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. 2014 Apr 10;142(1):67–77. doi: 10.1111/imm.12197

Tryptase is a candidate autoantigen in rheumatoid arthritis

Yanyan Guo 1, Qiao Wu 1, Bing Ni 1, Zhirong Mou 1, Qiong Jiang 1, Yi Cao 1, Hui Dong 1, Yuzhang Wu 1,✉
PMCID: PMC3992049  PMID: 24433328

Abstract

Autoimmune processes have been implicated in the development of rheumatoid arthritis (RA); however, specific autoantigens that play a role in the aetiology of RA have been lacking. In this study, we found that sera from RA patients were particularly immunoreactive against the protein tryptase. Compared with osteoarthritis (OA) patients and healthy controls, RA patients had relatively higher levels of tryptase and concomitant anti-tryptase antibodies in their synovial tissues and sera. Similarly, synovial fluid from RA patients, but not from OA patients, contained antibodies that recognized tryptase in vitro. In addition, serum tryptase levels in both early and late RA patients significantly correlated with clinical indices usually used to diagnose RA, such as rheumatoid factor, Disease Activity Score using 28 joint counts and autoantibodies against cyclic citrullinated peptide. Our results identify tryptase as a candidate autoantigen involved in the pathogenesis of RA and monitoring its levels may have diagnostic and prognostic value.

Keywords: autoantibody, autoantigen, rheumatoid arthritis, tryptase

Introduction

Rheumatoid arthritis (RA) affects the overall health and quality of life of 0·5–1% of adults worldwide. RA is a complex autoimmune disease that can cause synovial inflammation, joint destruction and damage to blood vessels and organs.1,2 A normal synovium is composed of an intimal lining layer and a synovial sublining. However, in RA patients, CD4+ T cells, B cells and macrophages invade the synovium causing marked hyperplasia of the intimal lining and massive infiltration of the synovial sublining. Moreover, the immunogenic pathways that follow, such as secretion of inflammatory mediators and degradative enzymes, drive the destruction of local cartilage and bone.

Although the exact mechanism of RA pathogenesis is still not fully understood, autoimmune responses are considered to play an important role.3,4 It has been hypothesized that autoantigens, presented by RA-associated HLA-DR molecules, activate self-reactive antigen-specific immune cells to initiate synovial inflammation.5 Currently, the autoantigens purported to induce autoimmune responses in RA remain unclear.5 For the past few years, a variety of candidate autoantigens have been studied and categorized into three major groups: (i) antigens derived from the origin of the joint, such as type II collagen, human chondrocyte glycoprotein 39 and proteoglycan; (ii) highly conserved foreign antigens with human homologues, including heat-shock proteins, Epstein–Barr virus trans-acting factor and Escherichia coli dnaJ; and (iii) post-translationally modified proteins, such as citrullinated filaggrin and immunoglobulin G.6,7 Although these putative autoantigens are specifically detected in the joint, many of them lack a definite association with clinical RA pathogenesis. For example, while type II collagen is found in the joints of some RA patients, they generally lack anti-type II collagen antibodies.8,9 Furthermore, levels of type II collagen in the joints of antibody-positive patients do not correlate well with the duration, activity or severity of RA.8,9 Therefore, there is a need to identify novel RA-associated autoantigens that will not only inform mechanistic studies of RA pathogenesis, but also be of diagnostic value.

In this study, we identified tryptase as a candidate RA autoantigen by analysing proteins from synovial tissues of RA patients using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We detected high levels of tryptase protein and its cognate antibody in synovial tissues and sera of RA patients. Furthermore, tryptase in synovial tissues co-localized with IgG complexes as determined by co-immunofluorescence analysis. Importantly, tryptase levels were markedly associated with key indices for RA disease, including the Disease Activity Score using 28 joint counts (DAS28), rheumatoid factor (RF) and autoantibodies against cyclic citrullinated peptide (anti-CCP). Our results implicate tryptase in the pathogenesis of RA and suggest that its presence in serum or synovium may serve as a diagnostic indicator of RA.

