Clin Rheumatol https://doi.org/10.1007/s10067-017-3862-8
ORIGINAL ARTICLE
Autoantibodies against interleukin-21 correlate with disease activity in patients with rheumatoid arthritis Rui Xing 1,2 & Lin Sun 1 & Dong Wu 1 & Yinji Jin 1 & Changhong Li 1 & Xiangyuan Liu 1 & Jinxia Zhao 1
Received: 15 March 2017 / Revised: 29 August 2017 / Accepted: 1 October 2017 # International League of Associations for Rheumatology (ILAR) 2017
Abstract The objective of this study is to investigate the levels of interleukin (IL)-21 and autoantibodies (AAbs) against IL-21 and their association with clinical characteristics and laboratory parameters in patients with rheumatoid arthritis (RA). One hundred twenty-six patients with RA, 69 patients with osteoarthritis (OA), and 88 healthy controls (HC) were included in this study. The levels of IL-21 and AAbs against IL-21 in the serum were measured using enzyme-linked immunosorbent assay (ELISA). The correlation between the levels of IL-21 and anti-IL-21 AAbs with clinical and laboratory parameters was evaluated. The results showed that the concentration of IL-21 was significantly higher in the serum of patients with RA (15.58 ± 3.22 ng/ml) than OA (1.80 ± 0.99 ng/ml) and HC (0.07 ± 0.03 ng/ml, p < 0.01). The levels of IL-21 in the serum correlated with erythrocyte sedimentation rate (ESR) in patients with RA (r = 0.435, p < 0.01). Anti-IL-21 AAbs could be detected in RA patients. The median AU value of AAbs against IL-21 was significantly higher in serum of RA (47.90) than in that of OA (15.17) and HC (8.19, p < 0.01). The titers of AAbs against IL-21 correlated with Disease Activity Score-28 (DAS28) (r = 0.449, p < 0.001), ESR (r = 0.386, p < 0.001), and CRP (r = 0.241, p = 0.03). Both IL-21 and AAbs against IL-21 are elevated in RA. The levels of AAbs against IL-21 correlate with disease activity, which suggests that anti-IL-21 AAbs may play a role in the pathogenesis of RA. * Jinxia Zhao [email protected] 1
Department of Rheumatology and Immunology, Peking University Third Hospital, No. 49, North Garden Road, Beijing 100191, China
2
Department of Rheumatology and Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430000, China
Keywords Autoantibody . IL-21 . Rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by synovial inflammation, hyperplasia of synovial tissues, and cartilage and bone destruction. Multiple pro-inflammatory cytokines and chemokines play critical roles in the onset and progression of RA [1]. Regulating the pro-inflammatory cytokines may prevent disease progression, which has been regarded as optimal therapy strategy for RA. In recent years, biologics targeting various pro-inflammatory cytokines have been used to treat RA, such as anti-TNF-α and anti-IL-6 receptor antagonists [2]. Due to the complex networks of cytokines in RA, not one single biologic agent targeting specific cytokine could cure the disease. In recent years, more biologic agents targeting different cytokines have been under clinical trials. Among these, interleukin-21 (IL-21) is one of the therapeutic targets with anti-IL-21 monoclonal antibody having been investigated [3]. IL-21 is a member of the IL-2 family of cytokines and belongs to the type I cytokine family, which is produced primarily by CD4+ T cells and natural killer (NK) T cells [4]. IL-21 possesses widely pleiotropic functions, by regulating both adaptive and innate immune responses, exerting key functions in controlling and directing the T and B cell responses leading to the formation of antibodies [4]. IL-21 has been found to be involved in some autoimmune diseases including RA [5]. Previous studies show that the increased levels of IL-21 in the serum are positively associated with disease activity and radiographic progression in early RA [6]. Decrease in serum IL-21 levels is associated with disease activity improvement and better outcome in early phase of RA [7]. IL-21 induces migration and invasion of fibroblastlike synoviocytes (FLS) of RA and promotes secretion of pro-
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inflammatory cytokines in RA-FLS [8, 9]. In addition, IL-21 shows osteoclastogenic potential both in RA and in mice with collagen-induced arthritis (CIA) [10, 11]. Inhibition of IL-21 with the IL-21 receptor Fc fusion protein (IL-21R.Fc) attenuates progression of arthritis in CIA [12]. All of these studies suggest that IL-21 plays important roles in regulating the inflammation and bone erosions in RA. It has been shown that AAbs against cytokines can be detected in some autoimmune diseases [13]. Whether there exist AAbs against IL-21 in RA remains unknown. Studies on AAbs against IL-21 may help to understand the precise role of IL-21 in disease progression, and whether targeting against IL-21 would be a proper way to treat RA. In this study, the levels of IL-21 and AAbs against IL-21 in the serum of RA, osteoarthritis (OA), and healthy controls (HC) were measured. In addition, the correlations of IL-21 and AAbs against IL-21 with disease activity were investigated.
