Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membranes of multiple affected joints. Interleukin-1 (IL-1) and IL-1 receptor families play important roles in inflammatory and immunological responses. IL-33 and its receptor ST2 belong to the IL-1 and IL-1 receptor families, respectively. The pathogenic role of IL-33 in RA has been widely studied, but there are few studies about its receptor ST2, especially the soluble form, in RA.
The ST2 gene produces two forms: soluble ST2 and transmembrane ST2 (ST2L). Soluble ST2 lacks the intracellular and transmembrane domains, but both isoforms have the same extracellular domain. ST2L is selectively expressed on Th2 but not on Th1 T-cells, which indicates that it might be involved in Th2 cell-mediated immunological responses. Evidence has been provided that treatment with an anti-ST2 antibody attenuates the severity of joint arthritis in mice with collagen-induced arthritis (CIA). This finding might be due to the blocking of the IL-33/ST2L axis by an anti-ST2 antibody.
Elevated levels of soluble ST2 have been found in many diseases, such as asthma, dengue virus infection, sepsis, and RA. Our previous studies have found elevated soluble ST2 in the serum of RA patients, which is consistent with other studies. However, to date, only one study has reported an association between soluble ST2 and RA disease activity, but it is limited in early RA. In addition, the results of the former studies were limited in terms of information about the function of soluble ST2 in RA. Most of the studies focused on other diseases instead of RA. A recent study on allergic airway inflammation demonstrated that soluble ST2 blocked IL-33 signaling. In addition, an in vitro study demonstrated that soluble ST2 could suppress lipopolysaccharide (LPS)-induced IL-6 production in THP-1 cells, and whether this function was restricted to the inhibition of the IL-33/ST2 axis was unknown. Furthermore, there was no report of the function of soluble ST2 in RA.
In this study, we therefore investigated the correlation between soluble ST2 levels and disease activity and explored the function of soluble ST2 in RA.
The study protocols were approved by the Institutional and Medical Ethics Review Board of Peking University International Hospital. Informed consent was obtained from all patients.
Patients and healthy controls
From 2014 to 2015, a total of 56 RA patients (51 women and 5 men) were enrolled in this study; all of them met the 1987 revised classification criteria of the American College of Rheumatology. All patients had active disease, which was defined by a Disease Activity Score with C-reactive protein (DAS28-CRP) in 28 joints of >2.6. Thirty-eight age-matched healthy controls (HCs) were recruited from staff of our hospital, and all of them did not have any rheumatologic conditions.
The 56 enrolled RA patients who received disease-modifying antirheumatic drugs (at least two of methotrexate, sulfasalazine, hydroxychloroquine, and leflunomide) were evaluated for 24 weeks. The sera from the patients who had a lower disease activity than at baseline were collected and soluble ST2 levels were evaluated.
Synovial fluid (SF) samples were collected from another 30 RA patients and 20 osteoarthritis (OA) patients and were stored at −70°C for <6 months.
Clinical and laboratory assessments
All the 56 RA patients underwent clinical assessments at baseline. The patients who had a lower disease activity after treatment also underwent clinical assessments at the end of the follow-up period. The assessment consisted of the number of swollen joints (28 joints were evaluated) and the number of tender joints (28 joints were evaluated). Cut points of DAS28 for low (<3.2), moderate (3.2–5.1), and high (>5.1) disease activity were used.
Laboratory assessments included complete blood counts, the levels of rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), CRP, and the erythrocyte sedimentation rate (ESR) of the patients.
Cell culture and cell treatment
The synovial tissue specimens were minced into small pieces and incubated with 1 mg/ml Type I collagenase (Sigma-Aldrich, Bornem, Belgium, Germany) in Dulbecco's Modified Eagle's Medium (DMEM) at 37°C for 2 h. The cells were collected by filtering the suspension through nylon mesh (70 μm), followed by extensive washing with phosphate-buffered saline. Then, RA synovial fibroblasts (RASFs) were cultured in complete high-glucose DMEM (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 6-well plates for 24 h. The concentration of the cells was 5 × 104 cells per well in 1 ml medium.
Peripheral blood mononuclear cells (PBMCs) were isolated from 20 RA patients and then cultured in RPMI 1640 (HyClone, Logan, UT, USA) with 10% FBS and 1% antibiotics. PBMCs were cultured in 24-well plates at a concentration of 1 × 106/well with 1 ml medium (or medium with soluble ST2 1 μg/ml) for 24 h. Every sample was divided into two groups: control or soluble ST2 treated. After 24 h of treatment, we collected the PBMCs and the supernatants and then determined the protein or mRNA levels of cytokines, including IL-1β, IL-33, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). We detected the above cytokines in supernatants with ELISA, and if the cytokine was undetectable, we then evaluated the cytokine mRNA levels by real-time-quantitative polymerase chain reaction (RT-qPCR).