Materials and methods

Sera and synovium samples

We collected samples from patients and healthy volunteers in the Department of Traditional Chinese Medicine of Southwest Hospital in Chongqing. Synovial samples were collected from patients with RA and osteoarthritis (OA), and serum samples were collected from patients with early RA (< 6 months), late RA (> 6 months), OA, systemic lupus erythematosus (SLE) as well as from normal controls (healthy volunteers). The samples were assigned codes to maintain patient anonymity. The institutional review board of the Third Military Medical University approved this study, and written consents were obtained from all participants. Diagnoses of RA were carried out according to the 1987 classification criteria of the American College of Rheumatology.10

Two-dimensional electrophoresis and Western blot assays

Two-dimensional electrophoresis and Western blots assays were performed as described previously.11,12 Briefly, the synovial tissue samples from RA patients were harvested and washed in PBS and then homogenized with an HG30 homogenizer (Hitachi Koki Co., Ltd, Tokyo, Japan) in lysis buffer containing 40 mm Tris–HCl, 8 m urea, 4% CHAPS, 60 mm dithiothreitol, 0·8% immobilized pH gradient buffer (pH 3–11), and protease inhibitor cocktail (Roche, Mannheim, Germany) on ice. Next, the samples were frozen and thawed five times consecutively and then centrifuged at 12 000 g, at 4° for 30 min. Protein concentration in the supernatant was determined using a Bradford Protein Assay Kit (Beyotime, Shanghai, China). Total protein (800 μg) was analysed by isoelectric focusing assays, which were run using the following conditions: 200 V for the first hour, 500 V for the next hour, 1000 V for another hour, and then gradual increase to 8000 V during a 5–6 hr period. Focusing was carried out at 35 000 V/h. The second electrophoresis was the denatured 12·5% SDS–PAGE After the two-dimensional electrophoresis, the proteins were transferred to a PVDF membrane or visualized by Coomassie brilliant blue G250 staining. Images were digitalized using a GS-800 Calibrated Densitometer (BioRad, Hercules, CA) and analysed by pd quest 2-d analysis software (BioRad). For Western blot assays, the membranes were incubated overnight at 4° with 1 : 200 diluted sera, which were pooled from five patients with RA, OA or SLE, or healthy controls, respectively. After washing three times with Tris-buffered saline containing 0·01% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated anti-human IgG antibodies (Beijing Zhongshan, China). Immunodetection was performed by enhanced chemiluminescence reagent (Roche, Indianapolis, IN) followed by exposure to Kodak film. A monoclonal antibody against tryptase (Abcam, Cambridge, UK) and an horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Beijing) were used.

Protein identification with MALDI-TOF-MS

The proteins on the Coomassie brilliant blue stained gel that were positive by Western blot were cut out and dried with a SpeedVac plus SC1 10 (Savant, Holbook, NY) centrifuge. The dried gel was rehydrated in trypsin solution (Promega, Sunnyvale, CA) at 37° overnight. After rehydration, peptides were first eluted with 5% trifluoroacetic acid (TFA) at 40° for 1 hr and then with 2·5% TFA/50% acetonitrile at 30° for 1 hr. Acetonitrile was removed by centrifugation in a vacuum centrifuge. The peptides were concentrated using C18 matrix embedded pipette tips (Millipore, Bedford, MA). Analysis was performed primarily using a MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Peptide mixtures were analysed using a saturated solution of alpha-cyano-4-hydroxy-cinnamic acid (CHCA) (Sigma, St. Louis, MO) in acetone containing 1% TFA. Peptides were selected in the molecular weight range of 800–4000. The peptide sequence was determined with the mascot software (Matrix Science Inc., Boston, MA). Sequence homology was analysed using the MASCOT program and the NCBI BLAST online search service.

Isolation and culture of synovial fibroblasts

The isolation and culture of RA synovial fibroblasts (RASFs) were conducted by referring to a published method.13 Briefly, samples of RA synovial tissues were minced and digested for 30 min at 37° in 20 ml PBS containing 0·1% trypsin (Sigma). After removal of trypsin/PBS, the samples were digested in 20 ml of 0·1% collagenase P (Roche) in Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum (FCS) for 2 hr at 37° and 5% CO2. The cell suspension was then filtered through a sterile sieve (Sigma), washed twice with serum-free DMEM, and subsequently maintained in DMEM/10% FCS, 25 mm HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin and 2·5 μg/ml amphotericin B (all from Gibco BRL, Shanghai, China).

Immunohistochemistry and laser scanning confocal microscopy

Primary RASFs or serial cryosections from the synovium of patients with either active RA synovitis or osteoarthritis were fixed in acetone and blocked with 5% BSA. To detect tryptase, indirect immunofluorescence was carried out with anti-tryptase primary antibodies (1 : 100) (MAB1222; Millipore) and Cy5-conjugated secondary antibodies (A0539; Beyotime). Concurrently, FITC-conjugated anti-CD55 (ab25634; Abcam) antibodies were used to mark fibroblast-like synoviocytes. Nuclei were stained with DAPI. Cells were examined by a laser scanning confocal microscope (Leica, Heidelberg, Germany). For co-localization experiments, freshly frozen synovial tissue samples were stained with mouse monoclonal anti-tryptase and FITC-labelled IgG (F9512; Sigma-Aldrich).

