International Journal of Rheumatic Diseases 2016; 19: 644–650
ORIGINAL ARTICLE
Elevated autoantibodies against interleukin-17F correlate with disease activity in patients with early rheumatoid arthritis Lin YANG,1 Li BAI,2 Feifei WEI,2 Yuan LIU,2 Lin SUN,1 Wenming WANG,3 Xiangyuan LIU1 and Yongfu WANG2 1
Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing, 2Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, Baotou, and 3Department of Hematology, Peking University Third Hospital, Beijing, China
Abstract Aim: To investigate the presence of autoantibodies (aAbs) against interleukin (IL)-17A and IL-17F and observe whether anti-IL-17A or IL-17F aAbs are associated with disease activity in patients with early rheumatoid arthritis (ERA). Methods: At present, 60 patients with ERA, 72 patients with osteoarthritis (OA) and 61 healthy controls (HC) have been included in a database. Clinical assessment and laboratory data were recorded. We detected the titer of aAbs against IL-17A and IL-17F using enzyme-linked immunosorbent assay and analyzed the correlation of these aAbs in patients with ERA. Results: Our results showed that the levels of aAbs against IL-17A and IL-17F were significantly higher in ERA OA and HC (P < 0.0001). The level of aAbs against IL-17F was correlated with Disease Activity Score-28 erythrocyte sedimentation rate (ESR) (P = 0.0457) and ESR alone (P = 0.0032) in patients with ERA. In addition, in the ERA group, the level of C-reactive protein and rheumatoid factor immunoglobulin M was lower in patients with aAbs against IL-17F than patients without aAbs (P = 0.0247; P = 0.0439). No significant correlation was observed between the clinical characteristics and level of aAbs against IL-17A in patients with ERA except ESR (P = 0.0239). Conclusions: Elevated aAbs against IL-17F correlate with disease activity in patients with ERA. This evidence suggests that anti-IL-17F aAbs may have a protective role in the pathogenesis of ERA. Key words: autoantibody, C-reactive protein, DAS-28, erythrocyte sedimentation rate, IL-17, rheumatoid arthritis.
INTRODUCTION Correspondence: Yongfu Wang, MD, Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, No.41, LinYin Road, Baotou 014010, China. Email: [email protected]. and Xiangyuan Liu, MD, Department of Rheumatology and Immunology, Peking University Third Hospital, No.49, HuaYuan (North) Road, Beijing 100191, China. Email: [email protected].
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by abnormal immune responses which lead to synovial hyperplasia and ultimately progressive destruction of cartilage and bone. The altered activation of innate or adaptive immune mechanisms contributes to the development of this autoimmune disease. Among great progress in underlying the pathogenesis of RA, defective regulation of
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Autoantibodies against IL-17A and IL-17F
inflammatory cytokines has been reported as a central component in synovial hyperplasia and joint destruction.1,2 At present, more and more evidence suggests that defective regulation of interleukin (IL)-17 may be a central component in the pathogenesis of RA. In human RA, IL-17-producing T cells have been identified3 and IL-17A has been found to be elevated in the synovial fluid of RA patients.4,5 In animal models of RA, overexpression of IL-17 promotes extensive inflammatory cell migration and induces bone erosion and cartilage degradation.6 In contrast, inhibition of IL-17 has been demonstrated to ameliorate collagen-induced arthritis (CIA).7,8 Compared with IL-17A, IL-17F is a less potent activator of synoviocytes9 and plays a minimal role in an arthritis animal model10 and evidence exists that IL-17F has the potential to destroy cartilage in vitro.11 An IL-17F variant has been reported to be associated with an increased disease activity in Polish patients with RA.12 The level of 17F is higher in plasma and synovial fluid in RA patients than controls and IL-17F, but not IL17A decreases with multiple classes of disease-modifying anti-rheumatic drugs (DMARDs).13 Recently, higher levels of IL-17A and IL-17F were detected in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from patients with RA; meanwhile, IL-17A and IL-17F were also increased in the CIA model.14 However, the presence of autoantibodies (aAbs) against IL-17A and IL-17F in patients with RA and whether aAbs are associated with disease activity are less known. In the current study, we detected the presence of aAbs against and IL-17A and IL-17F in serum samples of patients with early RA (ERA) and analyzed the association with the clinical manifestations and laboratory parameters of ERA disease.
PATIENTS AND METHODS Patients From 2006 to 2013, 683 Chinese patients with RA were admitted when they first visited the Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College. The ERA group was defined as disease duration < 12 months.15 Sixty patients with ERA, 72 patients with osteoarthritis (OA) and 61 healthy controls (HC) were included in a database. ERA patients underwent extensive serological evaluations. All serum samples were obtained from the patients when they were first diagnosed as RA and frozen at 80°C immediately after collection. Ethics approval (no. EC-
International Journal of Rheumatic Diseases 2016; 19: 644–650
BMC1209007) was obtained by the ethics committee of Baotou Medical College.
