Monocyte Chemoattractant Protein-1 (CCL-2) Integrates
Mechanical and Endocrine Signals That Mediate Term and
Oksana Shynlova,2* Prudence Tsui,*†Anna Dorogin,* and Stephen J. Lye*†‡
Recent evidence suggests that leukocytes infiltrate uterine tissues at or around the time of parturition, implicating inflammation
as a key mechanism of human labor. MCP-1 (also known as C-C chemokine motif ligand 2, CCL-2) is a proinflammatory cytokine
that is up-regulated in human myometrium during labor. Myometrium was collected from pregnant rats across gestation and at
labor. Total RNA and proteins were subjected to real-time PCR and ELISA, respectively. Ccl-2 gene and protein expression was
significantly up-regulated in the gravid rat myometrium before and during labor, which might suggest that it is regulated posi-
tively by mechanical stretch of the uterus imposed by the growing fetus and negatively by physiological withdrawal of progesterone
(P4). We confirmed in vivo that: 1) administration of P4 receptor antagonist RU486 induced an increase in Ccl-2 mRNA and
preterm labor, whereas 2) artificial maintenance of elevated P4 levels at late gestation caused a significant decrease in gene
expression and blocked labor; 3) Ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that
mechanical strain imposed by the growing fetus controls its expression in the myometrium; 4) in vitro static mechanical stretch
of primary rat myometrial smooth muscle cells (25% elongation) induced a release of Ccl-2 protein, which was repressed by
pretreatment with P4 (1 ?M); and 5) stretch enhanced their monocyte chemoattractant activity. These data indicate that Ccl-2
protein serves to integrate mechanical and endocrine signals contributing to uterine inflammation and the induction of labor and
thus may represent a novel target for therapeutic prevention of preterm labor in humans. The Journal of Immunology, 2008, 181:
hibit many characteristics of a systemic inflammatory response,
which can be as strong as the response seen in patients with
sepsis, but it does not seem to harm the mother in any way (1).
Many researchers have shown that leukocytes infiltrate uterine
tissues at or around the time of parturition (2, 3). These leuko-
cytes are the primary cellular source of cytokine production in
gestational tissues (4). Spontaneous labor at term is associated
with the infiltration of inflammatory cells in the cervix, myo-
metrium, chorioamniotic membranes, amniotic cavity of labor-
ing women. This activity is paralleled by increases in the pro-
duction of proinflammatory cytokines (IL-1?, IL-6, TNF-?, and
IL-8) and chemokines (GRO?, G-CSF/G-CSF, GM-CSF, neu-
trophil-activating peptide-1/IL-8, MCP-1/MCP-1) (5). In the
myometrium there is a massive influx of macrophages, neutro-
phils, and T lymphocytes with the onset of labor at term (6).
Studies over the last decade report a significant percentage of
ur understanding of the regulation of the events pre-
ceding human parturition is still incomplete. Recent
evidence suggests that normal pregnant women ex-
preterm births is associated with enhanced immune cell activity
within the womb. Uterine tissues from preterm deliveries (with
and without intrauterine infection) show a correlation between
cytokine levels and the extent of leukocyte infiltration, suggest-
ing a direct link between the host response to infection and the
onset of preterm labor (7, 8). Hence, it is generally accepted that
proinflammatory cytokines play a central role in the mecha-
nisms of term and inflammation/infection-induced preterm par-
Macrophages are known to be commonly present and are the
most widely distributed immune cells in the uterus contributing to
the development of inflammatory response. They account for 10%
and 22% of cells in virgin and pregnant murine uteri, respectively
(9, 10). Macrophages are abundant in the human deciduas during
the first and last trimester of pregnancy (11). In the rodent, uterine
macrophages are distributed throughout the pregnant endo-
metrium, as well as in stroma and connective tissue around muscle
bundles in the myometrium (9, 12). These specialized immune
cells reside and traffic within discrete regions of the pregnant
uterus and may be a source of the cytokine production that
contributes to the local immune process that initiates parturi-
tion. Among proinflammatory cytokines that participate in the
development of inflammatory reaction in myometrium, MCP-1
(also known as chemokine C-C motif ligand 2, CCL-2) expres-
sion was found to be markedly increased in labor as compared
with quiescent pregnant human myometrium (13). CCL-2 levels
are also increased in amniotic fluid and within cervical secre-
tions of women experiencing term and preterm delivery (14–
16). CCL-2 is a member of a large chemokine family of soluble
chemoattractant cytokines, which locally mediate leukocyte
migration into various tissues (17, 18). CCL-2 is produced by a
number of cell types including endothelial cells, fibroblasts,
*Samuel Lunenfeld Research Institute, Mount Sinai Hospital,†Department of Phys-
iology, and‡Department of Obstetrics & Gynecology, University of Toronto, To-
ronto, Ontario, Canada
Received for publication December 27, 2007. Accepted for publication May 9, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by Grant 37775 from the Canadian Institutes of Health
2Address correspondence and reprint requests to Dr. Oksana Shynlova, Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Suite
870, Toronto, Ontario M5G 1X5, Canada. E-mail address: email@example.com
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
monocytes, lymphocytes, smooth muscle cells (SMCs),3and
selected tumor cell lines (19–21). The source and mechanisms
