Identification of a novel autoantibody against self-vimentin specific in secondary Sjögren’s syndrome
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Sjögren’s syndrome (SS) is a primary autoimmune disease (pSS) or secondarily associated with other autoimmune diseases (sSS). The mechanisms underlying immune dysregulation in this syndrome remain unknown, and clinically it is difficult to diagnose owing to a lack of specific biomarkers.
We extracted immunoglobulins (Igs) from the sera of patients with sSS associated with rheumatoid arthritis (RA) and used them to screen a phage display library of peptides with random sequences.
Our results show that an sSS-specific peptide, designated 3S-P, was recognized by sera of 68.2% (60 of 88) patients with sSS, 66.2% of patients with RA-sSS, and 76.5% of patients with systemic lupus erythematosus (SLE)-sSS. The anti-3S-P antibody was scarcely found in patients with pSS (1.8%), RA (1.3%), SLE (4.2%), ankylosing spondylitis (0%), and gout (3.3%), as well as in healthy donors (2%). The 3S-P-binding Igs (antibodies) were used to identify antigens from salivary glands and synovial tissues from patients with sSS. A putative target autoantigen expressed in the synovium and salivary gland recognized by anti-3S-P antibody was identified as self-vimentin.
This novel autoantibody is highly specific in the diagnosis of sSS, and the underlying molecular mechanism of the disease might be epitope spreading involved with vimentin.
KeywordSjögren’s syndrome Biomarker Autoantigen Vimentin
Secondary Sjögren’s syndrome-associated peptide
Bovine serum albumin
Enzyme-linked immunosorbent assay
Healthy control subject
Heat shock protein 60
Keyhole limpet hemocyanin
National Center for Biotechnology information
PBS with Tween 20
Primary Sjögren’s syndrome
Rabbit-anti-secondary Sjögren’s syndrome-associated peptide
Sodium dodecyl sulfate
Systemic lupus erythematosus
Secondary Sjögren’s syndrome
Time of flight
Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by marked reduction of exocrine glandular secretions, and it progresses to lymphocytic infiltration and destruction of exocrine glands [1, 2, 3]. In addition to arising as a primary disease (primary Sjögren’s syndrome [pSS]), pSS occurs in association with other autoimmune diseases (secondary Sjögren’s syndrome [sSS]), particularly rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [4, 5, 6, 7]. The impact of sSS on RA is illustrated by a twofold increased risk of non-Hodgkin’s lymphoma compared with patients with RA without sSS, and there is a tendency toward increased mortality in patients with RA with sSS compared with those without sSS [8, 9, 10]. Patients with SLE and SS more commonly had renal, peripheral vascular, and musculoskeletal damage than those with SLE without SS .
Evidence indicates that the clinical and immunological features of patients with sSS differ from those of patients with pSS . Antinuclear antibody, anti-SSA, and anti-SSB have been applied to the diagnosis of SS, but none of these autoantibodies provides sufficient specificity and sensitivity [13, 14, 15]. The molecular mechanism of sSS is unknown, and the lack of specific biomarkers constrains efforts to establish the clinical diagnosis of and intervention for the disease. Therefore, the aim of the present study was to identify potential biomarkers and molecules that are specifically associated with sSS. We employed a strategy successfully applied to collagen-induced arthritis, autoimmune pancreatitis, and other autoimmune diseases using a library of peptides with random sequences [16, 17, 18, 19]. We report the identification of a novel autoantibody that was specifically associated with sSS and recognized vimentin as an autoantigen of the disease.
Clinical characteristics of patients
No. of subjects
Mean age, years (range)
Female/male sex (no.)
