Monocytes from Pregnant Women with Pre-Eclampsia arePolarized to a M1 PhenotypeLeonardo T. L. Medeiros1, Jos�e C. Perac�oli1, Camila F. Bannwart-Castro1, Mariana Rom~ao2, Ingrid C.Weel2, Marjorie A. Golim3, Leandro G. de Oliveira4, Cilmery S. Kurokawa1, Vera T. Medeiros Borges1,Maria T. S. Perac�oli2
1Department of Gynecology and Obstetrics, Botucatu Medical School, S~ao Paulo State University, Botucatu, Brazil;2Department of Microbiology and Immunology, Institute of Biosciences, S~ao Paulo State University, Botucatu, Brazil;3Division of Hemocenter, Botucatu Medical School, S~ao Paulo State University, Botucatu, Brazil;4Department of Gynecology and Obstetrics, Federal University of Sao Paulo, S~ao Paulo, Brazil
Keywords
Cytokines, monocyte subsets, pre-eclampsia,
surface receptors
Correspondence
Maria Terezinha S. Perac�oli, Department of
Microbiology and Immunology, Institute of
Biosciences, Rubi~ao Junior s/n, UNESP,
Botucatu, Sao Paulo, CEP 18618-970, Brazil.
E-mail: [email protected]
Submission November 22, 2013;
accepted February 3, 2014.
Citation
Medeiros LTL, Perac�oli JC, Bannwart-CastroCF, Rom~ao M, Weel IC, Golim MA, de Oliveira
LG, Kurokawa CS, Medeiros Borges VT,
Perac�oli MTS. Monocytes from pregnant
women with pre-eclampsia are polarized to a
M1 phenotype. Am J Reprod Immunol 2014;
72: 5–13
doi:10.1111/aji.12222
Problem
This study evaluated whether the monocyte inflammatory state in pre-
eclampsia (PE) might be associated with polarization to either M1 classi-
cally or M2 alternatively activated monocyte subsets.
Method of Study
Eighty-five women with (PE) and 52 normotensive (NT) pregnant
women matched for gestational age were included. Expression of surface
receptors characteristic of M1, such as Toll-like receptor (TLR)2, TLR4,
and CD64, or M2, such as CD163 and CD206 monocyte subsets were
evaluated in peripheral blood monocytes by flow cytometry. Tumour
necrosis factor-alpha (TNF-a), interleukin-(IL)-12p40, IL-12p70, and IL-
10 were evaluated in the supernatant of monocyte cultures by ELISA.
Results
Expression of TLR4 and CD64 by monocytes from pre-eclamptic women
was significantly higher, while the expression of CD163 and CD206
expression was significantly lower compared with NT pregnant women.
Endogenous production of TNF-a, IL-12p40, and IL-12p70 by monocytes
was increased, while synthesis of IL-10 was lower in women with PE
than in NT pregnant women.
Conclusions
Monocytes from women with PE are classically activated, producing
higher levels of pro-inflammatory cytokines, and express surface recep-
tors characteristic of the M1 subset. These results provide evidence that
the systemic inflammatory environment in PE may differentiate and
polarize these cells to the M1 phenotype.