that regulate myometrial CCL-2 expression are unknown. We
hypothesized that CCL-2 may contribute to the initiation or
propagation of normal labor by serving as a chemoattractant for
macrophages in the myometrium and that mechanical stretch of
the uterus imposed by the growing fetus as well as physiolog-
ical withdrawal of progesterone (P4) contribute to its expres-
sion. Our study used a well-characterized in vivo rat model to
test this hypothesis. In this study, we 1) investigated the ex-
pression profile of Ccl-2 in the rat myometrium during normal
pregnancy, spontaneous term labor, and postpartum using real-
time PCR, ELISA, and immunohistology techniques; 2) defined
the role of P4 on the expression of Ccl-2 gene using models of
P4-delayed labor and RU486-induced (P4 antagonist) preterm
labor; 3) examined the effect of gravidity on the expression of
Ccl-2 using a unilateral tubal ligation rat model; 4) investigated
whether mechanical stretch of myometrial SMCs induced Ccl-2
expression in vitro and whether this response is modulated by
P4; and 5) accessed whether stretch-induced Ccl-2 production
by myometrial SMCs resulted in enhanced monocyte chemo-
Materials and Methods
Wistar rats (Charles River Breeding Laboratories) were housed individu-
ally under standard environmental conditions (12 h light/dark cycle) and
fed Purina Rat Chow (Ralston Purina) and water ad libitum. Female virgin
rats were mated with male Wistar rats. Day 1 of gestation was designated
as the day a vaginal plug was observed. The average time of delivery under
these conditions was during the morning of day 23 (between 8 a.m. and
noon). Our criteria for labor were based on delivery of at least 1 pup from
an average number of 16 pups in two uterine horns. The Samuel Lunenfeld
Research Institute Animal Care Committee approved all animal
Normal pregnancy and term labor. Animals were killed by carbon dioxide
inhalation, and myometrial samples were collected on gestational days 0
(nonpregnant), 6, 8, 10, 12, 14, 15, 17, 19, 21, 22, and 23 (labor) or days
1 and 4 postpartum. Tissue was collected at noon on all days with the
exceptions of the labor sample (day 23) that was collected once the animals
(n ? 4) had delivered at least one pup. The part of uterine horn close to
cervix from where fetus was already expelled was removed and discarded;
the remainder was collected for analysis. Postpartum samples were col-
lected at noon at least 24 h after delivery (for day 1 postpartum) or 4 days
after delivery (for day 4 postpartum).
Progesterone-delayed labor. To determine whether maintenance of high
plasma levels of progesterone (P4) might modulate the expression of the
MCP-1 gene near term, pregnant rats were randomized to receive daily s.c.
injections of either P4 (medroxyprogesterone acetate, MPA) at 16 mg/kg in
0.4 ml of sterile saline (Pharmacia) or vehicle starting on day 20 of ges-
tation. Our data indicate similar effects of P4 and MPA on labor prevention.
In our preliminary experiments, we confirmed that both drugs are equally
effective in blocking term labor for at least 24 h, preventing increases in the
expression of myometrial genes (22–25). Animals (n ? 4 at each time point
for each treatment) were killed on days 21–23 during labor in the vehicle-
treated group or days 21–24 in the P4-treated group.
RU486-induced preterm labor. On day 19 of gestation, two groups of rats
were treated with either RU486 (mifepristone 17?-hydroxy-11?-[4-di-
methylaminophenyl]-17-[1-propynyl]-estra-4,10-dien-3-one (Biomol), 10
mg/kg, s.c. at 10 a.m., in 0.5 ml of corn oil containing 10% ethanol) or with
vehicle. Myometrial samples were collected from RU486-treated animals
after delivery of at least one pup on day 20, or at the equivalent gestational
day 20 in control rats (n ? 4 for control and RU486-treated group).
Unilaterally pregnant rats. Under general anesthesia, virgin female rats
underwent tubal ligation through a flank incision to ensure that they sub-
sequently became pregnant in only one horn (26). Animals were allowed to
recover from surgery for at least 7 days before mating. Pregnant myome-
trial samples from empty and gravid horns were collected on days 6, 12, 14,
15, 17, 19, 21, 22, and 23 or day 1 postpartum (n ? 4 animals at each time
point for each treatment).
Animals were killed by carbon dioxide inhalation. For RNA and protein
extraction, the uterine horns were placed into ice-cold PBS, bisected lon-
gitudinally, and dissected away from both pups and placenta. The endo-
metrium was carefully removed from the myometrial tissue by mechanical
scraping on ice, which we have previously shown removes the entire lu-
minal, glandular epithelium, and the majority of the uterine stroma (27).
The myometrial tissue was flash-frozen in liquid nitrogen and stored at
?70°C. The whole uterus (both horns) used in each specific experiment
was crushed under liquid nitrogen, and the whole RNA or protein were
extracted from every myometrial sample to prevent any intra-animal vari-
ations. For each day of gestation, tissue was collected from four different
Real-time PCR analysis
Total RNA was extracted from the frozen rat tissues using TRIzol (Life
Technologies) according to the manufacturer’s instructions. RNA samples
were column purified using RNeasy Mini kit (Qiagen), and treated with 2.5
?l of DNase I (2.73 Kunitz unit/?l; Qiagen) to remove genomic DNA
contamination. Reverse transcription and real-time PCR were performed to
detect the mRNA expression of MCP-1 in rat myometrium using specific
set of primers (see Fig. 1) as described earlier (22). Real-time PCR was
3Abbreviations used in this paper: SMC, smooth muscle cell; Ct, cycle threshold;
MPA, medroxyprogesterone acetate.
