The aim of this study is to evaluate serum levels of tumor necrosis factor-alpha (TNF-α) in rheumatoid arthritis patients and to assess the correlations of this cytokine with clinical and laboratory parameters.
Journal of military pharmaco-medicine n06-2018 SERUM LEVEL TNF-ALPHA IN PATIENTS WITH RHEUMATOID ARTHRITIS Hoang Trung Dung*; Doan Van De**; Vien Van Doan* SUMMARY Objectives: To evaluate serum levels of tumor necrosis factor-alpha (TNF-α) in rheumatoid arthritis patients and to assess the correlations of this cytokine with clinical and laboratory parameters Subjects and methods: 122 patients with rheumatoid arthritis and 51 healthy volunteers were enrolled in the study Disease activity was determined by disease activity score (DAS28) in patients with rheumatoid arthritis The serum levels of TNF-α cytokine was measured by chemiluminescent immune assay Results: We found that rheumatoid arthritis patients had significantly higher levels of serum TNF-α (p < 0.01) as compared to healthy controls Serum TNF-α showed no significant correlations with mesurements of disease activity Conclusions: This study showed that patients with rheumatoid arthritis had a significantly higher TNF-α cytokine than that of healthy controls, and serum TNF-α cytokine was not associated with disease activity mesurements * Keywords: Rheumatoid arthritis; TNF-α; Biomarkers INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory disorder that is characterised by polyarthritis with often progressive joint damage and disability, immunological abnormalities, systemic inflammation, increased co-morbidity, and premature mortality [1] It affects 1% of the adult population worldwide and also occurs among one in a thousand children as juvenile RA RA is much more common in women and affects women - times more frequently than men The aetiology of RA is not known, but it is classified as one of the autoimmune diseases It is associated with reduced life expectancy and a major cause of chronic disability and handicap, and conditions become more dangerous with time Many studies have shown that advance therapy including the use of early, aggressive therapy, and the introduction of anti-cytokines agent have improved patient’s quality of life, eased clinical symptoms, retarded the progression of joint destruction, and delayed disability Cytokine networks play a fundamental role in the processes that cause inflammation, articular destruction of RA [2] TNF-α is one of the pivotal pro-inflammatory cytokines responsible for inflammation and joint destruction in RA TNF-α is readily detected in both synovial fluid and serum of patients with RA TNF-α is a key cytokine in the pathogenesis of RA that involved in chronic synovial inflammation andarticular destruction * Bachmai Hospital ** 103 Military Hospital Corresponding author: Hoang Trung Dung (dungbsbm@gmail.com) Date received: 15/05/2018 Date accepted: 20/06/2018 162 Journal of military pharmaco-medicine n06-2018 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), 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) [3, 4] The aim of this study was: To evaluate serum levels of TNF-α in RA patients and to assess the correlations of this cytokine with clinical and laboratory parameters SUBJECTS AND METHODS Subjects * Patients: This study was carried out at Bachmai Hospital between October 2014 and April 2018 122 patients (103 women and 19 men) with the diagnosis of RA fulfilled the ACR/EULAR 2010 RA classification criteria [1] 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: Fifty one sex-matched healthy controls (43 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 (DAS28CRP) [5] in RA patients Based on the DAS28CRP, 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 provides global assessment of disease activity were evaluated using a 10 cm horizontal visual analog scale (VAS) Erythrocyte sedimentation rate (ESR) and CRP were recorded * Laboratory analysis: Blood samples of patients and controls were collected and put in sterile plain tubes and stored frozen at -80oC until analysis Serum TNF-α was assayed by chemiluminescent immune assay (CLIA) 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 Mann-Whitney U test for 163 Journal of military pharmaco-medicine n06-2018 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 RESULTS Patients and demographic, clinical characteristics Table 1: Demographic and clinical characteristics of RA patients and control RA patients (n = 122) Controls (n = 51) 48.9 ± 11.3 48.1 ± 11.7 103/19 43/8 Mean age ± SD (years) Sex, n (female/male) Mean tender joint count ± SD (range - 28) 13.30 ± 4.34 Mean swollen joint count ± SD (range - 28) 9.95 ± 3.71 Mean morning stiffness ± SD (minutes) 61.48 ± 27.64 Mean ESR ± SD (mm/h) 43.89 ± 22.76 Mean DAS28 CRP ± SD (Median; min-max) 5.77 ± 0.94; 6.02; 2.85 - 7.86 DAS28 CRP Low and moderat (n; %) ≤ 5,1 31; 25.4% High (n; %) > 5,1 91; 74.6% 16.14 ± 12.70 (Abbreviations: ESR: Erythrocyte sedimentation rate; DAS28 CRP: Disease activity score c-reactive protein) The mean age of the 122 patients with RA was 48.9 ± 11.3 years and the patient group was comprised of 19 males and 103 females Patients and controls did not significantly differ in age