Materials and methods Patients and serum samples Serum samples were obtained from 126 RA patients recruited from the Department of Rheumatology and Immunology, Peking University Third Hospital, from 2012 to 2016. All the RA patients (93 women, 33 men, mean age 57.30 ± 15.27 years, disease duration 8.03 ± 9.83 years) fulfilled the 1987 American College of Rheumatology (ACR) classification criteria of RA. Forty-six patients were early RAwith disease duration less than 1 year. The patients who had infectious diseases were not included in this study. Meanwhile, serum samples from 69 patients with OA and 88 with HC were used as controls. Written informed consent was obtained from individual patients. The study was approved by the ethics committees of Peking University Third Hospital. The clinical characteristics of RA patients were collected from the medical documents, including age, gender, disease duration, number of swollen joints, number of tender joints, disease activity score (DAS) 28, extra-articular manifestations, and bone erosions on radiograph. The laboratory parameters including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, and immunoglobulin (Ig) G, IgM, and IgA were tested. Measurement of IL-21 The concentration of IL-21 in all the serum samples was measured using enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ protocol. Human IL-21 ELISA kits were obtained from eBioscience (eBioscience, San Diego, CA, USA).
Detection of anti-IL21 AAbs Anti-IL21 AAbs in the serum samples from 86 RA patients, 50 OA patients, and 60 HC were detected using ELISA. Briefly, 96well ELISA plates (Nunc; Thermo Fisher Scientific, Denmark) were coated with 1 μg/ml recombinant human IL-21 by incubation over night at 4 °C (Peprotech, Rocky Hill, NJ, USA). The plates were washed (phosphate-buffered saline/Tween 0.05%) and blocked by incubation with the same buffer supplemented with 5% skimmed milk powder for 2 h at room temperature, then washed and incubated with 1:200 dilutions of serum samples from RA patients or controls for 2 h at room temperature. The plates were washed and incubated with 1:4000 dilutions of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Kangwei, Beijing, China) for 1 h at room temperature. Bound antibodies were detected with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Kangwei, Beijing, China) as the substrate. Absorbance was read at a wavelength of 405 nm (optical density [OD] 405 nm) with an ELISA reader (Thermo Fisher Scientific). Each serum sample was assayed in duplicate. A positive serum sample was included on each plate as a positive control. The titer of AAbs against IL-21 was expressed as arbitrary units (AU) and calculated as follows: AU = [ODIL-21 − ODbackground] sample/ [ODIL-21 − ODbackground] positive control × 100. Cut-off value for positivity of anti-IL-21 AAbs was defined as the 95th percentile of the values in healthy serum. Statistical analysis Statistical analyses were carried out using SPSS software (ver. 16.0 for Windows; SPSS, Chicago, IL). Frequency distributions between two groups were compared with the Pearson chi-squared test (χ2). The Mann-Whitney U test or t test was used to unpaired comparisons between two groups. Correlations were determined by computing Spearman rank correlation coefficients. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for the statistical comparisons of AAbs in different groups. p values less than 0.05 were considered statistically significant.
Results Serum IL-21 levels in patients with RA, OA, and HC The IL-21 levels in the serum of patients with RA, OA, and HC are shown in Fig. 1. The levels of IL-21 (means ± SEM) were significantly elevated in RA patients (15.58 ± 3.22 ng/ ml) than in OA patients (1.80 ± 0.99 ng/ml, p < 0.001) and HC group (0.07 ± 0.03 ng/ml, p < 0.001). If we defined the cut-off value for positivity of IL-21 as the 95th percentile of the values in healthy serum, the positive rates of IL-21 in RA,
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corticosteroid therapy than those treatment-naïve patients (13.96 ± 3.09 vs 22.16 ± 10.42 ng/ml, p = 0.018).
Distribution of AAbs against IL-21 in patients with RA, OA, and HC
Fig. 1 Levels of IL-21 in rheumatoid arthritis (RA), osteoarthritis (OA), and healthy controls (HC). Serum levels of IL-21 in patients with RA, OA, and HC. The levels of IL-21 were measured by enzyme-linked immunosorbent assay (ELISA) in patients with RA (n = 126), OA (n = 69), and HC (n = 88). The results showed that the level of IL-21 was significantly higher in the serum of patients with RA compared with OA and HC. ***p < 0.001 vs RA group
OA, and HC were 73.8% (93/126), 17.4% (12/69), and 4.5% (4/88), respectively.
Association of IL-21 with clinical and laboratory parameters in patients with RA As shown in Table 1, IL-21 positively correlated with RF (r = 0.435, p < 0.01). Meanwhile, there were weak positive correlations between IL-21 and the disease course (r = 0.324, p < 0.01), and ESR (r = 0.237, p < 0.01). No correlation was found between the level of IL-21 and DAS28 (r = 0.150, p = 0.109), even in the early RA patients (r = 0.261, p = 0.125). The IL-21 level was lower in the patients with disease-modifying antirheumatic drugs (DMARDs) or Table 1 Correlation of IL-21 with clinical characteristics and laboratory parameters in patients with RA Parameters
r
p
Age Disease course DAS28 ESR CRP RF Anti-CCP antibodies IgG IgA IgM
− 0.09 0.324 0.15 0.237 0.097 0.435 0.152 0.123 0.158 0.173
0.314 < 0.001*** 0.109 0.01* 0.293 < 0.001*** 0.118 0.214 0.11 0.08
DAS28, disease activity score of 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A *p < 0.05, ***p < 0.001
ELISA was used to test AAbs against IL-21 in the serum from RA patients (n = 86), OA patients (n = 50), and HC (n = 60). The presence of anti-21 AAbs in the serum of patients with RA, OA, and HC are shown in Fig. 2. The median AU values of anti-21 AAbs were 47.9 for RA, 15.17 for OA, and 8.19 for HC. The cut-off AU value for positivity was 48.06. The titer of the anti-IL-21 AAbs was significantly higher in the serum of patients with RA compared to OA and HC (p < 0.001). The positive rates of anti-IL-21 AAbs in the patients with RA, OA, and HC were 50% (43/86), 12% (6/50), and 5% (3/60), respectively. There is no difference in anti-21 AAbs in early RA and longstanding RA (57.12 ± 4.52 and 66.49 ± 8.53, p = 0.335).
