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BOR - Papers in Press, published online ahead of print December 6, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.055376
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BIOLOGY OF REPRODUCTION 76, 448–454 (2007)
DOI: 10.1095/biolreprod.106.055376
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Coordinated Regulation of Human Trophoblast Invasiveness by Macrophages and Interleukin 101

Stephen J. Renaud , Shannyn K. Macdonald-Goodfellow , and Charles H. Graham 2

Department of Anatomy and Cell Biology and Research Group in Reproduction, Development and Sexual Function, Queen's University, Kingston, Ontario, Canada, K7L 3N6

ABSTRACT

Trophoblast invasion and modification of the spiral arterioles are essential for the establishment of adequate uteroplacental blood flow during pregnancy. However, such vascular remodeling is deficient in preeclampsia. This disease is also associated with increased maternal levels of circulating proinflammatory cytokines such as tumor necrosis factor (TNF) and reduced levels of immunoregulatory cytokines such as interleukin 10 (IL10). We have previously shown that activated macrophages inhibit trophoblast invasiveness in vitro. The present study demonstrates that IL10 interferes with the invasion-inhibitory effect that activated macrophages exert on trophoblast cells. Co-culture experiments revealed that human lipopolysaccharide (LPS)-activated macrophages inhibited the ability of immortalized HTR-8/SVneo human trophoblast cells to invade through reconstituted extracellular matrix. This effect of activated macrophages on trophoblast invasiveness was paralleled by decreased expression of urokinase plasminogen activator receptor (PLAUR) on the surface of trophoblast cells, and by increased secretion of plasminogen activator inhibitor type 1 (SERPINE1). Exposure of LPS-treated macrophages to IL10 prior to co-culture prevented their ability to inhibit trophoblast invasion, PLAUR expression, and to stimulate SERPINE1 secretion. Interleukin 10 prevented macrophage activation by LPS as determined by the lack of secretion of TNF in the culture medium, and a neutralizing TNF antibody completely blocked the effect of macrophages on trophoblast invasion. These results indicate that decreased circulating levels of IL10 associated with preeclampsia may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles.

cytokines, immunology, placenta, pregnancy, trophoblast

INTRODUCTION

Preeclampsia is one of the most common causes of morbidity and mortality during pregnancy, and is associated with decreased trophoblast invasion and remodeling of the uterine spiral arterioles during the first half of pregnancy [1–3]. Because of this structural maladaptation, the spiral arterioles in pregnancies complicated with preeclampsia have reduced diameters and, due to the persistence of an intact endothelium and tunica media, they maintain their responsiveness to vasoactive molecules [4]. This results in decreased and intermittent uteroplacental blood flow, which is thought to cause oxidative damage to the chorionic villi and maternal endothelium in the second half of pregnancy.

Although the etiology of preeclampsia is unknown, increasing evidence suggests that some form of maternal immune maladaptation plays a key role. For example, studies have provided evidence that an excessive maternal inflammatory response is causally linked to the clinical manifestation of preeclampsia [5, 6]. Associated with this maternal inflammatory response is an increased secretion of proinflammatory cytokines such as interferon gamma (IFNG) and tumor necrosis factor (TNF) in peripheral blood [7–9] and at the fetal–maternal interface [10]. Studies by Reister et al. showed increased macrophage infiltration of the spiral arterioles of women with preeclampsia [11], and recent studies have shown that activated macrophages, as well as low concentrations of the proinflammatory cytokine TNF, can decrease the invasiveness of trophoblast cells in vitro without inducing apoptosis [12, 13]. Conversely, studies have revealed that uncomplicated pregnancies had significantly higher levels of immunoregulatory cytokines such as interleukin (IL) 4, IL5, and IL10 than pregnancies complicated with preeclampsia [9].