ELISA analysis

Sera from 20 early RA patients, 28 late RA patients, 10 OA patients, 12 SLE patients and 30 healthy controls were tested for immunoreactivity to purified tryptase (20 μg/ml), which was coated on the plates overnight at 4°. After washing with PBS–0·05% Tween-20, the plates were blocked with 10% BSA. Sera were diluted at a ratio of 1 : 100 and incubated with the test antigen for 2 hr at 37°. After washing, horseradish peroxidase-labelled goat anti-human IgG antibody (1 : 4000) was added. After washing with PBS–1% Tween-20 ten times, bound antibodies were visualized with tetramethyl benzidine substrate and measured at 450 nm. To assure standardized conditions for the anti-tryptase ELISA, analyses of two standard control sera were always included. All serum samples were tested at least three times. Statistical analysis for significant differences between samples was performed using the unpaired Student's t-test.

To determine serum tryptase levels in RA patients, sera from 20 patients with early RA, 28 patients with late RA, 10 patients with OA, 15 patients with SLE and 52 normal controls were tested by ELISA using anti-tryptase (1 : 200) (MAB1222; Millipore). Sera were diluted at a ratio of 1 : 100 and incubated with anti-tryptase for 2 h at 37° and detected as stated above. To assure standardized conditions for all anti-tryptase ELISAs in the study, analyses of two standard control sera were always included. All serum samples were tested at least three times. Statistical analysis for significant differences between samples was performed using the unpaired Student's t-test.

Statistical analysis

Each experiment was performed at least three times. In our studies, significance was determined by Student's t-test and one-way analysis of variance using graphpad prism software version 5.02 (GraphPad Software, Inc., La Jolla, CA). P value < 0·05 was considered significant.

Results

Tryptase is a candidate autoantigen detected in the synovial tissues of RA patients

To find novel RA-associated autoantigens, synovial proteins from RA patients were resolved by two-dimensional electrophoresis and either stained with Coomassie brilliant blue (Fig. 1a) or subjected to Western blot analysis using patient sera (Fig. 1a,b). Using this strategy, we found several immunoreactive proteins against sera from RA patients (Fig. 1b), OA patients (Fig. 1c), SLE patients (Fig. 1d) or healthy controls (Fig. 1e). Notably, we found that one protein spot, No. 12, was specifically recognized by RA sera only (Fig. 1b, arrow). Analysis by MALDI-TOF-MS identified this protein as tryptase (Fig. 1f). Western blot analysis using a monoclonal antibody specific for tryptase confirmed our MS analysis (Fig. 1g). Intriguingly, tryptase levels in synovial tissues of RA patients were significantly higher than those of OA patients (Fig. 1h). Taken together, our results show that RA patients not only have anti-tryptase antibodies in their sera, but also express high levels of tryptase in synovial tissues, suggesting that tryptase might be involved in the pathogenesis of RA.

Figure 1.

Figure 1

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Identification of candidate synovial autoantigens in rheumatoid arthritis (RA) patients. (a) Proteins extracted from synovial tissues of five RA patients were separated by two-dimensional electrophoresis and stained with Coomassie brilliant blue. In parallel, proteins were transferred onto PVDF membranes and probed with pooled sera samples (diluted 1 : 200) from five patients with (b) RA, (c) osteoarthritis (OA), (d) systemic lupus erythematosus (SLE), or (e) normal controls. (f) The No. 12 protein spot was specific to RA patient sera [indicated in (a) and (b) with arrow] and was analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. (g) Synovial proteins from RA patients were separated by two-dimensional electrophoresis and analysed by Western blotting using anti-tryptase. (h) The expression levels of tryptase in RA and OA synovial tissues as detected by Western blotting using anti-tryptase.

Unique distribution of tryptase in synovial fibroblasts of RA patients

Our results above indicated that tryptase is highly expressed in synovial fibroblasts of RA patients. To investigate the distribution of tryptase in synovial tissues, serial cryosections of RA or OA patient tissues were stained with antibodies against tryptase (red) or CD55 (green), a marker for synovial fibroblast cells.14,15 The images, captured by laser-scanning confocal microscopy, initially revealed that the synovial layers of the inflamed joints were thicker in RA patients (Fig. 2a,c), as compared to those of OA patients (Fig. 2b,d). Importantly, almost all tryptase-positive cells were also positive for CD55 (Fig. 1a,c), suggesting that synovial fibroblasts are the likely source of tryptase in the joints of RA patients. In contrast, hardly any tryptase staining was observed in the synovial tissue of OA patients (Fig. 2b,d). We further investigated the expression of tryptase in primary cultured RASFs and detected high protein levels in these cells (Fig. 2e), but not in primary synovial fibroblasts from OA patients (data not shown). Overall, these ex vivo and in vitro assays not only confirmed that tryptase expression is specific to synovial tissues of RA patients, but also showed that tryptase is mainly expressed in CD55-positive fibroblasts, further suggesting that tryptase may function as an autoantigen in RA patients.

