Elevated IL-6 Receptor Expression on CD4+ T Cells contributes to the increased Th17 Responses in patients with Chronic Hepatitis B
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 15 February 2011
Accepted: 3 June 2011
Published: 3 June 2011
Increased numbers of Interleukin-17-producing CD4+ T cells (Th17) have been found in association with hepatitis B virus (HBV)-induced liver injury. However, the mechanism underlying the increase of Th17 responses in patients with HBV infection remains unclear. In this study, we investigate the possible regulatory mechanisms of increased Th17 responses in patients with chronic hepatitis B(CHB).
Th17 response and IL-6R expression on CD4+ T cells in peripheral blood samples were determined by flow cytometry. Cytokines TGF-β, IL-1β, IL-6 and IL-17 in plasma and/or supernatant samples were determined by ELISA and the IL-17 and IL-6R mRNA levels were quantified by quantitative real-time reverse polymerase chain reaction.
All these data indicated that the frequency of periphery Th17 cells is significantly correlated with the percentage of CD4 + T cells expressing IL-6R in CHB patients. CD4+ T cells from patients with CHB, but not those from healthy donors, produced higher levels of IL-17 and had more IL-6R expression upon stimulation with the HBV core antigen (HBcAg) in vitro. The PMA/ionomycin and HBcAg -stimulated up-regulation of IL-17 production by CD4+ T cells could be reversed by a neutralizing antibody against IL-6R.
we showed that enhancement of IL-6R expression on CD4+ T cells upon HBV infection contributes to increased Th17 response in patients with CHB.
Hepatitis B virus (HBV) infection is a major public health problem worldwide, especially in China, where nearly 7.18% of the population is persistently infected with HBV and most of them develop chronic hepatitis B [1, 2]. Previous studies have revealed that cellular immunity is critical for the outcome of HBV infection . While HBV-specific CD4 + T cells and CD8+ T cells have been demonstrated to be essential for the control of HBV infection[4–7], antigen non-specific T cells infiltrating the liver are reported to be involved in the live injury[8, 9].
Th17 cells are a new lineage of peripheral CD4 + T cells which have been identified as a proinflammatory T cell subset [10, 11]. In human, IL-6 in combination with TGF-β and IL-1β drive naive CD4 + T cell to differentiate into Th17 cells [10, 12–14]. Th17 cells can produce multiple cytokines including IL-17A (also known as IL-17), IL-17F, IL-21 and IL-22[12, 15]. IL-17, a major effector cytokine of Th17 cells, is involved in mobilizing, recruiting, and activating neutrophils and leads to massive tissue inflammation. Recent studies suggested that Th17 cells also play a central role in the immune-mediated liver injury [17–20]. In particular, Zhang et al reported that antigen non-specific Th17 response was increased in patients with chronic hepatitis B (CHB) and the peripheral Th17 frequency in CHB patients was closely associated with the degree of liver damage which determined by serum alanine amino-transferase (ALT) levels and liver histological activity index(HAI) scores. Consistently, Ge et al demonstrated that the frequency of Th17 cells was positively correlated with serum ALT levels in CHB patients . However, the regulatory mechanism of Th17 responses in patients with HBV infection remains unclear.
In this study, we have found that the increased Th17 response in patients with CHB is correlated with the enhanced IL-6 receptor (IL-6R) expression on CD4+ T cells. In addition, our results indicated that up-regulation of IL-6R expression on CD4+ T cells is important for increased Th17 responses in patients with CHB. These findings suggest that IL-6R can be a novel target for immunotherapy of hepatitis induced by HBV infection.