throughout gestation. A, Total RNAs were extracted from frozen myome-
trial tissues, single-stranded cDNAs were synthesized as described in Ma-
terials and Methods, and mRNA levels were analyzed on the indicated
days of gestation by real-time PCR. Specific forward and reverse primers
were designed using Primer Express software (version 2.0.0; Applied Bio-
systems) as follows: MCP-1 mRNA, 5?-CTCTTGAGCTTGGTGACA
AATACT-3? (sense) and 5?-CGGCTGGAGAACTACAAGAGA-3? (anti-
sense) (GenBank accession no. NM_031530); and 18S, 5?-GCGA
GTCGGAAC-3? (antisense) (GenBank accession no. X01117). MCP-1
mRNA levels were normalized to 18S mRNAs and expressed as fold
change relative to a corresponding nonpregnant (NP) sample. Data are
mean ? SD (n ? 4 rats at each time point). Results labeled with different
letters are significantly different from each other. ???, p ? 0.001. B, Im-
munoreactive (IR) Ccl-2 protein levels were measured by ELISA within
nonpregnant, pregnant, and postpartum (PP) rat myometrium as expressed
in picograms per milliliter. Data shown are mean ? SD (n ? 3–4 rats at
each time point). A significant difference is indicated. ?, p ? 0.05.
Ccl-2 transcript and protein levels in the rat myometrium
1471 The Journal of Immunology
performed with an ABI PRISM 7900 HT Sequence Detection System (Ap-
plied Biosystems), using the SYBR Green detection chemistry. A cycle
threshold (Ct) value was recorded for each sample. PCR were set up in
triplicates and the mean of the three Cts was calculated. Relative quanti-
tation of gene expression was the approach to compare differences of gene
expression across gestation. An arithmetic formula from the comparative
Ct method (per Applied Biosystems User Bulletin No. 2) was applied to the
raw Ct values to extract relative gene expression data. mRNA level from
each rat myometrial sample was normalized to ribosomal 18 S mRNA. All
rat normal gestational mRNA levels were expressed as a fold change rel-
ative to the nonpregnant mRNA level. Gene expression for tubal-ligated
animals was shown as the fold change relative to day 6 gravid horn mRNA
level and gene expression for MPA- and RU486-treated animals was
shown as the fold change relative to the vehicle.
MCP-1 protein measurement (ELIZA)
Frozen myometrial tissue was crushed under liquid nitrogen using a mortar
and pestle. Crushed tissue was homogenized in ELISA lysis buffer (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl), supplemented with 100 ?M sodium
orthovanadate and protease inhibitor cocktail tablets (Complete Mini;
Roche). Samples were spun at 12,000 ? g for 15 min at 4°C, the super-
natant was transferred to a fresh tube to obtain a crude protein lysate and
stored at ?20°C until assayed. Protein concentrations were determined
using the Bio-Rad protein assay buffer (Bio-Rad). Culture supernatants
from stretched and control (nonstretched) cells were collected and stored at
?20°C for assay. MCP-1 protein concentration was measured in duplicate
in myometrial tissue supernatants and in medium conditioned by primary
rat myometrial cells using an ELISA for rat MCP-1 (Endogen rat MCP-1
ELISA kit; Pierce), according to the manufacturer’s protocol (range 0 to
1500 pg/ml). The optimal sample protein content for the measurement of
immunoreactive MCP-1 was established by serial dilution. A total of 100
?g of protein from tissue homogenates of each gestational day or 1 ?l of
stretch-conditioned medium were used for MCP-1 assay.
The formalin-fixed myometrial tissues were gradually dehydrated in etha-
nol and embedded in paraffin. Sections of 5-?m thickness were collected
on Superfrost Plus slides (Fisher Scientific). Paraffin sections were depar-
affinized and rehydrated. After immersion in 3% hydrogen peroxide (Fisher
Scientific) the sections were retrieved in 0.125% trypsin solution at room
temperature for 10 min for CD68 Ab or microwaved for 10 min in 0.001
M sodium citrate for MCP-1 Ab. Abs were blocked with Protein Serum-
Free Blocking solution (DAKO-Cytomation) and incubated with primary
Abs overnight. Primary Abs used to label macrophages were mouse anti-rat
CD68 (1/1000; Serotec) and goat polyclonal anti-MCP-1 (1/100; Santa
Cruz Biotechnology). For the negative controls, ChromPure nonspecific
mouse IgGs and nonspecific goat IgGs (Santa Cruz Biotechnology)
were used at the same concentration as primary Abs. Secondary Abs
rat myometria during gestation. Immunohistochemical
examination was performed on sections of uterus from
nonpregnant (NP) (A), 8 days (B), 15 days (C), or 20
days (D) pregnant, laboring (day 23) (E) and 1 day post-
partum (F) animals. Expression of Ccl-2 protein (ar-
rows) increased significantly during late gestation (D
and E). The lack of immunostaining after incubation of
myometrial tissue with nonspecific rabbit IgG on gesta-
tional day 14 (G) or with anti-rabbit secondary Abs in
the absence of primary Ab on laboring day 22 (H) is
shown. Magnification is at ?200. Scale bar represents
MCP-1 immunolocalization in pregnant
1472 MCP-1 IN PREGNANT MYOMETRIUM
used for detection of CD68 were biotinylated anti-mouse (1/300; DAKO-
Cytomation) and donkey anti-sheep HRP-conjugated (1/1000; Serotec)
for MCP-1. Final visualization was achieved using Vectastain Elite
kit (Vector Laboratories). Counterstaining with Harris’ hematoxylin
(Sigma-Aldrich) was conducted before slides were mounted with Cy-
toseal XYL (Richard-Allan Scientific). For the assessment of staining
intensity, myometrial cells from each of the two sets of tissues were
observed on a Leica DMRXE microscope (Leica Microsystems). A
minimum of five fields were examined for each gestational day and
uterine horn for each set of tissue, and representative tissue sections
were photographed with Sony DXC-970 MD 3CCD color video camera.