Figure 2.

Figure 2

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Distribution of tryptase in synovial tissue of rheumatoid arthritis (RA) patients. Serial cryosections from the synovium of patients with RA or osteoarthritis (OA) were co-stained with mouse anti-tryptase (red) and anti-CD55 (green), and analysed by laser scanning confocal microscopy. Tryptase and CD55 (a marker for synovial fibroblast cells) were co-localized in the synovial lining cells of (a) RA patients and (b) OA patients. (c, d) Enlarged images of the (a) and (b), respectively. (e) Tryptase and CD55 were co-localized in primary cultured RA synovial fibroblasts.

Tryptase protein levels are elevated in RA patient sera

Given that tryptase is expressed in the synovial tissues of RA patients, we next tested whether tryptase levels are also elevated in the sera of RA patients. To address this, we measured serum tryptase levels in 20 early RA patients, 28 late RA patients, 25 patients with other autoimmune diseases (10 with OA and 15 with SLE) and 52 normal healthy individuals. We found that the serum levels of tryptase in both early RA patients and late RA patients were significantly higher than those in OA and SLE patients and in healthy controls (Fig. 3). We also observed higher serum tryptase levels in early RA patients compared with its levels in late RA patients (Fig. 3).

Figure 3.

Figure 3

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Significantly increased tryptase levels in sera from rheumatoid arthritis (RA) patients. Serum tryptase levels of 20 early RA patients, 28 late RA patients, 10 osteoarthritis (OA) patients, 15 systemic lupus erythematosus (SLE) patients and 52 healthy individuals were measured by ELISA using anti-tryptase (1 : 200) for the primary antibody. All serum samples were tested at least three times. Statistical analysis for significant differences between samples was performed using the unpaired Student's t-test. The P-values for the corresponding statistical comparisons are indicated.

High concentration of anti-tryptase IgG in RA sera correlates with high levels of serum tryptase

A candidate autoantigen for RA pathogenesis should have high levels of corresponding antibodies in RA patient sera. Therefore, we examined patient sera for levels of anti-tryptase antibodies. Initially, we expressed and purified recombinant human tryptase (rhTryptase) in E. coli (data not shown), and showed that it is recognized by commercial antibodies (Fig. 4a). Next, we sought out antibodies against rhTryptase in sera from RA, OA and SLE patients and from normal controls by Western blot. We found that all RA patients were positive for anti-tryptase antibodies, which were lacking in OA and SLE patients and healthy controls (Fig. 4a).

Figure 4.

Figure 4

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Increased tryptase-specific antibody levels in sera of rheumatoid arthritis (RA) patients. (a) Western blot analysis of recombinant human (rh) Tryptase detected with a commercially available antibody or sera from RA, osteoarthritis (OA), or systemic lupus erythematosus (SLE) patients and normal controls. (b) Tryptase-specific antibody levels determined by indirect ELISA. The 96-well plates were coated with rhTryptase (20 μg/ml) overnight at 4°. The sera from 20 patients with early RA, 28 patients with late RA, 10 OA patients, 12 SLE patients and 30 healthy controls were diluted at a ratio of 1 : 100 and incubated with the test antigen for 2 hr at 37°. Horseradish peroxidase-labelled goat anti-human IgG antibody (1 : 4000) was used as the secondary antibody and the optical density at 450 nm (OD450) was measured for each well. All serum samples were tested at least three times. The P-values for the corresponding statistical comparisons are indicated. (c) Correlation between serum tryptase levels and serum anti-tryptase levels in early RA and late RA patients. The correlation coefficient and P-values are shown.

To investigate the possible correlation between anti-tryptase antibody levels and RA disease, serum anti-tryptase levels were examined in 20 early RA patients, 28 late patients, 10 OA patients, 12 SLE patients and 20 healthy controls. As shown in Fig. 4(b), early RA patients (0·78 ± 0·2) had significantly higher levels of antibodies against tryptase than OA patients (0·4 ± 0·08, P < 0·05), SLE patients (0·37 ± 0·2, P < 0·001) and healthy controls (0·25 ± 0·1, P < 0·001). Although late RA patients (0·52 ± 0·1) had significantly higher levels of anti-tryptase antibodies compared with SLE patients (0·37 ± 0·2, P < 0·05) and healthy controls (0·25 ± 0·1, P < 0·05), there was no significant difference with OA patients (0·4 ± 0·08, P > 0·05). We also found that serum tryptase levels were significantly correlated with serum anti-tryptase IgG levels in early and late RA patients (P < 0·01, Fig. 4c).

