Serum level TNF-alpha in patients with rheumatoid arthritis

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 2 - 3 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 ()
Date received: 15/05/2018
Date accepted: 20/06/2018

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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
1. 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 8 men) were included in
the study.
2. 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 2 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
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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
1. 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 0 - 28)

13.30 ± 4.34

Mean swollen joint count ± SD (range 0 - 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 min.
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.
2. 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).
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Journal of military pharmaco-medicine n06-2018
3. 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 do 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.
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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
1. 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.
2. Brennan F.M, I.B. McInnes. Evidence
that cytokines play a role in rheumatoid arthritis.
J Clin Invest. 2008, 118 (11), pp.3537-3345.
3. Gabay C, I. Kushner. Acute-phase proteins
and other systemic responses to inflammation.
N Engl J Med. 1999, 340 (6), pp.448-454.
4. 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.
5. 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.
6. do 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.
7. 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.