Determination of Interleukin-6 and Tumor Necrosis Factor-alpha concentrations in Iranian-Khorasanian patients with preeclampsia.

Abstract

Our objective was to determine the role of Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-alpha), markers of immune activation and endothelial dysfunction, in patients with preeclampsia.
Twenty four women with preeclampsia and eighteen antepartum normotensive pregnant women were recruited as controls. Serum levels of IL-6 and TNF-alpha were measured by enzyme-linked immunosorbent assay. We used independent-samples t test to assess the differences in the concentration of cytokines in preeclamptic patients and control subjects.
IL-6 levels [mean (S.D.)] were significantly higher in preeclamptic women [5.8 (4.85) pg/ml] compared to normal pregnant women [3.01 (2.45) pg/ml] (p = 0.02). There was no significant change in concentration of TNF-alpha in preeclamptic women [53.8 (30.0) pg/ml] compared to normal pregnant women [51.9 (33.8) pg/ml] (p > 0.1).
The results of this study show that IL-6 as a pro-inflammatory cytokine is present in higher concentration in women with preeclampsia. The study was undertaken in women with established preeclampsia and it is not possible to determine whether the increased concentration of IL-6 is a cause or consequence of the disease. Furthermore, these findings suggest that serum TNF-alpha level is not associated with preeclampsia.