Clinical assessment and laboratory data Clinical data were recorded as the following index: age, sex, disease duration, number of swollen joints, number of tender joints and other clinical characteristics. General health status reported by patients on a 100mm visual analogue scale (VAS) was also recorded. Erythrocyte sedimentation rate (ESR) was evaluated by the Westergren method. C-reactive protein (CRP) and rheumatoid factor (RF) – immunoglobulin M (IgM) were examined by immunonephelometry method. Anti-citrullinated peptide (CCP) antibodies, RF-IgA and RF-IgG were tested by enzyme-linked immunosorbent assay (ELISA). The 28-joint count Disease Activity Score (DAS28-ESR) was calculated.16 The data of the ERA patients are summarized in Table 1. Auto-Abs detection ELISA was performed as previously described17 with some modifications. Briefly, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific, Shanghai, China) were coated by incubation overnight at 4°C with 1 lg/mL rIL-17A or rIL-17F (Sigma, St Louis, MO, USA). The plates were then washed (phosphate-buffered slaine/ Tween 0.05%), blocked by incubation with the same buffer supplemented with 5% nonfat milk powder, washed and incubated with 1 : 500 dilutions of plasma
Table 1 Characteristics of enrolled patients with early rheumatoid arthritis Patient numbers Age (year, mean SD) Sex, n (%) Female Male Disease duration (years, mean SD) Swollen joints (number, mean SD) Tender joints (number, mean SD) CRP (mg/L, mean SD) ESR (mm/h, Mean SD) DAS-28-ESR (score, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
60 58.55 12.51 43 (71.66) 17 (28.33) 0.37 0.25 12.90 9.05 20.61 7.89 33.32 37.56 47.94 44.16 6.29 1.20 233.92 245.61 98.43 122.72 111.72 96.95 529.66 611.78
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IgM, immunoglobulin M; RF, rheumatoid factor; RU, relative units of serum concentration; SD, standard deviation.
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samples from the patients or controls for 2 h at room temperature. The plates were again thoroughly washed. Horseradish peroxidase (HRP)-conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG (Abcam, Hongkong, China) were added to a final concentration of 0.5 lg/mL. The plates were incubated for 1 h at room temperature and then washed. Bound antibodies were detected with 2,20 -azinobis [3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt (ABTS) (Sigma, MO, USA) 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-17A and IL-17F was expressed as arbitrary units (AU) and calculated as follows: AU ¼
[ODil17 - ODbackground]sample 100 [ODil - ODbackground]positive control
Cut-off level for positivity was defined as the 95th percentile of healthy sera. The threshold for positivity is based on the cut-off value.
Statistical analysis One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for the statistical comparisons of aAb detection. Frequency distributions between two groups
(a)
were compared with the Chi squared test. Unpaired comparisons between two groups were performed using the Mann–Whitney U test. Correlation coefficients were calculated as Spearman’s correlation (rs). These tests were performed using GRAPHPAD PRISM 5.0 (GraphPad Software, San Diego, CA, USA).
RESULTS Distribution of aAbs against IL-17F in patients with ERA, OA and HC The presence of anti-IL-17A and F aAbs in the sera of patients with ERA (n = 60), OA (n = 72) and HC (n = 61) are shown in Fig. 1. The median AU of antiIL-17A was 27.09 in RA, 16.47 in OA and 13.74 in HC. The cut-off value for positivity for the anti-IL-17A aAbs was 27.08. The median AU of anti-IL-17F was 37.44 in RA, 16.23 in OA and 15.47 in HC. The cut-off value for positivity for the anti-IL-17F aAbs was 32.62. The titer of the anti-IL-17A and IL-17F aAbs was significantly higher in the sera of patients with ERA compared with those in OA and HC (P < 0.0001).
Association of aAbs against IL-17A and IL-17F with disease activity in patients with ERA It is shown in Table 2 that ESR was positively correlated with the titer of anti-IL-17A aAbs (r = 0.3928, P = 0.0293) and both ESR and DAS28-ESR were positively correlated with the titer of anti-IL-17F aAbs (r = 0.4909, P = 0.0293; r = 0.3450, P = 0.0457).
(b)
Figure 1 Distribution of auto-antibodies (aAbs) against interleukin (IL)-17A and IL-17F in patients with early rheumatoid arthritis (ERA), osteoarthritis (OA) and healthy controls (HC). The titers of anti-IL-17A and IL-17F aAbs were measured by enzymelinked immunosorbent assay in patients with ERA (n = 60), OA (n = 72) and HC (n = 61). Our results showed that the arbitrary unit (AU) value of anti-IL-17A (a) and IL-17F (b) aAbs were both significantly higher in the sera of patients with ERA compared with those in OA and HC (mean SEM, ***P < 0.0001). Detection was performed in duplicate.