Interleukin 10 is an immunoregulatory cytokine that is secreted by various cells of the immune system, including monocytes and macrophages as well as by invading trophoblast cells [14]. It is capable of inhibiting the synthesis of proinflammatory molecules in mononuclear cells, such as interleukin 1 beta, interleukin 8, macrophage inflammatory protein 2 alpha, and TNF. It can also suppress the secretion of interleukin 2 and IFNG in T cells, and stimulates natural killer (NK) cell activation [15, 16]. Studies have shown that serum levels of IL10 are decreased in women with preeclampsia, likely due to reduced secretion of this cytokine by peripheral blood mononuclear cells [17] and trophoblast cells [18]. Since cells of the immune system play an important role in regulating trophoblast invasion and placental development [11, 19], we sought to determine the effect of IL10 on the ability of macrophages to regulate trophoblast invasiveness in vitro. In addition, since an important component of trophoblast invasion involves the expression of the urokinase plasminogen activator receptor (PLAUR; also referred to as uPAR), we also measured the cell surface expression of this receptor, and the secretion of plasminogen activator inhibitor 1 (SERPINE1, formerly known as PAI1), a physiological inhibitor of the urokinase plasminogen activator (PLAU) proteolytic pathway.

MATERIALS AND METHODS

Cells

The human HTR-8/SVneo trophoblast cell line was used in these experiments. We previously established this cell line from explant cultures of first trimester (weeks 8–10) chorionic villi, and immortalized it by transfection with a cDNA encoding the Simian Virus-40 large T antigen [20]. The HTR-8/SVneo cells proliferate and are invasive in vitro, but are not tumorigenic when injected into nude mice. They possess several properties of invasive extravillous cytotrophoblasts as described previously [20–22]. HTR-8/SVneo cells were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS).

Human mononuclear leukocytes were isolated from 75–100 ml of peripheral blood from healthy male and female donors. Informed consent was obtained from all volunteer donors and the use of human blood was approved by the Queen's University Research Ethics Board. Whole blood was placed directly into heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) to prevent clotting, and diluted with an equal volume of PBS. Isolation of mononuclear leukocytes was achieved by density gradient centrifugation for 30 min at 900 x g after layering over Lymphoprep (Nycomed Pharma As, Oslo, Norway). Mononuclear leukocytes were removed, washed twice using Hanks Balanced Salt Solution (Gibco BRL), and resuspended in Dulbecco Modified Eagle Medium (DMEM; Gibco BRL) at a concentration of 2 x 106 cells/ml. Cells were incubated for 1 h in a 100-mm tissue culture plate at 37°C, 5% CO2. The medium was then removed and the plates rinsed twice to remove nonadherent cells. Approximately 5%–10% of the cells (1 million to 2 million) remained attached to the surface of the plates. This panning method is a well-established procedure that yields macrophage cultures of >90% purity [23, 24]. To activate macrophages, plates were incubated overnight at 37°C, 5% CO2 with 100 ng/ml lipopolysaccharide (LPS; Sigma Chemical Co., St. Louis, MO). Parallel plates were incubated overnight with 50 ng/ml IL10 (Sigma), in the presence or absence of LPS. Medium was removed the next day, centrifuged (300 x g, 10 min), aliquoted and placed at –20°C for short-term storage. The adhering macrophages were harvested using 1 ml of 0.05% trypsin (Sigma) and a plastic scraper (Sarstedt, Newton, NC). Nonactivated and LPS-activated macrophages incubated with and without IL10 were >95% viable after 20 h of plastic adherence as determined by trypan blue exclusion. In addition, there was no evident disparity between donor leukocytes as measured by viability of the macrophages and by the ability of LPS to induce cytokine secretion by these cells. The desired concentration of macrophages was obtained by diluting in appropriate volumes of RPMI 1640 medium containing 5% FBS.