Introduction
Pre-eclampsia (PE) is a specific syndrome of human
pregnancy affecting between 5 and 7% of primigr-
avid women1,2 and is characterized by new-onset
hypertension and proteinuria after 20 weeks of ges-
tation.3,4
Clinically, PE is classified as mild or severe,
according to patients’ signs and symptoms,4 and
more recently into early- or late-onset PE, depend-
ing on the time of appearance of clinical manifesta-
tions: early-onset PE tends to develop before
34 weeks of gestation, and late-onset PE develops
from 34 weeks of gestation.5,6 According to Hup-
American Journal of Reproductive Immunology 72 (2014) 5–13
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5
ORIGINAL ARTICLE
pertz,6 early- and late-onset PE are two entities that
differ in aetiology and disease manifestation. Early-
onset PE is considered a foetal disorder that is typi-
cally associated with placental dysfunction, abnormal
uterine and umbilical artery Doppler evaluation, and
foetuses with growth restriction, intrauterine foetal,
or maternal complications.7,8 On the other hand,
late-onset PE is considered a maternal disorder more
frequently associated with a normal placenta, nor-
mal uterine and umbilical artery Doppler evaluation,
a low rate of foetal compromise, and more favour-
able perinatal outcome.1,8,9
Association between increased levels of serum
pro-inflammatory cytokines and chemokines as well
as enhanced leucocyte activation and oxidative stress
has been reported in PE.10–14 This systemic inflam-
matory reaction is similar to that observed in normal
pregnancy, but shows greater intensity.15,16 There-
fore, this disease might be resulting from an exacer-
bated maternal inflammatory response that includes
activation of inflammatory cells such as monocytes
and granulocytes, and endothelial cells.17,18 Thus, an
abnormal production of cytokines as well as imbal-
ance between pro-inflammatory cytokines, such as
tumour necrosis factor-alpha (TNF-a), interleukin-
(IL)-12, interferon-gamma (IFN-c), and IL-2 as well
as anti-inflammatory cytokines (IL-4, IL-10, and IL-
13),is involved in the vascular damage observed in
PE.19
The excessive activation of intravascular mono-
cytes and granulocytes as well as macrophages in PE
suggests that innate immune system activation can
cause problems in pregnancy progression. However,
the significance of these immunological changes in
the pathophysiology of PE is still unknown. In previ-
ous studies, we demonstrated that monocytes from
pregnant women with PE were endogenously acti-
vated and released high levels of reactive oxygen
intermediates associated with high production of
TNF-a and might be the major source of this cyto-
kine detected in patients’ plasma.13,20,21
Monocytes and macrophages are cells belonging to
the monocytic–macrophage lineage and are consid-
ered cell populations that may be adapted and
respond to a wide variety of signs in their environ-
ment.22 Macrophages can be classified into at least
two major subpopulations with distinct phenotypes
and functions, that is, classically activated/inflamma-
tory (M1) and alternatively activated/regenerative
(M2).22–25 This M1 and M2 classification refers to
the two extremes of a macrophage activation spec-
trum. These polarized cells differ by surface receptor
expression, cytokines and chemokines production, as
well as transcription and epigenetic pathways.25 M1
macrophages, activated by IFN-c, TNF-a, or lipopoly-saccharide (LPS), express opsonin Fcc-type receptors
RI (CD64), RII (CD32), and RIII (CD16), toll-like
receptor 2 (TLR2) and toll-like receptor 4 (TLR4),
inflammatory cytokines such as TNF-a, IL-6, IL-12,
and IL-23, as well as reactive oxygen and nitrogen
intermediates.22,26 On the other hand, activation by
IL-4 and IL-13 polarizes to the M2 profile, character-
ized by increased expression of CD163 scavenger
receptor for haemoglobin,27 which also has anti-
inflammatory and immunoregulatory functions,28
mannose receptor (CD206), as well as increased pro-
duction of IL-10 and transforming growth factor beta
(TGF-b1).22 This macrophage plasticity allows the
cell change from an activated state or M1 to an M2
regulatory state and vice versa, upon the influence
of specific environmental signals.29,30
The M1 and M2 classification, initially proposed
for macrophages, can be extended to human periph-
eral blood monocytes in diseases such as sepsis,31
infections,32 type 2 diabetes,33 and studies employ-
ing modulatory agents on the inflammatory response
to in vitro treatment of these cells.34,35 Considering
that peripheral blood monocytes are activated in PE
and produce higher levels of inflammatory cyto-
kines,13,20,21 in this study, we evaluated the two M1
and M2 monocyte phenotypes as well as their cyto-
kine profile in pre-eclamptic women.