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Autoantibodies against IL-17A and IL-17F
DISCUSSION
Table 2 Correlation of clinical characteristics and laboratory parameters with titers of aAbs against IL-17A and IL-17F in patients with ERA Anti-IL-17A positive (30)
Tender joints Swollen joints RF-IgM RF-IgA RF-IgG ESR CRP Anti-CCP DAS28-ESR
The presence of aAbs against cytokines has been reported in various autoimmune diseases. Neutralizing aAbs against IL-1a have been demonstrated in the sera from patients with RA18 and correlated with a mild disease course in patients with RA.19 Auto-Abs against IL-6 was found in patients with systemic sclerosis (SSc) and associated with the cutaneous form of the disease.20 Auto-Abs against human neutrophil-activating IL-8 are involved in synovial tissue inflammation.21,22 High levels of aAbs against IL-17 and IL-22 have been detected to cause chronic mucocutaneous candidiasis (CMC) in patients with autoimmune polyendocrine syndrome type I (APS-I).17 Anti-interferon (IFN) aAbs were also reported in systemic lupus erythematosus (SLE).23–25 In the current study, we demonstrated that the titers of aAbs against IL-17A and IL-17F were significantly higher in the sera of patients with ERA compared with those in OA and HC, which suggests that anti-IL-17A and IL-17F aAbs may play an important role in pathogenesis of RA. Then we investigated the correlation between the disease activity of patients with the titer of anti-IL-17A and IL-17F aAbs. Our results showed that ESR and DAS28-ESR were positively correlated with the titer of anti-IL-17F aAbs, and ESR was also positively correlated with the titer of anti-IL-17A aAbs. This evidence suggested that anti-IL-17F aAbs might be more positively correlated with disease activity in ERA than anti-IL-17A aAbs. Then we compared the disease activity between the patients with or without aAbs against IL-17A and IL17F. The results showed that CRP and RF-IgM were significantly lower in ERA patients with aAbs against IL-17F than those without aAbs against IL-17F. Meanwhile, the level of RF-IgM was lower in patients with
Anti-IL-17F positive (34)
r
P
r
P
0.1386 0.1063 0.2976 0.2597 0.2417 0.3982 0.0411 0.0285 0.3220
0.4651 0.5760 0.1103 0.3141 0.3671 0.0293* 0.8292 0.8811 0.0884
0.1158 0.0653 0.1123 0.4774 0.1121 0.4909 0.2678 0.011 0.3450
0.5144 0.7136 0.5273 0.0843 0.7028 0.0032** 0.1257 0.8724 0.0457*
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IL, interleukin; RF, rheumatoid factor; IgM, immunoglobulin M. (*, P < 0.05; **, P < 0.005)
Comparison of disease activity in ERA patients with or without aAbs against IL-17A and IL-17F As shown in Tables 3 and 4, we found that CRP and RF-IgM were significantly lower in ERA patients with aAbs against IL-17F than those without aAbs against IL17F (P = 0.0247; P = 0.0439). However, no significant difference was observed on disease activity in ERA patients with or without aAbs against IL-17A.
Association of aAbs against an IL-17A and IL17F with other clinical characteristics in patients with ERA It is shown in Tables 5 and 6 that in the ERA group, no significant correlation was observed between the clinical characteristics and titer of aAbs against IL-17A or IL-17F.
Table 3 Comparison of disease activity between the ERA groups with or without anti-IL-17A aAbs Anti-IL-17A positive (30) DAS28-ESR (score, mean SD) Tender joints (number, mean SD) Swollen joints (number, mean SD) ESR (mm/h, mean SD) CRP (mg/dL, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
6.33 20.69 11.54 48.73 23.52 174.41 75.73 124.91 548.21
1.29 9.01 9.47 30.21 23.18 196.21 85.58 117.11 622.71
Anti-IL-17A negative (30)
P
0.9164 0.6058 0.2014 0.2384 0.1336 0.0621 0.4795 0.9652 0.6172
6.28 20.56 13.94 47.35 40.82 279.51 121.11 97.64 514.21
1.16 7.19 8.87 53.43 45.00 274.91 154.41 76.02 622.51
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; RU, relative units of serum concentration; IgM, immunoglobulin M; SD, standard deviation;.
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Table 4 Comparison of disease activity between the ERA groups with or without anti-IL-17F aAbs Anti-IL-17F positive (34) DAS28-ESR (score, mean SD) Tender joints (number, mean SD) Swollen joints (number, mean SD) ESR (mm/h, mean SD) CRP (mg/dL, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
6.13 19.82 11.62 41.44 23.24 183.11 98.36 141.81 587.11
Anti-IL-17F negative (26)
P
0.1173 0.4625 0.1728 0.5066 0.0247* 0.0439* 0.5995 0.3119 0.2615
1.18 8.23 8.93 30.57 21.91 199.71 102.61 113.51 649.71
6.52 21.27 14.58 55.15 49.81 343.31 104.4 88.93 439.3
1.23 7.55 9.31 57.77 49.67 332.91 148.01 75.82 578.01
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IgM, immunoglobulin M; RF, rheumatoid factor; RU, relative units of serum concentration; SD, standard deviation.(*, P < 0.05)
