J Clin Immunol (2013) 33:947–953 DOI 10.1007/s10875-013-9885-2
ORIGINAL RESEARCH
Serum Levels of Calreticulin in Correlation with Disease Activity in Patients with Rheumatoid Arthritis Min Ni & Wei Wei & Yichao Wang & Na Zhang & Hongmei Ding & Chen Shen & Fang Zheng
Received: 17 December 2012 / Accepted: 13 March 2013 / Published online: 27 March 2013 # Springer Science+Business Media New York 2013
Abstract Objective The aim of our study was to investigate the contribution of serum calreticulin (CRT) in the assessment of disease activity in rheumatoid arthritis (RA). Methods Serum CRT levels were measured by ELISA in 70 patients with established RA, 30 systemic lupus erythematosus (SLE), 25 other autoimmune diseases, 20 osteoarthritis (OA), and 35 of healthy controls (HC). Correlations of CRT serum levels with disease activity [Disease Activity Score for 28 joints (DAS28)], erythrocyte sedimentation rate(ESR) and C-reactive protein (CRP) were assessed. Serum CRT levels were also detected in RA patients whose RF, anti-CCP and anti- MCV antibodies were positive and negative. Results Serum CRT levels in RA patients (4.817±2.425 ng/ml) was significantly higher (P <0.05) compared with those in the serum of OA (3.574±0.942 ng/ml), SLE (4.013±1.536 ng/ml), other autoimmune diseases (3.882±0.837 ng/ml) and HC (3.726±0.627 ng/ml). Significant positive correlation of CRT with DAS28, ESR and CRP was found in RA patients. Furthermore, RA patients whose anti-CCP and anti-MCV antibodies were positive had higher levels of CRT (P<0.01). Min Ni, and Wei Wei contributed equally to this manuscript. M. Ni : Y. Wang : H. Ding : C. Shen : F. Zheng (*) School of Laboratory Medicine, Tianjin Medical University, 1 Guangdong Road, Tianjin 300203, China e-mail:
[email protected] M. Ni : F. Zheng Department of Immunology, Tianjin Medical University, Tianjin, China W. Wei : N. Zhang Department of Rheumatology, General Hospital, Tianjin Medical University, No. 154 Anshan Road, Heping District, Tianjin 300070, China
Conclusion Serum CRT levels were increased in patients with RA compared with those controls. Moreover, a significant correlation was observed between serum CRT levels and disease activity in RA. It might be used as a potential biomarker for clinical diagnosis and provide additional information regarding disease activity along with the traditional indices such as ESR and CRP. Keywords Calreticulin . rheumatoid arthritis . disease activity . DAS-28 score
Introduction Rheumatoid arthritis (RA) is the most common inflammatory joint disease, characterized by uncontrolled proliferation of synovial tissue and a wide array of multisystem comorbidities, affecting 0.5 %–1 % of the world population [1, 2]. The main goal of therapy in patients with RA is to achieve remission which is defined as no active joint inflammation and no erosive or functional deterioration [3, 4]. Therefore, in order to monitor disease progression and assess the effects of therapies in RA, rheumatologists need to be able to measure disease activity and damage. The Disease Activity Score (DAS) is an assessment used to measure the level of disease activity in people with RA. It is useful for guiding treatment and monitoring patients to assess treatment effectiveness [5]. DAS 28 is a widely accepted tool to measure disease activity and it can be regarded as the gold standard for this kind of assessment [6–9]. What’s more, currently, inflammatory biomarkers like erythrocyte sedimentation rate (ESR) and high sensitivity C-reactive protein (hs-CRP) may serve as helpful indicators for evaluation of disease activity [10]. Moreover, rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) and anti-mutated citrullinated