Full text

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BioMed Central
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BMC Pregnancy and Childbirth
Open Access
Research article
Determination of Interleukin-6 and Tumor Necrosis Factor-alpha
concentrations in Iranian-Khorasanian patients with preeclampsia
J Tavakkol Afshari*1, N Ghomian†2, A Shameli†2, MT Shakeri†3,
MA Fahmidehkar†1, E Mahajer1, R Khoshnavaz4 and M Emadzadeh4
Address: 1Immunogenetics department, Immunology Research Group, Bu-Ali Research Institute, Bu-Ali Sq., Mashhad University of Medical
Sciences (MUMS), Mashhad, Iran, 2Obstetrics & Gynecology department, Imam Reza Hospital, Imam Reza Sq., Mashhad University of Medical
Sciences (MUMS), Mashhad, Iran, 3Community medicine department, Medical school, Daneshgah St., Mashhad University of Medical Science
(MUMS), Mashhad, Iran and 4Department of Cardiology, Ghaem Hospital, Ahmad Abad St., Mashhad University of Medical Sciences (MUMS),
Mashhad, Iran
Email: J Tavakkol Afshari* - jtavakkol@yahoo.com; N Ghomian - N-ghomian@mum.ac.ir; A Shameli - Ahmad-shameli@mums.ac.ir;
MT Shakeri - m_T_shakeri@yahoo.com; MA Fahmidehkar - mafahmidehkar@yahoo.com; E Mahajer - mmahajer@yahoo.com;
R Khoshnavaz - Rasoulkhoshnavazmd@yahoo.com; M Emadzadeh - M-emadzadeh@mums.ac.ir
* Corresponding author †Equal contributors
Abstract
Background: Our objective was to determine the role of Interleukin-6 (IL-6) and Tumor Necrosis
Factor-alpha (TNF-alpha), markers of immune activation and endothelial dysfunction, in patients
with preeclampsia.
Methods: Twenty four women with preeclampsia and eighteen antepartum normotensive
pregnant women were recruited as controls. Serum levels of IL-6 and TNF-alpha were measured
by enzyme-linked immunosorbent assay. We used independent-samples t test to assess the
differences in the concentration of cytokines in preeclamptic patients and control subjects.
Results: IL-6 levels [mean (S.D.)] were significantly higher in preeclamptic women [5.8 (4.85) pg/
ml] compared to normal pregnant women [3.01 (2.45) pg/ml] (p = 0.02). There was no significant
change in concentration of TNF-alpha in preeclamptic women [53.8 (30.0) pg/ml] compared to
normal pregnant women [51.9 (33.8) pg/ml] (p > 0.1).
Conclusion: The results of this study show that IL-6 as a pro-inflammatory cytokine is present in
higher concentration in women with preeclampsia. The study was undertaken in women with
established preeclampsia and it is not possible to determine whether the increased concentration
of IL-6 is a cause or consequence of the disease. Furthermore, these findings suggest that serum
TNF-alpha level is not associated with preeclampsia.
Background
Preeclampsia is a critically important disease of preg-
nancy, one of the major causes of fetal and maternal mor-
bidity and mortality throughout the world. In spite of its
importance for public health, the etiology of preeclampsia
has not yet been fully elucidated. Although the patho-
physiology of preeclampsia is not understood completely,
there is an interest in a possible link between inflamma-
tion and endothelial dysfunction [1] and between
endothelial dysfunction and preeclampsia [2]. Cytokines
Published: 01 November 2005
BMC Pregnancy and Childbirth 2005, 5:14doi:10.1186/1471-2393-5-14
Received: 25 February 2005
Accepted: 01 November 2005
This article is available from: http://www.biomedcentral.com/1471-2393/5/14
© 2005 Afshari et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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have been described to play a major role in the pathogen-
esis of preeclampsia. Recent studies have demonstrated
that cytokines – mediators of inflammatory response-may
cause endothelial dysfunction through different mecha-
nisms such as oxidative stress [3] and endothelial cell
damage [4].
IL-6 is a proinflammatory cytokine produced by mononu-
clear phagocytes, endothelial cells, fibroblasts and T cells
and has many functions and effects [5]. IL-6 is involved in
immune activation, vascular wall function and modula-
tion of TNF- production.
Recent studies have shown that amniotic fluid IL-6 is
decreased in pregnancies complicated by preeclampsia
and placental IL-6 production is decreased in these
patients. These findings suggest a role for this cytokine in
the pathophysiology of this disease. TNF-α is a potent
modulator of immune and inflammatory responses that
are produced by macrophages, lymphocytes and trophob-
lasts and contribute to the trophoblast growth and inva-
sion. Its role in different pathologic conditions of
pregnancy has been shown in recent studies. Increased
amniotic fluid concentrations of TNF-α in patients with
severe preeclampsia suggest a role for this cytokine in the
pathophysiology of this disease. There are some studies in
different populations that demonstrated the possible role
of these cytokines in the pathophysiology of preeclampsia
[6-10]. Therefore in the present study we measured their
concentrations in serum of Khorasanian women (north-
east of Iran) with preeclampsia, and compared them with
concentrations found in normotensive control women.
Both IL-6 and TNF-α are expressed in adipose tissue
[11,12] and in vitro release of TNF-α by adipocytes has
been reported [13]. Among the known effects of these
cytokines are inhibition of insulin signaling [14] and
induction of both hypertriglyceridemia [15] and endothe-
lial activation [16].
Methods
This study was approved by the ethical committee of the
Mashhad University of Medical Sciences (MUMS) and
was conducted at Imam-Reza Hospital and Bu-Ali
Research Institute. It was a cross-sectional study and
preeclamptic patients were selected from those admitted
to Imam-Reza Hospital between February 2004 and
December 2004. Written informed consent was obtained
from participating women. Twenty four patients with
preeclampsia at 26–41 weeks' gestation were enrolled in
this study. Preeclampsia was diagnosed according to the
clinical criteria defined by the "International Society for
the study of Hypertension in Pregnancy" [17]. All preec-
lamptic patients had blood pressure of at least 140/90
mmHg and proteinuria greater than (+) as assessed by a
dipstick or 300 mg or greater in a 24-hour urine sample.
None of the preeclamptic patients had underlying dis-
eases such as chronic hypertension, diabetes mellitus,
renal disease or urinary infection. None of these patients
were in labor at the time of sampling. Some patients were
prescribed hydralazine, dexamethazone or magnesium
sulphate before sampling.
Eighteen antepartum normotensive healthy women at
26–39 weeks' gestation were included as control group.
Maternal venous blood samples (5 ml) were collected
from patient and control groups and were centrifuged at
3500 RPM for 10 minutes. Serum specimens were stored
at -70°C in aliquots to avoid possible interference with
assay results due to repeated freeze-thaw cycles.
Serum IL-6 and TNF-α concentrations were determined by
commercially available high sensitivity indirect sandwich
enzyme-linked immunosorbent assay (Bender MedSys-
tems, Austria). Briefly, IL-6 or TNF-α present in the sam-
ples or standard binds to anti-IL-6 or anti-TNF-α
monoclonal antibody adsorbed to the microwells. A
biotin-conjugated monoclonal anti-IL-6 or anti-TNF-α
antibody was added and binds to IL-6 or TNF-α captured
by the first antibody. Following incubation unbound
biotin-conjugated anti-IL-6 or anti-TNF-α is removed dur-
ing a wash step. Streptavidin-HRP was added and binds to
the biotin-conjugated anti-IL-6 or anti-TNF-α; following
incubation unbound Streptavidin-HRP was removed dur-
ing a wash step, and substrate solution reactive with HRP
was added to the wells. A colored product was formed in
proportion to the amount of IL-6 or TNF-α present in the
sample. The reaction was terminated by addition of acid
and absorbance was measured at 450 nm. A standard
curve was prepared from seven IL-6 and TNF-α standard
dilutions and IL-6 or TNF-α sample concentrations deter-
mined. The detection limit for IL-6 and TNF-α were 1.4
pg/ml and 3.83 pg/ml respectively. The overall inter-assay
coefficient of variation for IL-6 and TNF-α were 5.2% and
6.9% respectively. All serum IL-6 and TNF-α analyses were
performed at the same time, in the same batch, and in
duplicate according to manufacturer's instructions. IL-6
and TNF-α serum levels are given as normal distribution.
Independent-samples t test was used to compare the mean
levels between patient and control groups. P-value < 0.05
were considered statistically significant.
Results
A total of 24 women with preeclampsia and 18 women as
controls were examined. The characteristics of women
included in the study are presented in Table 1. There is no
significance difference between two groups. Therefore the
two groups were match for age, gestational age and grav-
ity. IL-6 serum concentrations are depicted in Figure 1. IL-
6 serum levels [mean (S.D.)] were significantly higher (p
α .