Materials and methods
Patients and Controls
The study comprised 137 pregnant women without
previous history of hypertension or obstetric and
medical complications, admitted to the Obstetric Unit
of Botucatu Medical School, Botucatu, SP, Brazil
between March 2009 and October 2011. Eight-five
women were diagnosed with PE, defined as a persis-
tent increased blood pressure value of 140 9
90 mm Hg and proteinuria (≥300 mg in urine col-
lected during 24 hr) after the 20th week of gesta-
tion.36 Pre-eclamptic pregnant women were
classified according to the onset of clinical manifesta-
tions into early-onset (24–33 weeks of gestation,
n = 26) and late-onset (34–40 weeks of gestation,
n = 59) pre-eclampsia, according to the criteria sug-
gested by von Dadelszen et al.5 A group of 52 preg-
American Journal of Reproductive Immunology 72 (2014) 5–13
6 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
MEDEIROS ET AL.
nant women (24–33 weeks of gestation, n = 26),
and (34–40 weeks of gestation, n = 26) with an
uncomplicated pregnancy that remained normoten-
sive and non-proteinuric throughout pregnancy,
were recruited as controls and matched for gesta-
tional age with the pre-eclamptic group. Gestational
age was calculated from the last menstrual period
and confirmed by ultrasound dating. Blood samples
were collected from pre-eclamptic women when
diagnosed with early- or late-onset PE and from nor-
motensive women when they were paired with pre-
eclamptic women according to gestational age. Data
on the participating women are shown in Table I.
Proteinuria in 24-hr urine was measured by a calori-
metric method, the Technicon RAXT automation
system, and uric acid was assessed by uric acid enzy-
matic Trinder (Biotrol Diagnostic, Bridgewater, NJ,
USA) in the Clinical Laboratory, Hospital das Clini-
cas, Faculdade de Medicina de Botucatu – UNESP.
Exclusion criteria included multiple gestation, prior
PE, pregnant women in labour, illicit drug use, and
pre-existing medical conditions such as chronic
hypertension, diabetes, infectious and renal diseases.
The study was approved by the Ethics Committee of
the Botucatu Medical School, and informed consent
was obtained from all women involved in the study.
Isolation of Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMC) were
isolated from heparinized venous blood by density
gradient centrifugation on Histopaque [density
(d) = 1.077] (Sigma–Aldrich, Chemical Co., St Louis,
Missouri, USA). Briefly, 10 mL of heparinized blood
was mixed with an equal volume of RPMI 1640 tis-
sue culture medium (Gibco Laboratories, Grand
Island, NY, USA) containing 2 mM L-glutamine,
10% heat-inactivated foetal calf serum, 20 mM 4-(2-
Hydroxyethyl)piperazine-1-ethanesulfonic acid (HE-
PES-Sigma-Aldrich), and 40 lg/mL gentamicin
(complete medium). Samples were layered over
5 mL Histopaque in a 15-mL conical plastic centri-
fuge tube. After centrifuging at 400 g for 30 min at
room temperature, the interface layer of PBMCs was
carefully aspirated and washed twice with phos-
phate-buffered saline (PBS) containing 0.05 mM
ethylenediaminetetraacetic acid (PBS-EDTA) and
once with complete medium, with centrifugation in
between washes at 300 g for 10 min. Cell viability,
as determined by 0.2% trypan blue dye exclusion,
was >95% in all experiments. Monocytes were
counted using neutral red (0.02%) in the PBMC sus-
pension, and a suspension of 5 9 105 monocytes/mL
in complete medium was employed for cytokine
determination and flow cytometry studies.
Monocyte Culture Supernatants
Monocyte suspensions (5 9 105/mL) were distrib-
uted (1 mL/well) in 24-well flat-bottomed plates
(Nalge Nunc, Rochester, NY, USA). After incubation
for 2 hr at 37°C in a humidified 5% CO2 atmo-
sphere, non-adherent cells were removed by aspira-
tion, and each well was rinsed twice with complete
medium. Monocyte preparations routinely contained
>90% monocytes, as determined by morphological
examination and staining for non-specific esterase.37
Monocytes were incubated with complete medium,
Table I Characteristics of Normotensive and Pre-eclamptic Pregnant Women
Groups
Normotensive Pre-eclamptic
Variable <34 weeks n = 26 ≥34 weeks n = 26 <34 weeks n = 26 ≥34 weeks n = 59
Age (years) 22 (17–40) 23 (15–40) 23 (14–43) 25 (15–43)
Gestational age (weeks) 29 (24–33) 37 (34–40) 31 (24–33) 37 (34–42)
Systolic blood pressure (mmHg) 105 (95–110) 100 (95–110) 160* (140–210) 150* (140–210)
Diastolic blood pressure (mmHg) 65 (60–70) 60 (60–70) 110* (90–140) 100* (90–130)
Proteinuria (mg/24 hr) <300 <300 1550* ** (320–20,160) 720* (300–17,550)
Uric acid (mg/dL) 3.2 (1.6–4.1) 3.4 (1.8–3.9) 5.9* (3.6–10.2) 5.0* (2.4–8.2)
Results are expressed as median (range).