A random dodecamer peptide library that expresses peptides on a phage virion was purchased from New England Biolabs (Ipswich, MA, USA). The peptide library was screened against three pooled IgG fractions. Each fraction was purified from the pooled sera of ten patients with RA-sSS. To enrich for specific binding phage clones (putatively disease-related), IgGs from ten patients with RA and ten HCs were employed to subtract nonspecific binding clones. Aliquots of 2 × 1011 plaque-forming units in 0.05% PBS with Tween 20 (PBST) were added to IgG-coated wells of a polyvinyl chloride microtiter plate and incubated for 60 minutes at room temperature. The wells were washed ten times with 0.1% PBST, and bound phages were eluted with acidic elution buffer (20 mmol/L Gly-HCl, pH 2.2). Low-pH eluate was immediately neutralized with 1 mol/L Tris-HCl, pH 9.1. Eluates were amplified by infecting Escherichia coli ER2738 host cells (New England Biolabs). After 4.5 h of vigorous shaking at 37 °C, bacteria were removed by centrifugation. Phage particles were purified by two consecutive precipitations with polyethylene glycol (PEG)/NaCl (20% PEG-8000, 2.5 mol/L NaCl) and then resuspended in PBS. These amplified eluates were titered to determine phage concentration and then used as the input phage for the next selection round. Finally, unamplified eluate from the third round of biopanning was used to infect plated bacterial host cells. Single phage clones were assayed by enzyme-linked immunosorbent assay (ELISA). DNA was extracted from positive clones and sequenced.
Analysis of antibody binding
Antibody reactivity against peptides was measured using a solid-phase ELISA. Synthetic purified peptides were coated overnight at 4 °C at 2 μg/well in 0.1 M sodium hydrogen carbonate buffer, pH 9.6. Unbound antibodies were discarded, and wells were blocked with 5% bovine serum albumin (BSA) for 1 h at 37 °C. Serum samples diluted 1:25 in 1% BSA were added to the wells and incubated for 1 h. After a washing step with PBS containing 0.5% (vol/vol) Tween 20, antibody binding was detected by incubation with a goat antihuman IgG horseradish peroxidase-conjugated antibody, followed by color development using 3,3′,5,5′-tetramethylbenzidine as a substrate. The reaction was stopped by adding 1 mol/L H2SO4, and the color intensity was measured at 450 nm. Absorbance values greater than 3 SDs above the mean for each serum dilution of the control group were considered positive.
Preparation of polyclonal anti-3S-P antibodies
The peptide YSLHNAGPWSLHQ, designated 3S-P (secondary Sjögren’s syndrome-related peptide), was manually synthesized using a standard method  for solid-phase peptide synthesis. Polyclonal antibodies against the 3S-P were generated in New Zealand white rabbits using standard techniques (R-anti-3S-P) . R-anti-3S-P antibodies were purified using an immunoaffinity column, which was prepared by conjugating the peptide to SulfoLink Coupling Resin (Pierce Biotechnology/Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions .
Human synovial tissue, salivary gland biopsies, and cells
Synovial tissue specimens and fluid were obtained from patients with RA-sSS during joint replacement surgery at People’s Hospital Peking University in China. Salivary gland specimens were obtained from patients with sSS during salivary gland biopsies. All the methods were carried out in accordance with the guidelines and regulations issued by the Ethics Committee of People’s Hospital Peking University. Human primary fibroblast-like synoviocyte (FLS) cultures were established as previously described with some modifications . Briefly, tissue pieces were minced, treated with 1 mg/ml collagenase for 1 h at 37 °C, and passed through a cell strainer. The cell suspension was incubated at 37 °C in complete DMEM in an atmosphere containing 5% CO2. Cells were harvested at passages 3 through 6.
Sections of the synovial or salivary gland were mounted on glass slides. Immunohistochemical analysis was performed as previously described . R-anti-3S-P and anti-keyhole limpet hemocyanin (anti-KLH) antibodies were used as primary antibodies (25 μg/ml each). The sections were subsequently stained with horseradish peroxidase-conjugated antirabbit antibody (Abcam, Cambridge, UK). Detection was accomplished using diaminobenzidine, and sections were counterstained with hematoxylin.