Myometrial SMC isolation and culture
Primary rat myometrial SMC isolation was performed as previously de-
scribed (28). Briefly, cells were harvested from rat uteri by collagenase
treatment and subjected to a differential attachment technique to select for
SMCs. Freshly isolated myometrial SMCs were directly seeded on to
6-well flexible-bottom culture plates coated with collagen I (Flexcell In-
ternational) at a plating density of 3 ? 106cells/well. The cells were grown
to confluence within 3–4 days in phenol red-free DMEM (Life Technol-
ogies) supplemented with 10% FBS (CanSera), 25 mM HEPES, 100 U/ml
penicillin/streptomycin (Life Technologies), and 2.5 ?g/ml amphotericin B
(Sigma-Aldrich). We have previously shown that uterine cells maintain a
smooth muscle phenotype beyond 4 days in culture (28). Therefore, all
stretch experiments conducted in this study were performed on day 4.
Application of static stretch
Static stretch was applied using a Flexcell Strain Unit (FX-3000; Flexcell
International). The stretch unit consists of a computer-controlled vacuum
unit and a base plate to hold the 6-well culture dishes, which is placed in
a humidified incubator with 5% CO2at 37°C. Static strain was applied by
deforming the membrane with 150 mm Hg of vacuum pressure, which
produces a maximal 25% stretch equal to the average 10% elongation (29).
Control cells were cultured under identical conditions but remained sta-
tionary. Before exposure to mechanical stretch, the confluent cells were
incubated for 24 h in serum-free DMEM. We have previously reported that
1 ?M P4, a dose mimicking the levels present during late pregnancy, both
delayed and reduced the stretch induction of several members of the AP-1
family of genes in uterine SMCs (30). Thus, for the P4 studies, cell mono-
layers were preincubated with hormone (1 ?M; Sigma-Aldrich) for 24 h
before the application of stretch. Myometrial cells were then stretched for
2–24 h, and the supernatants were collected. Immunoreactive CCl-2 pro-
tein content in stretched-conditioned medium with and without P4 was
measured by ELISA.
metrium before the onset of parturition. Photomicro-
graph of macrophages in the nonpregnant (A), pregnant
(B–E), and postpartum rat uterus. Macrophages (arrows)
stained dark brown in paraffin-embedded tissue sections
that were counterstained a blue with hematoxylin. Mac-
rophage infiltrated into the vascular plexus between cir-
cular and longitudinal myometrial layers in nonpregnant
(A) and day 8 (B) groups and in the circular myome-
trium (M) of day 15 (C) and day 20 (D) groups. Mac-
rophages were evident in the myometrium-decidua junc-
tion (MDJ) peripartum (day 23) (E) and postpartum (day
1) (F). The lack of immunostaining after incubation of
myometrial tissue with nonspecific goat IgG on gesta-
tional day 22 (G) or with anti-goat secondary Abs in the
absence of primary Ab on day 1 postpartum (H) is
shown. Magnification is at ?200. Scale bar represents
Macrophage infiltration into the rat myo-
1473 The Journal of Immunology
Isolation of rat macrophages
Primary rat macrophage isolation was performed as previously described
(31). Wistar rats (12- to 20-wk-old males) were injected with 20 ml of 2%
glycogen (Sigma-Aldrich). After 4 days, macrophages were harvested from
the peritoneal cavity by washing with ice-cold PBS containing heparin (0.5
U/ml; Sigma-Aldrich) and centrifuged at 1000 rpm for 10 min. The pellet
was immediately suspended in sterile water and centrifuged. The pellet was
then resuspended in incubation medium DMEM (Life Technologies) sup-
plemented with 0.5% BSA. The cells obtained were ?95% macrophages
according to CD68 staining, and viability was found to be 93% by trypan
blue dye exclusion. The cells at a density 1 ? 106cells/ml were used in the
migration assay or plated on culture plates (4 million/plate) in DMEM
containing 10% heat-deactivated FBS to adhere overnight. The cells were
washed next day, and medium was replaced with DMEM containing 10%
heat-deactivated FBS with PMA (100 nM) to induce adherence and dif-
ferentiation. After 3 days, medium was changed to fresh PMA plus M-CSF
(100 ng/ml) (32).
Determination of chemotactic activity
Chemotaxis assay was performed on freshly isolated rat monocytes using
Fluorometric Cell Migration Assay kit with polycarbonate membrane in-
serts (5 ?m pore size; Cell Biolabs). In pilot studies, primary rat monocyte
migration was low in serum-free medium (negative control) and was in-
duced by medium with 10% FBS (positive control). To determine whether
stretch-induced myometrial cell Ccl-2 production resulted in enhanced
monocyte chemoattractant activity, serum-deprived SMCs were exposed to
stretch for 24 h, and the conditioned medium was collected. Aliquots of
monocyte suspension (2 ? 105cells) were placed inside the insert and
stretched conditioned medium to the outside. To determine whether Ccl-2
secreted from mechanically stimulated SMCs induced chemotaxis of rat
monocytes, we incubated stretch-conditioned medium with general viral
C-C chemokine-binding protein inhibitor vCCI (500 ng/ml; BD Bio-
sciences) at room temperature for 2 h. Cells were allowed to migrate for
mRNA expression. Real-time PCR analysis of Ccl-2 mRNA levels in preg-
nant rat myometrium during MPA-blocked (A) and RU486-induced (B)
preterm labor normalized vs 18 S mRNA. Levels expressed in fold change
relative to a vehicle day 21 (A) or day 20 (B) sample. Shown are vehicles
(f), MPA-treated (?), or RU486-treated (vertical hatched) samples. Data
represent mean ? SD (n ? 4 rats in each group at each time point). A
significant difference is indicated. ?, p ? 0.05 or ???, p ? 0.001.
