proteins on the nitrocellulose blots was detected using the anti-modified citrulline (AMC) detection kit (Upstate, Charlottesville, VA, USA) according to the manufacturer's protocol. Each AMC detection was accompanied with a positive control, as indicated in the manufacturer's protocol.

Visualization of proteins in the gels was performed using Sypro Ruby Protein Gel staining (Invitrogen, Carlsbad, CA, USA) for at least three hours after a 30-minute fixation in a 10% MeOH, 7% acetic acid solution. After staining, the gel was washed twice with a 10% MeOH, 7% acetic acid solution. Proteins of interest were excised from the gel and digested with modified sequence grade porcine trypsin (Promega, Madison, WI, USA) as described earlier [30]. Proteins were analyzed and identified by LC-MSMS, using a Q-TOF Ultima Mass spectrometer (Waters, Milford, MA, USA) combined with ESI source. The data were processed using Mascot Distiller and searched against the Swissprot human database, using the in-house mascot daemon searching algorithm. Identification was considered positive with a P-value < 0.05.

Before immunodetection, each blot was blocked for one hour in 0.3% Tween-20 in PBS. Vimentin was detected with the mouse anti-human vimentin antibody (clone V9, Sigma, St. Louis, MI, USA) at a concentration of 1/400 in 0.3% Tween-20 in PBS. After overnight incubation, vimentine was detected with HRP labelled goat anti-mouse IgG followed by ECL detection. Detection of Fibβ, Fibγ and fibronectin was performed using respectively rabbit anti-human Fibβ, rabbit anti-human Fibγ and rabbit anti-human fibronectin. Anti-rabbit HRP labelled antibody was used as a secondary antibody. ECL detection was carried out by means of Supersignal West Dura Extended Duration Substrate (Pierce).

Following each immunodetection, the blot was stripped for 30 minutes at 50°C with stripping buffer (2% SDS, 0.1 M β-mercapto-ethanol, 0.05 M Tris pH 6.8) and washed three times with 0.3% Tween20 in PBS. To check the stripping efficiency, the blot was re-incubated with secondary antibody and detected with ECL. Afterwards the blot was stripped for another 15 minutes, before incubation with a new primary antibody. Additionally, the sequence of antibodies used for immunodetection varied throughout the different experiments in order to exclude false positive results. Protein patterns were scanned and digitized using the VersaDoc Imaging System (Biorad).

Immunoprecipitation (IP) was used in order to isolate the IC from serum and synovial fluid. Because of the high viscosity of SF, a hyaluronidase treatment was necessary. Both, the flow-through and the eluted IC fraction from a pool of RA SF (SF2; SF13; SF15; SF26) were subjected to 1 D gel electrophoresis (20 μg/lane). In order to identify potential autoantigens in the eluted IC fractions from SF, each lane (20 μg) from the gel was divided in 30 different plugs and analysed separately by mass spectrometry after in gel digestion. Mass spectrometric analysis revealed that the eluted IC fraction from RA SF contained mainly immunoglobulins, while almost none were detected in the flow-through fraction (data not shown). Additionally, Fibβ (at MW 50 kDa, Figure 1a box (x)) could be identified in the SF IC fraction as well as in the flow-through. At this MW, a clear positive AMC staining was detected in the isolated IC fraction from RA SF (Figure 1a lane 2), while no citrullinated proteins could be detected in the flow through of RA SF (Figure 1a lane 1). On the contrary, when the same setup was repeated with a pool of RA sera no citrullinated proteins were detected in the IC from RA sera (RA1 to RA3) (Figure 1b lane 2), while the positive control for AMC staining was explicit. In order to confirm these findings, a set of immunoblotting experiments was performed.

First, a pool of serum obtained from healthy persons (n = 4), a pool of serum from RA patients (RA1 to RA3) and a pool of SF from RA patients (SF2; SF13; SF14; SF15) were used to isolate IC by IP. Subsequent identification of potential antigens in the isolated IC was performed by sequential immunodetection with anti-vimentin, anti-Fibβ, anti-Fibγ and anti-fibronectin on a 2D-Western blot. Between the different immunodetections, the blot was carefully stripped and adequate stripping was checked each time before subsequent primary antibody addition. The results of these experiments are summarized in Figure 2.

Fibβ was detected in IC from healthy serum and RA serum and from RA SF at a molecular weight of 50-60 kDa and pI 5-6. However in IC from RA SF, and not in IC from RA or healthy serum, some extra spots that reacted with anti-Fibβ could be detected at MW 37-50 kDa and pI 6-7. These are probably processed isoforms of Fibβ (Figure 2a, box x), which are specifically found in IC from SF.

