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Objectives: To evaluate serum levels of tumor necrosis factor (TNF)-α in rheumatoid arthritis (RA) patients and to assess the correlations of this cytokine with clinical and laboratory parameters.
Journal of military pharmaco-medicine n08-2017 THE CHANGES IN SERUM TUMOR NECROSIS FACTOR ALPHA IN PATIENTS WITH RHEUMATOID ARTHRITIS Nguyen Huy Thong*; Doan Van De*; Nguyen Dang Dung** SUMMARY Objectives: To evaluate serum levels of tumor necrosis factor (TNF)-α 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 The serum levels of TNF-α cytokine was measured by fluorescence covalent microbead immunosorbent assay (FCMIA) Results: Serum TNF-α levels was significantly decreased in RA patients comparing with controls (p < 0.001) Serum TNF-α showed no significant correlations with mesurements of disease activity Conclusions: Patients with RA had a significantly lower TNF-α cytokine than that of healthy controls, and serum TNF-α cytokine was not associated with disease activity mesurements However, further follow-up studies involving larger samples are required to clarify precise role of this cytokine in development and progress of disease * Keywords: Rheumatoid arthritis; TNF-α; Biomarkers INTRODUCTION Rheumatoid arthritis (RA) 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 play a fundamental role in the processes that cause inflammation, articular destruction of RA [2] TNF-α is a key cytokine in the pathogenesis of RA that involved in chronic synovial inflammation and articular destruction TNF-α induces the production of other proinflammatory cytokines, including IL-1 and IL-6 It also induces the production and release of chemokines, hepcidine, acute phase response as well as endothelial cell activation, angiogenesis, activation of chondrocyte of metalloproteinase production, osteoclast activation [2], thus it may be related to disease activity of RA 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 influeced by aging, sex and conditions other than RA (eg., osteoarthritis, fibromyalgia, anemia) [3, 4] * 103 Military Hospital ** Vietnam Military Medical University Corresponding author: Nguyen Huy Thong (bsthong103(@gmail.com) Date received: 10/07/2017 Date accepted: 26/09/2017 180 Journal of military pharmaco-medicine n08-2017 The aim of this study was: To evaluate serum levels of TNF-α in RA patients and its role in assessing disease activity SUBJECTS AND METHODS Subjects * Patients: This study was carried out 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 concomitant other rheumatic disease, severe infection, chronic autoimmune disease, and/or taking bio-DMARDs which may effect 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) [5] 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 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) Continuous 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 and MannWhitney 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 181 Journal of military pharmaco-medicine n08-2017 RESULTS Patients and demographic, clinical characteristics Table 1: Demographic and clinical characteristics of RA patients and control Mean age ± SD, - max (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 Sex, n (female/male) Mean disease duration ± SD (years) Mean tender joint count ± SD (range - 28) 4.29 ± 5.34 14.13 ± 9.08; 13.00 Mean swollen joint count ± SD (range - 28) Mean morning stiffness ± SD (minutes) 10.52 ± 7.38; 9.0 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 7.63 ± 3.92 Mean plasma CRP ± SD (mg/L) 68.37 ± 47.24 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 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 182 Journal of military pharmaco-medicine n08-2017 Comparison of laboratory parameters among patients and healthy subjects Figure 1: The comparison of serum TNF-α level between RA patients and controls (p, test Mann - Whiney) The mean and median of serum TNF-α of RA patients and controls was 2.37 ± 2.69; 1.68 and 3.87 ± 2.11; 3.69 pg/mL, respectively Median of serum TNF-α concentrations in RA patients was significantly lower than that in controls group (p < 0.001) Figure 2: The correlation of serum IL-6 levels and serum TNF-α levels in RA patients (numbers are Spearman correlation coefficients) Figure 3: The correlation of serum TNF-α levels and serum IL-17 levels in RA patients (numbers are Spearman correlation coefficients) Serum TNF-α had a possitive correlation with serum IL-6 and IL-17 in RA patients (r = 0.233; p = 0.035 and r = 0.25; p = 0.023, respectively) 183 Journal of military pharmaco-medicine n08-2017 Correlation between serum TNF-α and clinical, laboratory variables in RA patients group Table 2: The comparision of serum TNF-α based on measurements of disease activity Serum TNF-α levels (pg/ml) Mean ± SD Median - (n = 13) 2.72 ± 3.22 2.24 ≥ (n = 68) 2.33 ± 2.62 1.64 - (n = 20) 2.34 ± 2.84 2.24 ≥ (n = 61) 2.41 ± 2.68 1.64 Low & moderate (n = 14) 2.51 ± 3.17 1.94 High (n = 65) 2.40 ± 2.65 1.72 Joint tender count 28 p 0.694 Joint swollen count 28 0.784 DAS28 CRP 0.944 Table 3: The correlation of serum TNF-α levels in RA patients with measurements of disease activity TJC28 SJC28 MS PtGA PGA CRP ESR r 0.105 0.040 0.050 -0.062 -0.131 -0.183 -0.065 p 0.352 0.725 0.664 0.585 0.245 0.102 0.600 Serum TNF-α (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 TNF-α levels with composite indices in RA patients DAS28 CRP DAS28 ESR SDAI CDAI r 0.001 0.090 -0.009 0.024 p 0.995 0.472 0.938 0.832 Serum TNF-α (Abbreviations: SDAI: Simplified disease activity index, CDAI: Clinical disease activity index) There were not associations between the serum TNF-α