Anti-tryptase antibodies are highly concentrated in synovial fluids of RA patients and IgGs co-localize with tryptase in synovial tissues of RA patients

As the deposition of immune complexes in affected tissues is a common mechanism for the pathogenesis of various autoimmune diseases,16 we tested whether anti-tryptase-associated immune complexes are present in the synovial tissues of RA patients. Hence, we initially investigated whether anti-tryptase antibodies were present in the synovial fluids of RA patients. The results showed that the synovial fluids of RA patients, but not OA patients, were positive for anti-tryptase antibodies (Fig. 5a).

Figure 5.

Figure 5

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Tryptase-specific antibodies are present in synovial fluids and possibly co-localize with tryptase protein in synovial tissues of rheumatoid arthritis (RA) patients. Recombinanat human Tryptase was separated by SDS–PAGE and analysed by Western blotting using synovial fluids from RA and osteoarthritis (OA) patients diluted at a ratio of 1 : 200 (a). Serial cryosections of synovial tissues from RA patients (b) or OA patients (c) were stained with mouse anti-tryptase and anti-IgG antibodies, and analysed by laser scanning confocal microscopy. (d) and (e) are enlarged images of (b) and (c), respectively.

Given that anti-tryptase antibodies are present in synovial fluids in RA patients, we next investigated whether tryptase co-localizes with IgGs in the synovial tissues of RA patients. Serial cryosections from the synovium of patients with either RA synovitis or OA were co-stained with anti-tryptase and anti-IgG antibodies and analysed by laser scanning confocal microscopy. We found that tryptase and IgG were co-localized in the synovial lining and underlining cells of RA patients (Fig. 5b,d), but not in OA patients (Fig. 5c,e). Although we could not detect tryptase co-localization with anti-tryptase antibodies due to technical limitations, these results suggest that anti-tryptase antibodies may mediate the formation of immune complexes associated with tryptase in synovial tissues.

Serum tryptase levels correlate with clinical data of RA patients

There are several clinical parameters required to efficiently evaluate the disease activity of RA patients, including DAS28, RF anti-CCP, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and glucose-6-phosphate isomerase (GPI).17,18 In order to understand the relationship between serum tryptase levels and RA disease activity, we performed a correlation analysis of clinical indices and serum tryptase levels in early and late RA patients. The results showed that serum tryptase levels in both early and late RA patients were correlated with DAS28, RF and anti-CCP, but not with CRP, ESR and GPI indices (Fig. 6).

Figure 6.

Figure 6

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Correlation analysis between clinical parameters and serum tryptase levels in rheumatoid arthritis (RA) patients. The correlations between clinical indices including disease activity score using 28 joint counts (DAS28), rheumatoid factor (RF), autoantibodies against cyclic citrullinated peptide (anti-CCP), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and glucose-6-phosphate isomerase (GPI) and serum tryptase levels in early RA (a) and late RA (b) patients were analysed. The corresponding P-values are indicated.

Discussion

Autoantigens probably play pivotal roles in the development of RA.5 However, although several candidate autoantigens have been reported, their functional link to RA pathogenesis has remained elusive.5 To identify RA-specific autoantigens, we probed the synovial proteome of RA patients with RA patient sera. We identified tryptase by MALDI-TOF-MS as a candidate RA autoantigen. Both tryptase protein and anti-tryptase antibody levels were significantly elevated in RA patient sera and synovial tissues. In contrast, almost no tryptase or anti-tryptase antibodies were detected in healthy controls, OA patients, and SLE patients. Interestingly, tryptase co-localized with immune complexes, presumably through its interaction with anti-tryptase antibodies, in synovial tissues of RA patients. Finally, we found that key diagnostic indices of RA including DAS28, RF and anti-CCP were positively correlated with serum tryptase levels, indicating that tryptase might not only be involved in the pathogenesis of RA, but also might serve as a specific biomarker for RA.

It has been known that tryptase is a trypsin-like serine protease, which is stored in the secretory granules of mast cells. When mast cells are triggered, e.g. during allergic conditions, tryptase is released together with other inflammatory mediators. Accordingly, tryptase has been implicated in many diseases linked to inflammation, such as asthma, sudden infant death syndrome,19 arthritis,20 multiple sclerosis/experimental autoimmune encephalomyelitis,21,22 psoriasis,23 fibrosis24 and atopic dermatitis.25 In patients with RA, the number of mast cells and tryptase protein levels increase in synovial fluids.26,27 Tryptase has an anti-apoptotic effect on RASFs via the activation of Rho, which promotes proliferation of RASFs and hyperplasia of synovial tissues in RA patients.28 These results suggest that tryptase from mast cells acts as an effector molecule in inflammatory diseases such as RA. However, in this study, we found that fibroblasts, including RASFs, may constitute a novel source of tryptase in synovial tissues.

