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 positively 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 μM); 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.
Our understanding of the regulation of the events preceding human parturition is still incomplete. Recent evidence suggests that normal pregnant women exhibit 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 leukocytes 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, myometrium, chorioamniotic membranes, amniotic cavity of laboring women. This activity is paralleled by increases in the production of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-8) and chemokines (GROα, G-CSF/G-CSF, GM-CSF, neutrophil-activating peptide-1/IL-8, MCP-1/MCP-1) (5). In the myometrium there is a massive influx of macrophages, neutrophils, and T lymphocytes with the onset of labor at term (6). Studies over the last decade report a significant percentage of 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, suggesting 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 mechanisms of term and inflammation/infection-induced preterm parturition (5).
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 endometrium, 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 parturition. 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) expression 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 secretions of women experiencing term and preterm delivery (14, 15, 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, monocytes, lymphocytes, smooth muscle cells (SMCs),3 and selected tumor cell lines (19, 20, 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 physiological withdrawal of progesterone (P4) contribute to its expression. Our study used a well-characterized in vivo rat model to test this hypothesis. In this study, we 1) investigated the expression 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 chemotactic activity.
Materials and Methods
Wistar rats (Charles River Breeding Laboratories) were housed individually 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 experiments.
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 collected at noon at least 24 h after delivery (for day 1 postpartum) or 4 days after delivery (for day 4 postpartum).
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 gestation. 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, 23, 24, 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 17β-hydroxy-11β-[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 subsequently became pregnant in only one horn (26). Animals were allowed to recover from surgery for at least 7 days before mating. Pregnant myometrial 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 longitudinally, and dissected away from both pups and placenta. The endometrium was carefully removed from the myometrial tissue by mechanical scraping on ice, which we have previously shown removes the entire luminal, 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 variations. For each day of gestation, tissue was collected from four different animals.
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 μl of DNase I (2.73 Kunitz unit/μl; 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 performed with an ABI PRISM 7900 HT Sequence Detection System (Applied 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 quantitation 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 relative 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 μM sodium orthovanadate and protease inhibitor cocktail tablets (Complete Mini; Roche). Samples were spun at 12,000 × g for 15 min at 4°C, the supernatant 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 μg of protein from tissue homogenates of each gestational day or 1 μl of stretch-conditioned medium were used for MCP-1 assay.
The formalin-fixed myometrial tissues were gradually dehydrated in ethanol and embedded in paraffin. Sections of 5-μm thickness were collected on Superfrost Plus slides (Fisher Scientific). Paraffin sections were deparaffinized 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 used for detection of CD68 were biotinylated anti-mouse (1/300; DAKOCytomation) 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 Cytoseal 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 described (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 International) at a plating density of 3 × 106 cells/well. The cells were grown to confluence within 3–4 days in phenol red-free DMEM (Life Technologies) supplemented with 10% FBS (CanSera), 25 mM HEPES, 100 U/ml penicillin/streptomycin (Life Technologies), and 2.5 μg/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% CO2 at 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 stationary. Before exposure to mechanical stretch, the confluent cells were incubated for 24 h in serum-free DMEM. We have previously reported that 1 μM 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 monolayers were preincubated with hormone (1 μM; 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 protein content in stretched-conditioned medium with and without P4 was measured by ELISA.
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) supplemented 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 × 106 cells/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 differentiation. 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 inserts (5 μm pore size; Cell Biolabs). In pilot studies, primary rat monocyte migration was low in serum-free medium (negative control) and was induced 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 × 105 cells) 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 Biosciences) at room temperature for 2 h. Cells were allowed to migrate for 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 multiple comparison procedures (Student-Newman-Keuls 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 pairwise 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 appropriate method to obtain a normal distribution. Statistical analysis was conducted using SigmaStat (version 2.01; Jandel) with the level of significance for comparison set at p < 0.05.
Ccl-2 expression in nonpregnant, pregnant, and postpartum rat myometrium
Fig. 1,A illustrates the expression of Ccl-2 gene throughout pregnancy 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 samples, 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 nonpregnant, 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. 1 B, the immunoreactive Ccl-2 protein was significantly up-regulated in term pregnant and laboring rat myometrium compared with nonpregnant sample (p < 0.05) and decreased abruptly postpartum.
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 pregnant, 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. 2,D). Consistent with our gene and protein expression results, the most intense staining was found in labor samples (Fig. 2 E). Ccl-2 protein was always detected in the cytoplasm of myometrial SMCs and this spatial distribution was similar in both longitudinal and circular uterine muscle layers.