Materials and methods
Clinical Characteristics of the Populations Enrolled in the Study
24.5 ± 7.5
29.3 ± 9.2
30.6 ± 7.7
25.5 ± 4.0
21.9 ± 8.4
264.2 ± 171.2
1169.8 ± 617.8
13.7 ± 4.2
27.3 ± 19.3
214.7 ± 116.6
3.8 ± 3.6
6.0 ± 1.4
5.1 ± 1.4
Intracellular staining and flow cytometry analysis
Fluorescence-conjugated antibodies against CD3, CD4, CD8, IFN-γ, IL-17, CD126 (IL-6R) were purchased from BD Biosciences (San Jose, CA). For intracellular staining, fresh heparinized peripheral blood (250 μL) was incubated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich, St. Louis, MO), ionomycin (1 μg/ml; Sigma-Aldrich), and BFA (0.4 μM, BD PharMingen) in 750 μL RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) for 6 hours, 37°C. Cells were stained for cell surface markers first, and then the blood was lysed with fluorescence-activated cell sorting (FACS) lysing solution (BD PharMingen) and further permeabilized, stained with the corresponding intracellular antibody. Flow cytometry analysis was performed on cells acquired using a FACSCalibur (BD Biosciences, San Jose, CA) and data were analyzed using FACSDiva (BD Biosciences)or FlowJo software (Tristar, San Carlos, CA).
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Total CD4+ T cell were purified by positive selection using microbeads according to the manufacturer's instructions (BD Biosciences). The purity of the CD4+ T cell was > 90%. Freshly purified cells were incubated in complete RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.
To analyze the effect of HBcAg on IL-6R and IL-17 expression by CD4+ T cells, PBMCs were cultured in the absence or presence of HBcAg (10 μg/mL; ARP, Belmont, MA), or LPS(1 μg/ml; Peprotech, Rocky Hill, NJ). At different time points after incubation as indicated in legends, cells were harvested and CD4+ T cells were isolated for quantification of IL-17/IL-6R messenger RNA (mRNA) expression. Aliquot of cultured cells were also subjected flow cytometry analysis of IL-6R expression on CD4+ T cells. Supernatant were collected for cytokines measurement.
IL-6R blockade treatment
Purified CD4+ T cells from CHB were incubated in medium alone or stimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) for 6 h in the presence of 5 μg/ml of brefeldin A(0.4 μM, BD PharMingen) or with HBcAg (10 μg/mL, ARP) for 24 h, neutralizing antibody against IL-6R(R&D systems, Minneapolis, MN) were added at a concentration of 20 μg/ml 30 min before the addition of PMA or HBcAg. Cells were harvested for intracellular cytokine staining and analyzed by flow cytometry. Cell culture supernatants were harvested and the concentration of IL-17, IFN-γ was determined by ELISA.
RNA extraction and real-time RT-PCR
Total RNA was extracted from purified CD4+ T cells with RNeasy Mini Kit (Qiagen, Santa Clarita, CA) according to the manufacturer's protocol. The RNA was reverse-transcribed to complementary DNA (cDNA) using oligo (dT) primers at 37°C for 15 minutes and at 85°C for 5 seconds. Quantitative real-time PCR was performed using Applied Biosystems 7500 sequence detection system. The primers and probes of IL-17(cat:Hs00936345) and inner control GAPDH(cat:Hs02758991) for real-time PCR were purchased from Applied Biosystems (Foster City, CA). IL-6R primer and probe(Takara, China) sequences were as follows:
forward primer: 5-AAGACAATG CCACTGTTCACTG-3;
reverse primer: 5-GGTAGCATGAATAGTTTCCAGAGTC-3;
Results are expressed in terms of relative mRNA quantification calculated by using the arithmetic formula 2-ΔCtΔCt.
The concentrations of IL-17, TGF-β, IL-6, and IL-1β in culture supernatants and TGF-β, IL-6, IL-1β in plasma were determined by ELISA (R&D Systems, Minneapolis, MN) following manufacturer's guidelines.