or sex The mean value of morning stiffness was 61.48 ± 27.64 The mean DAS28 CRP was 5.77 ± 0.94 (range 2.85 - 7.86) Thirty one (25.4%) and ninety one (74.6%) patients had low-moderate and high DAS28 CRP, respectively Comparison of laboratory parameters among patients and healthy subjects Table 2: Mean values of laboratory variables in RA patients and controls Parameters RA patients (n = 122) Controls (n = 51) p Serum TNF-α (pg/mL) 15.32 ± 7.37 8.84 ± 2.17 < 0.01 Plasma CRP (mg/dL) 2.56 ± 2.81 0.12 ± 0.12 < 0.01 (Abbreviations: TNF: Tumour necrosis factor; p: Test Mann-Whiney was used Data is expressed as mean ± standard deviation (SD)) We found that the mean level of TNF-α was highly, significantly increased (p < 0.01) in RA cases (15.32 ± 7.37 mg/dL) compared to the healthy controls (8.84 ± 2.17 pg/mL) There were highly significant increases in CRP (2.56 ± 2.81 vs 0.12 ± 0.12 mg/dL) levels in patients with RA compared to the control group (p < 0.001) 164 Journal of military pharmaco-medicine n06-2018 Correlation between serum TNF-α and clinical, laboratory variables in RA patients group Table 3: The comparison of serum TNF-α based on measurements of disease activity Serum TNF-α levels (pg/mL) Low and moderate (n = 31) DAS28 CRP High (n = 91) Mean ± SD Median 13.26 ± 6.64 11.30 16.02 ± 7.50 14.10 p > 0,05 Table 4: The correlation of serum TNF-α levels in RA patients with measurements of disease activity TJC28 SJC28 MS CRP ESR r 0.077 0.016 0.067 0.136 0.186 p > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 Serum TNF-α (Abbreviations: TJC: Tender joint count; SJC: Swollen joint count: MS: Morning stiffness, r: Spearman’s correlation coefficient) There were no differences according to joint tender count 28, joint swollen count 28, morning stiffness, C reactive protein and erythrocyte sedimentation rate Table 5: The correlation of serum TNF-α levels with composite indices in RA patients DAS28 CRP DAS28 ESR r 0.113 0.160 p > 0.05 > 0.05 Serum TNF-α There were not associations between the serum TNF-α levels of RA patients with measurements of disease activity DISCUSSION In the present study, we evaluated serum levels of TNF-α cytokines in patients with established RA, and associations of these cytokines with clinical and laboratory parameters TNF-α is one of the key cytokines in the pathogenesis of RA, and TNF inhibitors are major biologics in the treatment of RA In our study, we found significant increased levels of TNF-α in RA patients as compared to the healthy controls (table 2) A study by Prado A.D et al (2016) observed serum TNF-α was increased in RA patients compared to healthy controls (p < 0.001) [6] However, Kokkonen H et al (2010) found serum TNF-α had no differences between RA patients and healthy controls [7] This condition may be caused by RA patients who were first diagnosed and not treated, TNF-α levels were high These findings suggest that TNF-α is important mediators of inflammation in RA and play a pivotal role in the development and progression of RA 165 Journal of military pharmaco-medicine n06-2018 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, MS, CRP, ESR, DAS28 CRP, DAS28 ESR However, we did not find differences based on these parameters Consistantly with the present study, Prado A.D et al (2016) observed serum TNF-α had no associations with joint tender count 28, joint swollen count 28, DAS28 CRP, DAS28 ESR [6] Keiko Shimamoto et al (2013) found serum TNF-α was not related to DAS28 CRP and DS28 ESR Our study has some limitations The sample size of patients was relatively small, and the patients were on drug treatment including DMARDs Treatment regimes might influence on the serum expression of cytokines In fact, our study had a cross-sectional design, and cytokines profile could not evaluate compared to patients with early treatment naive RA CONCLUSION Our study demonstrated a significantly higher 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 166 REFERENCES Aletaha D, T.Neogi, A.J Silman et al Rheumatoid arthritis 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 rheumatoid arthritis J Clin Invest 2008, 118 (11), pp.3537-3345 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 rheumatoid arthritis and disease assessment Rheumatology (Oxford) 2010, 49 (5), pp.924-928 Wells G, J.C Becker, J Teng et al Validation of the 28-joint Disease Activity Score (DAS28) and European League Against Rheumatism response criteria based on Creactive protein against disease progression in patients with rheumatoid arthritis, and comparison with the DAS28 based on erythrocyte sedimentation rate Ann Rheum Dis 2009, 68 (6), pp.954-960 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, pp.27-32 Kokkonen H, I Soderstrom, J Rocklov et al Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis Arthritis Rheum 2010, 62 (2), pp.383-3891 ... associations between the serum TNF-α levels of RA patients with measurements of disease activity DISCUSSION In the present study, we evaluated serum levels of TNF-α cytokines in patients with established... 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 rheumatoid arthritis J Clin Invest 2008,... pharmaco-medicine n06-2018 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