Cell Culture

Since early trophoblast invasion may occur in an environment characterized by low O2 concentrations (intraplacental pO2 ~ 17 mmHg) [25], in most experiments, 24-h incubations were conducted in an atmosphere of 2% O2, which is equivalent to a pO2 value of approximately 15 mmHg. However, interstitial trophoblast invasion takes place in the decidua basalis, where pO2 values may be higher than in the placenta [26]. Therefore, additional experiments were conducted at 10% O2. Upregulation of PLAUR expression has been shown to be inversely related to the O2 concentrations to which cells are exposed [27, 28], with maximal expression by human breast carcinoma cells exposed to 0.2% O2 [28]. Therefore, to determine the effect of macrophages on PLAUR expression under conditions that maximize surface PLAUR levels, trophoblast cells were incubated in 0.2% O2. To determine whether the effects of macrophages on PLAUR expression depend on O2 concentrations, additional experiments were conducted at 10% O2. Similarly, our previous studies revealed that SERPINE1 secretion by HTR-8/SVneo cells is stimulated by culture in low O2 concentrations [29]. Therefore, to determine whether macrophages affect SERPINE1 secretion in an O2-dependent manner, HTR-8/SVneo cells were incubated in 0.5%, 2%, and 10% O2 in the absence or presence of macrophages pretreated with LPS or LPS + IL10. To establish an atmosphere of ≤ 2% O2, cells were placed in airtight chambers (Bellco Biotechnology, Vineland, NJ) that were flushed with a gas mixture of 5% CO2/95% N2 until the desired O2 concentration was reached. To obtain an atmosphere of 10% O2 and 5% CO2, the chambers were flushed with a gas mixture of 10% CO2/90% N2. Chambers were then placed in a 37°C incubator. The levels of O2 in the chambers were monitored and maintained using Pro-Ox model 110 O2 regulators (Biospherix Co., Redfield, NY).

Invasion Assay

Transwell inserts (6.5-mm diameter polycarbonate membrane, 8-µm pore; Corning Costar Corp., Cambridge, MA) were coated with 100 µl of 1 mg/ml reconstituted basement membrane (Growth Factor Reduced Matrigel; Collaborative Biomedical Products, Bedford, MA) in serum-free RPMI 1640 medium (Gibco BRL). The Matrigel was allowed to solidify for 3 h at room temperature. A total of 800 µl of medium containing 5% FBS was placed in the compartment below the Transwell chamber, i.e., a well in a 24-well tissue culture plate. Subsequently, 4 x 104 HTR-8/SVneo cells suspended in 100 µl of FBS-supplemented RPMI 1640 (Gibco BRL) were overlaid on the surface of the Matrigel-coated polycarbonate membrane of the Transwells, in the presence or absence of 2 x 104 control (LPS-untreated) macrophages or LPS-treated macrophages previously incubated with or without 50 ng/ml IL10. In parallel invasion assays, 10 µg/ml of a neutralizing antibody against TNF (anti-TNF; Sigma) was added to trophoblast cells incubated with LPS-activated macrophages. Cells were allowed to invade through the Matrigel for 24 h. A 2:1 trophoblast:macrophage ratio was chosen based on our previous observations in which a similar co-culture ratio resulted in the greatest decrease in trophoblast invasiveness [13].

After the incubation, excess cells and Matrigel on the top surface of the polycarbonate membrane of the Transwell were removed using a cotton swab. To fix the cells that invaded through the Matrigel, methanol (800 µl) prechilled at –20°C was added to the lower compartment and left for 7–10 min with the Transwell insert in place. Methanol was then removed and replaced with 0.3% H2O2 (800 µl; Fisher Scientific, Fair Lawn, NJ) in methanol for 30 min to block endogenous peroxidase activity. Following several washes with PBS, the membranes were removed using a scalpel and transferred with the upper surface facing down into a well of a 24-well plate.

Cells on the membranes were stained with a pan-cytokeratin antibody (DAKO Corp., Carpinteria, CA) that recognizes trophoblast cells and not macrophages, as previously shown [13]. After mounting the membrane on a microscope slide using aqueous mounting medium, all the trophoblast cells that invaded completely under the membrane were counted at 40x magnification.

Determination of SERPINE1 Secretion

To determine the levels of SERPINE1 secreted by trophoblast cells, 1.6 x 105 HTR-8/SVneo cells suspended in 250 µl were incubated in 24-well plates with various numbers (1 x 104–8 x 104) of LPS-treated and control macrophages preincubated with or without 50 ng/ml IL10. As additional controls, HTR-8/SVneo cells were preincubated with 50 ng/ml of exogenous IL10. To determine the contribution of SERPINE1 secretion by macrophages, LPS-treated macrophages were incubated without trophoblast cells. After 24 h, media were collected and centrifuged at 300 x g for 10 min. For ELISA, media were diluted 1/200 with 1% BSA in wash buffer. The amount of SERPINE1 secreted into the medium by the various treatment groups was determined by use of an ELISA kit from American Diagnostica according to the manufacturer's instructions.