Importantly, in addition to increased tryptase levels, we detected higher concentrations of tryptase-specific antibodies in RA patient sera and synovial fluids, compared with samples from OA, SLE and healthy controls. We also observed a marked correlation between serum tryptase levels and anti-tryptase antibody levels in early and late RA patients. These results implicate tryptase as a candidate RA-specific autoantigen. It has been suggested that immune complexes form in inflamed joints of patients with RA.29 In this study, we observed that tryptase was co-localized with IgGs in synovial lining tissues, suggesting that anti-tryptase antibodies may mediate the formation of immune complexes in the synovial tissues of RA patients. These results further implicate tryptase in the immunopathology of joint inflammation in RA. The tryptase-containing immune complexes may activate the complement pathway of inflammatory arthritis, depending on FcγR and constituents of the alternative complement pathway, similar to that observed in K/BxN arthritis mouse models.30

Rheumatoid arthritis is a comprehensive disease with a highly variable and unpredictable course of progression. Consequently, there have been several clinical parameters developed to monitor RA, such as RF, DAS28, CCP, CRP, ESR and GPI.17,18 In this study, we analysed the correlation between these clinical parameters and tryptase levels in RA patient sera. We found that RF, DAS28 and anti-CCP were significantly correlated with serum tryptase levels. Among these parameters, serological RF is currently used for RA diagnosis because it is detected in 70–80% of RA patients.31 Our results showed that there is significant correlation between high serum tryptase levels and RF, indicating that serum tryptase levels might serve as a novel and specific diagnostic marker for RA. The DAS28 index is generally used to estimate disease activity in RA patients.32 DAS28 is a complex index that provides clinicians with an objective evaluation of disease activity and progression.32 As a result of its correlation with DAS28, high serum tryptase levels may reflect progressive disease activity of RA. Recently, it was shown that serum anti-CCPs are highly specific and predictive for RA.33 Anti-CCPs can be observed many years before initiation of disease34 and are associated with joint destruction.35 Our results show that high serum tryptase levels were significantly correlated with anti-CCP concentrations in RA patients. Taken together, the correlation analysis revealed that serum tryptase might serve as a novel diagnostic marker of RA.

In conclusion, we identified tryptase as a novel candidate autoantigen present in a large proportion of patients with RA and provided evidence potentially linking tryptase to the pathogenesis of RA. We conclude that monitoring tryptase levels may serve as a helpful diagnostic or prognostic tool for RA disease. However, the specific molecular mechanisms underlying tryptase-mediated RA pathogenesis remain to be elucidated in RA animal models.

Acknowledgments

YG carried out the two-dimensional electrophoresis, Western blots, immunohistochemistry and ELISAs. QW performed the clinical correlation analysis. ZM carried out the immunoglobulin experiment and participated in the drafting of the manuscript. QJ collected the patients' samples and participated in the analysis of the related results. YC carried out the ELISA experiments. HD participated in the immunohistochemistry assays. BN helped draft the manuscript. YW designed the study and drafted the manuscript. All authors read and approved the final manuscript. This study was supported by grants from the Major State Basic Research Development Programme of China (973 Programme) (No.2007CB512401) and the Programme for Changjiang Scholars and Innovative Research Team in University (PCSIRT10521).

Glossary

anti-CCP

autoantibodies against cyclic citrullinated peptide

CRP

C-reactive protein

DAS28

Disease Activity Score using 28 joint counts

DMEM

Dulbecco's modified Eagle's medium

ESR

erythrocyte sedimentation rate

FCS

fetal calf serum

GPI

glucose-6-phosphate isomerase

ICs

immune complexes

MALDI-TOF-MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

OA

osteoarthritis

RA

rheumatoid arthritis

RASFs

rheumatoid arthritis synovial fibroblasts

RF

rheumatoid factor

rhTryptase

recombinant human tryptase

SLE

systemic lupus erythematosus

TFA

trifluoroacetic acid

Disclosures

All authors declare no financial or commercial conflict of interest.