All statistical tests were performed with SPSS 13.0(Chicago, IL) or Prism 3.0 (GraphPad, La Jolla, CA). The one-way analysis of variance/Newman-Keuls multiple comparison test was used for statistical analysis to compare the differences among multiple groups. The unpaired t test was used to analyze the difference between two groups. The Wilcoxon matched pair t test was used to analyze the effect of antigens on IL-6R and IL-17 expression by CD4+ T cells in vitro. Correlation analysis was performed by Pearson's t test. For all test, two-sided P < 0.05 was considered statistically significant.
Increased Th17 responses were associated with liver injury in patients with HBV infection
Cytokines related to Th17 differentiation were increased in HBV infected subjects
Elevated IL-6R expression on CD4 + T cells correlated with an increased Th17 response in HBV infected individuals
HBcAg enhanced IL-17 production and IL-6R expression by CD4 + T cells from patients with chronic hepatitis B in vitro
To determine whether in vivo chronic exposure to HBV infection influences the capability of CD4+ T cells to express IL-6R and IL-17 in response to HBcAg stimulation in vitro, we stimulated CD4+ T cells from CHB patients with HBcAg and compared their responsiveness with those from HD individuals. We found that, upon HBcAg stimulation, IL-6R expression on CD4+ T cells from CHB patients was significantly increased. In contrast, IL-6R expression on CD4+ T cells from HD was decreased upon HBcAg stimulation (Figure 4E). Consistently, we found that PBMCs from CHB patients up-regulated IL-17 production upon HBcAg stimulation to a higher percentage than those from HD (P < 0.001, Figure 4F).
Taken together, these data suggested that modulation of IL-6R expression on CD4+ T cells is an important mechanism underlies the enhanced Th17 responses in patients with chronic hepatitis B.
An anti-IL-6R antibody inhibited PMA/ionomycin and HBcAg induced IL-17 production by CD4 + T cells
HBcAg and LPS Induced Th17 differentiation related cytokines production by PBMCs in vitro
Recent studies have shown that Th17 responses were significantly increased in patients with CHB. Correlation analysis also suggested that Th17 cells play an important role in inflammatory response and cell mediated liver injury of CHB patients [17–20]. In this study, we extended these observations by finding that Th17 responses were even significantly higher in AHB patients than CHB patients. In contrast, Th17 responses were not different between asymptomatic HBV carriers and healthy donors. Taken together, these data provided substantial evidences that Th17 cells contribute to inflammatory responses and cell-mediated liver injuries in individuals with HBV infection. Accordingly, understanding the regulatory mechanism of Th17 responses in HBV infected individuals is of particular importance, considering it may provide novel strategies for the treatment of patients with hepatitis B.
In attempt to investigate the mechanism underlying the increased Th17 response in patients with CHB, we first looked at the cytokine milieu related to the Th17 differentiation and found that TGF-β, IL-6 and IL-1β were increased in plasma of HBV infected individuals, irrespective their clinical manifestation. Consistent with these findings, in vitro experiments showed that PBMCs produced TGF-β, IL-1β, and IL-6 upon stimulation with HBcAg. However, correlation analysis indicated that only the concentration of plasma TGF-β, but not IL-6 nor IL-1β, significantly correlated with increased Th17 responses in HBV infected patients. Thus, unlike the inhibitory role TGF-β plays during the HCV-specific Th17 response , TGF-β may facilitate the development of Th17 responses in HBV infected subjects. Nevertheless, because TGF-β is also involved in CD4+ CD25+ regulatory T cells (Treg) differentiation and our previous study have found that the concentration of plasma TGF-β in CHB patients also correlated with the frequency of Treg , the exact role of TGF-β in regulating Th17 response as well as balancing between Th17 and Treg in HBV infected individuals remains to be elucidated.