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vimentin (anti-MCV) antibodies positive patients are more likely to develop joint erosions than negative patients, though they do not reliably change with disease activity [11]. It seems reasonable to determine more specific biomarkers in correlation with the RA activity. Recently, researchers had demonstrated that several serological markers were associated with disease activity of RA, such as higher serum adenosine deaminase (ADA) and interleukin-17 (IL-17) [12, 13]. Calreticulin (CRT) has been identified as an autoantigen of RA [14, 15]. The full-length CRT is of 46 kDa, an endoplasmic reticulum (ER) chaperone responsible for Ca2+ homeostasis and glycoprotein folding [14]. CRT, expressed on the surface of various types of cells such as T and B lymphocytes, and macrophages et al. [16], acts an important innate immune system receptor. It has been shown that the intracellular CRT can be secreted from cells [17]. New information has revealed that CRT is implicated in a number of autoimmune processes, including molecular mimicry, epitope spreading, complement inactivation and stimulation of inflammatory mediators, such as nitric oxide production. These findings suggest that CRT is not just an autoantigen, but plays an active role in the pathology of various autoimmune diseases. Recently, Holoshitz J et al. [18, 19] indicated that cell surface CRT (csCRT) acted as a signal transducing receptor for the RA shared epitope. Furthermore, Tarr JM et al. [20] demonstrated that extracellular CRT was present in the joints of patients with RA and inhibited FasL mediated apoptosis of T Cells. Although CRT had been shown previously to be associated with RA in related studies [16–21], the serum concentration of CRT has seldom been reported, and its association with disease activity and individual clinical manifestations has not been established. The present study was performed to evaluate the serum levels of CRT in RA and the correlations between CRT levels and RA disease activity, various clinical and laboratory parameters.
Patients and Methods Patients and Samples Serum samples of patients were obtained from 70 patients of RA, 30 patients of systemic lupus erythematosus (SLE), and 25 patients of other autoimmune disease (12 Sjogren’s syndrome, 5 ankylosing spondylitis, 4 systemic scleroderma, 2 spondyloarthropathy, 1 psoriatic arthritis, and 1 chondritis), and from 20 cases of osteoarthritis (OA). Thirty-five age and gender-matched healthy control subjects were recruited (Table I). Synovial fluid (SF) samples of patients were obtained from 10 patients of RA, 16 patients of OA. All patients with RA fulfilled the American College of Rheumatology (ACR) 1987 criteria for RA. All patients with other disease fulfilled their corresponding diagnostic criteria. Local ethics approval was provided (by the Medical Ethics and Human Clinical Trial Committee of Tianjin Medical University) for all experiments. The informed consent was obtained from all patients and the control subjects. Sample Preparation All serum samples and SF samples were collected and kept at 4 °C for 1 h for clotting, then centrifuged at 4,000 rpm/min for 5 min, immediately aliquot and stored at −80 °C. All samples were only allowed to thaw once. Measurement of CRT Levels in Serum and Synovial Fluid Samples by ELISA The concentration of CRT in serum and SF was detected by sandwich ELISA (Xin Yue, Shanghai, China). The ELISA assay was performed strictly according to the standard protocol of manufacturer. Briefly, serum and SF samples were diluted 1:15. Next, forty microliters of sample, 10 μl of rabbit anti-human CRT and 50 μl of HRP-labeled streptavidin were
Table I Clinical characteristics of patients in each group Group
n
Gender (F/M)
Age (years)
ESR(mm/h)
CRP (mg/dl)
RA SLE
70 30
51/19 27/3
57±13 50±17
43.1±18.3* 42.7±16.4
5.7±6.1* 5.6±5.7
Others OA HC
25 20 35
23/2 15/5 26/9
53±13 56±14 55±7
42.2±11.2 26.5±27.1 12.3±13.3
5.5±6.2 3.6±2.4 0.6±0.4
Values are means±SD RA rheumatoid arthritis; SLE systemic lupus erythematosus; Others: other autoimmune disease; OA: osteoarthritis; HC healthy control; ESR erythrocyte sedimentation rate, CRP C-reactive protein *P<0.05 RA vs OA, RA vs HC