References

  • 1.Firestein GS. Evolving concepts of rheumatoid arthritis. Nature. 2003;423:356–61. doi: 10.1038/nature01661. [DOI] [PubMed] [Google Scholar]
  • 2.Silman AJ, Pearson JE. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res. 2002;4(Suppl. 3):S265–72. doi: 10.1186/ar578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Cronstein BN, Weissmann G. The adhesion molecules of inflammation. Arthritis Rheum. 1993;36:147–57. doi: 10.1002/art.1780360204. [DOI] [PubMed] [Google Scholar]
  • 4.Jalkanen S. Leukocyte–endothelial cell interaction and the control of leukocyte migration into inflamed synovium. Springer Semin Immunopathol. 1989;11:187–98. doi: 10.1007/BF00197188. [DOI] [PubMed] [Google Scholar]
  • 5.Corrigall VM, Panayi GS. Autoantigens and immune pathways in rheumatoid arthritis. Crit Rev Immunol. 2002;22:281–93. [PubMed] [Google Scholar]
  • 6.Auger I, Balandraud N, Rak J, Lambert N, Martin M, Roudier J. New autoantigens in rheumatoid arthritis (RA): screening 8268 protein arrays with sera from patients with RA. Ann Rheum Dis. 2009;68:591–4. doi: 10.1136/ard.2008.096917. [DOI] [PubMed] [Google Scholar]
  • 7.Hoffmann MH, Tuncel J, Skriner K, et al. The rheumatoid arthritis-associated autoantigen hnRNP-A2 (RA33) is a major stimulator of autoimmunity in rats with pristane-induced arthritis. J Immunol. 2007;179:7568–76. doi: 10.4049/jimmunol.179.11.7568. [DOI] [PubMed] [Google Scholar]
  • 8.Conrad K, Roggenbuck D, Reinhold D, Dorner T. Profiling of rheumatoid arthritis associated autoantibodies. Autoimmun Rev. 2010;9:431–5. doi: 10.1016/j.autrev.2009.11.017. [DOI] [PubMed] [Google Scholar]
  • 9.Mottonen T, Hannonen P, Oka M, Rautiainen J, Jokinen I, Arvilommi H, Palosuo T, Aho K. Antibodies against native type II collagen do not precede the clinical onset of rheumatoid arthritis. Arthritis Rheum. 1988;31:776–9. doi: 10.1002/art.1780310612. [DOI] [PubMed] [Google Scholar]
  • 10.Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315–24. doi: 10.1002/art.1780310302. [DOI] [PubMed] [Google Scholar]
  • 11.Bruschi M, Musante L, Candiano G, Ghiggeri GM, Herbert B, Antonucci F, Righetti PG. Soft immobilized pH gradient gels in proteome analysis: a follow-up. Proteomics. 2003;3:821–5. doi: 10.1002/pmic.200300361. [DOI] [PubMed] [Google Scholar]
  • 12.Tsuruha J, Masuko-Hongo K, Kato T, Sakata M, Nakamura H, Nishioka K. Implication of cartilage intermediate layer protein in cartilage destruction in subsets of patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum. 2001;44:838–45. doi: 10.1002/1529-0131(200104)44:4<838::AID-ANR140>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 13.Zimmermann T, Kunisch E, Pfeiffer R, et al. Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture – primary culture cells markedly differ from fourth-passage cells. Arthritis Res. 2001;3:72–6. doi: 10.1186/ar142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Schwartz LB, Bradford TR. Regulation of tryptase from human lung mast cells by heparin. Stabilization of the active tetramer. J Biol Chem. 1986;261:7372–9. [PubMed] [Google Scholar]
  • 15.Smith MD, Barg E, Weedon H, et al. Microarchitecture and protective mechanisms in synovial tissue from clinically and arthroscopically normal knee joints. Ann Rheum Dis. 2003;62:303–7. doi: 10.1136/ard.62.4.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Nowling TK, Gilkeson GS. Mechanisms of tissue injury in lupus nephritis. Arthritis Res Ther. 2011;13:250. doi: 10.1186/ar3528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Korganow AS, Ji H, Mangialaio S, et al. From systemic T cell self-reactivity to organ-specific autoimmune disease via immunoglobulins. Immunity. 1999;10:451–61. doi: 10.1016/s1074-7613(00)80045-x. [DOI] [PubMed] [Google Scholar]
  • 18.Schaller M, Burton DR, Ditzel HJ. Autoantibodies to GPI in rheumatoid arthritis: linkage between an animal model and human disease. Nat Immunol. 2001;2:746–53. doi: 10.1038/90696. [DOI] [PubMed] [Google Scholar]