*P < 0.05 compared with both groups of normotensive women (Kruskal–Wallis test).
**P < 0.05 compared with pre-eclamptic women with gestation age ≥34 weeks (Mann–Whitney U-test).
American Journal of Reproductive Immunology 72 (2014) 5–13
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M1 MONOCYTE POLARIZATION IN PRE-ECLAMPSIA
without stimulus, for 18 hr at 37°C in 5% CO2. Cul-
ture supernatants were harvested and stored at
�80°C until assayed for cytokine determination.
Cytokine Determination
Cytokine concentrations in monocyte culture super-
natants were determined by enzyme-linked immu-
nosorbent assay (ELISA), using Quantikine ELISA
kits (R&D Systems, Minneapolis, MN, USA) for TNF-
a and IL-10, and ELISA MAX kits (Biolegend, Inc,
San Diego, CA, USA) for IL-12p40 and IL-12p70
according to the manufacturer’s instructions. Assay
sensitivity limits were 1.6 pg/mL for TNF-a, 3.9 pg/
mL for IL-10, 6.8 pg/mL for IL-p40, and 2 pg/mL for
IL-12p70.
Flow Cytometric Analysis of TLR2, TLR4, CD64,
CD163, and CD206 on Monocyte Surface
Monocyte surface expression of TLR2, TLR4, CD64,
CD163, and CD206 was assessed by flow cytometry
using a FACScalibur flow cytometer with Cell Quest
software (Becton Dickinson, Franklin Lakes, NJ,
USA). PBMCs containing 5 9 105 monocytes/mL
from normotensive and pre-eclamptic women were
distributed into non-adherent polystyrene tubes
(Becton Dickinson-ref 352054) and incubated for
18 hr at 37°C in a humidified 5% CO2 atmosphere
with complete medium. Cells were washed and
incubated with the following monoclonal antibodies
according to the manufacturer’s instructions: R-phy-
coerythrin/CyanineTM7 (PECy7)-labelled anti-CD14
(Biolegend), R-phycoerythrin (PE)-labelled anti-
TLR2 (Biolegend), fluorescein isothiocyanate (FITC)-
labelled anti-TLR4 (Biolegend), FITC-labelled anti-
CD64, PE-labelled anti-CD163 (Biolegend), or FITC-
labelled anti-CD206 (Biolegend). The cells were
incubated for 30 min in the dark at room tempera-
ture, washed, and then fixed with 2% paraformalde-
hyde in PBS. Background staining was determined
by staining cells for 30 min with FITC, PE, or
PECy7-labelled control isotype antibodies at room
temperature in the dark. All samples were then
washed twice with wash buffer containing 10%
endotoxin-free foetal bovine serum (Sigma-Aldrich),
fixed with saline buffer plus 2% paraformaldehyde
in PBS (Sigma-Aldrich) at room temperature for
20 min, and analysed by flow cytometry. Ten thou-
sand monocyte events, defined as cells with respec-
tive side scatter (SSC) and CD14 staining
characteristics, were acquired, and corresponding
levels of TLR2, TLR4, CD64, CD163, and CD206
were obtained from the CD14 cell gate. The results
are expressed as median fluorescence intensity (MFI)
in CD14+ cells analysed.
Statistical Analysis
Comparisons between characteristics of the PE and
NT groups, such as maternal age, gestational age,
serum uric acid, cytokine production by monocytes,
as well as monocyte surface markers, were analysed
by the nonparametric Kruskal–Wallis test. Protein-
uria concentration in early- and late-onset PE was
compared by the nonparametric Mann–Whitney U-
test. All analyses were carried out using INSTAT
3.05 software (GraphPad, San Diego, CA, USA). Sta-
tistical significance was accepted at P < 0.05.