Immunofluorescence and confocal microscopy
FLSs obtained from patients with RA were immobilized, fixed, and permeabilized as previously described . Cells were incubated with 1% BSA for 30 minutes before incubation. The fixed cells were incubated with the purified antibodies (25 μg/ml each) for 60 minutes. After a washing step, the cells were incubated with fluorescein isothiocyanate-conjugated goat antihuman IgG diluted 1:200, and nuclei were stained with 4′,6-diamidino-2-phenylindole.
Western blot analysis
Proteins extracted from FLSs were electrophoresed and transferred onto a Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences, Marlborough, MA, USA). The membranes were incubated with affinity-purified polyclonal R-anti-3S-P antibodies (5 μg/ml) and then with horseradish peroxidase-conjugated goat antirabbit IgG.
Two-dimensional gel electrophoresis
Proteins obtained from FLSs were separated by two-dimensional gel electrophoresis. Protein samples were loaded onto immobilized pH gradient gel strips (GE Healthcare Life Sciences). After rehydration for 14 h, isoelectric focusing (IEF) was carried out for 1 h at 200 V, 1 h at 500 V, and 1 h at 1000 V continuously, then a gradient was applied from 1000 to 8000 for 1 h and finally at 8000 V for 8 h to reach a total of 72 KVh at 20 °C. Following IEF separation, gel strips were incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate [SDS]) with 10 mg/ml dithiothreitol for 15 minutes, followed by equilibration buffer with 25 mg/ml iodoacetamide for 15 minutes. Then strips were loaded onto 12.5% SDS-PAGE gels and electrophoresed for 20 minutes at a constant current of 10 mA and then at 30 mA per gel until the bromophenol blue reached the bottom of the gels. Subsequently, the two-dimensional gel was silver-stained and scanned. The digital images were analyzed using the GS-800 image scanner (Bio-Rad Laboratories, Hercules, CA, USA) at 300-dpi resolution. Spot detection, quantification, and analyses of two-dimensional protein patterns were done using PDQuest 2-D Analysis software version 7.2 (Bio-Rad Laboratories). Then the report of quantitative differences between two gel images was generated. The t test was performed to compare the relative volume of spots in gels. Significant spots were selected for protein identification.
MS analysis and data interpretation
The bands of interest were excised from the gels and digested with trypsin. The trypsin-digested samples were initially transferred with an aqueous 0.1% formic acid solution to the precolumn at a flow rate of 5 μl/minute for 4 minutes. Mobile phase A was water with 0.1% formic acid, and mobile phase B was 0.1% formic acid in acetonitrile. The peptides were separated with a linear gradient of 10–40% mobile phase B over 60 minutes at 200 nl/minute, followed by 10 minutes at 85% mobile phase B. The column was re-equilibrated at initial conditions for 10 minutes. The column temperature was maintained at 35 °C. The lock mass was delivered from the auxiliary pump of the nanoACQUITY pump (Waters, Milford, MA, USA) with a constant flow rate of 400 nl/minute at a concentration of 100 fmol/μl [Glu1]-fibrinopeptide B. Analysis of trypsin peptides was performed using a SYNAPT high-definition mass spectrometer (Waters). For all measurements, the mass spectrometer was operated in V-mode with a typical resolving power of at least 10,000 FWHM. The time-of-flight (TOF) analyzer of the mass spectrometer was calibrated with the MS/MS fragmentations of [Glu1]-fibrinopeptide B from 50 to 1600 mass/charge ratio. The reference sprayer was sampled with a frequency of 30 seconds. Accurate LC-MS and MS/MS data were collected in high definition data-directed analysis mode. LC-MS/MS data were processed using ProteinLynx Global SERVER version 2.3 (PLGS 2.3; Waters), and the resulting peak lists were subjected to a search against the National Center for Biotechnology information (NCBI) protein database of with the Mascot search engine.