  • 19.Buckley MG, Variend S, Walls AF. Elevated serum concentrations of β-tryptase, but not α-tryptase, in Sudden Infant Death Syndrome (SIDS). An investigation of anaphylactic mechanisms. Clin Exp Allergy. 2001;31:1696–704. doi: 10.1046/j.1365-2222.2001.01213.x. [DOI] [PubMed] [Google Scholar]
  • 20.Buckley MG, Walters C, Wong WM, Cawley MI, Ren S, Schwartz LB, Walls AF. Mast cell activation in arthritis: detection of α-and β-tryptase, histamine and eosinophil cationic protein in synovial fluid. Clin Sci (Lond) 1997;93:363–70. doi: 10.1042/cs0930363. [DOI] [PubMed] [Google Scholar]
  • 21.Pedotti R, DeVoss JJ, Youssef S, et al. Multiple elements of the allergic arm of the immune response modulate autoimmune demyelination. Proc Natl Acad Sci USA. 2003;100:1867–72. doi: 10.1073/pnas.252777399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rozniecki JJ, Hauser SL, Stein M, Lincoln R, Theoharides TC. Elevated mast cell tryptase in cerebrospinal fluid of multiple sclerosis patients. Ann Neurol. 1995;37:63–6. doi: 10.1002/ana.410370112. [DOI] [PubMed] [Google Scholar]
  • 23.Naukkarinen A, Harvima IT, Aalto ML, Horsmanheimo M. Mast cell tryptase and chymase are potential regulators of neurogenic inflammation in psoriatic skin. Int J Dermatol. 1994;33:361–6. doi: 10.1111/j.1365-4362.1994.tb01069.x. [DOI] [PubMed] [Google Scholar]
  • 24.Kondo S, Kagami S, Kido H, Strutz F, Muller GA, Kuroda Y. Role of mast cell tryptase in renal interstitial fibrosis. J Am Soc Nephrol. 2001;12:1668–76. doi: 10.1681/ASN.V1281668. [DOI] [PubMed] [Google Scholar]
  • 25.Jarvikallio A, Naukkarinen A, Harvima IT, Aalto ML, Horsmanheimo M. Quantitative analysis of tryptase-and chymase-containing mast cells in atopic dermatitis and nummular eczema. Br J Dermatol. 1997;136:871–7. [PubMed] [Google Scholar]
  • 26.Crisp AJ, Chapman CM, Kirkham SE, Schiller AL, Krane SM. Articular mastocytosis in rheumatoid arthritis. Arthritis Rheum. 1984;27:845–51. doi: 10.1002/art.1780270802. [DOI] [PubMed] [Google Scholar]
  • 27.Nakano S, Mishiro T, Takahara S, et al. Distinct expression of mast cell tryptase and protease activated receptor-2 in synovia of rheumatoid arthritis and osteoarthritis. Clin Rheumatol. 2007;26:1284–92. doi: 10.1007/s10067-006-0495-8. [DOI] [PubMed] [Google Scholar]
  • 28.Sawamukai N, Yukawa S, Saito K, Nakayamada S, Kambayashi T, Tanaka Y. Mast cell-derived tryptase inhibits apoptosis of human rheumatoid synovial fibroblasts via rho-mediated signaling. Arthritis Rheum. 2010;62:952–9. doi: 10.1002/art.27331. [DOI] [PubMed] [Google Scholar]
  • 29.Zvaifler NJ. The immunopathology of joint inflammation in rheumatoid arthritis. Adv Immunol. 1973;16:265–336. doi: 10.1016/s0065-2776(08)60299-0. [DOI] [PubMed] [Google Scholar]
  • 30.Matsumoto I, Maccioni M, Lee DM, Maurice M, Simmons B, Brenner M, Mathis D, Benoist C. How antibodies to a ubiquitous cytoplasmic enzyme may provoke joint-specific autoimmune disease. Nat Immunol. 2002;3:360–5. doi: 10.1038/ni772. [DOI] [PubMed] [Google Scholar]
  • 31.Egeland T, Munthe E. The role of the laboratory in rheumatology. Rheumatoid factors. Clin Rheum Dis. 1983;9:135–60. [PubMed] [Google Scholar]
  • 32.Prevoo ML, van‘t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum. 1995;38:44–8. doi: 10.1002/art.1780380107. [DOI] [PubMed] [Google Scholar]
  • 33.van Gaalen FA, Linn-Rasker SP, van Venrooij WJ, de Jong BA, Breedveld FC, Verweij CL, Toes RE, Huizinga TW. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum. 2004;50:709–15. doi: 10.1002/art.20044. [DOI] [PubMed] [Google Scholar]
  • 34.Rantapaa-Dahlqvist S, de Jong BA, Berglin E, Hallmans G, Wadell G, Stenlund H, Sundin U, van Venrooij WJ. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum. 2003;48:2741–9. doi: 10.1002/art.11223. [DOI] [PubMed] [Google Scholar]
  • 35.Meyer O, Labarre C, Dougados M, et al. Anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage. Ann Rheum Dis. 2003;62:120–6. doi: 10.1136/ard.62.2.120. [DOI] [PMC free article] [PubMed] [Google Scholar]

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