Results
Clinical Characteristics of Pre-eclamptic and
Normotensive Pregnant Women
Characteristics of pre-eclamptic and NT pregnant
women showed no significant differences in relation
to age and maternal age. The concentration of pro-
teinuria was significantly higher in early-onset PE
than in late-onset PE. Serum uric acid levels were
similar in both groups of PE women and showed sig-
nificant differences compared with both groups of
NT pregnant women (Table I).
Production of Pro-Inflammatory Cytokines is
Augmented in Pre-Eclamptic Pregnant Women
Endogenous production of pro-inflammatory cyto-
kines TNF-a, IL-12p40, and IL-12p70 was signifi-
cantly higher while production of the anti-
inflammatory cytokine IL-10 by monocytes from
pre-eclamptic women was lower than in NT preg-
nant women (Fig. 1). Similar results were observed
when the groups with PE were distributed according
to gestational age in early- and late-onset PE. Both
groups of pre-eclamptic women showed significantly
higher levels of TNF-a, IL-12p40, and IL-12p70 com-
pared with NT pregnant women classified by gesta-
tional age (Fig. 2a–c). On the other hand, IL-10
production was significantly lower in both groups of
early- and late-onset PE than in NT pregnant
women (Fig. 2d). There was no significant difference
American Journal of Reproductive Immunology 72 (2014) 5–13
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MEDEIROS ET AL.
between the two groups of pregnant women with
PE, or between the two groups of NT pregnant
women with regard to this basal cytokine produc-
tion.
Expression of Surface Markers on Monocytes of
Pre-eclamptic Pregnant Women
Monocytes from pregnant women with PE expressed
TLR4 and CD64 surface markers with higher expres-
sion than monocytes from NT pregnant women. No
significant differences were observed between these
groups in relation to TLR2 expression. On the other
hand, the MFI of CD163 and CD206 was lower in
the PE group than in the NT group. A representative
histogram with MFI of TLR2, TLR4, CD64, CD163,
and CD206, as well as the results of MFI expressed
by monocytes clearly, shows the differences between
pre-eclamptic and NT pregnant groups (Fig. 3).
When the pre-eclamptic group was classified as
early- and late-onset PE, similar results were
obtained. There were no statistically significant dif-
ferences in TLR2 expression between PE and NT
pregnant women with the same gestational age
(Fig. 4a). The expression of TLR4 and CD64 on the
monocyte surface, both in early- and late-onset PE,
was significantly higher than in the NT group with
equivalent gestational age. TLR4 was significantly
higher in early-onset PE compared with late-onset
PE. (Fig. 4b, c). Although there was a tendency to
higher expression of CD64 in the early-onset PE
group, the values were not significant different in
relation to the late-onset PE group.
The MFI of CD163 expression was significantly
lower in cells from pregnant women with early-
onset PE compared with NT women with gestational
age <34 weeks. No significant differences were
observed between late-onset PE and NT pregnant
women with the same gestational age (Fig. 4d).
Fig. 1 Cytokines produced by monocytes from patients with pre-
eclampsia (PE n = 85) and from normotensive (NT n = 52) pregnant
women cultured for 18 hr without stimulus. The results are expressed
as median (horizontal line), 25th and 75th percentile (box), and range
(whisker). *(P < 0.05) versus normotensive (Mann–Whitney U-test).
(a) (b)
(c) (d)
Fig. 2 Endogenous production of tumour necrosis factor-alpha (TNF-a) (a), interleukin (IL)-12p40 (b), IL-12p70 (c), and IL-10 (d) by monocytes from
patients with pre-eclampsia (PE) classified as early-onset (<34 weeks of gestation, n = 26) or late-onset (≥34 weeks of gestation, n = 59) pre-
eclampsia, and by monocytes from normotensive (NT) pregnant women (<34 weeks of gestation, n = 26) and (≥34 weeks of gestation, n = 26)
cultured for 18 hr without stimulus. The results are expressed as median (horizontal line), 25th and 75th percentile (box), and range (whisker). *
(P < 0.05) versus NT <34 weeks; +(P < 0.05) versus NT ≥34 weeks (Kruskal–Wallis test).