The effects of MPA and RU486 on myometrial Ccl-2
ing and macrophage infiltration is up-regulated during
RU486-induced preterm labor. Immunohistochemical
examination was performed on sections of uterus from
normal 20 days pregnant vehicle (A, C, and E) and 20
days preterm laboring (B, D, and F) animals. Expression
of Ccl-2 protein increased considerably in the myome-
trium and uterine epithelium (arrows) during RU486
preterm labor (A and B); it was associated with massive
macrophage infiltration (arrows) in preterm laboring
myometrium (C and D). The negative controls were per-
formed on vehicle day 20 (E) and RU486-treated (F)
uterine tissue samples. Magnification is at ?200. Scale
bar represents 50 ?m.
Myometrial Ccl-2 protein immunostain-
1474MCP-1 IN PREGNANT MYOMETRIUM
1–3 h in a cell culture incubator. Solution containing cells that migrated
through the membrane and into the medium, and migratory cells detached
from the bottom side of the membrane was lysed and detected by the
patented CyQuant GR dye (Invitrogen). Fluorescence measurement was
performed in a Wallace Victor-2 1420 Multilabel counter (PerkinElmer)
with a 490/535 nm filter set.
Gestational profiles were subjected to a one-way ANOVA followed by
pairwise multiplecomparison procedures
method) to determine differences between groups. Data from in vivo MPA
(days 21–23) and tubal ligation study as well as in vitro migration, stretch,
and progesterone were analyzed by two-way ANOVA followed by pair-
wise multiple comparison procedures as described. The day 24 MPA-
treated group was compared with the day 23 vehicle group using a t test.
RU486 and vCCI inhibitor results were compared with vehicle using a
one-way ANOVA. Where required the data were transformed by the ap-
propriate method to obtain a normal distribution. Statistical analysis was
conducted using SigmaStat (version 2.01; Jandel) with the level of signif-
icance for comparison set at p ? 0.05.
Ccl-2 expression in nonpregnant, pregnant, and postpartum rat
Fig. 1A illustrates the expression of Ccl-2 gene throughout preg-
nancy and postpartum in the rat. Relative abundance of the Ccl-2
mRNA was low at early gestation, significantly increased at late
gestation (15.6 ? 1.1 fold increase on day 21 vs nonpregnant sam-
ples, n ? 4; p ? 0.05), before labor (41.1 ? 3.3 fold increase on
day 22, n ? 4; p ? 0.001), and at labor (48.9 ? 9.8 fold increase
on day 23 vs nonpregnant, n ? 4; p ? 0.001). Transient increase
in Ccl-2 was followed by a quick decrease in postpartum period
(12.7 ? 1.1 fold increase on day 1 postpartum vs nonpregnant; p ?
0.05 and 4.0 ? 0.4 fold change on day 4 postpartum vs nonpreg-
nant, n ? 4). We confirmed that the induction of Ccl-2 gene during
labor was correlated with the increase in the immunoreactive Ccl-2
protein content in myometrial tissue supernatant. As shown on Fig.
1B, the immunoreactive Ccl-2 protein was significantly up-regu-
lated in term pregnant and laboring rat myometrium compared
with nonpregnant sample (p ? 0.05) and decreased abruptly
In situ localization of Ccl-2 protein revealed that this chemokine
was expressed by myometrial SMCs (Fig. 2). Immunostaining of
Ccl-2 protein in uterine smooth muscle of nonpregnant, early preg-
nant, and mid-pregnant animals was weak (Fig. 2, A–C). However,
after gestational day 20, Ccl-2 protein immunoreactivity of the rat
myometrium increased dramatically (Fig. 2D). Consistent with our
gene and protein expression results, the most intense staining was
found in labor samples (Fig. 2E). Ccl-2 protein was always de-
tected in the cytoplasm of myometrial SMCs and this spatial dis-
tribution was similar in both longitudinal and circular uterine mus-
We also attempted to examine whether Ccl-2 protein expression
indeed correlates with macrophage infiltration into the rat myome-
trium before the onset of parturition. Uterine tissue sections
throughout gestation were immunostained with specific anti-CD68
Abs for rat macrophage (Fig. 3). According to our observation in
the nonpregnant uterus, CD68-positive cells were diffusely present
pregnant rats during gestation. mRNA levels were analyzed on the indi-
cated days of gestation by real-time PCR using specific primers (see Fig.
1). Ccl-2 gene expression levels in empty (?) and gravid (f) were nor-
malized to 18 S mRNAs and expressed in fold changes relative to a day 6
gravid sample. Data represent mean ? SD (n ? 4 rats per group at each
time point). A significant difference between gravid and empty horn of the
same gestational day is indicated. ?, p ? 0.05.