Real time-quantitative polymerase chain reaction assays
Total RNA was extracted from the RASF cells with an RNeasy mini kit (QIAGEN, USA). Reverse transcription was performed with the RevertAid First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer's instructions. The resulting cDNA was subjected to PCR and real-time PCR analyses. RT-qPCR analyses were then performed with SYBR Green Master mix kit (Applied Biosystems, Foster City, CA, USA) and primers for GAPDH, IL-1β, TNF-α, IL-6, IL-8, and IL-33. The primer sequences were as follows: GAPDH forward, 5'-AAGGTGAAGGTCGG AGTCAA-3', GAPDH reverse, 5'-AATGAAGGGGTCATTGATGG-3'; IL-1β forward, 5'-GCTGAGGAAGATGCTGGTTC-3', IL-1β reverse, 5'-GTGATCGTACAGGTGCATCG-3'; TNF-α forward, 5'-CGAGTGACAAGCCTGTAGC-3', TNF-α reverse, 5'-GGTGTGG GTGAGGAGCACAT-3'; IL-6 forward, 5'-GCCCAGCTATGAACTCCTTCT-3', IL-6 reverse, 5'-GAAGGCAGCAGGCAACAC-3'; IL-8 forward, 5'-AACTTCTCCAC AACCCTCTG-3', IL-8 reverse, 5'-TTGGCAGCCTTCCTGATTTC-3'; and IL-33 forward, 5'-GGTGTTACTGAGTTACTATGAA-3', IL-33 reverse, 5'-GGAGCTCCACAGAGTGTTCCTTG-3'.
Serum samples and SF were obtained and stored at −70°C until analysis. Soluble ST2 was quantified with an R and D Systems human IL-1R4/ST2 DuoSet ELISA. IL-1β was measured with a human IL-1 beta ELISA Ready-SET-Go! kit (eBioscience, USA). IL-6 and IL-8 ELISA kits were purchased from Neobioscience Technology Co, Ltd., Beijing, China. The ELISA procedure was performed according to the instructions of the manufacturer.
Statistical analyses were performed with SPSS 17.0 (SPSS, Chicago, Illinois, USA). Continuous variables from the study were analyzed by the Student's t test with a parametric distribution or the Mann-Whitney U test with a nonparametric distribution. The association between clinical and laboratory parameters was evaluated with Spearman's correlation coefficients. Differences between cell groups were evaluated by the Mann-Whitney U test or the Wilcoxon signed-rank test, when appropriate. A value of P < 0.05 was considered statistically significant.
Soluble ST2 levels in serum of rheumatoid arthritis patients and healthy controls and in synovial fluid of rheumatoid arthritis and osteoarthritis patients
We first assessed the soluble ST2 levels in the serum of 56 RA patients and 38 HCs. The characteristics of RA patients are shown in Table 1. RA patients were found to have significantly higher serum levels of soluble ST2 (127.14 ± 61.43 pg/ml) than that of HCs (78.37 ± 41.93 pg/ml, P < 0.01; Figure 1a). To extend the study, we then measured the soluble ST2 levels in the SF of another thirty RA patients and twenty OA patients. SF levels of soluble ST2 tended to be higher in RA patients (41.90 ± 33.58 pg/ml) than that in OA patients (19.71 ± 16.72 pg/ml, P < 0.05; Figure 1b).
Association of soluble ST2 with laboratory parameters and disease activity
The 56 RA patients were included in a follow-up study. Of all the 56 RA patients who received DMARD therapy, 24 patients had a lower disease activity than at baseline. We detected soluble ST2 levels at the end of the follow-up period and compared these with baseline values. We found that soluble ST2 levels (113.01 ± 53.90 pg/ml) were lower than baseline values (139.59 ± 68.36 pg/ml) and the difference was statistically significant (P = 0.01, Figure 2a).
To further confirm the result, we compared the soluble ST2 levels in patients with different disease activities. We found that RA patients with high disease activity had higher soluble serum ST2 levels (162.02 ± 56.78 pg/ml) than those with low disease activity (94.67 ± 40.27 pg/ml, P = 0.001; Figure 2b).