Association of AAbs against IL-21 with disease activity and laboratory parameters in patients with RA As shown in Table 2, the titer of anti-IL-21 AAbs positively correlated with DAS28, ESR, and CRP (r = 0.449, p < 0.001; r = 0.386, p < 0.001; r = 0.241, p = 0.032). In the early RA, the positive correlation between anti-IL-21 AAbs and DAS28 is r = 0.494 (p = 0.009). Meanwhile, there were positive correlations between the titers of anti-21 AAbs and IgG (r = 0.571, p < 0.001), IgA (r = 0.348, p = 0.004), and IgM (r = 0.273, p = 0.026). There was no correlation between the levels of IL21 and anti-IL-21 AAbs (r = − 0.088, p = 0.423), even in the early non-treated RA patients (r = − 0.119, p = 0.589).
Fig. 2 Distribution of anti-IL-21 AAbs in patients with RA, OA, and HC. The titers of anti-IL-21 AAbs were measured using ELISA in patients with RA (n = 86), OA (n = 50), and HC (n = 60). The arbitrary unit (AU) values of anti-IL-21 AAbs were significantly higher in the serum of patients with RA than OA and HC. ***p < 0.001 vs RA group
Clin Rheumatol Table 2 Correlation of anti-IL-21AAbs with clinical characteristics and laboratory parameters in patients with RA Parameters
r
p
Age
− 0.182
0.094
Disease course
− 0.114
0.297
DAS28 ESR
0.449 0.386
< 0.001*** < 0.001***
CRP
0.241
0.032*
RF Anti-CCP antibodies
0.099 − 0.065
0.409 0.585
IgG IgA
0.571 0.348
< 0.001*** 0.004**
IgM
0.273
0.026*
IL-21
0.088
0.423
DAS28, disease activity score of 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A *p < 0.05, **p < 0.01, ***p < 0.001
Comparison of clinical and laboratory features in RA patients with or without anti-IL-21 AAbs It was shown in Table 3 that the proportion of patients who were on the therapy of DMARDs in anti-IL-21 AAbsnegative patients was relatively higher than that in anti-IL-21 AAbs-positive patients. But no difference was found in the titer of anti-IL-21 AAbs between patients using DMARDs or corticosteroid therapy or treatment-naïve patients (64.58 ± 4.51 vs 61.69 ± 6.97, p = 0.809). The gender ratio (female/ male) was different between anti-IL-21 AAbs-positive and anti-IL-21 AAbs-negative patients, with more female in antiIL-21 AAbs-positive patients. No significant differences were observed in other clinical characteristics. ESR and IgG were significantly higher in RA patients with positive anti-IL-21 AAbs than those without anti-IL-21 AAbs (p < 0.05). There was no difference of IL-21 concentration between anti-IL-21 AAbs positive and anti-IL-21 AAbs-negative patients.
Discussion Cytokines play important roles in the development of various autoimmune diseases, including RA [14]. It is interesting that anti-cytokine antibodies response can be detected in healthy subjects and some diseases [15]. AAbs against cytokines detected in healthy individuals could act as physiological regulators of many cytokine homeostasis [16]. In some immunodeficiency diseases and autoimmune disease, the existence of AAbs against cytokines may influence the progression and the severity of diseases.
AAbs against IL-17A, IL-17F, and IL-22 have been demonstrated in the patients with autoimmune polyendocrine syndrome type I (APS-I), which may impair immune protection against specific microbes and induce secondary immune deficiency [17]. AAbs against TNF-α, interferon-α (IFN-α), IFN-γ, GM-CSF, and BAFF were found in patients with SLE [18–21]. These anti-cytokine AAbs may contribute to the pathophysiology and modulate the clinical course of SLE [22]. For example, the presence of anti-IFN-γ AAbs was found to correlate with more severe disease activity [23]. The presence of neutralizing anti-IFN-α AAbs in SLE patients was associated with a lower disease severity [19]. In RA, the presence of AAbs against IL-1α, IL-8, G-CSF, IL17, and osteopontin has been reported [24–29]. The serum levels of these anti-cytokine AAbs positively or negatively correlated with disease activity or extra-articular manifestations. For instance, high levels of neutralizing anti-IL-1α AAbs correlated with low disease activity in RA and were negatively related to the radiographic joint damage. It was found that anti-IL-1α AAbs were more commonly found in RA with interstitial lung disease (ILD) than those without ILD [30]. IL-8 is a kind of chemokine with high concentration in synovial fluids of RA. Anti-IL-8 autoantibodies are frequently detected in RA and are associated with disease severity and extra-articular features [27]. As a pro-inflammatory cytokine, IL-21 plays an important role in the pathogenesis of RA. IL-21R is expressed in RA synovium by synovial fibroblasts and synovial macrophages [31]. IL-21 induces T cell activation and synovial inflammation in RA [32]. In the current study, we found that the serum level of IL-21 was higher in RA than OA and HC. The concentration of IL-21 in RA was associated with inflammatory marker of ESR, but no correlations were found between IL-21 and DAS28 or CRP, which was different from previous study [6, 7]. In addition, in the cohort of early RA patients, no correlation was found between DAS28 and IL-21. These inconsistent results may be due to different patient cohorts in various studies, since the clinical characteristics such as ethnic, disease course, and other baseline characteristics were variable in different studies. The sensitivity of IL-21 ELISA kit used in different studies may also explain the inconsistent result partially. Prospective study to compare the level of IL-21 before and after treatment in RA patients may provide us more valuable information about the role of IL-21 in the disease. It was shown that anti-IL-21 AAbs could be detected in RA, and the levels of anti-IL-21 AAbs in RAwere higher than those in OA and HC. Anti-IL-21 AAbs positively correlated with DAS28, ESR, and CRP. It is possible that anti-IL-21 AAbs may play a role in the pathogenesis of RA and may serve as a biomarker in monitoring disease activity. The existence of antiIL-21 AAbs raises the concern that monoclonal antibodies targeting IL-21 might not be a good therapeutic method for RA. The exact mechanisms of anti-cytokine AAbs production in the healthy individuals and various autoimmune diseases have not been fully understood by now. The role of AAbs
Clin Rheumatol Table 3 Comparison of clinical and laboratory parameters between anti-IL-21 AAbspositive and anti-IL-21 AAbsnegative patients in RA
Parameters
Anti-IL-21 positive (n = 43)
Anti-IL-21 negative (n = 43)
p
Age (years, mean ± SD) Gender ratio (female/male)
54.70 ± 16.38 36/7
59.93 ± 14.28 26/7
0.118 0.016*
Disease course (years, mean ± SD)
6.20 ± 9.42
8.79 ± 10.37
0.23
Extra-articular manifestation Bone erosion on radiograph
16.3% (7/43) 27.9% (12/43)
20.9% (9/43) 23.3% (10/43)
0.579 0.621
DMARDs therapy Corticosteroid therapy
60.5% (26/43) 25.6% (11/43)
86% (37/43) 27.9% (12/43)
0.007** 0.808
DAS28 (score, mean ± SD)
6.05 ± 4.29
4.55 ± 3.47
0.105
ESR (mm/h, mean ± SD) CRP (mg/dl, mean ± SD)
53.14 ± 46.65 3.07 ± 3.92
30.93 ± 30.61 2.94 ± 4.19
0.013* 0.889
RF (IU/ml, mean ± SD)
446.83 ± 824.84
288.55 ± 370.02
0.285
Anti-CCP antibodies (RU/ml, mean ± SD) IgG (g/L, mean ± SD)
481.95 ± 713.65
2622.22 ± 7285.05
0.088
18.29 ± 7.31
12.25 ± 4.68
< 0.001***
IgA (g/L, mean ± SD)
3.48 ± 1.63
3.12 ± 2.02
0.423
IgM (g/L, mean ± SD) IL-21 (ng/ml, mean ± SD)
1.48 ± 0.86 24.61 ± 54.74
1.16 ± 0.59 12.16 ± 18.70
0.081 0.162
DAS28, disease activity score of 28 joints; CRP, C-reactive protein; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A; SD, standard deviation *p < 0.05, **p < 0.01, ***p < 0.001
against cytokines may be determined by the function of the antibodies. In healthy subjects, anti-cytokine AAbs may show function in the regulation of cytokine homeostasis. Some anticytokine AAbs may repress the function of cytokines by preventing them to contact with their receptors. Anticytokine AAbs may have neutralizing activity and inhibit the function of targeting cytokines, which may induce acquired immunodeficiency leading to specific infectious phenotypes. Some anti-cytokine AAbs may behave as carrier proteins, increasing half-life and modulating bioactivity of the cytokines [22]. If the combination of anti-cytokine AAbs and targeted cytokines stabilized the cytokines from degradation, the increased accumulation of cytokines may promote the progression of some diseases. In this study, the positive correlation of anti-IL-21 AAbs with disease activity indicated that these AAbs might play a pathogenic instead of protective role in RA. It was supposed that the combination of anti-IL-21 AAbs and IL-21 might retard the degradation of the cytokine and facilitate the continuous pathological effect of IL-21 in RA. Since no correlations between IL-21 and anti-IL-21 AAbs were found in this study, the exact mechanism can hardly be explained based on the current data. The limitation of this study is that the traditional ELISA detection for autoantibodies may sometimes lead to a false-positive reaction. We detected anti-IL-21 AAbs using western blot in one serum sample with high AU value of anti-IL-21 AAbs by ELISA. The negative result of western blot may suggest the possibility of
false-positive reaction of ELISA. It may also indicate that the AAbs against IL-21 might target conformational determinants of IL-21. Further researches about the functional analysis of anti-IL-21 AAbs need to be carried out in the future. In addition, long-term follow-up study should be performed to further evaluate the relationship of anti-IL-21 AAbs and disease progression. In summary, IL-21 and anti-21 AAbs can be detected in RA, and anti-IL-21 AAbs correlate with disease activity. More studies are needed to further understand the mechanism of anti-IL-21 AAbs in the pathogenesis and progression of disease. Funding information This work was supported by the National Natural Science Foundation of China (Grant No. 81571573, 81471599). Compliance with ethical standards Disclosures None.