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added to the wells. After that, the plate was left to incubate at 37 °C for 1 h. Wells were then washed for five times for 30 s for each rinse. The chromogenic agent was applied and the reaction was developed for 10 minnutes at 37 °C in the dark. After adding the stop solution, the wells were assessed by ELISA plate reader at 450 nm. CRT Expression in Sera by Western Blot Analysis All serum samples were diluted 1:4 with PBS and denatured at 95 °C for 5 min after addition of loading buffer. Then each well was added with 8 μl serum sample. The serum proteins were separated by SDS-PAGE and subsequently were transferred to the polyvinylidene difluoride (PVDF) for 1 h at a constant current of 250 mA. Two membranes were blocked for 1 h at room temperature in 5 % skim milk/TBST (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.05 % Tween 20)and incubated with rabbit anti-human CRT polyclonal antibody (PA3-900, Thermo) and rabbit anti-human serum albumin antibody (Abcam83465, Cambridge, UK), respectively for 1 h at room temperature (1: 2,500 in 5 % skim milk/TBST). After washing for 30 min with TBST three times, the membranes was incubated with HRP-conjugated goat anti-rabbit IgG (Bioword, St. Louis Park, MN) for 1 h (1: 1,000 in 5 % skim milk/TBST). After washing proteins were detected with an enhanced chemiluminescence system (Solarbio, Shanghai, China). Measurement of Disease Activity Disease activity was calculated using DAS28-ESR. Visual analogue scales (VAS) is a straight horizontal line of fixed length, usually 100 mm. The ends are defined as the extreme limits of the parameter to be measured (symptom, pain, health) orientated from the left (worst) to the right (best). DAS28-ESR is a validated scoring system for prediction of disease activity and is calculated by the following formula: DAS28 ESR ¼ 0:56*sqrtðnumber tender jointsÞ þ 0:28*sqrtðnumber swelling jointsÞ þ 0:70*LnðESRÞ þ 0:014*VAS
ESR should be measured in mm/hr, and VAS should be measured in mm in this formula. The range of the test varies from 0 to 10. DAS28 scores less than 2.6 indicate patients in remission (comparable to American College of Rheumatology remission criteria). Scores between 2.6 and 3.2 indicate patients with low disease activity. Scores between 3.2 and 5.1 indicate patients with moderate disease activity. Scores above 5. 1 indicate patients with high disease activity [6–9].
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Clinical and Laboratory Measurements When the serum samples were collected, the following clinical and laboratory data were collected: age, sex, disease duration, number of swollen joints, number of tender joints, ESR, CRP, RF,anti-CCP, anti-MCV antibodies. Regarding the laboratory features, ESR was measured by the Westergren method; values<15 mm/h for men and<20 mm/h for women were considered normal. CRP was examined by immunonephelometry method. The values above 0.8 mg/dl for CRP were considered positive. RF (IgA, IgG) in serum was measured by the rate nephelometry (Immage; Beckman Coulter, Fullerton, CA, USA); values above 18 U/ml for RF were considered positive. The second-generation anti-CCP2 antibody reactivity was tested using the second-generation ELISA kit (Fuchun-Zhongnan Biotech Co. Shanghai, China); values above 18 U/ml for anti-CCP were considered positive. Anti-MCV antibodies were performed using a commercial ELISA kit (Orgentec Diagnostica GmbH, Mainz, Germany); values above 20 U/ml for anti-MCV were considered positive. Statistical Analysis Data were expressed as mean±standard deviation (SD). Data were processed with SPSS software 11.0. (SPSS Inc., USA). Differences among groups were analyzed with oneway ANOVA. Student-Newman-Keuls test was used for a comparison between two groups. A P value of less than 0.05 was considered statistically significant. Correlations were determined by computing Spearman rank correlation coefficients.