levels of RA patients with measurements of disease activity 184 Journal of military pharmaco-medicine n08-2017 DISCUSSION In the present study, level of serum TNF-α cytokine was evaluated in patients with RA, and associations of its with clinical and laboratory parameters In accordance with other study [6], we found that serum TNF-α was significantly lower in RA patients compared to healthy subjects (figure 1) However, Kokkonen H et al (2010) found serum TNF-α had no differrences between RA patients and healthy controls [7] By contrast our results, Prado A.D et al (2016) observed serum TNF-α increased in RA patients compared to healthy controls (p < 0.001) [8] This condition may be caused by treatment before, this study including 50.6% of patients treated by glucocorticoid Glucocorticoids exert potent inhibitory effects on the transcription and action of a large variety of cytokines with pivotal importance in the pathogenesis of RA Most T helper type (Th1) proinflammatory cytokines are inhibited by glucocorticoids, including interleukin (IL)-1β, IL-2, IL-3, IL-6, TNF, interferon-γ [9] In the current study, serum TNF-α had a significantly positive correlation with serum IL-6 and serum IL-17 (figure and figure 3) In consistent of our observation, Manicourt D.H et al (1993) [10] and Zhao P.W et al (2014) [11] also reported that serum TNF-α had a positive correlation with serum IL-6 and serum IL-17 These studies supports the concept that TNF-α plays a key role in pathogenesis of RA by stimulating pro-inflammation cytokines including IL-6, IL-17 [2] TNF-α is a key cytokine in the pathogenesis of RA that involved in chronic synovial inflammation and articular destruction, thus it may influence disease activity of RA patients We assessed the change of serum TNF-α according to measurements of disease activity including TJC28, SJC28 and DAS28 CRP, however we did not found differences based on these parameters In the present study, we also did not observe the correlation between serum IL-6 with measurements of disease activity such as TJC28, SJC28, morning stiffness, PtGA, PGA, ESR, plasma CRP levels as well as composite index DAS28 CRP, DAS28 ESR, SDAI and CDAI Consistantly with the present study, Prado A.D et al (2016) observed that serum TNF-α had no associations with joint tender count 28, joint swollen count 28, DAS28 CRP, DAS28 ESR [8] Keiko Shimamoto et al (2013) found serum TNF-α was not related to DAS28 CRP and DS28 ESR [12] By contrast these results, Reham Dwedar A.R.A et al (2015) reported serum TNF-α had a negative relation to DAS28 (r = -0.404, p = 0.045) [13] Thus, there are many controversial studies regarding the relationship between serum TNF-α as an assessing role of disease activity and measurements of disease activity in RA patiets, so we need more studies with larger sample number to discover this interesting correlation 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 185 Journal of military pharmaco-medicine n08-2017 CONCLUSION Our study demonstrated a significantly lower of serum TNF-α in RA patients comparing with healthy controls However, we did not find any associations between serum TNF-α levels and measurements of disease activity in RA patients REFERENCES Aletaha D, T Neogi, A.J Silman et al RA classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative Arthritis Rheum 2010, 62 (9), pp.2569-2581 Brennan F.M, I.B McInnes Evidence that cytokines play a role in RA J Clin Invest 2008, 118 (11), pp.3537-3545 Gabay C, I Kushner Acute-phase proteins and other systemic responses to inflammation N Engl J Med 1999, 340 (6), pp.448-454 Pollard L.C, G.H Kingsley, E.H Choy et al Fibromyalgic RA 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 Selaas O, H.H Nordal, A.K Halse et al Serum markers in RA: A longitudinal study of patients undergoing infliximab treatment 2015, p.276815 186 Kokkonen H, I Soderstrom, J Rocklov et al Up-regulation of cytokines and chemokines predates the onset of RA Arthritis Rheum 2010, 62 (2), pp.383-391 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 2016, 83 Ultrasound power Doppler synovitis is associated with plasma IL-6 in established RA pp.27-32 Gary S Firestein Kelley’s Textbook of th Rheumatology ed Glucocorticoid therapy, ed Johannes W.G Jacobs Johannes W.J Bijlsma Elsevier Saunders Philadelphia 2013 10 Manicourt D.H, R Triki, K Fukuda et al Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with RA Relationship to serum levels of hyaluronan and antigenic keratan sulfate Arthritis Rheum 1993, 36 (4), pp.490-499 11 Zhao P.W, W.G Jiang, L Wang et al Plasma levels of IL-37 and correlation with TNF-alpha, IL-17A, and disease activity during DMARD treatment of RA PLoS One 2014, (5), p.e95346 12 Shimamoto K., T Ito, Y Ozaki et al Serum interleukin before and after therapy with tocilizumab is a principal biomarker in patients with RA J Rheumatol 2013, 40 (7), pp.1074-1081 13 Reham Dwedar A.R.A, Hala A Raafat Does novel IL-33 correlates with TNF-α in RA and SLE? Egyptian Journal of Medical Microbiology 2015, 24, pp.13-20 ... pharmaco-medicine n08-2017 DISCUSSION In the present study, level of serum TNF-α cytokine was evaluated in patients with RA, and associations of its with clinical and laboratory parameters In accordance with. .. et al Levels of circulating tumor necrosis factor alpha and interleukin-6 in patients with RA Relationship to serum levels of hyaluronan and antigenic keratan sulfate Arthritis Rheum 1993, 36... importance in the pathogenesis of RA Most T helper type (Th1) proinflammatory cytokines are inhibited by glucocorticoids, including interleukin (IL)-1β, IL-2, IL-3, IL-6, TNF, interferon-γ [9] In the current