Flow Cytometry

To determine trophoblast cell surface expression of PLAUR, HTR-8/SVneo cells (5 x 105 cells/ml) were plated for 24 h with culture medium conditioned by control or LPS-treated macrophages in the presence or absence of 50 ng/ml IL10. Subsequently, cells were incubated with a mouse monoclonal antihuman PLAUR antibody (1/50 dilution; American Diagnostica, Stanford, CT) for 1 h on ice, followed by FITC-conjugated polyclonal goat antimouse immunoglobulin (1/10; DakoCytomation, Glostrup, Denmark) for 1 h on ice. Finally, cells were fixed in 2% paraformaldehyde. Controls consisted of cells incubated with mouse IgG2a (1/10; DakoCytomation) at the same concentration as the anti-PLAUR antibody as well as cells incubated without primary or secondary antibodies. Samples were analyzed using a Coulter Elite Flow Cytometer (Beckman-Coulter Corp., Miami, FL).

TNF Secretion by Macrophages

The levels of TNF secreted into the medium by LPS-treated and control macrophages were determined by ELISA using a kit purchased from Chemicon (Temecula, CA). Following isolation, 2 x 107 macrophages were incubated in 10 ml DMEM in the presence or absence of LPS (100 ng/ml) for 20 h at 37°C, 5% CO2, with or without 50 ng/ml of IL10. The culture medium was then centrifuged at 300 x g for 10 min, aliquoted and stored at –80°C prior to TNF analysis by ELISA, which was performed according to the manufacturer's instructions. This ELISA kit has a detection limit of 4.8 {rho}g/ml.

Statistical Analysis

All data were normalized using values obtained from control cultures with only HTR-8/SVneo cells, or co-cultures with control macrophages not treated with LPS. This normalization was performed to facilitate comparisons between experiments. Experiments were repeated at least three times. All data except for the PLAUR flow cytometry results are presented as normalized means ± SEMs. Statistical analysis for flow cytometry was performed using relative fluorescence indices (RFI), whereby the median value for each sample was divided by the negative control value for that sample. The mean percent difference of RFI for each sample ± SEM relative to RFI obtained from the control macrophage sample was then determined. Statistical analysis was performed using the Statview statistical software package (Abacus Concepts Inc., Berkeley, CA). Statistical significance was determined by one-way analysis of variance, followed by a Fischer post hoc analysis. All statistical tests were two-sided and differences were considered significant at P < 0.05.

RESULTS

Role of IL10 on the Anti-Invasive Effect of LPS-Treated Macrophages

To determine the effect of IL10 on the ability of macrophages to regulate trophoblast invasiveness, HTR-8/SVneo cells were co-cultured with LPS-treated or control macrophages that had been preincubated with or without IL10. The total number of trophoblast cells that successfully penetrated the reconstituted extracellular matrix (ECM) after 24 h was counted, and the results are presented in Figure 1. LPS-activated macrophages significantly inhibited the ability of trophoblast cells to invade through the reconstituted ECM by about 50% (P < 0.05, N = 3 independent assays). Activation of macrophages was required for this inhibitory effect on the invasiveness of HTR-8/SVneo cells, as macrophages that were not exposed to LPS (–LPS) were unable to inhibit trophoblast invasiveness (Fig. 1). Preexposure of LPS-treated macrophages to IL10 completely prevented their ability to inhibit trophoblast invasiveness. In contrast, trophoblast invasiveness was unaffected by co-incubation with control (LPS-untreated) macrophages pre-incubated with or without IL10 (Fig. 1).


Figure 01
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  		FIG. 1. Effect of IL10 on the regulation of trophoblast invasiveness by activated macrophages. HTR-8/SVneo cells were co-cultured with or without LPS-treated (+LPS) or untreated (–LPS) macrophages that had been pre-incubated with or without IL10. Values represent normalized means ± SEM. Values significantly different from those obtained by measuring the invasiveness of trophoblast cells alone are represented by an asterisk (*P < 0.05).

Effect of IL10 on the Macrophage-Mediated Stimulation of SERPINE1 Secretion by Trophoblast Cells

To determine the effect of LPS-activated macrophages on the secretion of SERPINE1 by trophoblast cells, HTR-8/SVneo cells were cultured with LPS-activated macrophages at various ratios for 24 h. Compared with trophoblast-only controls, LPS-activated macrophages induced trophoblast secretion of SERPINE1 in a dose-dependent manner (Fig. 2; n = 4; P < 0.01). Activated macrophages alone did not secrete detectable levels of SERPINE1.