Expression of Ccl-2 gene in the myometrium of unilaterally
cells. Myometrial SMCs were grown on collagen-coated Flexcell plates until confluent. After 24 h of serum starvation, SMCs were stretched for different
time intervals (2–24 h), and conditioned medium was collected from stretched (SCM, stretch-conditioned medium (f) and static (NSM, nonstretched
medium) (?) cultures. ELISA showed that 24 h of static mechanical stretch caused a significant accumulation in immunoreactive (IR) Ccl-2 (45% increase
in stretched compared with nonstretched medium cultures; p ? 0.05). The immunoreactive Ccl-2 content in nonstretched control medium cultures pretreated
with 1 ?M P4 for 24 h (o) and medium from SMCs pretreated with P4 and stretched for 24 h (p) was significantly lower than nonstretched and stretched
control medium from SMCs at all experimental time points. Data represent mean ? SD (n ? 3–4 at each time point). Data labeled with different letters
are significantly different from each other. ?, p ? 0.05; ??, p ? 0.01; or ???, p ? 0.001.
The effect of static mechanical stretch and P4 on immunorreactive Ccl-2 protein levels in medium conditioned by primary rat myometrial
1475The Journal of Immunology
in the densely packed tissue (Fig. 3A). During pregnancy, labeled
cells were sparsely distributed in both myometrial layers but were
concentrated along the vascular plexus (between longitudinal and
circular muscle layers), often near blood vessels. The number of
macrophages was increased in the rat myometrium at term (Fig. 3,
D and E). CD68-labeled cells appeared mostly to surround the
circular muscle bundles and clustered between longitudinal muscle
fascicles. Importantly, macrophage numbers remained elevated in
the postpartum group within 24 h; macrophage infiltration was
apparent within the myometrium, along myometrium-decidua
junction and in decidual stroma (Fig. 3F).
Modulation of Ccl-2 gene and protein expression by
Animals treated from day 20 with daily injections of stable syn-
thetic analog of progesterone, MPA failed to initiate labor on day
23 and did not show the expected increase in expression of Ccl-2
mRNA (Fig. 4A). Although there was no significant difference
between the control and MPA-treated groups on days 21 and 22 (1
or 2 days after injection), Ccl-2 mRNA levels in rats treated with
hormone were significantly lower on day 23 (p ? 0.001) com-
pared with the control group. Moreover, transcript levels of Ccl-2
in MPA-treated rats remained low on day 24 (1 day after normal
time of delivery) compared with that in vehicle-treated animals on
day 23 (laboring sample) (p ? 0.001). On the contrary, the ad-
ministration of P4 receptor antagonist, RU486 at day 19 of gesta-
tion induces preterm labor within 24 h and significant 8.8-fold
increase (p ? 0.05) in Ccl-2 mRNA levels (Fig. 4B), enhanced
Ccl-2 protein immunostaining (Fig. 5B) and was associated with
substantial macrophage infiltration (Fig. 5D). Our results demon-
strate that a sudden artificial blockade of P4 signaling during late
pregnancy caused by RU486 led to Ccl-2 gene and protein induc-
tion, whereas maintenance of elevated plasma P4 levels prevented
this induction and normal term labor.
Gravidity modulates the expression of Ccl-2 in unilaterally
pregnant rat myometrium
Because increases in Ccl-2 gene expression occurred immediately
before labor when mechanical stretch of uterine walls imposed by
growing fetuses is maximal, we decided to investigate whether
myocyte stretch could modulate Ccl-2 transcript levels. We used
an in vivo unilateral pregnant rat model that enabled us to assess
interactions between endocrine and mechanical stimuli because
both gravid and empty horns were subjected to the same endocrine
environment (Fig. 6). Ccl-2 gene in the empty horn was expressed
at very low levels throughout gestation. In contrast, Ccl-2 tran-
script levels were dramatically increased at term in the gravid uter-
ine horns compared with the empty horn (days 21–23; p ? 0.05),
showing a profile similar to that of normal pregnant animals.
Stretch induces Ccl-2 protein secretion in medium conditioned
by primary rat myometrial cells
To further determine whether mechanical forces modulated Ccl-2
expression, we used an in vitro model, in which primary rat myo-
metrial SMCs are subjected to stretch through the use of a com-
puter-driven vacuum system. Analysis of medium conditioned by
SMCs revealed that 24-h static mechanical stretch caused a sig-
nificant accumulation (p ? 0.05) in immunoreactive Ccl-2 (45%
increase compared with nonstretch control medium) (Fig. 7). The
rise in Ccl-2 protein levels was preceded by a transient increase on
Ccl-2 mRNA (data not shown). Our in vivo experiments revealed
that treatment of pregnant rats with P4 prevents the induction of
Ccl-2; stretch-induced Ccl-2 expression was blocked in vitro by
pretreatment of SMCs with P4 (p ? 0.05 and p ? 0.001) (Fig. 7).
Progesterone also reduced basal Ccl-2 expression in nonstretched
(control) SMCs (Fig. 7).
Stretch enhances monocyte chemotaxis via a Ccl-2-dependent
We next asked whether the increased secretion of immunoreactive
Ccl-2 protein in response to stretch is capable of stimulating che-
motaxis of monocyte cells. The migration of primary rat mono-
cytes in response to conditioned medium from stretched myome-
trial SMCs was much greater than the migration of conditioned
medium from nonstretched cells or negative control (50–70% in-
duction, p ? 0.05) (Fig. 8). Monocyte chemotaxis in response to
stretch-conditioned medium was blocked by vCCI, a CCR2 che-
mokine receptor antagonist, demonstrating involvement of this
Ccl-2 receptor (p ? 0.05) (Fig. 8B). These results suggest that the
stimulation of chemotaxis by the conditioned medium was caused,
at least in part by biologically active Ccl-2 synthesized and se-
creted by myometrial SMCs following mechanical stimulation. It
monocytes. A, Conditioned medium from primary rat SMCs stretched for
24 h (SCM, stretch-conditioned medium) (f) and static cultures (NSM,
nonstretched medium) (?) were collected. Serum-free medium was used
as a negative (NEG) control (vertical hatched). Negative control, stretched,
or nonstretched medium from primary rat SMCs was then placed in the
bottom chamber of the migration plate. A total of 200,000 primary mono-
cytes were placed to the inside of 5-?m pore insert and migrated through
the pores for an indicated time. Migrated monocytes were lysed and de-
tected. The number of migrating cells was calculated by relative fluorescent
units. Data are expressed as the mean ? SE of at least three separate
experiments. ?, p ? 0.05 significant difference from negative control. B,
MCP-1 secreted from SMCs in response to stretch is biologically active.