Results Clinical Characters of the Participants A total of 180 participants were involved in this study, including of 70 RA, 30 SLE, 25 other autoimmune disease, 20 OA and 35 HC. The detailed clinical status of these participants was shown in (Table I). There was no statistical difference in gender and age constitute among all groups (P>0.05). The levels of CRP and ESR were significantly higher in the RA group (P<0.05) compared to that in OA and HC groups. Increased Levels of CRT in Sera from Patients with RA Serum CRT levels were measured by ELISA. Data of CRT levels from five groups was analyzed by one-way ANOVA. The difference was statistically significant (F = 3.864, P<0.005). Further, pair comparisons were performed using
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Table II The concentrations of serum CRT in each group Group
n
x s (ng/ml)
RA SLE Others OA HC
70 30 25 20 35
4.817±2.425* 4.013±1.536 3.882±0.837 3.574±0.942 3.726±0.627
F
P
Association Between CRT and Clinical Features in RA 3.864
0.005
*P<0.05
Student-Newman-Keuls test, our results shown that CRT levels in RA [(4.817±2.425) ng/ml] were significantly higher than that in SLE, other autoimmune diseases, OA, and HC. And there were no significant differences among the other groups (Table II). Though there was increased CRT in synovial fluid of RA, no significance diffience between RA and OA group was found (mean 3.700±0.220 ng/ml and 3.563±0.153 ng/ml; p=0.601) (Fig. 1a). Western blot was used to detect the serum CRT in RA, SLE, OA and HC simultaneously when CRT levels were detected using ELISA. CRT levels were increased in patients with RA compared with SLE, OA and HC (Fig. 1b). Correlation of Serum CRT Levels with RA Disease Activity, ESR and CRP CRT levels in RA serum were correlated with disease activity. Spearman’s rank correlation analyses were performed to assess correlations between the levels of CRT in RA patients’ sera and DAS28 (Fig. 2a). A significant correlation was observed between CRT levels in sera and DAS28 in RA patients (r2 =0.445, P <0.001). Comparison of the mean serum CRT levels in different disease activity categories and the frequencies of the patients based on DAS28-ESR scores are shown in (Table III). ESR and CRP levels were positively correlated with DAS28 (R2 =0.253, p<0.0001),
Fig. 1 Expression of CRT in serum and synovial fluid of RA and controls. a CRT levels in serum and synovial fluid from patients of RA and OA Concentration of CRT in sera from RA (n=70), OA (n=20), and synovial fluid (SF) samples from patients with RA (n=10) and OA (n=16); b Representative CRT expression in sera of RA, SLE, OA and HC by western blot
R2 =0.113, p<0.0001) (Fig. 2b, c). It was found that CRT levels positively correlated with ESR (R2 =0.567, p<0.0001) and CRP (R2 =0.255, p<0.0001) (Fig. 2d, e).
Patients with RA who were positive for RF had increased levels of CRT compared to seronegative patients but with no significance (mean 5.152 ± o.467 ng/ml and 4.157 ± o. 603 ng/ml; p=0.192; Fig. 3a). Patients with RA who were positive for anti-CCP and anti-MCV had significantly increased levels of CRT compared to seronegative patients (mean 5.387±o.468 ng/ml and 2.928±o.143 ng/ml Fig. 3b; p=0.003; 5.568±0.510 ng/ml and 3.091±o.144 ng/ml; p=0.001 Fig. 3c).
Discussion RA is a chronic inflammatory autoimmune disease of unknown etiology. It causes persistent synovitis, pain, joint destruction, and functional disability. Thus, in order to monitor disease progression and assess the effects of therapies in RA, it is necessary to identify more biomarkers for measuring disease activity and damage [3, 4, 22]. In our study, serum CRT levels were increased in patients with RA compared with those controls. Moreover, a significant correlation was observed between serum CRT levels and disease activity in RA. We showed that CRT levels were increased in patients with RA compared with other autoimmune diseases, OA and HC. And our results were also consistent with another study recently which described increased plasma levels of CRT in RA patients [20]. The accumulated data indicated that CRT played pathophysiological role in RA [16–21], such as extracellular CRT is present in the joints of patients with RA and inhibits FasL mediated apoptosis of T cells. Previous studies by Hong C et al. [16] reported that serum CRT levels were increased in patients with RA compared
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Fig 2 Scatter Plot demonstrating correlation of a serum CRT levels with DAS28 score; b ESR (mm/h) with DAS28 score; c CRP (mg/dl) with DAS28 score d serum CRT levels with ESR (mm/h); e serum CRT levels with CRP (mg/dl) of RA patients