Figure 02
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  		FIG. 2. Effect of LPS-treated macrophages on trophoblast SERPINE1 secretion. HTR-8/SVneo cells were co-cultured with LPS-treated macrophages at various ratios. Values represent normalized SERPINE1 secretion ± SEM. Values significantly different from those obtained from cultures of trophoblast cells without macrophages are represented by an asterisk (*P < 0.01).

To determine the effect of IL10 on the ability of macrophages to regulate SERPINE1 secretion by trophoblast cells, HTR-8/SVneo cells were co-cultured for 24 h in various O2 concentrations (Fig. 3, A = 10% O2; B = 0.5% O2; C = 2% O2) with LPS-treated or untreated macrophages that had been pre-incubated with or without IL10. Compared with controls, LPS-treated macrophages significantly increased SERPINE1 secretion by trophoblast cells (P < 0.01, n = 4; Fig. 3, A–C). Prior exposure of LPS-treated macrophages to IL10 completely inhibited their ability to stimulate trophoblast SERPINE1 secretion (Fig. 3, A–C). In contrast, secretion of SERPINE1 by trophoblast cells was unaffected by co-incubation with control (LPS-untreated) macrophages preincubated with or without IL10 (Fig. 3, A and C). The effects of activated macrophages and IL10 on SERPINE1 secretion by trophoblast cells were observed at all O2 concentrations used, and were consistent with those obtained by incubating trophoblast cells in culture medium derived from macrophages (Fig. 3D). The addition of exogenous IL10 directly to trophoblast cells had no effect on SERPINE1 secretion (not shown).


Figure 03
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  		FIG. 3. Effect of IL10 treatment on the ability of macrophages to regulate SERPINE1 secretion by trophoblast cells. Trophoblast cells were co-cultured with or without LPS-treated (+LPS) or control (–LPS) macrophages that had or had not received prior exposure to IL10, or incubated with medium conditioned by macrophages. A–C) Normalized SERPINE1 secretion from trophoblast/macrophage co-culture experiments at different O2 concentrations (10%, 0.5%, and 2% O2, respectively). D) Secreted SERPINE1 protein by trophoblast cells incubated with culture media derived from macrophages. All values represent normalized SERPINE1 secretion ± SEM. Values significantly different from controls are indicated with an asterisk (*P < 0.01).

Effect of Macrophages on PLAUR Expression by Trophoblast Cells

To determine the effect of macrophages preincubated with or without IL10 on the expression of PLAUR by trophoblast cells, HTR-8/SVneo cells were placed for 24 h in culture medium conditioned by macrophages that were preexposed to LPS in the absence or presence of 50 ng/ml IL10. Compared with medium conditioned by control macrophages, medium conditioned by activated macrophages significantly decreased the expression of PLAUR (P < 0.01, n = 4) as determined by flow cytometry. This effect was observed at 0.2% O2 (34% decrease) and 10% O2 (27% decrease). Conversely, preexposure of LPS-treated macrophages to IL10 prevented their ability to inhibit trophoblast PLAUR expression. A representative experiment is shown in Figure 4. In addition, compared with controls, PLAUR expression was decreased by 20% in trophoblast cells incubated with TNF (1 ng/ml) for 24 h (P < 0.01; not shown).


Figure 04
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  		FIG. 4. Effect of IL10 treatment on the ability of macrophages to regulate PLAUR expression by trophoblast cells. Cells were incubated with culture medium derived from control (–LPS) macrophages, or LPS-treated macrophages with or without exposure to IL10. A representative of four experiments is shown.

IL10-Mediated Inhibition of TNF Secretion by Activated Macrophages

To determine the effect of IL10 on the secretion of TNF by LPS-treated macrophages, culture medium was collected and analyzed by ELISA. Compared with controls, the presence of LPS significantly enhanced TNF secretion by macrophages (Figure 5A). Preexposure of LPS-treated macrophages to IL10 inhibited the secretion of TNF by 83% (P < 0.01, n = 3; Fig. 5A).