Table 5 Comparison of other clinical characterizations between ERA patients with or without anti-IL-17A aAbs
Anti-IL-17A positive (30)
Anti-IL-17A negative (30)
P-value
Gender (male), n (%) Age, mean SD, years Dry eye, n (%) Dry mouth, n (%) Rheumatoid nodules, n (%) Cardiac involvement, n (%) Renal lesion, n (%) Pulmonary fibrosis, n (%) Leucopenia, n (%) Anemia, n (%) Thrombocytosis, n (%) IgM, mean SD, g/L IgA, mean SD, g/L IgG, mean SD, g/L
7 (23.33)
12 (40.00)
0.4302
61.54 11.48
56.26 13.15
0.1354
6 (20.00) 10 (33.34) 1 (3.33)
8 (26.66) 12 (40.00) 0 (0.0)
0.7690 0.8053 1.0000
1 (3.33)
1 (3.33)
1.0000
1 (3.33)
3 (10.00)
0.6136
4 (13.33)
5 (16.66)
1.0000
2 (6.66)
0 (0.0)
0.4923
9 (30.00) 8 (26.66)
10 (33.33) 12 (40.00)
1.0000 0.6059
1.53 0.91
1.30 0.53
0.8016
3.27 1.90
3.36 2.75
0.6360
15.03 6.66
15.02 5.36
0.7427
Table 6 Comparison of other clinical characterizations between ERA patients with or without anti-IL-17F aAbs
Gender (male), n (%) Age, mean SD, years Dry eye, n (%) Dry mouth, n (%) Rheumatoid nodules, n (%) Cardiac involvement, n (%) Renal lesion, n (%) Pulmonary fibrosis, n (%) Leucopenia, n (%) Anemia, n (%) Thrombocytosis, n (%) IgM, mean SD, g/L IgA, mean SD, g/L IgG, mean SD, g/L
Anti-IL-17F positive (34)
Anti-IL-17F negative (26)
P-value
9 (26.47)
11 (42.30)
0.4412
58.32 11.31
58.27 14.70
0.6979
9 (26.47) 12 (35.29) 0 (0.0)
8 (23.07) 11 (42.30) 1 (3.84)
1.0000 0.8607 0.4426
0 (0.0)
0 (0.0)
–
4 (11.76)
0 (0.0)
0.1397
4 (11.76)
4 (11.76)
0.7243
4 (11.76) 10 (29.41) 11 (32.35)
1 (3.84) 9 (34.61) 6 (23.07)
0.3926 0.7958 0.5911
1.27 0.58
1.65 0.92
0.1943
3.48 2.63
3.01 1.72
0.8287
15.57 7.03
14.02 4.36
0.6652
IgM, immunoglobulin M; SD, standard deviation.
IgM, immunoglobulin M; SD, standard deviation.
aAbs against IL-17A, although the difference was not significant. Hsu et al.26 reported that in vivo neutralization of IL-17 by anti–IL-17 treatment in older BXD2 mice does not suppress IgM autoantibody-forming B cells, but does suppress IgG autoantibody-forming B
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cells. However, according to our findings, IgM autoantibody-forming B cells but not IgG autoantibodyforming B cells induced by IL-17 might decrease in ERA patients with anti-IL-17A and IL-17F aAbs, so the mechanism of the suppression of Ig autoantibody by anti–IL-17 neutralization needs further study in RA.
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Autoantibodies against IL-17A and IL-17F
IL-6, IL-1b and tumor necrosis factor (TNF)-a have been reported to increase CRP production,27 and IL17 can increase CRP production by promoting IL-6 production directly28 or indirectly via a synergistic effect with IL-1b and TNF-a.6,29 In the ERA group, we assumed that high aAbs titers against IL-17F may prevent the cytokines from binding to their receptors, thereby inhibiting their biological functions,30 which could explain why the levels of CRP were lower in the patients with aAbs and suggests that IL-17F seems to be more effective in promoting CRP production than IL-17A. Although aAbs against cytokines have been described in autoimmune diseases, their frequency and role have been debated long-term. Auto-Abs against IL-1a have been demonstrated to neutralize IL-1a activity and are correlated with a mild disease course in patients with RA,18,19 but anti-IFN aAbs were reported as having an aggressive role in SLE.24,25 High aAbs titer may inhibit biological functions of cytokines by preventing cytokines binding to their receptors, but it also could lead to a high accumulation of cytokines in a form of stable immune complexes. For example, it was reported that anti-IL-6 antibody could not neutralize but induces high amounts of IL-6 in some patients with multiple myeloma.31 In our study, although results suggested that anti-IL-17F aAbs may be a protective role in ERA, further study on mechanisms needs to be performed to directly prove the role of aAbs against IL-17A and IL17F in ERA. In conclusion, our study demonstrated that the levels of aAbs against IL-17A and IL-17F were higher in ERA patients and had a correlation with ESR and DAS28ESR in ERA. Furthermore, elevated aAbs against IL-17F play a more important role than those against IL-17A to reduce the levels of CRP and RF-IgM, suggesting that anti-IL-17F aAbs may have a protective role.
ACKNOWLEDGEMENTS This work was supported by the First Affiliated Hospital of Baotou Medical College. We would like to thank other clinicians: Fengfeng Lv, Jianbo Zhao, Chunyan Pang, Wei Zhang, Zhifang Chang, Hui Wang, Dongmei Bao, Xiuyuan Feng, Shu Zhang, Kangkai Luo and Guoan Yang for providing us with samples. The authors declare that they have no competing interests. No financial or other relationships could lead to a conflict of interest. This work was supported by the National Nature Science Foundation of China (no. 81260461 and no. 81102255).