We also attempted to examine whether Ccl-2 protein expression indeed correlates with macrophage infiltration into the rat myometrium 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 in the densely packed tissue (Fig. 3,A). 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. 3 F).
Modulation of Ccl-2 gene and protein expression by progesterone
Animals treated from day 20 with daily injections of stable synthetic 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. 4,A). 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) compared 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 administration of P4 receptor antagonist, RU486 at day 19 of gestation induces preterm labor within 24 h and significant 8.8-fold increase (p < 0.05) in Ccl-2 mRNA levels (Fig. 4,B), enhanced Ccl-2 protein immunostaining (Fig. 5,B) and was associated with substantial macrophage infiltration (Fig. 5 D). Our results demonstrate that a sudden artificial blockade of P4 signaling during late pregnancy caused by RU486 led to Ccl-2 gene and protein induction, 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 transcript levels were dramatically increased at term in the gravid uterine 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 myometrial SMCs are subjected to stretch through the use of a computer-driven vacuum system. Analysis of medium conditioned by SMCs revealed that 24-h static mechanical stretch caused a significant 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 mechanism
We next asked whether the increased secretion of immunoreactive Ccl-2 protein in response to stretch is capable of stimulating chemotaxis of monocyte cells. The migration of primary rat monocytes in response to conditioned medium from stretched myometrial SMCs was much greater than the migration of conditioned medium from nonstretched cells or negative control (50–70% induction, p < 0.05) (Fig. 8). Monocyte chemotaxis in response to stretch-conditioned medium was blocked by vCCI, a CCR2 chemokine receptor antagonist, demonstrating involvement of this Ccl-2 receptor (p < 0.05) (Fig. 8 B). 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 secreted by myometrial SMCs following mechanical stimulation. It 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 inflammation, which is a characteristic feature of parturition. We provide in this study substantial evidence that myometrial cells can actively participate in the inflammatory process in the uterus by the release of proinflammatory mediator CCL-2. Firstly, we have demonstrated that myometrial SMCs in vivo are able to actively synthesize Ccl-2 protein during gestation and that its expression was significantly up-regulated before and at labor. Secondly, the increased 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 mechanical 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 medium was able to increase migratory ability of primary rat macrophages, 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 parturition (5). Leukocytes are known to infiltrate uterine tissue and their migration is regulated by chemokines, an ever-growing family of chemotactic cytokines. CCL-2 is a prototypical CC chemokine that recruits monocytes (but not neutrophils), and also T lymphocytes 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, 35, 36). Endometrial production of Ccl-2 by eosinophils during early gestation may play a central role in implantation or placentation that is crucial for successful establishment of ovine pregnancy (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 regression (38). There is very limited information about Ccl-2 expression in the myometrium. Sozen et al. (39) have shown that Ccl-2 transcript 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 chemokine in healthy myometrium. Esplin and colleagues (13) also reported 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 predominantly 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 myometrial 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 stimulation. Mechanical stress has been demonstrated to induce cortical expression of Ccl-2 as well as renal cortical macrophage infiltration 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 migration.
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 mechanisms such as changes in progesterone receptor isoforms (45), reduction in progesterone receptor transactivation (46), or increases 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 expression 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 infiltration during RU486-induced preterm labor. It is plausible to speculate that rapid development of uterine inflammation facilitated 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 myometrium. They observed that an absence of sex steroids led to a pronounced elevation of CCL-2 within nonpregnant human myometrium 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 myometrium.
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-regulation of Ccl-2 levels during normal term labor coincide with increased infiltration of CD68-positive immune cells in the rat myometrium. 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). Activated macrophages are able to release 1) matrix metalloproteinases likely contributing to cervical ripening and to the rupture of the gestational membranes; 2) PGs, histamine, or serotonin, capable of exerting a direct uterotonic effect; and 3) cell adhesion molecules (6, 33, 52, 53). Other proinflammatory cytokines released 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-regulated during active labor in women (54), contributing to an inflammatory 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 junction. 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 reorganization, matrix metalloproteinase induction, extracellular matrix degradation, and apoptosis. CCL-2 production might greatly enhance these processes. However, we cannot exclude the possibility that other chemokines shown to be critical for inflammatory endometrial destruction during menstruation (MCP-3, Eotaxin, FNK, MIP-1β (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 consequently 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 assistance of Julie Wright for chemotaxis assay.
The authors have no financial conflict of interest.
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.
This study was supported by Grant 37775 from the Canadian Institutes of Health Research.
Abbreviations used in this paper: SMC, smooth muscle cell; Ct, cycle threshold; MPA, medroxyprogesterone acetate.