The mouse antivimentin antibody was incubated with the FLS protein extract (400 μl) for 3 h at 4 °C. Next, protein G-conjugated magnetic beads (100 μl) were added and incubated for 1 h at 4 °C, and the beads were collected and washed as instructed by the manufacturer. Laemmli sample buffer was added next, and the mixture was heated for 5 minutes at 95 °C. The supernatants were collected after centrifugation and subjected to SDS-PAGE and Western blot analysis. To detect the 3S-P antigen, the anti-3S-P serum was diluted to 1:800, and peroxidase-conjugated goat antirabbit IgG was used as the secondary antibody. An antivimentin antibody was used at 1:1000 dilution.
We evaluated the sensitivity and specificity of the tests using the ROC curve and estimated the AUC at 95% CIs. Statistical analysis was performed using SPSS software version 16 (SPSS, Chicago, IL, USA).
Identification of a peptide specifically recognized by sera from patients with sSS
Autoantigen is expressed in synovial membrane and salivary gland
Vimentin is autoantigen in sSS
In clinical practice, there is no specific serologic marker for sSS, and the immunopathology of this condition is poorly understood. Diagnosis relies on salivary gland biopsy, which is invasive, requires evaluation by an expert histopathologist, and may have adverse sequelae . We report the detection of an antibody against 3S-P in 60 of 88 of patients with sSS in the present study. This assay was highly sensitive and specific for distinguishing sSS from pSS. Furthermore, the profile of reactivity of anti-3S-P differs from those reported for anti-SSA and anti-SSB [14, 15], which suggests that the antigen recognized by anti-3S-P differs from those recognized by anti-SSA and anti-SSB. Taken together, these results indicate that the immunopathogenesis of sSS differs from that of pSS.
In the present study, we determined that vimentin is an autoantigen recognized by anti-3S-P. In humans, vimentin is expressed by a wide variety of mesenchymal cell types, such as fibroblasts and endothelial cells, and is easily detected in the synovium [27, 28]. Antibodies against citrullinated vimentin are prominent in the development of RA [29, 30, 31, 32]. Our results show that the antibody present in the majority of patients with sSS tested, which we designated as anti-3S-P, reacted with noncitrullinated recombinant human vimentin. This indicates that the production of anti-3S-P antibodies in patients with sSS may result from epitope spreading, which occurs in several models of autoimmunity [33, 34, 35, 36]. There is no evidence, to our knowledge, that antivimentin antibodies are present in patients with SLE. Therefore, the occurrence and clinical correlates of anti-3S-P antibodies in patients with SLE should be investigated further.
In the present study, we identified an antibody that is highly specific and sensitive for sSS. Further studies are required to determine whether this peptide antibody is useful for clinical diagnosis of sSS and involved in disease pathogenesis. We have demonstrated a novel autoantibody that is highly specific in the diagnosis of sSS and that vimentin is an autoantigen recognized by anti-3S-P. The underlying molecular mechanism of the disease might be epitope spreading involved with vimentin.
The authors thank all the patients who participated in this study. Furthermore, the authors acknowledge C. Liu, Dr. Z. Pei, and Dr. J. X. Zhu for their contributions to clinical data collection.
This work was supported by the National Natural Science Foundation of China (81671602 and 31530020) and the Beijing Nova Program (Z171100001117025)
Availability of data and materials
The dataset generated during the present study is available from the corresponding author on reasonable request.
YHL and JD performed the majority of the experiments and drafted the manuscript. YPG screened the peptide library. XLS, RL, XWZ, YL, and QJZ obtained patients’ synovial tissues and clinical data at Peking University People’s Hospital. LL and JH carried out the cell cultures. LJS contributed to data analysis. EM interpreted data and wrote the paper. ZGL and GY designed the study. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All the procedures involving specimens obtained from human subjects were performed under protocols approved by the Human Research Protective Committee of People’s Hospital Peking University, and all the methods were carried out in accordance with the guidelines and regulations of the Ethics Committee of People’s Hospital Peking University. Informed consent was also provided by all subjects before the study protocol was initiated.
Consent for publication
The authors declare that they have no competing interests.
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