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ORIGINAL ARTICLE
Autoantibodies against interleukin-21 correlate with disease activity in patients with rheumatoid arthritis Rui Xing 1,2 & Lin Sun 1 & Dong Wu 1 & Yinji Jin 1 & Changhong Li 1 & Xiangyuan Liu 1 & Jinxia Zhao 1
Received: 15 March 2017 / Revised: 29 August 2017 / Accepted: 1 October 2017 # International League of Associations for Rheumatology (ILAR) 2017
Abstract The objective of this study is to investigate the levels of interleukin (IL)-21 and autoantibodies (AAbs) against IL-21 and their association with clinical characteristics and laboratory parameters in patients with rheumatoid arthritis (RA). One hundred twenty-six patients with RA, 69 patients with osteoarthritis (OA), and 88 healthy controls (HC) were included in this study. The levels of IL-21 and AAbs against IL-21 in the serum were measured using enzyme-linked immunosorbent assay (ELISA). The correlation between the levels of IL-21 and anti-IL-21 AAbs with clinical and laboratory parameters was evaluated. The results showed that the concentration of IL-21 was significantly higher in the serum of patients with RA (15.58 ± 3.22 ng/ml) than OA (1.80 ± 0.99 ng/ml) and HC (0.07 ± 0.03 ng/ml, p < 0.01). The levels of IL-21 in the serum correlated with erythrocyte sedimentation rate (ESR) in patients with RA (r = 0.435, p < 0.01). Anti-IL-21 AAbs could be detected in RA patients. The median AU value of AAbs against IL-21 was significantly higher in serum of RA (47.90) than in that of OA (15.17) and HC (8.19, p < 0.01). The titers of AAbs against IL-21 correlated with Disease Activity Score-28 (DAS28) (r = 0.449, p < 0.001), ESR (r = 0.386, p < 0.001), and CRP (r = 0.241, p = 0.03). Both IL-21 and AAbs against IL-21 are elevated in RA. The levels of AAbs against IL-21 correlate with disease activity, which suggests that anti-IL-21 AAbs may play a role in the pathogenesis of RA. * Jinxia Zhao [email protected] 1
Department of Rheumatology and Immunology, Peking University Third Hospital, No. 49, North Garden Road, Beijing 100191, China
2
Department of Rheumatology and Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430000, China
Keywords Autoantibody . IL-21 . Rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by synovial inflammation, hyperplasia of synovial tissues, and cartilage and bone destruction. Multiple pro-inflammatory cytokines and chemokines play critical roles in the onset and progression of RA [1]. Regulating the pro-inflammatory cytokines may prevent disease progression, which has been regarded as optimal therapy strategy for RA. In recent years, biologics targeting various pro-inflammatory cytokines have been used to treat RA, such as anti-TNF-α and anti-IL-6 receptor antagonists [2]. Due to the complex networks of cytokines in RA, not one single biologic agent targeting specific cytokine could cure the disease. In recent years, more biologic agents targeting different cytokines have been under clinical trials. Among these, interleukin-21 (IL-21) is one of the therapeutic targets with anti-IL-21 monoclonal antibody having been investigated [3]. IL-21 is a member of the IL-2 family of cytokines and belongs to the type I cytokine family, which is produced primarily by CD4+ T cells and natural killer (NK) T cells [4]. IL-21 possesses widely pleiotropic functions, by regulating both adaptive and innate immune responses, exerting key functions in controlling and directing the T and B cell responses leading to the formation of antibodies [4]. IL-21 has been found to be involved in some autoimmune diseases including RA [5]. Previous studies show that the increased levels of IL-21 in the serum are positively associated with disease activity and radiographic progression in early RA [6]. Decrease in serum IL-21 levels is associated with disease activity improvement and better outcome in early phase of RA [7]. IL-21 induces migration and invasion of fibroblastlike synoviocytes (FLS) of RA and promotes secretion of pro-
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inflammatory cytokines in RA-FLS [8, 9]. In addition, IL-21 shows osteoclastogenic potential both in RA and in mice with collagen-induced arthritis (CIA) [10, 11]. Inhibition of IL-21 with the IL-21 receptor Fc fusion protein (IL-21R.Fc) attenuates progression of arthritis in CIA [12]. All of these studies suggest that IL-21 plays important roles in regulating the inflammation and bone erosions in RA. It has been shown that AAbs against cytokines can be detected in some autoimmune diseases [13]. Whether there exist AAbs against IL-21 in RA remains unknown. Studies on AAbs against IL-21 may help to understand the precise role of IL-21 in disease progression, and whether targeting against IL-21 would be a proper way to treat RA. In this study, the levels of IL-21 and AAbs against IL-21 in the serum of RA, osteoarthritis (OA), and healthy controls (HC) were measured. In addition, the correlations of IL-21 and AAbs against IL-21 with disease activity were investigated.
Materials and methods Patients and serum samples Serum samples were obtained from 126 RA patients recruited from the Department of Rheumatology and Immunology, Peking University Third Hospital, from 2012 to 2016. All the RA patients (93 women, 33 men, mean age 57.30 ± 15.27 years, disease duration 8.03 ± 9.83 years) fulfilled the 1987 American College of Rheumatology (ACR) classification criteria of RA. Forty-six patients were early RAwith disease duration less than 1 year. The patients who had infectious diseases were not included in this study. Meanwhile, serum samples from 69 patients with OA and 88 with HC were used as controls. Written informed consent was obtained from individual patients. The study was approved by the ethics committees of Peking University Third Hospital. The clinical characteristics of RA patients were collected from the medical documents, including age, gender, disease duration, number of swollen joints, number of tender joints, disease activity score (DAS) 28, extra-articular manifestations, and bone erosions on radiograph. The laboratory parameters including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, and immunoglobulin (Ig) G, IgM, and IgA were tested. Measurement of IL-21 The concentration of IL-21 in all the serum samples was measured using enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ protocol. Human IL-21 ELISA kits were obtained from eBioscience (eBioscience, San Diego, CA, USA).