Figure 05
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  		FIG. 5. IL10-mediated inhibition of TNF secretion by LPS-treated macrophages. A) Culture medium was collected from control (–LPS) or LPS-treated (+LPS) macrophages incubated with or without IL10, and analyzed for the presence of TNF by ELISA. Values represent mean TNF secretion ± SEM. Values significantly different from medium derived from cultures of LPS-untreated macrophages alone are represented by an asterisk (*P < 0.01). B) Trophoblast cells were co-cultured with control and LPS-treated macrophages in the presence or absence of a neutralizing antibody against TNF. Values represent normalized means ± SEM. Values significantly different from control are indicated with an asterisk (*P < 0.05).

Role of TNF on the Macrophage-Mediated Inhibition of Trophoblast Invasiveness

To determine whether the invasion-inhibitory effect of activated macrophages is mediated by TNF, trophoblast-macrophage co-culture invasion assays were performed in the presence or absence of neutralizing anti-TNF antibody (10 µg/ml). Results shown in Figure 5B reveal that LPS-treated macrophages significantly inhibited trophoblast invasion through Matrigel by about 50% (P < 0.01). The addition of TNF neutralizing antibody completely prevented this effect.

DISCUSSION

The findings from this study provide evidence that IL10 promotes trophoblast invasiveness indirectly by interfering with a macrophage-mediated inhibition of trophoblast invasiveness. It has been well documented that pregnancies complicated by preeclampsia are characterized by decreased trophoblast invasion of the spinal arterioles during the first half of pregnancy [2, 3] and by decreased levels of circulating immunoregulatory cytokines, including IL10 [17, 18]. However, it was previously reported that when IL10 is administered directly to villous explants, it functions as a negative regulator of trophoblast invasiveness, and that it decreases MMP9 expression in these tissues [30]. In contrast to those studies, which examined the direct effects of IL10 on trophoblast cell invasiveness, the present study determined the effect of IL10 on the ability of macrophages to regulate trophoblast invasiveness. Macrophages have been shown to infiltrate in and around the spiral arterioles in pregnancies complicated by preeclampsia [11].

We previously showed that LPS-activated macrophages are capable of inhibiting trophoblast invasiveness in vitro [13]. In the present study, this inhibitory effect of LPS-treated macrophages was prevented by preexposure to IL10. This cytokine is capable of inhibiting the synthesis of various proinflammatory factors in monocytic cells, including TNF. It has been previously shown that TNF inhibits trophoblast invasiveness [12, 13]. The present study showed that IL10 was able to block the LPS-mediated stimulation of TNF secretion by macrophages. In addition, neutralizing TNF prevented the inhibitory effect of LPS-treated macrophages on trophoblast invasion. Therefore, IL10 may regulate trophoblast invasion indirectly by a mechanism that likely involves inhibition of TNF secretion by activated macrophages. Interestingly, abnormally high levels of serum TNF and low levels of IL10 have been found in women with preeclampsia [7–9, 17, 18].

Trophoblast invasion and remodeling of the spiral arterioles occurs early in pregnancy and continues until about the middle of the second trimester [31]. During much of this time, intraplacental O2 levels are relatively low [25, 32], and it has been suggested that such O2 concentrations may provide optimal conditions for adequate placentation and trophoblast invasion [27, 33]. Trophoblast cells migrate from the anchoring villi of the placenta toward the uterine spiral arterioles in the decidua and inner myometrium, where interactions with macrophages occur. As the trophoblast cells migrate from the placenta to the spiral arterioles, the O2 levels progressively increase. However, the O2 concentration at the site of interaction between invading trophoblast cells and macrophages is unknown. Nevertheless, our results revealed that the effect of macrophages on HTR-8/SVneo cells was independent of O2 concentrations.