with SLE patients. Consistent with it, our results demonstrated that serum CRT levels were significant increased not only compare with SLE group but also OA, HC, and other autoimmune diseases, such as Sjogren’s syndrome, ankylosing spondylitis et al., which may imply its potential value for RA diagnosis. In our study, western blot was also used to measure the amount of serum CRT to see whether it is comparable to that of ELISA, and to detect any CRT forms which may exist in sera such as dimer, or combined CRT. We demonstrated that the result was consistent with that in ELISA, and other CRT forms were not observed. Recent studies [16] also indicated that similar levels of serum antiCRT autoantibodies were detected in RA, SLE and also in healthy subjects, while elevated levels of CRT Abs in SLE were showed in previous investigations [20]. The above might imply that result of CRT levels in each group were comparable by ELISA. In the present study, we further measured the levels of CRT in synovial fluid of RA patients. There was increased CRT in synovial fluid of RA, while no significance
diffidence was found between RA and OA group. In contrast, a previous study by Tarr JM et al. indicated that extracellular CRT was present at a significantly higher concentration in the synovial fluid of RA compared with psoriatic arthritis [20]. Except for the different reference group, a possible reason of the distinguishing result might be the limited number of samples in the present study. In the later
Table III Mean serum CRT levels and frequency in different disease activity categories based on Disease Activity Score 28
DAS28-ESR scores Score <2.6 2.6≤Score <3.2 3.2≤Score ≤5.1 Score>5.1
CRT (ng/ml) Mean (±SD)
Frequency
2.77±0.23 2.87±0.21 5.29±1.72 12.61±1.76
21(30 16(23 26(37 7(10
%) %) %) %)
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Fig. 3 Levels of CRT vs a RF; b anti-CCP antibodies; and c anti-MCV antibodies positive in RA patients
study, a larger scale of samples involved in the research group was recommended. Herein, we also found striking correlation of CRT with laboratory parameters of disease activity. DAS28 is now the most common index in the DAS for assessing the level of disease activity in people with RA [5]. Our date indicated a significant correlation between CRT levels in sera and disease activity (by DAS28) in RA patients. In the present study, we showed the comparison of the mean serum CRT levels in different disease activity categories and the frequencies of the patients based on DAS28. Patients with scores above 5.1 have highest levels of CRT, which further suggested that CRT may serve as a helpful indicator of disease activity of RA patients. The data demonstrated by Tarr JMet et al. were in agreement with our results [20]. Furthermore, significant positive correlation of CRT with ESR and CRP were found in RA patients. ESR and CRP are inflammatory markers, which can indicate disease activity in RA. Recently researchers found that higher serum and synovial fluid levels of IL-17 [13] and serum IL-22 levels [23] correlated with DAS28 in RA. Both IL-17 and IL-22 can be produced by Th17 cells. A pioneer work by Holoshitz J et al. pointed that csCRT acted as a signal transducing receptor for the RA SE and the SE ligand increased secretion of IL-6 and IL-23 and facilitated generation of Th17 cells. These results also suggested a possible role of CRT in disease activity of RA. Autoantibodies in RA are useful not only for diagnosis but also prognosis. It is accepted that the presence of anti-
CCP antibodies is associated with presence of deformities [24]. Some researchers reported that comparing with anti-CCP, anti-MCV has superior sensitivity and RA patients who were anti-MCV-positive showed more severe bone erosion and joint space narrowing according to the Sharp-van der Heijde method [11]. In our study, there was significant correlation between higher levels of CRT and positive anti-CCP and anti-MCV, which may indicate that CRT is associated with presence of deformities. While when samples from RA patients were classified into RF-negative and RF-positive groups, there was a high level of CRT in RF-positive groups but with no significant differences. The results may be related with the low specificity of RF, since RF can also be detected in other infectious diseases, other autoimmune diseases and even healthy people. And it is reported that IgM RF, are associated with severe erosive diseases but no association of IgA RF was seen with erosions, deformities, or extra-articular features [24]. While, we only detected IgA RF and IgG RF in our study, it could explain the results. We conclude that the elevated serum CRT levels in RA patients parallel the degree of disease activity. This may identify patients prone to more severe disease, who might benefit from more aggressive treatment. Our observations provide further evidence for a possible role of CRT in RA. Greater understanding of the intra- and extracellular roles of CRT may provide further insight into its role in RA.
J Clin Immunol (2013) 33:947–953 Acknowledgments The authors thank all the subjects for their participation in this study. Funding statement This study was supported by the Specialized Research Fund for the Doctoral Program of Higher education funded by the Ministry of Education (MOE, 20101202110008).
Conflict of interest The authors declare no conflicts of interest.
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