Consistent with our previous finding in patients with tuberculosis, we found IL-6R expression on CD4+ T cells, but not plasma IL-6, correlated with the Th17 response in HBV infected individuals . However, unlike Mycobacterium tuberculosis antigens which down-regulated IL-6R expression on CD4 + T cells from patients with tuberculosis, HBV antigen (HBcAg) up-regulated IL-6R expression on CD4+ T cells from patients with CHB. More importantly, our in vitro data validated that blockade of IL-6R signaling on CD4+ T cells significantly inhibited antigen non-specific (PMA/Ionomycin-stimulated) and HBcAg antigen-specific IL-17, but not IFN-γ, production by CD4+ T cells, which was not surprised considering the fact that no requirement of IL-6 for Th1 differentiation. Thus, while both infections modulate IL-6R expression on CD4+ T cells, the effect of Mycobacterium tuberculosis infection was markedly different from that of HBV infection. In addition, the difference was likely due to in vivo "tuning" of CD4+ T cells by chronic exposure to inflammatory environment superimposed by HBV infection (including circulating HBV antigens), since HBcAg, similar to Mycobacterium tuberculosis antigens, down-regulated IL-6R expression on CD4+ T cells in healthy donors. While further investigations are warranted to identify the exact mechanisms that account for "tuning" CD4+ T cells in vivo, circulating HBV antigens as well as the cytokines induced by HBV infection might be most potential candidates that are responsible for such different effect. For example, elevated TGF-β in patients with hepatitis B might "tune" CD4+ T cells with increased sensitivity to IL-6R signaling through inhibition of SOCS3[30, 31].
In summary, our present study provided substantial evidences that enhancement of IL-6R expression on CD4+ T cells by HBV is an important mechanism for increased Th17 responses in patients with CHB. First, the percentage of IL-6R expression on CD4+ T cells correlated with the frequency of Th17 cells in peripheral blood in vivo. Second, HBcAg up-regulated IL-6R and IL-17 expression by CD4+ T cells from CHB patients in vitro. Third, the blockade of IL-6R signaling reversed the increase of IL-17 production by CD4+ T cells. Combined with previous reports about the usefulness of neutralizing anti-IL-6R monoclonal antibody to treat autoimmune diseases via inhibiting Th17 responses[32–34], our finding suggests a novel strategy for treating CHB patients, e.g. inhibiting IL-6R expression by anti-IL-6R antibodies to restrain Th17-mediated liver damage.
List of Abbreviations
Interleukin-17-producing CD4+ T cell
hepatitis B virus
chronic hepatitis B
histological activity index
Hepatitis B core antigen
Hepatitis B surface antigen
human immunodeficiency virus
asymptomatic HBV carrier
acute hepatitis B
Peripheral blood mononuclear cells
This study was funded in full by Natural Science Foundation of China, grant number: 30872238. We declare that we have no conflict of interest to disclose. We would like to thank Cheng Xu, Song Wang, Weilong Liu, Qianting Yang, Lantian Wang from Shenzhen Third People's Hospital for collection of specimens and technical assistance.
- China MOHotPsRo: Sero-epidemiological Study on Hepa titis B Virus Infection in China. Beijing, the Minister Of Health 2008.Google Scholar
- Rehermann B, Nascimbeni M: Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 2005, 5: 215-229. 10.1038/nri1573View ArticlePubMedGoogle Scholar
- Ganem D, Prince AM: Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med 2004, 350: 1118-1129. 10.1056/NEJMra031087View ArticlePubMedGoogle Scholar
