Objectives: To evaluate serum levels of interleukin (IL)-6 in rheumatoid arthritis (RA) patients and to assess the correlations of this cytokine with clinical and laboratory parameters. Subjects and methods: 86 patients with RA and 30 healthy volunteers were enrolled in the study.
Journal of military phrmaco-medicine nO7-2017 THE CHANGES IN SERUM INTERLEUKIN-6 IN PATIENTS WITH RHEUMATOID ARTHRITIS Nguyen Huy Thong*; Doan Van De*; Nguyen Dang Dung** SUMMARY Objectives: To evaluate serum levels of interleukin (IL)-6 in rheumatoid arthritis (RA) patients and to assess the correlations of this cytokine with clinical and laboratory parameters Subjects and methods: 86 patients with RA and 30 healthy volunteers were enrolled in the study Disease activity was determined by disease activity score (DAS28) in patients with RA Patients with RA were categorized as low and moderate (DAS28 ≤ 5.1) and high (5.1 > DAS28) according to DAS28 The serum levels of IL-6 cytokine was measured by Fluorescence Covalent Microbead Immunosorbent Assay (FCMIA) Results: Serum IL-6 levels was significantly elevated in RA patients comparing with controls (p = 0.042) Serum IL-6 showed no significant correlations with mesurements of disease activity Conclusions: This study showed that patients with RA had a significantly increased cytokine level for IL-6, but high level of serum IL-6 cytokine was not associated with disease activity measurements However, further follow-up studies involving large samples are required to clarify precise role of this cytokine in development and progress disease * Keywords: Rheumatoid arthritis; IL-6; Disease activity INTRODUCTION Rheumatoid arthritis is a chronic inflammatory disease characterized by joint swelling, joint tenderness, and destruction of synovial joints, leading to severe disability and premature mortality [1] Cytokine networks, including IL-6, are critical for the initiation and perpetuation of both systemic and local inflammatory responses seen in chronic inflammatory arthritis [2] IL-6 may also be mediating many of the systematic manifestations of RA including inducing the acute-phase reaction [including C-reactive protein (CRP)], anaemia through hecipidin production, fatigue via the hypothalmic - pituitary adrenal (HPA) axis and osteoporosis from its effect on osteoclasts [3], thus it may influence on levels of disease activity in RA patients Several disease activity indices based on different clinical, laboratory, and physical measures have been introduced Most of these, including the Disease Activity Score (DAS), the modified DAS in 28 joints (DAS28), the Simplified Disease Activity Index (SDAI), the Clinical Disease Activity Index (CDAI), rely on either quantitative joint counts, patient-reported outcomes or both, and erythrocyte sedimentation rate (ESR) and serum CRP, those have some limitations and can be influenced by aging, sex and conditions other than RA (eg., osteoarthritis, fibromyalgia, anemia) [4, 5] ** 103 Hospital ** Vietnam Military Medical University Corresponding author: Nguyen Huy Thong (bsthong103@gmail.com) Date received: 10/07/2017 Date accepted: 08/08/2017 151 Journal of military parmaco-medicine n07-2017 The aim of this study was: To evaluate serum levels of IL-6 in RA patients and its role in assessing of disease activity SUBJECTS AND METHODS Subjects This study was conducted at Department of Rheumatology and Endocrinology of 103 Military Hospital between May, 2012 and June, 2015 Eighty six patients, 75 women and 11 men, with the diagnosis of RA fulfilled the ACR/EULAR 2010 RA classification criteria [1] Before entering study, 43 and patients were taking glucocorticoids and conventional synthetic disease-modifying antirheumatic drugs (DMARDs), respectively Patients with other concomitant rheumatic disease, severe infection, chronic autoimmune disease, and/or taking bio-DMARDs, which may affect laboratory and cytokine