Stretch-conditioned medium (f) was incubated with the MCP-1 receptor
antagonist vCCI (500 ng/ml) at room temperature for 2 h (horizontal
hatched). A total of 200,000 primary activated macrophages were placed to
the inside of 5-?m pore insert, migrated through the pores, and detected
fluorometrically after 1 h of incubation. The number of cells migrating
toward stretch-conditioned medium with and without vCCI were indicated
relative to nonstretch medium. Data are expressed as the mean ? SD of
three separate experiments. ?, p ? 0.05.
Mechanical stretch promotes chemotaxis of primary rat
1476MCP-1 IN PREGNANT MYOMETRIUM
also shows that increased migratory characteristics of primary rat
macrophages involved specific activation of the CCR2 receptor.
Our data support the hypothesis that the myometrium plays an
important role in the generation and regulation of uterine inflam-
mation, which is a characteristic feature of parturition. We provide
in this study substantial evidence that myometrial cells can ac-
tively participate in the inflammatory process in the uterus by the
release of proinflammatory mediator CCL-2. Firstly, we have dem-
onstrated that myometrial SMCs in vivo are able to actively syn-
thesize Ccl-2 protein during gestation and that its expression was
significantly up-regulated before and at labor. Secondly, the in-
creased accumulation of Ccl-2 mRNA and increased production of
immunoreactive Ccl-2 protein in term myometrium was associated
with uterine occupancy and regulated by P4, suggesting that me-
chanical and endocrine signals integrate to regulate expression of
this chemokine and the induction of labor in vivo. In support of
this assumption, we found that the release of Ccl-2 protein in the
cell culture supernatant by isolated myometrial SMCs in vitro was
stimulated by artificial mechanical stretch and suppressed by P4. In
addition, we confirmed that stretch-conditioned cell culture me-
dium was able to increase migratory ability of primary rat macro-
phages, suggesting a potential role of biological mechanical stretch
in the induction of leukocyte infiltration in term myometrium.
Inflammation has been implicated in the process of human par-
turition (5). Leukocytes are known to infiltrate uterine tissue and
their migration is regulated by chemokines, an ever-growing fam-
ily of chemotactic cytokines. CCL-2 is a prototypical CC chemo-
kine that recruits monocytes (but not neutrophils), and also T lym-
phocytes and NK cells from the bloodstream into sites of
inflammation (33). In the reproductive system, Ccl-2 protein is
produced by trophoblasts, decidual, endometrial, and myometrial
cells and is found elevated on the first day of pregnancy in the
mouse uterus and within the human late gestation myometrium
(19, 34–36). Endometrial production of Ccl-2 by eosinophils dur-
ing early gestation may play a central role in implantation or pla-
centation that is crucial for successful establishment of ovine preg-
nancy (37). Expression of Ccl-2 gene and accumulation of
monocytes/macrophages is also increased in the corpus lutea of
pregnant rats before parturition, suggesting a role in luteal regres-
sion (38). There is very limited information about Ccl-2 expression
in the myometrium. Sozen et al. (39) have shown that Ccl-2 tran-
script levels in human nonpregnant myometrium were higher in
the secretory than proliferative phase. Moreover, the same authors
demonstrated lower Ccl-2 mRNA expression in leiomyoma cells
than cells in controls, suggesting a protective role for this chemo-
kine in healthy myometrium. Esplin and colleagues (13) also re-
ported that human Ccl-2 gene and immunoreactive protein levels
were up-regulated in the term laboring myometrium as compared
with quiescent term pregnant myometrium. The present study is
the first to define the expression profile of Ccl-2 transcript in the
myometrium throughout gestation. Using an immunohistological
approach we confirmed that Ccl-2 protein was expressed predom-
inantly by rat myometrial SMCs. Because there is evidence that
chemokines from the MCP subfamily of cytokines are likely to
play a critical role in the regulation of the inflammatory response
in other type of SMCs (40), our findings demonstrate that myo-
metrial cells may contribute directly to the development of uterine
inflammation by promoting the recruitment of monocytes to term
myometrium, which is one of the hallmarks of parturition.