International Journal of Rheumatic Diseases 2016; 19: 644–650
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ORIGINAL ARTICLE
Elevated autoantibodies against interleukin-17F correlate with disease activity in patients with early rheumatoid arthritis Lin YANG,1 Li BAI,2 Feifei WEI,2 Yuan LIU,2 Lin SUN,1 Wenming WANG,3 Xiangyuan LIU1 and Yongfu WANG2 1
Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing, 2Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, Baotou, and 3Department of Hematology, Peking University Third Hospital, Beijing, China
Abstract Aim: To investigate the presence of autoantibodies (aAbs) against interleukin (IL)-17A and IL-17F and observe whether anti-IL-17A or IL-17F aAbs are associated with disease activity in patients with early rheumatoid arthritis (ERA). Methods: At present, 60 patients with ERA, 72 patients with osteoarthritis (OA) and 61 healthy controls (HC) have been included in a database. Clinical assessment and laboratory data were recorded. We detected the titer of aAbs against IL-17A and IL-17F using enzyme-linked immunosorbent assay and analyzed the correlation of these aAbs in patients with ERA. Results: Our results showed that the levels of aAbs against IL-17A and IL-17F were significantly higher in ERA OA and HC (P < 0.0001). The level of aAbs against IL-17F was correlated with Disease Activity Score-28 erythrocyte sedimentation rate (ESR) (P = 0.0457) and ESR alone (P = 0.0032) in patients with ERA. In addition, in the ERA group, the level of C-reactive protein and rheumatoid factor immunoglobulin M was lower in patients with aAbs against IL-17F than patients without aAbs (P = 0.0247; P = 0.0439). No significant correlation was observed between the clinical characteristics and level of aAbs against IL-17A in patients with ERA except ESR (P = 0.0239). Conclusions: Elevated aAbs against IL-17F correlate with disease activity in patients with ERA. This evidence suggests that anti-IL-17F aAbs may have a protective role in the pathogenesis of ERA. Key words: autoantibody, C-reactive protein, DAS-28, erythrocyte sedimentation rate, IL-17, rheumatoid arthritis.
INTRODUCTION Correspondence: Yongfu Wang, MD, Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College, No.41, LinYin Road, Baotou 014010, China. Email: [email protected]. and Xiangyuan Liu, MD, Department of Rheumatology and Immunology, Peking University Third Hospital, No.49, HuaYuan (North) Road, Beijing 100191, China. Email: [email protected].
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by abnormal immune responses which lead to synovial hyperplasia and ultimately progressive destruction of cartilage and bone. The altered activation of innate or adaptive immune mechanisms contributes to the development of this autoimmune disease. Among great progress in underlying the pathogenesis of RA, defective regulation of
© 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd
Autoantibodies against IL-17A and IL-17F
inflammatory cytokines has been reported as a central component in synovial hyperplasia and joint destruction.1,2 At present, more and more evidence suggests that defective regulation of interleukin (IL)-17 may be a central component in the pathogenesis of RA. In human RA, IL-17-producing T cells have been identified3 and IL-17A has been found to be elevated in the synovial fluid of RA patients.4,5 In animal models of RA, overexpression of IL-17 promotes extensive inflammatory cell migration and induces bone erosion and cartilage degradation.6 In contrast, inhibition of IL-17 has been demonstrated to ameliorate collagen-induced arthritis (CIA).7,8 Compared with IL-17A, IL-17F is a less potent activator of synoviocytes9 and plays a minimal role in an arthritis animal model10 and evidence exists that IL-17F has the potential to destroy cartilage in vitro.11 An IL-17F variant has been reported to be associated with an increased disease activity in Polish patients with RA.12 The level of 17F is higher in plasma and synovial fluid in RA patients than controls and IL-17F, but not IL17A decreases with multiple classes of disease-modifying anti-rheumatic drugs (DMARDs).13 Recently, higher levels of IL-17A and IL-17F were detected in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from patients with RA; meanwhile, IL-17A and IL-17F were also increased in the CIA model.14 However, the presence of autoantibodies (aAbs) against IL-17A and IL-17F in patients with RA and whether aAbs are associated with disease activity are less known. In the current study, we detected the presence of aAbs against and IL-17A and IL-17F in serum samples of patients with early RA (ERA) and analyzed the association with the clinical manifestations and laboratory parameters of ERA disease.
PATIENTS AND METHODS Patients From 2006 to 2013, 683 Chinese patients with RA were admitted when they first visited the Department of Rheumatology, the First Affiliated Hospital of Baotou Medical College. The ERA group was defined as disease duration < 12 months.15 Sixty patients with ERA, 72 patients with osteoarthritis (OA) and 61 healthy controls (HC) were included in a database. ERA patients underwent extensive serological evaluations. All serum samples were obtained from the patients when they were first diagnosed as RA and frozen at 80°C immediately after collection. Ethics approval (no. EC-
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BMC1209007) was obtained by the ethics committee of Baotou Medical College.