American Journal of Reproductive Immunology 72 (2014) 5–13
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 9
M1 MONOCYTE POLARIZATION IN PRE-ECLAMPSIA
The CD206 receptor was significantly higher
expressed in NT pregnant women in the two gesta-
tional age periods evaluated compared with women
with PE evaluated in the same period. There was no
significant difference between the two groups of NT
pregnant women, nor between those with early- or
late-onset PE (Fig. 4e).
Discussion
Pre-eclampsia is one of the major causes of both
maternal and foetal morbidity and mortality.1 It is
clear that an exaggerated inflammatory response
occurs in this gestational pathology, but until now,
the probable causes remain unknown. Early- and
late-onset PE may differ in disease manifestation,
but so far it is not clear whether these two PE classi-
fications are two distinct disorders or simply a spec-
trum of disease severity across a range of gestational
ages.9
In the present study, urinary protein excretion at
the time of diagnosis was significantly higher in
cases of early-onset PE compared with late-onset PE.
These results confirm that proteinuria together with
blood pressure levels, in addition to being funda-
mental criteria for the diagnosis of PE, are consid-
ered important markers of disease severity.38 Serum
levels of uric acid were similar in both early- and
late-onset PE and were significantly higher than in
NT pregnant women. Hyperuricaemia is a common
finding in PE and is associated with oxidative stress,
TNF-a production, and disease severity.13
Peripheral blood monocytes from pregnant women
with PE are endogenously activated and secrete high
levels of free radicals and inflammatory cytokines,
suggesting their involvement in the disease patho-
physiology.13,14 In the present study, monocytes
from both groups of pregnant women with PE pro-
duced endogenously higher levels of TNF-a, IL-
12p40, and IL-12 p70 compared with the NT group.
Similar results were obtained by Giorgi et al.14 who
studied the production of TNF-a and IL-1b in associ-
ation with activation of nuclear transcription factor-
kappa B (NF-kB) in pregnant women with PE. The
authors showed that both endogenous production of
TNF-a and activation of NF-kB were significantly
(a)
(b)
Fig. 3 (a) Expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), CD64, CD163, and CD206 by monocytes from patients with pre-
eclampsia (PE n = 85) and from normotensive pregnant women (NT n = 52). The results are expressed as median of fluorescence intensity (MFI)
(horizontal line), 25th and 75th percentile (box), and range (whisker). (b) Representative histogram profile of TLR2, TLR4, CD64, CD163, and CD206
expressed by monocytes from patients with pre-eclampsia and by monocytes from normotensive pregnant women. *(P < 0.05) versus
normotensive (Mann–Whitney U-test).
American Journal of Reproductive Immunology 72 (2014) 5–13
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MEDEIROS ET AL.
higher in pre-eclamptic pregnant women. The higher
levels of IL-12p40 and IL-12p70 in pre-eclamptic
pregnant women are in line with the results of Sakai
et al.,39,40 who suggested that increased production of
IL-12 and IL-18 by peripheral blood mononuclear
cells of pre-eclamptic pregnant women contributed to
the dominance of a Th1 profile in this pathology. It
has been reported that the exacerbated inflammatory
response in PE is associated with the release of sub-
stances with inflammatory activity, such as fragments
of the syncytiotrophoblast membranes, foetal DNA,
and soluble microparticles derived from leucocytes
and cytokines, which systemically activate inflamma-
tory cells such as monocytes, granulocytes, and endo-
thelial cells.10,15,18 Increased levels of heat-shock
protein 70 (Hsp70) associated with higher levels of
TNF-a, IL-1b, IL-12, and soluble TNF receptor-I (sTN-
FRI) have been described in serum from women with
early-onset PE compared with those with late-onset
PE.41 The higher levels of Hsp70 may originate from
the ischaemic and oxidatively stressed pre-eclamptic
placenta42 and are released into the circulation in
women with PE by increased shedding of syncytio-
trophoblast membrane microparticles, leading to
peripheral blood cell activation. These results support
a pro-inflammatory role of circulating Hsp70 in PE.43
Expression of TLR4 and CD64 on the surface of