- Boni C, Fisicaro P, Valdatta C, Amadei B, Di Vincenzo P, Giuberti T, Laccabue D, Zerbini A, Cavalli A, Missale G, Bertoletti A, Ferrari C: Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol 2007, 81: 4215-4225. 10.1128/JVI.02844-06PubMed CentralView ArticlePubMedGoogle Scholar
- Sobao Y, Tomiyama H, Sugi K, Tokunaga M, Ueno T, Saito S, Fujiyama S, Morimoto M, Tanaka K, Takiguchi M: The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication. J Hepatol 2002, 36: 105-115.View ArticlePubMedGoogle Scholar
- Maini MK, Boni C, Ogg GS, King AS, Reignat S, Lee CK, Larrubia JR, Webster GJ, McMichael AJ, Ferrari C, Williams R, Vergani D, Bertoletti A: Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology 1999, 117: 1386-1396. 10.1016/S0016-5085(99)70289-1View ArticlePubMedGoogle Scholar
- Jung MC, Spengler U, Schraut W, Hoffmann R, Zachoval R, Eisenburg J, Eichenlaub D, Riethmüller G, Paumgartner G, Ziegler-Heitbrock HW: Hepatitis B virus antigen-specific T-cell activation in patients with acute and chronic hepatitis B. J Hepatol 1991, 13: 310-317.View ArticlePubMedGoogle Scholar
- Wang FS: Clinical immune characterization of hepatitis B virus infection and implications for immune intervention: Progress and challenges. Hepatol Res 2007,37(Suppl 3):S339-346.View ArticlePubMedGoogle Scholar
- Rehermann B: Chronic infections with hepatotropic viruses: mechanisms of impairment of cellular immune responses. Semin Liver Dis 2007, 27: 152-160. 10.1055/s-2007-979468View ArticlePubMedGoogle Scholar
- Bettelli E, Oukka M, Kuchroo VK: T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol 2007, 8: 345-350.View ArticlePubMedGoogle Scholar
- Harrington LE, Mangan PR, Weaver CT: Expanding the effector CD4 T-cell repertoire: the Th17 lineage. Curr Opin Immunol 2006, 18: 349-356. 10.1016/j.coi.2006.03.017View ArticlePubMedGoogle Scholar
- Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, Basham B, Smith K, Chen T, Morel F, Lecron JC, Kastelein RA, Cua DJ, McClanahan TK, Bowman EP, de Waal Malefyt R: Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 2007, 8: 950-957.View ArticlePubMedGoogle Scholar
- Zhou L, Littman DR: Transcriptional regulation of Th17 cell differentiation. Semin Immunol 2007, 19: 409-417. 10.1016/j.smim.2007.10.011PubMed CentralView ArticlePubMedGoogle Scholar
- Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F: Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007, 8: 942-949.View ArticlePubMedGoogle Scholar
- Wei L, Laurence A, Elias KM, O'Shea JJ: IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. J Biol Chem 2007, 282: 34605-34610. 10.1074/jbc.M705100200PubMed CentralView ArticlePubMedGoogle Scholar
- O'Quinn DB, Palmer MT, Lee YK, Weaver CT: Emergence of the Th17 pathway and its role in host defense. Adv Immunol 2008, 99: 115-163.View ArticlePubMedGoogle Scholar
- Zhang JY, Zhang Z, Lin F, Zou ZS, Xu RN, Jin L, Fu JL, Shi F, Shi M, Wang HF, Wang FS: Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology 2010, 51: 81-91.View ArticlePubMedGoogle Scholar
- Ye Y, Xie X, Yu J, Zhou L, Xie H, Jiang G, Yu X, Zhang W, Wu J, Zheng S: Involvement of Th17 and Th1 Effector Responses in Patients with Hepatitis B. J Clin Immunol 2010, 30: 546-555. 10.1007/s10875-010-9416-3View ArticlePubMedGoogle Scholar
- Wu W, Li J, Chen F, Zhu H, Peng G, Chen Z: Circulating Th17 cells frequency is associated with the disease progression in HBV infected patients. J Gastroenterol Hepatol 2010, 25: 750-757. 10.1111/j.1440-1746.2009.06154.xView ArticlePubMedGoogle Scholar