Clinical assessment and laboratory data Clinical data were recorded as the following index: age, sex, disease duration, number of swollen joints, number of tender joints and other clinical characteristics. General health status reported by patients on a 100mm visual analogue scale (VAS) was also recorded. Erythrocyte sedimentation rate (ESR) was evaluated by the Westergren method. C-reactive protein (CRP) and rheumatoid factor (RF) – immunoglobulin M (IgM) were examined by immunonephelometry method. Anti-citrullinated peptide (CCP) antibodies, RF-IgA and RF-IgG were tested by enzyme-linked immunosorbent assay (ELISA). The 28-joint count Disease Activity Score (DAS28-ESR) was calculated.16 The data of the ERA patients are summarized in Table 1. Auto-Abs detection ELISA was performed as previously described17 with some modifications. Briefly, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific, Shanghai, China) were coated by incubation overnight at 4°C with 1 lg/mL rIL-17A or rIL-17F (Sigma, St Louis, MO, USA). The plates were then washed (phosphate-buffered slaine/ Tween 0.05%), blocked by incubation with the same buffer supplemented with 5% nonfat milk powder, washed and incubated with 1 : 500 dilutions of plasma
Table 1 Characteristics of enrolled patients with early rheumatoid arthritis Patient numbers Age (year, mean SD) Sex, n (%) Female Male Disease duration (years, mean SD) Swollen joints (number, mean SD) Tender joints (number, mean SD) CRP (mg/L, mean SD) ESR (mm/h, Mean SD) DAS-28-ESR (score, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
60 58.55 12.51 43 (71.66) 17 (28.33) 0.37 0.25 12.90 9.05 20.61 7.89 33.32 37.56 47.94 44.16 6.29 1.20 233.92 245.61 98.43 122.72 111.72 96.95 529.66 611.78
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IgM, immunoglobulin M; RF, rheumatoid factor; RU, relative units of serum concentration; SD, standard deviation.
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samples from the patients or controls for 2 h at room temperature. The plates were again thoroughly washed. Horseradish peroxidase (HRP)-conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG (Abcam, Hongkong, China) were added to a final concentration of 0.5 lg/mL. The plates were incubated for 1 h at room temperature and then washed. Bound antibodies were detected with 2,20 -azinobis [3-ethylbenzothiazoline-6-sulfonicacid]-diammonium salt (ABTS) (Sigma, MO, USA) 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-17A and IL-17F was expressed as arbitrary units (AU) and calculated as follows: AU ¼
[ODil17 - ODbackground]sample 100 [ODil - ODbackground]positive control
Cut-off level for positivity was defined as the 95th percentile of healthy sera. The threshold for positivity is based on the cut-off value.
Statistical analysis One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for the statistical comparisons of aAb detection. Frequency distributions between two groups
(a)
were compared with the Chi squared test. Unpaired comparisons between two groups were performed using the Mann–Whitney U test. Correlation coefficients were calculated as Spearman’s correlation (rs). These tests were performed using GRAPHPAD PRISM 5.0 (GraphPad Software, San Diego, CA, USA).
RESULTS Distribution of aAbs against IL-17F in patients with ERA, OA and HC The presence of anti-IL-17A and F aAbs in the sera of patients with ERA (n = 60), OA (n = 72) and HC (n = 61) are shown in Fig. 1. The median AU of antiIL-17A was 27.09 in RA, 16.47 in OA and 13.74 in HC. The cut-off value for positivity for the anti-IL-17A aAbs was 27.08. The median AU of anti-IL-17F was 37.44 in RA, 16.23 in OA and 15.47 in HC. The cut-off value for positivity for the anti-IL-17F aAbs was 32.62. The titer of the anti-IL-17A and IL-17F aAbs was significantly higher in the sera of patients with ERA compared with those in OA and HC (P < 0.0001).
Association of aAbs against IL-17A and IL-17F with disease activity in patients with ERA It is shown in Table 2 that ESR was positively correlated with the titer of anti-IL-17A aAbs (r = 0.3928, P = 0.0293) and both ESR and DAS28-ESR were positively correlated with the titer of anti-IL-17F aAbs (r = 0.4909, P = 0.0293; r = 0.3450, P = 0.0457).
(b)
Figure 1 Distribution of auto-antibodies (aAbs) against interleukin (IL)-17A and IL-17F in patients with early rheumatoid arthritis (ERA), osteoarthritis (OA) and healthy controls (HC). The titers of anti-IL-17A and IL-17F aAbs were measured by enzymelinked immunosorbent assay in patients with ERA (n = 60), OA (n = 72) and HC (n = 61). Our results showed that the arbitrary unit (AU) value of anti-IL-17A (a) and IL-17F (b) aAbs were both significantly higher in the sera of patients with ERA compared with those in OA and HC (mean SEM, ***P < 0.0001). Detection was performed in duplicate.