Fibγ was visualized as three spot trains at a molecular weight around 100 kDa. As Fibγ has a molecular weight of 56 kDa, the presence of spots at 100 kDa indicated the dimeric form of Fibγ. These dimers of Fibγ were only seen in IC from RA SF and not in IC from RA serum or healthy serum (Figure 2b).

Fibronectin could be detected in IC from healthy serum, RA serum and RA SF at a molecular weight of 150-250 kDa. However, fibronectin in IC from RA SF covered a wider range of isoforms in comparison with IC from RA serum and healthy serum. It is known that fibronectin is present in biological samples in many isoforms [31]. This explains the large spreading of fibronectin protein spots in molecular weight (150-250 kDa) and pI (pH 5-7). Many more isoforms of fibronectin were observed in IC from RA SF, in comparison to IC from RA serum and healthy RA (Figure 2c). Since these extra isoforms in IC from RA SF are located at a lower molecular weight range, we presume that these isoforms are cleavage products of fibronectin.

Vimentin was detected in IC from RA SF at MW 50-60 kDa and pI 4.6 (Figure 2d). On the contrary, in healthy serum and RA serum, no vimentin was detected in the IC pool (Figure 2d).

In order to reveal citrullinated proteins, AMC detection was performed after successful stripping. In IC from healthy serum and RA serum no citrullination was observed, confirming our prior 1 D analysis. In IC from RA SF on the contrary, spots corresponding to citrullinated proteins could be detected around 50 kDa and pI 4.5-6 (Figure 2e).

To make sure that the detection of these spots was due to binding of the primary antibody and not to aspecific binding, 2D-Western blotting and immunodetection were performed using only the secondary antibodies. The few spots detected on these blots did not correspond to the spots detected with anti-vimentin, anti-Fibβ, anti-Fibγ, or anti-fibronectin (data not shown).

In order to compare the AMC results with the immunodetection results, landmarks were positioned on the blot. Using these landmarks, accurate comparison between the different stainings was possible. This comparison revealed that vimentin and Fibβ were the citrullinated proteins in IC from RA SF. Remarkably, only a minor fraction of the detected Fibβ was also positive with AMC staining. In IC from RA serum no citrullinated Fibβ or citrullinated vimentin could be detected (Figure 2e).

The observed results were confirmed in a second pool of IC from RA SF. A different sequence of antibody staining was used to minimize technical variation and duplicate blots were run to make sure that the AMC detection was not influenced by previous detections or multiple stripping steps. Again, citrullinated vimentin and some citrullinated Fibβ isoforms were present in IC from RA SF (data not shown).

Immunoprecipitation and subsequent immunodetection and AMC staining were performed on individual SF samples of 24 RA patients (12 CCP- patients: SF1 to SF12 and 12 CCP+ patients: SF13 to SF25) and 12 SpA patients (SF27 to SF38). In all the SF samples (n = 36) from RA CCP+ patients as well as RA CCP- patients and SpA patients, Fibβ and/or the processed isoforms (Figure 2a box x) could be detected. Fibγ was found in the isolated IC from 9 out of 12 CCP+ patients, 10 out of 12 CCP- patients and 9 out of 12 SpA patients (Table 2). Interestingly, vimentin was detected in half of the CCP- patients (6 out of 12) and SpA patients (7 out of 12), while 11 out of 12 CCP+ patients were positive for vimentin. The 12^th CCP+ patient showed a weak signal for vimentin. Vimentin could be detected in two sets of spot clusters; between 50 and 60 kDa, at pI 5.3 and pI 4.6 (Figure 3). Strikingly, the acidic isoform (pI 4.6) could only be detected in 6 out of 12 CCP+ RA patients, while none of the CCP- RA patients or SpA patients possessed this acidic isoform of vimentin. AMC staining revealed that the detected vimentin at pI 4.6 was citrullinated in five of the six patients, who were positive for the acidic isoform of vimentin. In contrast, the vimentin at pI 5.3 was not citrullinated. No citrullinated vimentin could be detected in RA CCP- and SpA patients while 5 of the 12 RA CCP+ patients contained citrullinated vimentin in the IC from SF. Citrullinated Fibβ on the other hand could only be observed in SF from one RA CCP+ patient, while no citrullinated Fibβ was detected in RA CCP- patients and SpA patients. Additionally, this citrullinated Fibβ covered only a minor portion of the detected Fibβ in IC.

These results indicate that citrullinated antigens (mainly vimentin) present in IC of SF from RA patients can only be found in CCP+ patients and not in CCP- patients or SpA patients.