However, interestingly, we found that the serum levels of soluble ST2 had no correlation with CRP, ESR, DAS28-CRP, or antibodies, including anti-keratin antibody (AKA), RF, and ACPA (data not shown).
Soluble ST2 did not affect interleukin-1β, interleukin-6, interleukin-8, or tumor necrosis factor-α expression in peripheral blood mononuclear cells from rheumatoid factor patients
IL-33 was undetectable at both the mRNA and protein levels, and IL-1β and TNF-α were detectable only at the mRNA level. IL-6 and IL-8 were detectable in the culture supernatants. In some patients, soluble ST2 showed negative effects on IL-1β, TNF-α, IL-6, and IL-8 expressions, but in other patients, there was no suppressive function on these cytokines. There were no significant differences in IL-1β [Figure 3a], TNF-α [Figure 3b], IL-6 [Figure 3c], and IL-8 [Figure 3d] levels before and after soluble ST2 treatment.
Soluble ST2 suppressed interleukin-1β and interleukin-33 expressions in resting rheumatoid arthritis synovial fibroblasts
Soluble ST2 was higher in synovial fluid from RA patients than in that from patients with OA, but there was no information about the function of ST2 on RASFs. Therefore, we obtained RASF cells from three RA patients. Every sample was divided into two groups: control and soluble ST2 treated. After 24 h of culture, we collected the cells and the supernatants. We detected IL-1β, IL-33, IL-6, IL-8, and TNF-α. At the protein level, all of them were undetectable. At the mRNA level, IL-1β and IL-33 were suppressed by soluble ST2 [Figure 4a and 4b], but no suppression of IL-6, IL-8, or TNF-α was found.
Soluble ST2 suppressed interleukin-33, interleukin-1β, tumor necrosis factor-α, interleukin-6, and interleukin-8 expressions in activated rheumatoid arthritis synovial fibroblasts
Soluble ST2 exerted a suppressing role on IL-1β in RASFs but not in PBMCs, which may be attributed to the expression of IL-33 in RASFs. Therefore, if we increase the level of IL-33 in RASFs, soluble ST2 may play a negative role on more cytokines, such as IL-6, IL-8, and TNF-α. To examine this hypothesis, we activated RASF cells by exposure to TNF-α plus IL-1β for 24 h, and then RASFs showed higher IL-33 expression [Figure 5a]. The activated RASFs were treated with 1 μg/ml ST2 for 24 h. Our findings demonstrated that soluble ST2 suppressed IL-33 [Figure 5b], IL-1β [Figure 5c], TNF-α [Figure 5d], IL-6 [Figure 5e], and IL-8 [Figure 5f] expressions in RASFs.
In the present study, we demonstrated elevated levels of soluble ST2 both in serum and synovial fluid of RA patients and found that soluble ST2 negatively affected both IL-1β and IL-33 in RASF cells. The soluble ST2 levels in serum and synovial fluid were markedly elevated in RA patients compared to those in HCs and OA patients, respectively. The latter finding was not consistent with a recent study but was consistent with a former study. We thought that there might be two possible reasons for this discrepancy: (1) all our synovial fluid samples were fresh samples that had been acquired in the last 6 months and (2) the sample size was larger than in the recent study.
According to our knowledge, studies on the correlation between soluble ST2 and disease activity in RA were limited. Very recently, Shen et al. demonstrated that soluble ST2 was decreased after treatment and lower baseline soluble ST2 was an independent predictor of remission of early RA. However, their finding was limited in the early RA patients. Another study found that soluble ST2 was correlated with disease activity in patients with systemic juvenile idiopathic arthritis. We found that all patients in our follow-up who had a good response to the therapy had lower levels of soluble ST2 than at baseline. This result to a certain extent was consistent with a recent study that utilized seven naïve RA patients and compared the soluble ST2 levels before and after treatment. In our current study, there was no significant correlation between soluble ST2 and disease activity or laboratory parameters (including CRP, ESR, RF, ACPA, and AKA). Our findings indicated that the soluble ST2 was only an index associated with RA disease activity, but not directly correlated with the disease activity.