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DISCUSSION
Table 2 Correlation of clinical characteristics and laboratory parameters with titers of aAbs against IL-17A and IL-17F in patients with ERA Anti-IL-17A positive (30)
Tender joints Swollen joints RF-IgM RF-IgA RF-IgG ESR CRP Anti-CCP DAS28-ESR
The presence of aAbs against cytokines has been reported in various autoimmune diseases. Neutralizing aAbs against IL-1a have been demonstrated in the sera from patients with RA18 and correlated with a mild disease course in patients with RA.19 Auto-Abs against IL-6 was found in patients with systemic sclerosis (SSc) and associated with the cutaneous form of the disease.20 Auto-Abs against human neutrophil-activating IL-8 are involved in synovial tissue inflammation.21,22 High levels of aAbs against IL-17 and IL-22 have been detected to cause chronic mucocutaneous candidiasis (CMC) in patients with autoimmune polyendocrine syndrome type I (APS-I).17 Anti-interferon (IFN) aAbs were also reported in systemic lupus erythematosus (SLE).23–25 In the current study, we demonstrated that the titers of aAbs against IL-17A and IL-17F were significantly higher in the sera of patients with ERA compared with those in OA and HC, which suggests that anti-IL-17A and IL-17F aAbs may play an important role in pathogenesis of RA. Then we investigated the correlation between the disease activity of patients with the titer of anti-IL-17A and IL-17F aAbs. Our results showed that ESR and DAS28-ESR were positively correlated with the titer of anti-IL-17F aAbs, and ESR was also positively correlated with the titer of anti-IL-17A aAbs. This evidence suggested that anti-IL-17F aAbs might be more positively correlated with disease activity in ERA than anti-IL-17A aAbs. Then we compared the disease activity between the patients with or without aAbs against IL-17A and IL17F. The results showed that CRP and RF-IgM were significantly lower in ERA patients with aAbs against IL-17F than those without aAbs against IL-17F. Meanwhile, the level of RF-IgM was lower in patients with
Anti-IL-17F positive (34)
r
P
r
P
0.1386 0.1063 0.2976 0.2597 0.2417 0.3982 0.0411 0.0285 0.3220
0.4651 0.5760 0.1103 0.3141 0.3671 0.0293* 0.8292 0.8811 0.0884
0.1158 0.0653 0.1123 0.4774 0.1121 0.4909 0.2678 0.011 0.3450
0.5144 0.7136 0.5273 0.0843 0.7028 0.0032** 0.1257 0.8724 0.0457*
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IL, interleukin; RF, rheumatoid factor; IgM, immunoglobulin M. (*, P < 0.05; **, P < 0.005)
Comparison of disease activity in ERA patients with or without aAbs against IL-17A and IL-17F As shown in Tables 3 and 4, we found that CRP and RF-IgM were significantly lower in ERA patients with aAbs against IL-17F than those without aAbs against IL17F (P = 0.0247; P = 0.0439). However, no significant difference was observed on disease activity in ERA patients with or without aAbs against IL-17A.
Association of aAbs against an IL-17A and IL17F with other clinical characteristics in patients with ERA It is shown in Tables 5 and 6 that in the ERA group, no significant correlation was observed between the clinical characteristics and titer of aAbs against IL-17A or IL-17F.
Table 3 Comparison of disease activity between the ERA groups with or without anti-IL-17A aAbs Anti-IL-17A positive (30) DAS28-ESR (score, mean SD) Tender joints (number, mean SD) Swollen joints (number, mean SD) ESR (mm/h, mean SD) CRP (mg/dL, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
6.33 20.69 11.54 48.73 23.52 174.41 75.73 124.91 548.21
1.29 9.01 9.47 30.21 23.18 196.21 85.58 117.11 622.71
Anti-IL-17A negative (30)
P
0.9164 0.6058 0.2014 0.2384 0.1336 0.0621 0.4795 0.9652 0.6172
6.28 20.56 13.94 47.35 40.82 279.51 121.11 97.64 514.21
1.16 7.19 8.87 53.43 45.00 274.91 154.41 76.02 622.51
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; RU, relative units of serum concentration; IgM, immunoglobulin M; SD, standard deviation;.
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Table 4 Comparison of disease activity between the ERA groups with or without anti-IL-17F aAbs Anti-IL-17F positive (34) DAS28-ESR (score, mean SD) Tender joints (number, mean SD) Swollen joints (number, mean SD) ESR (mm/h, mean SD) CRP (mg/dL, mean SD) RF-IgM (IU/mL, mean SD) RF-IgA (IU/mL, mean SD) RF-IgG (IU/mL, mean SD) Anti-CCP (RU/mL, mean SD)
6.13 19.82 11.62 41.44 23.24 183.11 98.36 141.81 587.11
Anti-IL-17F negative (26)
P
0.1173 0.4625 0.1728 0.5066 0.0247* 0.0439* 0.5995 0.3119 0.2615
1.18 8.23 8.93 30.57 21.91 199.71 102.61 113.51 649.71
6.52 21.27 14.58 55.15 49.81 343.31 104.4 88.93 439.3
1.23 7.55 9.31 57.77 49.67 332.91 148.01 75.82 578.01
anti-CCP, anti-citrullinated peptide; CRP, C-reactive protein; DAS-28, Disease Activity Score of 28 joints; ESR, erythrocyte sedimentation rate; IgM, immunoglobulin M; RF, rheumatoid factor; RU, relative units of serum concentration; SD, standard deviation.(*, P < 0.05)