Detection of anti-IL21 AAbs Anti-IL21 AAbs in the serum samples from 86 RA patients, 50 OA patients, and 60 HC were detected using ELISA. Briefly, 96well ELISA plates (Nunc; Thermo Fisher Scientific, Denmark) were coated with 1 μg/ml recombinant human IL-21 by incubation over night at 4 °C (Peprotech, Rocky Hill, NJ, USA). The plates were washed (phosphate-buffered saline/Tween 0.05%) and blocked by incubation with the same buffer supplemented with 5% skimmed milk powder for 2 h at room temperature, then washed and incubated with 1:200 dilutions of serum samples from RA patients or controls for 2 h at room temperature. The plates were washed and incubated with 1:4000 dilutions of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Kangwei, Beijing, China) for 1 h at room temperature. Bound antibodies were detected with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Kangwei, Beijing, China) as the substrate. Absorbance was read at a wavelength of 405 nm (optical density [OD] 405 nm) with an ELISA reader (Thermo Fisher Scientific). Each serum sample was assayed in duplicate. A positive serum sample was included on each plate as a positive control. The titer of AAbs against IL-21 was expressed as arbitrary units (AU) and calculated as follows: AU = [ODIL-21 − ODbackground] sample/ [ODIL-21 − ODbackground] positive control × 100. Cut-off value for positivity of anti-IL-21 AAbs was defined as the 95th percentile of the values in healthy serum. Statistical analysis Statistical analyses were carried out using SPSS software (ver. 16.0 for Windows; SPSS, Chicago, IL). Frequency distributions between two groups were compared with the Pearson chi-squared test (χ2). The Mann-Whitney U test or t test was used to unpaired comparisons between two groups. Correlations were determined by computing Spearman rank correlation coefficients. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for the statistical comparisons of AAbs in different groups. p values less than 0.05 were considered statistically significant.
Results Serum IL-21 levels in patients with RA, OA, and HC The IL-21 levels in the serum of patients with RA, OA, and HC are shown in Fig. 1. The levels of IL-21 (means ± SEM) were significantly elevated in RA patients (15.58 ± 3.22 ng/ ml) than in OA patients (1.80 ± 0.99 ng/ml, p < 0.001) and HC group (0.07 ± 0.03 ng/ml, p < 0.001). If we defined the cut-off value for positivity of IL-21 as the 95th percentile of the values in healthy serum, the positive rates of IL-21 in RA,
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corticosteroid therapy than those treatment-naïve patients (13.96 ± 3.09 vs 22.16 ± 10.42 ng/ml, p = 0.018).
Distribution of AAbs against IL-21 in patients with RA, OA, and HC
Fig. 1 Levels of IL-21 in rheumatoid arthritis (RA), osteoarthritis (OA), and healthy controls (HC). Serum levels of IL-21 in patients with RA, OA, and HC. The levels of IL-21 were measured by enzyme-linked immunosorbent assay (ELISA) in patients with RA (n = 126), OA (n = 69), and HC (n = 88). The results showed that the level of IL-21 was significantly higher in the serum of patients with RA compared with OA and HC. ***p < 0.001 vs RA group
OA, and HC were 73.8% (93/126), 17.4% (12/69), and 4.5% (4/88), respectively.
Association of IL-21 with clinical and laboratory parameters in patients with RA As shown in Table 1, IL-21 positively correlated with RF (r = 0.435, p < 0.01). Meanwhile, there were weak positive correlations between IL-21 and the disease course (r = 0.324, p < 0.01), and ESR (r = 0.237, p < 0.01). No correlation was found between the level of IL-21 and DAS28 (r = 0.150, p = 0.109), even in the early RA patients (r = 0.261, p = 0.125). The IL-21 level was lower in the patients with disease-modifying antirheumatic drugs (DMARDs) or Table 1 Correlation of IL-21 with clinical characteristics and laboratory parameters in patients with RA Parameters
r
p
Age Disease course DAS28 ESR CRP RF Anti-CCP antibodies IgG IgA IgM
− 0.09 0.324 0.15 0.237 0.097 0.435 0.152 0.123 0.158 0.173
0.314 < 0.001*** 0.109 0.01* 0.293 < 0.001*** 0.118 0.214 0.11 0.08
DAS28, disease activity score of 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A *p < 0.05, ***p < 0.001
ELISA was used to test AAbs against IL-21 in the serum from RA patients (n = 86), OA patients (n = 50), and HC (n = 60). The presence of anti-21 AAbs in the serum of patients with RA, OA, and HC are shown in Fig. 2. The median AU values of anti-21 AAbs were 47.9 for RA, 15.17 for OA, and 8.19 for HC. The cut-off AU value for positivity was 48.06. The titer of the anti-IL-21 AAbs was significantly higher in the serum of patients with RA compared to OA and HC (p < 0.001). The positive rates of anti-IL-21 AAbs in the patients with RA, OA, and HC were 50% (43/86), 12% (6/50), and 5% (3/60), respectively. There is no difference in anti-21 AAbs in early RA and longstanding RA (57.12 ± 4.52 and 66.49 ± 8.53, p = 0.335).