The expression of PLAUR, a glycosyl phosphatidyl inositol (GPI)-anchored cell surface receptor, is present on the leading edge of invading extravillous trophoblast cells in situ [34] and has been shown to promote the invasion of various cell types including trophoblast cells [27, 35]. PLAUR binds to and activates pro-PLAU, which consequently promotes cellular invasion. The addition of an amino terminal fragment of PLAU, which is nonfunctional and therefore interferes with PLAU binding to PLAUR, significantly decreases the invasiveness of trophoblast cells in vitro [36]. The results of the present study indicate that trophoblast cell surface expression of PLAUR is decreased when trophoblast cells are cultured in medium derived from activated macrophages. Several inflammatory mediators have been shown to modulate PLAUR cell surface expression. For example, nitric oxide (NO) is an inflammatory mediator, and low concentrations of NO mimetics have been shown to reduce the levels of PLAUR mRNA and cell surface expression in trophoblast cells under low O2 conditions [37]. In addition, studies have shown increased GPI cleavage of PLAUR from the cell surface after the addition of exogenous TNF [38]. The present study revealed that the addition of exogenous TNF to trophoblast cells cultured under low O2 conditions results in a modest but significant decrease in PLAUR expression on the cell surface. Thus, it is possible that the more pronounced decrease of PLAUR expression by trophoblast cells in the presence of medium derived from activated macrophages is the result of synergistic effects by a variety of inflammatory mediators, including NO and TNF. Regardless, prior exposure of activated macrophages to IL10 prevented the downregulation of PLAUR expression on the surface of trophoblast cells. IL10 has been shown to inhibit the synthesis of various inflammatory mediators, including NO [39] and TNF [15].

The serine protease inhibitor SERPINE1 is an important physiological inhibitor of the PLAU proteolytic system. SERPINE1 is an ECM glycoprotein that inhibits free and PLAUR-bound PLAU through the formation of irreversible covalent complexes [40]. In addition, SERPINE1 is capable of forming a complex with active PLAU/PLAUR thereby inducing endocytosis and degradation of PLAU and SERPINE1 [41]. Previous studies have revealed that TNF increases SERPINE1 secretion by endothelial cells, chorionic villus explant cultures, and immortalized trophoblast cells [12, 13, 42]. In addition, SERPINE1 has been shown to inhibit the invasiveness of trophoblast cells in vitro [43]. Interestingly, SERPINE1 levels have been found to be increased in the serum of women with preeclampsia [44], and high SERPINE1/SERPINB2 ratios are characteristic of the disease [45]. In the present study, IL10 prevented the macrophage-mediated induction of trophoblast SERPINE1 secretion. Moreover, these effects were observed at 3 different levels of O2, suggesting that these effects are not limited to specific O2 concentrations. An increase in SERPINE1 secretion may be part of a mechanism by which activated macrophages inhibit trophoblast invasiveness.

In conclusion, the findings from this study provide further insight into the complexity of factors regulating trophoblast invasion and spiral arteriole remodeling. There is increasing evidence that the immune system plays a role in regulating trophoblast invasion and placental development during the first half of pregnancy, and it is widely accepted that inadequate endovascular invasion by trophoblast cells is an important aspect of the pathophysiology of preeclampsia. Trophoblast cells are semiallogeneic because they express maternal as well as paternal antigens. As they invade through the decidua, they encounter maternal immune cells such as macrophages, which would otherwise recognize trophoblast cells as foreign and initiate an immune response. We propose that trophoblast cells are able to invade unimpeded by maternal macrophages at least partly through the secretion of IL10. Interstitial and endovascular invasion by trophoblast cells occurs during the first half of pregnancy, and studies by Chaouat et al. have shown that murine placental expression of IL10 is highest in early and midpregnancy, and reduced at term [46]. Other studies reported that pregnant IL10 null mice were more susceptible to fetal resorption and intrauterine growth restriction and these effects were reversed with the administration of exogenous IL10 or a neutralizing antibody against TNF [47]. Together, these data provide evidence that the immune system plays a central regulatory role in the invasiveness of trophoblast cells as well as in the development of the placenta.

ACKNOWLEDGMENTS

We wish to thank Angela Black, Department of Urology at Kingston General Hospital for help in extracting blood. We also would like to thank Matt Gordon and Jeff Mewburn for FACS analysis, and Lori Maxwell, Colleen Schick, and Geneviève Paré for technical assistance.

FOOTNOTES

1Supported by a grant from the Heart and Stroke Foundation of Ontario awarded to C.H.G. (Grant no. T-5722). Back

Correspondence: 2Charles H. Graham, Department of Anatomy and Cell Biology, Botterell Hall Room 859, Queen's University, Kingston, ON, Canada K7L 3N6. FAX: 613 533 2566; e-mail: grahamc{at}post.queensu.ca

Received: 10 July 2006.

First decision: 4 August 2006.

Accepted: 1 December 2006.

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