Table 5 Comparison of other clinical characterizations between ERA patients with or without anti-IL-17A aAbs
Anti-IL-17A positive (30)
Anti-IL-17A negative (30)
P-value
Gender (male), n (%) Age, mean SD, years Dry eye, n (%) Dry mouth, n (%) Rheumatoid nodules, n (%) Cardiac involvement, n (%) Renal lesion, n (%) Pulmonary fibrosis, n (%) Leucopenia, n (%) Anemia, n (%) Thrombocytosis, n (%) IgM, mean SD, g/L IgA, mean SD, g/L IgG, mean SD, g/L
7 (23.33)
12 (40.00)
0.4302
61.54 11.48
56.26 13.15
0.1354
6 (20.00) 10 (33.34) 1 (3.33)
8 (26.66) 12 (40.00) 0 (0.0)
0.7690 0.8053 1.0000
1 (3.33)
1 (3.33)
1.0000
1 (3.33)
3 (10.00)
0.6136
4 (13.33)
5 (16.66)
1.0000
2 (6.66)
0 (0.0)
0.4923
9 (30.00) 8 (26.66)
10 (33.33) 12 (40.00)
1.0000 0.6059
1.53 0.91
1.30 0.53
0.8016
3.27 1.90
3.36 2.75
0.6360
15.03 6.66
15.02 5.36
0.7427
Table 6 Comparison of other clinical characterizations between ERA patients with or without anti-IL-17F aAbs
Gender (male), n (%) Age, mean SD, years Dry eye, n (%) Dry mouth, n (%) Rheumatoid nodules, n (%) Cardiac involvement, n (%) Renal lesion, n (%) Pulmonary fibrosis, n (%) Leucopenia, n (%) Anemia, n (%) Thrombocytosis, n (%) IgM, mean SD, g/L IgA, mean SD, g/L IgG, mean SD, g/L
Anti-IL-17F positive (34)
Anti-IL-17F negative (26)
P-value
9 (26.47)
11 (42.30)
0.4412
58.32 11.31
58.27 14.70
0.6979
9 (26.47) 12 (35.29) 0 (0.0)
8 (23.07) 11 (42.30) 1 (3.84)
1.0000 0.8607 0.4426
0 (0.0)
0 (0.0)
–
4 (11.76)
0 (0.0)
0.1397
4 (11.76)
4 (11.76)
0.7243
4 (11.76) 10 (29.41) 11 (32.35)
1 (3.84) 9 (34.61) 6 (23.07)
0.3926 0.7958 0.5911
1.27 0.58
1.65 0.92
0.1943
3.48 2.63
3.01 1.72
0.8287
15.57 7.03
14.02 4.36
0.6652
IgM, immunoglobulin M; SD, standard deviation.
IgM, immunoglobulin M; SD, standard deviation.
aAbs against IL-17A, although the difference was not significant. Hsu et al.26 reported that in vivo neutralization of IL-17 by anti–IL-17 treatment in older BXD2 mice does not suppress IgM autoantibody-forming B cells, but does suppress IgG autoantibody-forming B
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cells. However, according to our findings, IgM autoantibody-forming B cells but not IgG autoantibodyforming B cells induced by IL-17 might decrease in ERA patients with anti-IL-17A and IL-17F aAbs, so the mechanism of the suppression of Ig autoantibody by anti–IL-17 neutralization needs further study in RA.
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Autoantibodies against IL-17A and IL-17F
IL-6, IL-1b and tumor necrosis factor (TNF)-a have been reported to increase CRP production,27 and IL17 can increase CRP production by promoting IL-6 production directly28 or indirectly via a synergistic effect with IL-1b and TNF-a.6,29 In the ERA group, we assumed that high aAbs titers against IL-17F may prevent the cytokines from binding to their receptors, thereby inhibiting their biological functions,30 which could explain why the levels of CRP were lower in the patients with aAbs and suggests that IL-17F seems to be more effective in promoting CRP production than IL-17A. Although aAbs against cytokines have been described in autoimmune diseases, their frequency and role have been debated long-term. Auto-Abs against IL-1a have been demonstrated to neutralize IL-1a activity and are correlated with a mild disease course in patients with RA,18,19 but anti-IFN aAbs were reported as having an aggressive role in SLE.24,25 High aAbs titer may inhibit biological functions of cytokines by preventing cytokines binding to their receptors, but it also could lead to a high accumulation of cytokines in a form of stable immune complexes. For example, it was reported that anti-IL-6 antibody could not neutralize but induces high amounts of IL-6 in some patients with multiple myeloma.31 In our study, although results suggested that anti-IL-17F aAbs may be a protective role in ERA, further study on mechanisms needs to be performed to directly prove the role of aAbs against IL-17A and IL17F in ERA. In conclusion, our study demonstrated that the levels of aAbs against IL-17A and IL-17F were higher in ERA patients and had a correlation with ESR and DAS28ESR in ERA. Furthermore, elevated aAbs against IL-17F play a more important role than those against IL-17A to reduce the levels of CRP and RF-IgM, suggesting that anti-IL-17F aAbs may have a protective role.
ACKNOWLEDGEMENTS This work was supported by the First Affiliated Hospital of Baotou Medical College. We would like to thank other clinicians: Fengfeng Lv, Jianbo Zhao, Chunyan Pang, Wei Zhang, Zhifang Chang, Hui Wang, Dongmei Bao, Xiuyuan Feng, Shu Zhang, Kangkai Luo and Guoan Yang for providing us with samples. The authors declare that they have no competing interests. No financial or other relationships could lead to a conflict of interest. This work was supported by the National Nature Science Foundation of China (no. 81260461 and no. 81102255).
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