We also investigated the function of soluble ST2 in RA. As soluble ST2 is a decoy receptor for IL-33, several studies have focused on the function of IL-33 in heart disease or allergic airway inflammation. Hayakawa et al. have demonstrated that soluble ST2 can block IL-33 signaling in allergic airway inflammation. Ohto-Ozaki et al. demonstrated in vivo that ST2 can protect against the IL-33-induced Th2-type immune response in ST2 transgenic mice. All studies have consistently indicated that ST2 can selectively block the IL-33 response. Another study found that soluble ST2 suppressed LPS-induced IL-6 production in THP-1 cells. In our study, there were elevated ST2 levels in RA patients' serum and synovial fluid, and in the subsequent in vitro study, we found that soluble ST2 could suppress not only IL-33 but also IL-1β in resting RASFs. In activated RASFs, ST2 negatively affected IL-33, IL-1β, TNF-α, IL-6, and IL-8. In the PBMC culture study, soluble ST2 showed no effects on these inflammatory cytokines which might be attributed to the fact that there was no IL-33 expression in human PBMCs. Therefore, we thought that the negative effects of soluble ST2 on the above inflammatory cytokines might depend on IL-33 levels and these inflammatory cytokines might be downstream of IL-33.
IL-33, the ligand of ST2, plays a very important role in RA. The relationship between IL-1β and IL-33 has been explored in several studies. The finding that IL-1β could increase IL-33 expression is consistent with a recent study. In the present study, we also demonstrated the role of soluble ST2 on cytokines, including IL-33, IL-1β, TNF-α, IL-6, and IL-8. After soluble ST2 treatment, the expressions of both IL-1β and IL-33 were downregulated in resting RASFs, but the expressions of TNF-α, IL-6, and IL-8 were not affected. However, in the RASFs activated by TNF-α plus IL-1β, the negative effects of soluble ST2 extended to TNF-α, IL-6, and IL-8. In addition, Hayakawa et al. have reported that ST2 binds to IL-33, not IL-1β. It indicated that the extended effects of soluble ST2 on TNF-α, IL-6, and IL-8 might be attributed to the increased production of IL-33, not IL-1β, by TNF-α and IL-1β. Moreover, the findings also suggested that IL-33 could regulate TNF-α, IL-1β, and IL-6, as well as IL-8.
In an in vivo study, Leung et al. have demonstrated that soluble ST2 could attenuate CIA. Furthermore, Matsuyama et al. have found that patients with high levels of IL-33 in serum and synovial fluid are nonresponsive to antitumor necrosis factor therapy because of persistent IL-1β signaling. Based on our findings above, soluble ST2 could suppress not only IL-33 but also IL-33-induced inflammatory cytokines, so we speculated that ST2 might be a promising therapy choice in RA patients who had higher serum IL-33 level.
In summary, elevated and disease activity-associated soluble ST2 showed a negative regulatory function on IL-33 and inhibited expressions of IL-33-induced IL-1β, TNF-α, IL-6, and IL-8 in RASFs, which suggested that soluble ST2 might serve as a potential therapeutic candidate for RA.
Financial support and sponsorship
This work was supported by grants from the National Natural Science Foundation of China (No. 81501396), Peking University International Hospital Research Funds (No. YN2016QN01, YN2016QN10, and YN2017QX01), and China Postdoctoral Science Foundation (No. 2016 M600874).
Conflicts of interest
There are no conflicts of interest.
1. McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis
Nat Rev Immunol. 2007;7:429–42 doi: 10.1038/nri2094
2. Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family Annu Rev Immunol. 2009;27:519–50 doi: 10.1146/annurev.immunol.021908.132612
3. Lee JH, Wang LC, Yu HH, Lin YT, Yang YH, Chiang BL, et al Type I IL-1 receptor (IL-1RI) as potential new therapeutic target for bronchial asthma Mediators Inflamm. 2010;2010:567351 doi: 10.1155/2010/
4. Palmer G, Talabot-Ayer D, Lamacchia C, Toy D, Seemayer CA, Viatte S, et al Inhibition of interleukin-33
signaling attenuates the severity of experimental arthritis Arthritis Rheum. 2009;60:738–49 doi: 10.1002/art.24305
