The changes in serum tumor necrosis factor alpha in patients with rheumatoid arthritis

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.

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

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

1 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

4 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 4 men) were included

in the study

2 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 2 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

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

RESULTS

1 Patients and demographic, clinical characteristics

Table 1: Demographic and clinical characteristics of RA patients and control

Mean tender joint count ± SD (range 0 - 28) 14.13 ± 9.08; 13.00

Mean swollen joint count ± SD (range 0 - 28) 10.52 ± 7.38; 9.0

7.16 ± 2.25

Mean provider global assessment of disease

5.65 ± 1.92

DAS28 CRP

Pre-study

treatment

(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

2 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)

3 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)

p

Joint tender count 28

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

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

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

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,

do 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 1

(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 2 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, do 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

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

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