profile were excluded from the study - Healthy subject population: thirty sex-matched healthy controls (age mean 41.60 ± 4.57; range 35 - 50 years, 26 women and men) were included in the study Methods * Clinical assessment: Disease activity was assessed by the 28-joint disease activity score C-reactive protein (DAS28 CRP) [6] in RA patients Based on the DAS28 CRP, the patients were subdivided into subgroups: low and moderate group (DAS28 ≤ 5.1), and high group (DAS28 > 5.1) Patient global assessment of disease activity and provider global assessment of disease 152 activity were evaluated using a 10 cm horizontal visual analog scale (VAS) We also calculated SDAI (Simplified Disease Activity Index) and CDAI (Clinical Disease Activity Index) Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were recorded * Laboratory analysis: Blood samples of patients and controls were collected and put in a sterile plain tube and stored frozen at -80oC until analysis We used commercially available human fluorescence covalent microbead immunosorbent assay (FCMIA) kits for IL-6, IL-17 and TNF-α (R&D systems MN, USA) The procedure for the FCMIA method was performed according to the instructions provided by the manufacturer The levels of cytokines were recorded as a pg/mL * Statistical analysis: All statistical analyses were performed using the statistical package for the social sciences (SPSS), version 18.0, for Windows (SPSS, Chicago, IL, USA) Continuos variables are presented as the mean ± standard deviation or median The normality of the distribution for all variables was assessed by the Kolmogorov-Smirnov test Intergroup comparisons were made using the student’s t-test for normally distributed variables and Mann - Whitney U test for non-parametric variables To assess the correlations between variables, Sperman’s rank or Pearson’s correlation analysis were used according to data distribution Values of p < 0.05 were considered statistically significant Journal of military phrmaco-medicine nO7-2017 RESULTS Patients and demographic, clinical characteristics Table 1: Demographic and clinical characteristics of RA patients and control Mean age ± SD, - max (years) Sex, n (female/male) Mean disease duration ± SD (years) RA group (n = 86) Control group (n = 30) 53.44 ± 7.30; 35 - 66 41.60 ± 4.57; 35 - 50 75/11 26/4 4.29 ± 5.34 Mean tender joint count ± SD (range - 28) 14.13 ± 9.08; 13.00 Mean swollen joint count ± SD (range - 28) 10.52 ± 7.38; 9.0 Mean morning stiffness ± SD (minutes) 37.25 ± 33.82; 30.00 Mean patient global assessment of disease activity ± SD (cm) 7.16 ± 2.25 Mean provider global assessment of disease activity ± SD (cm) 5.65 ± 1.92 Mean ESR ± SD (mm/h) 79.68 ± 44.37; 85.50 7.66 ± 3.86 Mean plasma CRP ± SD (mg/L) 68.37 ± 47.24, 65.10 0.52 ± 0.36 Mean, DAS28 CRP DAS28 CRP Pre-study treatment 6.19 ± 1.36; 2.81 - 8.50 Low and moderate (n; %) 17; 20.5 High (n, %) 66 (79.5) Glucorticoids (n, %) 43 (50.6) DMARDs (n, %) (4.7) (DAS28 (CRP) is missing in three patients Abbreviations: anti-CCP: anti-cyclic citrulinated peptide; CRP: C-reative protein; DAS28: Disease Activity Score; ESR: Erythrocyte Sedimentation Rate) Patients and controls did not significantly differ in sex The mean age of controls was lower than that of RA patients The mean disease duration in RA patients was 4.29 ± 5.34 years The mean DAS28 CRP was 6.19 ± 1.36 (range 2.81 - 8.50) Seventeen (20.5%) and sixty six (79.5%) patients had low-moderate and high DAS28 CRP, respectively 153 Journal of military parmaco-medicine n07-2017 Comparison of laboratory parameters among patients and healthy subjects Figure 1: The comparision of serum interleukin Figure 2: The correlation of serum interleukin (IL)-6 levels between RA patients and controls (IL)-6 levels and serum tumor necrosis factor (p, test Mann - Whitney) (TNF)-α levels in rheumatoid arthritis patients (numbers are Spearman correlation coefficients) The mean