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= 0.02) in preeclamptic women [5.8 (4.85)] compared
with normal pregnant women [3.01 (2.45)].
TNF-α serum concentrations are depicted in figure 2.
There was no significant Change in concentration of TNF-
α serum levels in preeclamptic women [53.8 (30.0) pg/
ml] compared to normal pregnant women [51.9 (33.8)
pg/ml] (p > 0.1).
Discussion
Although the pathogenesis of preeclampsia is still
unknown, immunologic and inflammatory causes may
play an important role. IL-6 and other cytokines are
important components of immune response, and there-
fore can participate in the immune aspects of the patho-
physiology of this disease. Proinflammatory cytokines
appear to be involved in cellular events that establish and
maintain pregnancy [18]; however, their role has not yet
been well defined.
Our data indicate that IL-6 concentrations were increased
in the circulation of Iranian-Khorasanian preeclamptic
patients compared with control women. This finding is
consistent with reports by Greer et al in 1994 [6], Vince et
al in 1995 [7], Kupferminc et al in 1996 [8], Munno et al
in 1999 [9] and Teran et al in 2001 [10]. Endothelial dys-
function and increased endothelial permeability are the
characteristics of the pathophysiology of preeclampsia. IL-
6 may increase the permeability of endothelial cells [5] by
changing the cell shape and rearrangement of intracellular
actin fibers [19]. IL-6 can also reduce prostacyclin (PG I2)
synthesis by inhibiting the cyclooxygenase enzyme [20]. It
can increase the tromboxane A2 to prostacyclin ratio, an
abnormality that happens in preeclampsia. IL-6 can also
stimulate platelet-derived growth factor, a process seen in
preeclampsia [5]. Oxygen free radicals can induce synthe-
sis of IL-6 by endothelium [21]. Oxygen free radicals are
implicated in the pathogenesis of preeclampsia, because
they can cause endothelial damage, which leads to reduc-
tion in nitric oxide synthesis and prostaglandin balance
disturbance.
TNF-α is another pro-inflammatory cytokine that its con-
tribution in the pathogenesis of preeclampsia has been
suggested in recent studies [8,10]. In healthy pregnant
women, TNF-α is thought to modulate the growth and
invasion of tropoblasts in maternal spiral arteries [22].
TNF-α may contributes to abnormal placental invasion
[23], endothelial cell damage [4] and oxidative stress [3].
TNF-α can stimulates IL-6 production [24], since IL-6
inhibits TNF-α release [25]. In contrast to IL-6, no
increased serum concentration of TNF-α was found in
preeclamptic patients compared to control pregnant
women. Several investigators [26-28] have reported that
serum concentrations of TNF-α were significantly higher
in the first and second trimester among pregnant women
who subsequently developed preeclampsia compared to
those in the control group. This finding is consistent with
reports by Greer et al in1994 [6], Heyl et al in 1999 [28]
and Ellis et al in 2001 [29]. Furthermore, the levels of IL-
6 and TNF-α that we detected in the Khorsanian women
studied were significantly higher than those reported for
European and North American women [7,26,27].
Whether these differences are related to genetic (inflam-
Serum TNF-alpha concentration (pg/ml) measured by ELISA in normal pregnant (n-18) and preeclamptic (n = 24) women
Figure 2
Serum TNF-alpha concentration (pg/ml) measured by ELISA
in normal pregnant (n-18) and preeclamptic (n = 24) women.
Serum IL-6 concentration (pg/ml) measured by ELISA in nor-mal pregnant (n-18) and preeclamptic (n = 24) women
Figure 1
Serum IL-6 concentration (pg/ml) measured by ELISA in nor-
mal pregnant (n-18) and preeclamptic (n = 24) women.