We next addressed the mechanisms regulating CCL-2 induction
in the myometrium. Mechanical stretch of the uterus by growing
fetus has been shown earlier to stimulate the expression of genes
involved in the onset of labor (26, 41). Now we provide direct
evidence that Ccl-2 expression is stimulated by mechanical stretch
in vitro and that the increased expression of Ccl-2 in vivo in the
gravid uterine horn at term likely reflects this mechanical stimu-
lation. Mechanical stress has been demonstrated to induce cortical
expression of Ccl-2 as well as renal cortical macrophage infiltra-
tion in an experimental model of unilateral ureteral obstruction
(42). In vitro studies have also shown up-regulation of CCL-2 by
mechanical stretch in human mesangial and endothelial cells (17,
43). Importantly, we provide evidence that the CCR2 chemokine
receptor antagonist vCCI (44) inhibited the increased migration of
activated primary rat macrophages, suggesting a role for the MCP-
1/CCR2 signaling pathway in the stretch-induced macrophage
Progesterone is the major hormone of pregnancy. In virtually all
species a fall in tissue or plasma P4 levels is a critical event before
the onset of labor. In contrast, in the human there is no decrease in
circulating P4 levels with the onset of labor. However, multiple
studies suggest a functional withdrawal of P4 mediated by mech-
anisms such as changes in progesterone receptor isoforms (45),
reduction in progesterone receptor transactivation (46), or in-
creases in progesterone receptor repressors (47). Removal of the
source of P4 by ovariectomy or administration of a progesterone
receptor antagonist (e.g., RU486) causes termination of pregnancy
in animals (26, 48). We were able to modulate Ccl-2 gene expres-
sion in term pregnant rat myometrium by maintaining high levels
of stable synthetic analog of hormone (MPA) in maternal blood or
by inhibiting P4 receptor signaling by RU486. Blockade of P4
signaling on day 19 increases Ccl-2 transcript levels and Ccl-2
protein immunoreactivity, mimicking changes detected in term
myometrium. Importantly, we detected a massive macrophage in-
filtration during RU486-induced preterm labor. It is plausible to
speculate that rapid development of uterine inflammation facili-
tated the activation of the myometrium and subsequent preterm
labor contractions. We also found a direct inhibitory effect of P4
on the expression of Ccl-2 gene and immunoreactive Ccl-2 protein
in cultured primary myometrial SMCs. These data correspond well
with previous findings by Sozen and colleagues (39) reporting that
P4 may down-regulate Ccl-2 expression within nonpregnant myo-
metrium. They observed that an absence of sex steroids led to a
pronounced elevation of CCL-2 within nonpregnant human myo-
metrium and seemed to inhibit cell proliferation in the tissue while
attracting and activating macrophages. It was also reported that P4
was able to significantly down-regulate the expression of IL-8 in
amnion, chorion cells, and lower segment fibroblasts (49, 50). We
propose that the decrease in P4 signaling might be responsible for
an increase in the expression of CCL-2 in laboring human and rat
We suggest a hypothetical model of leukocyte recruitment into
pregnant myometrium. Thus during late gestation hormonal and
mechanical stimuli enhance Ccl-2 expression and secretion by
uterine SMCs. Increased chemokine levels in uterine tissue recruit
circulating monocytes from the local vasculature by chemotaxis
along a concentration gradient. We have confirmed that up-regu-
lation of Ccl-2 levels during normal term labor coincide with in-
creased infiltration of CD68-positive immune cells in the rat myo-
metrium. It is known that chemokines (including CCL-2) play key
roles in both homing of leukocytes to specific regions within a
tissue and activation of immune cells (reviewed in Ref. 51). Ac-
tivated macrophages are able to release 1) matrix metalloprotein-
ases likely contributing to cervical ripening and to the rupture of
the gestational membranes; 2) PGs, histamine, or serotonin, capa-
ble of exerting a direct uterotonic effect; and 3) cell adhesion mol-
ecules (6, 33, 52, 53). Other proinflammatory cytokines released
1477The Journal of Immunology
by the macrophages and other cell types may also contribute to this
process by promoting further leukocyte invasion (2). For instance
the prototypical CXC chemokine, IL-8, is significantly up-regu-
lated during active labor in women (54), contributing to an inflam-
matory reaction. Therefore, increased production of chemokines
by term myometrium could represent an initial step in the chain of
events preparing uterine tissue for labor by actively promoting the
chemotaxis of monocytes and other immune cells (neutrophils) for
the development of myometrial inflammation. We cannot rule out
the possibility that induction of Ccl-2 gene could be triggered by
other factors derived from maternal decidua or fetus itself. Further
studies are required to investigate this possibility.
We detected a very high level of macrophage infiltration into the
uterus during the early postpartum period. We speculate that in
addition to promoting labor, myometrial induction of CCL-2 may
also represent a mechanism regulating the process of postpartum
involution of uterine tissue. Increased numbers of CD68-positive
cells were localized close to the endometrium-myometrium junc-
tion. Postpartum uterine involution is a critical event because it
completes the reproductive cycle following pregnancy and labor
by returning the uterus to its nonpregnant state so that the females
can remain fertile. Uterine involution involves several processes
similar to wound healing, specifically substantial tissue reorgani-
zation, matrix metalloproteinase induction, extracellular matrix
degradation, and apoptosis. CCL-2 production might greatly en-
hance these processes. However, we cannot exclude the possibility
that other chemokines shown to be critical for inflammatory en-
dometrial destruction during menstruation (MCP-3, Eotaxin, FNK,
MIP-1? (51)) also play an important role in postpartum decidual
breakdown and involution of myometrium.
This study is the first to demonstrate the expression and release
of Ccl-2 from myometrial cells throughout gestation. Our findings
support the hypothesis that uterine SMCs may play an active role
in uterine inflammation by producing chemokines and promoting
the chemotaxis of immune cells into the myometrium conse-
quently generating and regulating an inflammatory response of
uterine tissue. Better understanding of the mechanisms directing
these inflammatory events might inform the development of new
therapeutic strategies for the management of preterm labor, which
remains a leading cause of neonatal morbidity and mortality.
We gratefully acknowledge Dr. B. L. Languille for his continuing scientific
input and help with the discussion of the manuscript as well as the assis-
tance of Julie Wright for chemotaxis assay.
The authors have no financial conflict of interest.
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1479 The Journal of Immunology