- Ge J, Wang K, Meng QH, Qi ZX, Meng FL, Fan YC: Implication of Th17 and Th1 cells in patients with chronic active hepatitis B. J Clin Immunol 2010, 30: 60-67. 10.1007/s10875-009-9328-2View ArticlePubMedGoogle Scholar
- Yan Z, Tan W, Zhao W, Dan Y, Wang X, Mao Q, Wang Y, Deng G: Regulatory polymorphisms in the IL-10 gene promoter and HBV-related acute liver failure in the Chinese population. Journal of Viral Hepatitis 2009, 16: 775-783. 10.1111/j.1365-2893.2009.01139.xView ArticlePubMedGoogle Scholar
- Vassilopoulos D, Rapti I, Nikolaou M, Hadziyannis E, Hadziyannis SJ: Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B. J Viral Hepat 2008, 15: 817-826.PubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25: 402-408. 10.1006/meth.2001.1262View ArticlePubMedGoogle Scholar
- Keller P, Penkowa M, Keller C, Steensberg A, Fischer CP, Giralt M, Hidalgo J, Pedersen BK: Interleukin-6 receptor expression in contracting human skeletal muscle: regulating role of IL-6. FASEB J 2005, 19: 1181-1183.PubMedGoogle Scholar
- Rose-John S: Coordination of interleukin-6 biology by membrane bound and soluble receptors. Adv Exp Med Biol 2001, 495: 145-151.View ArticlePubMedGoogle Scholar
- Taga T: IL6 signalling through IL6 receptor and receptor-associated signal transducer, gp130. Res Immunol 1992, 143: 737-739. 10.1016/0923-2494(92)80013-BView ArticlePubMedGoogle Scholar
- Rowan AG, Fletcher JM, Ryan EJ, Moran B, Hegarty JE, O'Farrelly C, Mills KH: Hepatitis C virus-specific Th17 cells are suppressed by virus-induced TGF-beta. J Immunol 2008, 181: 4485-4494.View ArticlePubMedGoogle Scholar
- Yang G, Liu A, Xie Q, Guo TB, Wan B, Zhou B, Zhang JZ: Association of CD4+CD25+Foxp3+ regulatory T cells with chronic activity and viral clearance in patients with hepatitis B. Int Immunol 2007, 19: 133-140.View ArticlePubMedGoogle Scholar
- Chen X, Zhang M, Liao M, Graner MW, Wu C, Yang Q, Liu H, Zhou B: Reduced Th17 response in patients with tuberculosis correlates with IL-6R expression on CD4+ T Cells. Am J Respir Crit Care Med 2010, 181: 734-742. 10.1164/rccm.200909-1463OCView ArticlePubMedGoogle Scholar
- Morishima N, Mizoguchi I, Takeda K, Mizuguchi J, Yoshimoto T: TGF-beta is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23. Biochem Biophys Res Commun 2009, 386: 105-110. 10.1016/j.bbrc.2009.05.140View ArticlePubMedGoogle Scholar
- Zhao S, Gu Y, Dong Q, Fan R, Wang Y: Altered interleukin-6 receptor, IL-6R and gp130, production and expression and decreased SOCS-3 expression in placentas from women with pre-eclampsia. Placenta 2008, 29: 1024-1028. 10.1016/j.placenta.2008.09.011PubMed CentralView ArticlePubMedGoogle Scholar
- Smolen JS, Beaulieu A, Rubbert-Roth A, Ramos-Remus C, Rovensky J, Alecock E, Woodworth T, Alten R: Effect of interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid arthritis (OPTION study): a double-blind, placebo-controlled, randomised trial. Lancet 2008, 371: 987-997. 10.1016/S0140-6736(08)60453-5View ArticlePubMedGoogle Scholar
- Maini RN, Taylor PC, Szechinski J, Pavelka K, Bröll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson D, Kishimoto T: Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate. Arthritis Rheum 2006, 54: 2817-2829. 10.1002/art.22033View ArticlePubMedGoogle Scholar
- Nishimoto N, Yoshizaki K, Miyasaka N, Yamamoto K, Kawai S, Takeuchi T, Hashimoto J, Azuma J, Kishimoto T: Treatment of rheumatoid arthritis with humanized anti-interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum 2004, 50: 1761-1769. 10.1002/art.20303View ArticlePubMedGoogle Scholar
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