and median of serum IL-6 of RA patients and controls was 19.06 ± 22.94; 10.49 and 9.19 ± 8.43; 7.18 (pg/mL), respectively Median of serum IL-6 concentrations in RA patients was significantly higher than that in controls group (p = 0.042) Serum IL-6 had a positive correlation with serum TNF-α in RA patients (r = 0.233, p = 0.035) Correlation between serum IL-6 and clinical, laboratory variables in RA patients group Table 2: The comparison of serum IL-6 based on measurements of disease activity Plasma IL-6 levels (pg/ml) Joint tender count 28 Mean ± SD Median - (n = 13) 26.20 ± 36.12 11.16 ≥ (n = 68) 17.57 ± 19.72 10.18 - (n = 20) 22.07 ± 33.49 5.74 ≥ (n = 61) 17.93 ± 18.70 12.14 Low and moderate (n = 14) 22.07 ± 34.46 7.23 High (n = 65) 16.27 ± 16.53 10.60 p 0.974 Joint swollen count 28 0.332 DAS28 CRP 0.581 (Abbreviations: DAS28 CRP: Disease Activity Score C-Reactive Protein) 154 Journal of military phrmaco-medicine nO7-2017 Table 3: The correlation of serum IL-6 levels in RA patients with measurements of disease activity IL-6 TJC28 SJC28 MS PtGA PGA CRP ESR r 0.035 0.033 -0.050 0.120 0.026 -0.028 -0.001 p 0.758 0.769 0.663 0.289 0.818 0.802 0.991 (Abbreviations: TJC: Tender joint count; SJC: Swollen joint count; MS: Morning stiffness; PtGA: Patient global assessment of disease activity’ PGA: Provider global assessement of disease activity; r: Spearman’s correlation coefficient) There were no differences according to joint tender count 28, joint swollen count 28 and DAS28 CRP Table 4: The correlation of serum IL-6 levels with composite indices in RA patients IL-6 DAS28 CRP DAS28 ESR SDAI CDAI r 0.101 0.107 0.063 0.055 p 0.374 0.392 0.581 0.627 (Abbreviations: DAS28 CRP: Disease Activity Score C-reactive protein; DAS28 ESR: Disease Activity Score erythrocyte sedimentation rate; SDA:, Simplified disease activity index; CDAI: Clinical disease activity index; r: Spearman’s correlation coefficient) There were no associations between the serum IL-6 levels of RA patients with measurements of disease activity DISCUSSION In the present study, we evaluated serum levels of IL-6 cytokine in patients with RA, and its associations with clinical and laboratory parameters IL-6 is a pleiotropic cytokine with diverse activities IL-6 plays an important role in inflammation, bone metabolic, haematopoiesis, immune regulation [7] These activities contribute to both systemic and local symptoms associated with RA [2] IL-6 is involved in pathology of chronic inflammation of synovium, joint damage as well as systemic symptoms such as anemia [8], fatigue [9], osteoporosis [9] In accordance with other authors [10, 11, 12], we found that serum IL-6 was significantly increased in RA patients compared to healthy subjects (figure 1) In the current study, serum IL-6 had a significantly positive correlation with serum TNF-α (figure 2) In consistent of our observation, Manicourt D.H et al (1993) also reported that serum IL-6 had a positive correlation with serum TNF-α (r = 0.487, p = 0.007) These studies supports the concept that TNF-α played a key role in pathogenesis of RA by stimulating pro-inflammation cytokines including IL-6 155 Journal of military parmaco-medicine n07-2017 IL-6 is a pleiotropic cytokine and contributes to both systemic and local symptoms associated with RA [2], so it may influence the disease activity of RA patients We assessed the change of serum IL-6 according to measurements of disease activity to value serum IL-6 in assessing levels of disease activities in RA patients In the present study, serum IL-6 median of low and moderate disease group lower than high group but it was not significant (p = 0.581) (table 2) Ibrahim Tekeog˘lu et al (2016) found a significant difference in serum IL-6 mean between high disease activity group and low group (p = 0.046), however there was no difference between patients had a moderate and low disease activity In the present study we