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matory response and L-arginine; NO pathway) and/or
environmental factors (e.g., infection) remains to be
determined. Several evidence links infection and inflam-
matory processes with preeclampsia [30,31]. The role of
infection in the pathogenesis of preeclampsia is particu-
larly relevant in developing countries, where the high inci-
dence of chronic subclinical infection may contribute to
the high incidence of preeclampsia.
Our findings support the hypothesis that immune activa-
tion is involved in preeclampsia and that IL-6 may partic-
ipate in the abnormal immune response. This study was
undertaken in women with established preeclampsia
therefore, it cannot be determined whether the increase
IL-6 was a cause or a consequence of the disease
[7,10,26,27]. We cannot determine whether IL-6 is an
active mediator in preeclampsia or a marker of immune
activation, and it seems necessary to perform further stud-
ies, including longitudinal studies before the onset of
preeclampsia, to elucidate the role of this cytokine in the
pathogenesis of preeclampsia.
Conclusion
The results of this study show that IL-6 as a pro-inflamma-
tory cytokine is present in higher concentration in women
with preeclampsia. The study was undertaken in women
with established preeclampsia and it is not possible to
determine whether the increased concentration of IL-6 is
a cause or consequence of the disease. Furthermore, these
findings suggest that serum TNF-α level is not associated
with preeclampsia.
Authors' contributions
JTA has participated in preparation and submission of the
manuscript and given final approval of the version to be
published. Also, conceived of the study, and participated
in its design and coordination. NG participated in the
diagnosis of patients with preeclampsia and in coordina-
tion of the study. AS participated in the diagnosis and
selection of patients with preeclampsia. MTS participated
in the design of the study and performed the statistical
analysis. MAF carried out the immunoassays. MM partici-
pated in preparation of samples and performing ELISA for
IL-6. RK: participated in drafting (English correction). ME
participated in revising the manuscript critically for
important intellectual content. All authors read and
approved the final manuscript.
Note
Table 1: Characteristics of women included in the study
Acknowledgements
This study was supported by the Vice Chancellor for Research, Mashhad
University of Medical Sciences (MUMS), Mashhad, Iran.
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Table 1:
Normal pregnant (n = 18)Preeclampsia (n = 24)t and p
Age (years)
Gestational age (weeks)
Gravidity
27.2 (5.8)
33.3 (3.7)
2.3 (1.4)
28.4 (4.9)
34.8 (4.3)
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0.76, p > 0.1
1.18, p > 0.1
0.65, p > 0.1
Data are presented as mean (S.D.).

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