Association of AAbs against IL-21 with disease activity and laboratory parameters in patients with RA As shown in Table 2, the titer of anti-IL-21 AAbs positively correlated with DAS28, ESR, and CRP (r = 0.449, p < 0.001; r = 0.386, p < 0.001; r = 0.241, p = 0.032). In the early RA, the positive correlation between anti-IL-21 AAbs and DAS28 is r = 0.494 (p = 0.009). Meanwhile, there were positive correlations between the titers of anti-21 AAbs and IgG (r = 0.571, p < 0.001), IgA (r = 0.348, p = 0.004), and IgM (r = 0.273, p = 0.026). There was no correlation between the levels of IL21 and anti-IL-21 AAbs (r = − 0.088, p = 0.423), even in the early non-treated RA patients (r = − 0.119, p = 0.589).
Fig. 2 Distribution of anti-IL-21 AAbs in patients with RA, OA, and HC. The titers of anti-IL-21 AAbs were measured using ELISA in patients with RA (n = 86), OA (n = 50), and HC (n = 60). The arbitrary unit (AU) values of anti-IL-21 AAbs were significantly higher in the serum of patients with RA than OA and HC. ***p < 0.001 vs RA group
Clin Rheumatol Table 2 Correlation of anti-IL-21AAbs with clinical characteristics and laboratory parameters in patients with RA Parameters
r
p
Age
− 0.182
0.094
Disease course
− 0.114
0.297
DAS28 ESR
0.449 0.386
< 0.001*** < 0.001***
CRP
0.241
0.032*
RF Anti-CCP antibodies
0.099 − 0.065
0.409 0.585
IgG IgA
0.571 0.348
< 0.001*** 0.004**
IgM
0.273
0.026*
IL-21
0.088
0.423
DAS28, disease activity score of 28 joints; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; RF, rheumatoid factor; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A *p < 0.05, **p < 0.01, ***p < 0.001
Comparison of clinical and laboratory features in RA patients with or without anti-IL-21 AAbs It was shown in Table 3 that the proportion of patients who were on the therapy of DMARDs in anti-IL-21 AAbsnegative patients was relatively higher than that in anti-IL-21 AAbs-positive patients. But no difference was found in the titer of anti-IL-21 AAbs between patients using DMARDs or corticosteroid therapy or treatment-naïve patients (64.58 ± 4.51 vs 61.69 ± 6.97, p = 0.809). The gender ratio (female/ male) was different between anti-IL-21 AAbs-positive and anti-IL-21 AAbs-negative patients, with more female in antiIL-21 AAbs-positive patients. No significant differences were observed in other clinical characteristics. ESR and IgG were significantly higher in RA patients with positive anti-IL-21 AAbs than those without anti-IL-21 AAbs (p < 0.05). There was no difference of IL-21 concentration between anti-IL-21 AAbs positive and anti-IL-21 AAbs-negative patients.
Discussion Cytokines play important roles in the development of various autoimmune diseases, including RA [14]. It is interesting that anti-cytokine antibodies response can be detected in healthy subjects and some diseases [15]. AAbs against cytokines detected in healthy individuals could act as physiological regulators of many cytokine homeostasis [16]. In some immunodeficiency diseases and autoimmune disease, the existence of AAbs against cytokines may influence the progression and the severity of diseases.
AAbs against IL-17A, IL-17F, and IL-22 have been demonstrated in the patients with autoimmune polyendocrine syndrome type I (APS-I), which may impair immune protection against specific microbes and induce secondary immune deficiency [17]. AAbs against TNF-α, interferon-α (IFN-α), IFN-γ, GM-CSF, and BAFF were found in patients with SLE [18–21]. These anti-cytokine AAbs may contribute to the pathophysiology and modulate the clinical course of SLE [22]. For example, the presence of anti-IFN-γ AAbs was found to correlate with more severe disease activity [23]. The presence of neutralizing anti-IFN-α AAbs in SLE patients was associated with a lower disease severity [19]. In RA, the presence of AAbs against IL-1α, IL-8, G-CSF, IL17, and osteopontin has been reported [24–29]. The serum levels of these anti-cytokine AAbs positively or negatively correlated with disease activity or extra-articular manifestations. For instance, high levels of neutralizing anti-IL-1α AAbs correlated with low disease activity in RA and were negatively related to the radiographic joint damage. It was found that anti-IL-1α AAbs were more commonly found in RA with interstitial lung disease (ILD) than those without ILD [30]. IL-8 is a kind of chemokine with high concentration in synovial fluids of RA. Anti-IL-8 autoantibodies are frequently detected in RA and are associated with disease severity and extra-articular features [27]. As a pro-inflammatory cytokine, IL-21 plays an important role in the pathogenesis of RA. IL-21R is expressed in RA synovium by synovial fibroblasts and synovial macrophages [31]. IL-21 induces T cell activation and synovial inflammation in RA [32]. In the current study, we found that the serum level of IL-21 was higher in RA than OA and HC. The concentration of IL-21 in RA was associated with inflammatory marker of ESR, but no correlations were found between IL-21 and DAS28 or CRP, which was different from previous study [6, 7]. In addition, in the cohort of early