5. Hu F, Shi L, Mu R, Zhu J, Li Y, Ma X, et al Hypoxia-inducible factor-1α and interleukin 33 form a regulatory circuit to perpetuate the inflammation in rheumatoid arthritis
PLoS One. 2013;8:e72650 doi: 10.1371/journal.pone.0072650
6. Mu R, Huang HQ, Li YH, Li C, Ye H, Li ZG, et al Elevated serum interleukin 33 is associated with autoantibody production in patients with rheumatoid arthritis
J Rheumatol. 2010;37:2006–13 doi: 10.3899/jrheum.100184
7. Takezako N, Hayakawa M, Hayakawa H, Aoki S, Yanagisawa K, Endo H, et al ST2 suppresses IL-6 production via the inhibition of ikappaB degradation induced by the LPS signal in THP-1 cells Biochem Biophys Res Commun. 2006;341:425–32 doi: 10.1016/j.bbrc.2005.12.206
8. Xu D, Chan WL, Leung BP, Huang Fp, Wheeler R, Piedrafita D, et al Selective expression of a stable cell surface molecule on type 2 but not type 1 helper T cells J Exp Med. 1998;187:787–94 doi: 10.1084/jem.187.5.787
9. Becerra A, Warke RV, de Bosch N, Rothman AL, Bosch I. Elevated levels of soluble ST2
protein in dengue virus infected patients Cytokine. 2008;41:114–20 doi: 10.1016/j.cyto.2007.11.001
10. Mok MY, Huang FP, Ip WK, Lo Y, Wong FY, Chan EY, et al Serum levels of IL-33 and soluble ST2
and their association with disease activity in systemic lupus erythematosus Rheumatology (Oxford). 2010;49:520–7 doi: 10.1093/rheumatology/kep402
11. Shen J, Shang Q, Wong CK, Li EK, Wang S, Li RJ, et al IL-33 and soluble ST2
levels as novel predictors for remission and progression of carotid plaque in early rheumatoid arthritis
: A prospective study Semin Arthritis Rheum. 2015;45:18–27 doi: 10.1016/j.semarthrit.2015.02.001
12. Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2
signaling in allergic airway inflammation J Biol Chem. 2007;282:26369–80 doi: 10.1074/jbc.M704916200
13. Talabot-Ayer D, McKee T, Gindre P, Bas S, Baeten DL, Gabay C, et al Distinct serum and synovial fluid interleukin (IL)-33 levels in rheumatoid arthritis
, psoriatic arthritis and osteoarthritis Joint Bone Spine. 2012;79:32–7 doi: 10.1016/j.jbspin.2011.02.011
14. Fraser A, Moore M, Jongbloed S, Gracie A, McInnes IB. Elevated soluble ST2
and cytokine levels in synovial fluids of patients with inflammatory synovitis Ann Rheum Dis. 2006;65(Suppl 1):A10
15. Ishikawa S, Shimizu M, Ueno K, Sugimoto N, Yachie A. Soluble ST2
as a marker of disease activity in systemic juvenile idiopathic arthritis Cytokine. 2013;62:272–7 doi: 10.1016/j.cyto.2013.03.007
16. Hong YS, Moon SJ, Joo YB, Jeon CH, Cho ML, Ju JH, et al Measurement of interleukin-33
(IL-33) and IL-33 receptors (sST2 and ST2L) in patients with rheumatoid arthritis
J Korean Med Sci. 2011;26:1132–9 doi: 10.3346/jkms.2011.26.9.1132
17. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines Immunity. 2005;23:479–90 doi: 10.1016/j.immuni.2005.09.015
18. Sanada S, Hakuno D, Higgins LJ, Schreiter ER, McKenzie AN, Lee RT, et al IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system J Clin Invest. 2007;117:1538–49 doi: 10.1172/JCI30634
19. Ohto-Ozaki H, Kuroiwa K, Mato N, Matsuyama Y, Hayakawa M, Tamemoto H, et al Characterization of ST2 transgenic mice with resistance to IL-33 Eur J Immunol. 2010;40:2632–42 doi: 10.1002/eji.200940291
20. Xu D, Jiang HR, Kewin P, Li Y, Mu R, Fraser AR, et al IL-33 exacerbates antigen-induced arthritis by activating mast cells Proc Natl Acad Sci U S A. 2008;105:10913–8 doi: 10.1073/pnas.0801898105
21. Matsuyama Y, Okazaki H, Hoshino M, Onishi S, Kamata Y, Nagatani K, et al Sustained elevation of interleukin-33
in sera and synovial fluids from patients with rheumatoid arthritis
non-responsive to anti-tumor necrosis factor: Possible association with persistent IL-1β signaling and a poor clinical response Rheumatol Int. 2012;32:1397–401 doi: 10.1007/s00296-011-1854-6
22. Leung BP, Xu D, Culshaw S, McInnes IB, Liew FY. A novel therapy of murine collagen-induced arthritis with soluble T1/ST2 J Immunol. 2004;173:145–50 doi: 10.4049/jimmunol.173.1.145
Edited by: Yi Cui
Keywords:© 2018 Chinese Medical Association
Inflammatory Cytokines; Interleukin-33; Rheumatoid Arthritis; Soluble ST2