also did not observe the correlation between serum IL-6 with measurements of disease activity such as joint tender count 28, joint swollen count 28, morning stiffness, PtGA, PGA, ESR, plasma CRP levels as well as composite index DAS28 CRP, DAS28 ESR, SDAI and CDAI In consistent of our observation, Soo-Jin Chung et al (2011) found that serum IL-6 was not associated with DAS28 ESR [11] However, contrary to our results, other studies found serum IL-6 had a positive association with DAS28 CRP and DAS28 ESR Prado et al found a positive correlation between IL-6 levels and TJC28 (r = 0.39; p < 0.01) [12] Thus, there are many controversial studies regarding the relationship between serum IL-6 as an assessing role of disease activity and measurements of disease activity in RA patients, so we need more studies with larger sample size to discover this interesting correlation 156 Our study has some limitations The sample size of patients was relatively small, and the patients were on drug treatment including glucorticoids DMARDs In fact, our study had a cross-sectional design, and cytokines profile had wide range CONCLUSION Our study demonstrated a significant higher increase of serum IL-6 in RA patients compared with healthy controls However, we did not find any associations between serum IL-6 levels and measurements of disease activity in RA patients REFERENCES Võ Tam, Phan Thị Thu Tâm Nghiên cứu nồng độ IL-6 huyết bệnh nhân viêm khớp dạng thấp Chương trình báo cáo Hội nghị Khoa học Công nghệ Tuổi trẻ Trường Đại học Huế lần thứ XVI 2016, tr.754-758 Aletaha D, T Neogi, A.J Silman et al 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative Arthritis Rheum 2010, 62 (9), pp.2569-2581 Cronstein B.N IL-6: a key mediator of systemic and local symptoms in rheumatoid arthritis Bull NYU Hosp Jt Dis 2007, 65, Suppl 1: pp.S11-5 Srirangan S, E.H Choy The role of IL-6 in the pathophysiology of rheumatoid arthritis Ther Adv Musculoskelet Dis 2010, (5), pp.247-256 Gabay C.I Kushner Acute-phase proteins and other systemic responses to inflammation N Engl J Med 1999, 340 (6), pp 448-454 Journal of military phrmaco-medicine nO7-2017 Pollard L.C, G.H Kingsley, E.H Choy et al Fibromyalgic rheumatoid arthritis and disease assessment Rheumatology (Oxford) 2010, 49 (5), pp.924-928 Prevoo M.L, M.A van 't Hof, H.H Kuper et al Modified disease activity scores that include twenty-eight-joint counts Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis Arthritis Rheum 1995, 38 (1), pp.44-48 Kishimoto T IIL-6: from basic science to medicine: 40 years in immunology Annu Rev Immunol 2005, 23, pp.1-21 Hashizume M, M Mihara The roles of iIL-6 in the pathogenesis of rheumatoid arthritis Arthritis 2011, p.765624 10 Gaffen S.L The role of IL-17 in the pathogenesis of rheumatoid arthritis Curr Rheumatol Rep 2009, 11 (5), pp.365-370 11 Chung S.J, Y.J Kwon, M.C Park et al The correlation between increased serum concentrations of IL-6 family cytokines and disease activity in rheumatoid arthritis patients Yonsei Med J 2011, 52 (1), pp.113-120 12 Prado A.D, M.C Bisi, D.M Piovesan et al Ultrasound power Doppler synovitis is associated with plasma IL-6 in established rheumatoid arthritis Cytokine Ultrasound power Doppler synovitis is associated with plasma IL-6 in established rheumatoid arthritis 2016, pp.27-32 157 ... associations between the serum IL-6 levels of RA patients with measurements of disease activity DISCUSSION In the present study, we evaluated serum levels of IL-6 cytokine in patients with RA, and its... healthy subjects Figure 1: The comparision of serum interleukin Figure 2: The correlation of serum interleukin (IL)-6 levels between RA patients and controls (IL)-6 levels and serum tumor necrosis... coefficient) There were no differences according to joint tender count 28, joint swollen count 28 and DAS28 CRP Table 4: The correlation of serum IL-6 levels with composite indices in RA patients