RA patients, no correlation was found between DAS28 and IL-21. These inconsistent results may be due to different patient cohorts in various studies, since the clinical characteristics such as ethnic, disease course, and other baseline characteristics were variable in different studies. The sensitivity of IL-21 ELISA kit used in different studies may also explain the inconsistent result partially. Prospective study to compare the level of IL-21 before and after treatment in RA patients may provide us more valuable information about the role of IL-21 in the disease. It was shown that anti-IL-21 AAbs could be detected in RA, and the levels of anti-IL-21 AAbs in RAwere higher than those in OA and HC. Anti-IL-21 AAbs positively correlated with DAS28, ESR, and CRP. It is possible that anti-IL-21 AAbs may play a role in the pathogenesis of RA and may serve as a biomarker in monitoring disease activity. The existence of antiIL-21 AAbs raises the concern that monoclonal antibodies targeting IL-21 might not be a good therapeutic method for RA. The exact mechanisms of anti-cytokine AAbs production in the healthy individuals and various autoimmune diseases have not been fully understood by now. The role of AAbs
Clin Rheumatol Table 3 Comparison of clinical and laboratory parameters between anti-IL-21 AAbspositive and anti-IL-21 AAbsnegative patients in RA
Parameters
Anti-IL-21 positive (n = 43)
Anti-IL-21 negative (n = 43)
p
Age (years, mean ± SD) Gender ratio (female/male)
54.70 ± 16.38 36/7
59.93 ± 14.28 26/7
0.118 0.016*
Disease course (years, mean ± SD)
6.20 ± 9.42
8.79 ± 10.37
0.23
Extra-articular manifestation Bone erosion on radiograph
16.3% (7/43) 27.9% (12/43)
20.9% (9/43) 23.3% (10/43)
0.579 0.621
DMARDs therapy Corticosteroid therapy
60.5% (26/43) 25.6% (11/43)
86% (37/43) 27.9% (12/43)
0.007** 0.808
DAS28 (score, mean ± SD)
6.05 ± 4.29
4.55 ± 3.47
0.105
ESR (mm/h, mean ± SD) CRP (mg/dl, mean ± SD)
53.14 ± 46.65 3.07 ± 3.92
30.93 ± 30.61 2.94 ± 4.19
0.013* 0.889
RF (IU/ml, mean ± SD)
446.83 ± 824.84
288.55 ± 370.02
0.285
Anti-CCP antibodies (RU/ml, mean ± SD) IgG (g/L, mean ± SD)
481.95 ± 713.65
2622.22 ± 7285.05
0.088
18.29 ± 7.31
12.25 ± 4.68
< 0.001***
IgA (g/L, mean ± SD)
3.48 ± 1.63
3.12 ± 2.02
0.423
IgM (g/L, mean ± SD) IL-21 (ng/ml, mean ± SD)
1.48 ± 0.86 24.61 ± 54.74
1.16 ± 0.59 12.16 ± 18.70
0.081 0.162
DAS28, disease activity score of 28 joints; CRP, C-reactive protein; RF, rheumatoid factor; ESR, erythrocyte sedimentation rate; Anti-CCP antibodies, anti-cyclic citrullinated peptide antibodies; IgG, IgM, IgA, immunoglobulin G, M, and A; SD, standard deviation *p < 0.05, **p < 0.01, ***p < 0.001
against cytokines may be determined by the function of the antibodies. In healthy subjects, anti-cytokine AAbs may show function in the regulation of cytokine homeostasis. Some anticytokine AAbs may repress the function of cytokines by preventing them to contact with their receptors. Anticytokine AAbs may have neutralizing activity and inhibit the function of targeting cytokines, which may induce acquired immunodeficiency leading to specific infectious phenotypes. Some anti-cytokine AAbs may behave as carrier proteins, increasing half-life and modulating bioactivity of the cytokines [22]. If the combination of anti-cytokine AAbs and targeted cytokines stabilized the cytokines from degradation, the increased accumulation of cytokines may promote the progression of some diseases. In this study, the positive correlation of anti-IL-21 AAbs with disease activity indicated that these AAbs might play a pathogenic instead of protective role in RA. It was supposed that the combination of anti-IL-21 AAbs and IL-21 might retard the degradation of the cytokine and facilitate the continuous pathological effect of IL-21 in RA. Since no correlations between IL-21 and anti-IL-21 AAbs were found in this study, the exact mechanism can hardly be explained based on the current data. The limitation of this study is that the traditional ELISA detection for autoantibodies may sometimes lead to a false-positive reaction. We detected anti-IL-21 AAbs using western blot in one serum sample with high AU value of anti-IL-21 AAbs by ELISA. The negative result of western blot may suggest the possibility of
false-positive reaction of ELISA. It may also indicate that the AAbs against IL-21 might target conformational determinants of IL-21. Further researches about the functional analysis of anti-IL-21 AAbs need to be carried out in the future. In addition, long-term follow-up study should be performed to further evaluate the relationship of anti-IL-21 AAbs and disease progression. In summary, IL-21 and anti-21 AAbs can be detected in RA, and anti-IL-21 AAbs correlate with disease activity. More studies are needed to further understand the mechanism of anti-IL-21 AAbs in the pathogenesis and progression of disease. Funding information This work was supported by the National Natural Science Foundation of China (Grant No. 81571573, 81471599). Compliance with ethical standards Disclosures None.
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