non-stimulated monocytes, both in early- and late-
onset PE, was significantly higher than in NT preg-
nant women with equivalent gestational age. The
expression of TLR4 was also significantly higher in
early-onset PE than in late-onset PE. The high
expression of CD64 and TLR4 on the monocyte sur-
face was associated with increased endogenous pro-
duction of pro-inflammatory cytokines TNF-a, IL-
12p40, and IL-12p70, showing M1 monocyte fea-
tures, in pre-eclamptic pregnant women. These
results confirm the activation state of monocytes in
Fig. 4 Expression of toll-like receptor (TLR)2 (a), TLR4 (b), CD64 (c), CD163 (d), and CD206 (e) by monocytes from patients with pre-eclampsia (PE)
classified as early-onset (<34 weeks of gestation, n = 26) or late-onset (≥34 weeks of gestation, n = 59) pre-eclampsia and by monocytes from
normotensive (NT) pregnant women (<34 weeks of gestation, n = 26 and ≥34 weeks of gestation, n = 26). The results are expressed as median of
fluorescence intensity (MFI) (horizontal line), 25th and 75th percentile (box), and range (whisker). *(P < 0.05) versus NT <34 weeks; +(P < 0.05)
versus NT ≥34 weeks; #(P < 0.05) versus PE ≥34 weeks (Kruskal–Wallis test).
American Journal of Reproductive Immunology 72 (2014) 5–13
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 11
M1 MONOCYTE POLARIZATION IN PRE-ECLAMPSIA
patients with PE. The TLR4 upregulation detected in
pre-eclamptic patients suggests that this pattern-rec-
ognition receptor plays an important role in the
exaggerated systemic inflammatory response seen in
PE.44
The association between increased endogenous
expression of TLR4 and CD64 and higher production
of TNF-a, IL-12p40, and IL-12p70 by monocytes
from patients with PE suggest that these cells can be
activated by endogenous products of maternal origin
and emphasize the participation of TLRs in the sys-
temic inflammatory response. These results imply
that the presence of an inflammatory environment
in PE can lead to monocyte polarization to an acti-
vated M1 profile, resulting in increased production
of pro-inflammatory cytokines by these cells.
Increased expression of mRNA for TLR2 and TLR4 in
neutrophils and increased production of IL-1b by
these cells, associated with higher serum levels of
TNF-a and IL-6, were observed in patients with PE,
suggesting that TLRs may modulate innate immunity
and contribute to the pathogenesis of this disease.45
On the other hand, the lower expression of
endogenous CD163 and CD206 in association with
low production of IL-10 by monocytes from pre-
eclamptic pregnant women compared with NT preg-
nant women confirms that monocytes from both
early- and late-onset PE are classically activated and
differentiated to the M1 phenotype. The expression
of classically activated monocytes or with an M1
profile is described in some diseases associated with
metabolic disorders such as type 2 diabetes33 or with
the involvement of a systemic inflammatory
response such as in sepsis.31
Although previous studies have reported an exces-
sive activation of monocytes in women with PE, as
evidenced by increased production of TNF-a, IL-6,
and IL-8 compared with NT pregnant
women13,20,44,46 and a high concentration of chemo-
kines CXCL10/IP10 in early-onset PE,12 this is the
first work that associates the expression of activation
markers and production of inflammatory cytokines
by monocyte subpopulations from early- and late-
onset pre-eclamptic pregnant women.
In conclusion, the results obtained in this study
confirm that monocytes from women with PE are
endogenously activated and polarized to an M1
inflammatory profile, by higher expression of TLR4,
CD64, and inflammatory cytokine production as well
as lower expression of surface markers of the M2
profile, CD163 and CD206, and IL-10 secretion.
Although monocytes from early-onset PE showed a
higher intensity of TLR4 expression than those from
late-onset PE, the great similarity among the several
parameters studied in both PE groups suggests that
the phenotypic characterization of monocytes cannot
be employed to differentiate these two forms of PE.
Acknowledgments
This work was supported by Fundac�~ao do Amparo �a
Pesquisa do Estado de S~ao Paulo, Brazil (FAPESP,
Grant No. 2009/11924-3).
Conflict of interest statement
The authors declare that there is no conflict of inter-
est.
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