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Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cytokines, immunology, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While the precise causes of pre-eclampsia remain unknown, increasing evidence suggests that an aberrant activation of the maternal immune system plays a role in the etiology of this disease [6–8]. For instance, long-term maternal exposure to seminal plasma, sperm antigens, or both, was shown to confer protection against pre-eclampsia [6, 8]. Hence, it has been hypothesized that induction of maternal tolerance to paternal molecules is important for normal placental development [9]. Evidence of abnormal activation of the maternal immune system has been provided by studies showing that, compared with normal pregnancy, there is increased infiltration of activated maternal macrophages around the nonremodeled spiral arterioles of women with pre-eclampsia [10]. Further studies have suggested that activated macrophages around the spiral arterioles secrete tumor necrosis factor (TNF)-alpha at levels that lead to the apoptotic death of the invading extravillous trophoblasts and therefore prevent them from invading and remodeling the vessels [11]. However, Bauer et al. have shown that TNF may induce the secretion of plasminogen activator inhibitor 1 (PAI1, also known as SERPINE1) in trophoblast cells without inducing apoptosis [12]. PAI1 is an extracellular matrix (ECM) glycoprotein of 52 kDa and 379 amino acids that is capable of inhibiting both free and bound urokinase plasminogen activator (uPA, also known as PLAU) through the formation of irreversible covalent complexes [13, 14]. Urokinase plasminogen activator is a serine proteinase that catalyzes the conversion of plasminogen into plasmin [15]. Because of its ability to break down various components of the ECM, plasmin plays an important role in cellular invasion [13].
TNF is a proinflammatory cytokine with pleiotropic effects on various cell types. It exerts its action by binding to the cell surface TNF type-1 or type-2 receptors (TNFRI [also known as TNFRSF1A], or TNFRII [also known as TNFRSF1B], respectively). Studies have shown that the TNFRI is present on invasive extravillous trophoblasts [16], whereas the TNFRII is absent from all trophoblast [12]. Binding of TNF to the TNFRI results in trimerization of the receptor, and can lead to either a pro-apoptotic signal, or a survival and activation signal, depending on the adaptor protein present [17]. Through its pro-apoptotic effects, TNF has been postulated to be necessary for trophoblast turnover during normal placental development [11]. In addition, TNF has been shown to regulate the expression of certain hormones such as hCG, progesterone, and estradiol [18, 19], as well as proteinases such as matrix metalloproteinase 9 (MMP9) [12, 20].
Abnormally high levels of TNF are found in the sera of women afflicted with pre-eclampsia [21, 22], preterm labor, and infection [23]. However, the role of TNF in the regulation of cytotrophoblast invasiveness has not been fully elucidated. Similarly, the role of activated macrophages in the regulation of trophoblast invasiveness remains unclear. Thus, the main objective of the present study was to determine the effect of activated macrophages and TNF on trophoblast invasion through ECM. Because trophoblastic invasion requires the participation of proteolytic enzyme systems, the present study also examined the effect of activated macrophages and TNF on the secretion of uPA and PAI1 by trophoblast cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The HTR-8/SVneo cell line was used in these experiments. This is an immortalized human trophoblast cell line established after transfection of first trimester primary trophoblasts with a cDNA that encodes the SV40 large T antigen [24]. Although the HTR-8/SVneo cells are nontumorigenic and nonmetastatic when transplanted into athymic mice, they are highly invasive in vitro and exhibit various properties of extravillous cytotrophoblasts, including the expression of cytokeratins 7, 8, and 18; placental type alkaline phosphatase; uPA receptor (PLAUR); HLA framework antigen W6/32; IGF2 mRNA and protein; as well as an integrin profile characteristic of invasive cytotrophoblasts [24–26]. When plated on Matrigel, HTR-8/SVneo cells express HLA-G [25], a nonpolymorphic HLA molecule expressed by extravillous cytotrophoblasts in situ. HTR-8/SVneo cells were maintained in RPMI medium (Gibco-BRL, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS).
Human mononuclear leukocytes were isolated from approximately 100 ml of peripheral blood collected from five healthy donors (three women and two men), and placed in heparinized tubes (Becton Dickinson, Franklin Lakes, NJ). Whole blood was diluted with an equal volume of PBS at room temperature. Mononuclear cells were isolated by centrifugation for 30 min at 900 x g after layering over Lymphoprep (Nycomed Pharma As, Oslo, Norway). Leukocytes were then removed, resuspended in 3x volume of Hanks balanced salt solution (HBSS; Gibco-BRL), and centrifuged twice at 400 x g for 10 min. The remaining pellet was resuspended in an appropriate volume of Dulbecco modified eagle medium (DMEM; Gibco-BRL) supplemented with 2 mM L-glutamine (Gibco-BRL) and 50 µg/ml gentamycin. Mononuclear leukocytes (2 x 107) were incubated for 1 h in a 100-mm tissue culture plate at 37°C in 5% CO2. The medium was then decanted and washed twice with serum-free DMEM to remove nonadherent cells, and replaced with fresh DMEM. Approximately 5%– 10% of the cells (1–2 million) remained attached to the surface of the plates. This panning method is a well-established procedure that yields monocyte cultures of >90% purity [27]. In addition, Tchou-Wong et al. found that a similar panning procedure resulted in cultures of 90%–95% macrophages as determined by nonspecific esterase activity [28]. To activate macrophages, 100 ng/ml lipopolysaccharide (LPS; Sigma Chemical Co., St. Louis, MO) were added to the fresh DMEM. Parallel plates were incubated without LPS (nonactivated macrophages). The medium was removed the next day from both LPS-activated and nonactivated macrophages, and the adhering macrophages were harvested using 1 ml of 0.05% trypsin (Sigma) and a plastic scraper (Sarstedt, Newton, NC) as described previously [29]. LPS-activated macrophages were >95% viable after 20 h of plastic adherence as determined by trypan blue-exclusion, and nonactivated macrophages were >94% viable after the same incubation. The desired numbers of macrophages were obtained by diluting with appropriate quantities of RPMI containing 5% FBS.
Cell Culture
Because normal trophoblastic invasion occurs in an environment characterized by relatively low O2 concentrations [30], 24-h incubations in the present study were conducted in an atmosphere of 0.5%–1% O2. To establish concentrations of 0.5%–1% 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. 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). As an additional control, cells were also incubated under standard (20% O2, 5% CO2) conditions in a Sanyo CO2 incubator.
Invasion Assay
Transwell inserts (6.5-mm diameter polycarbonate membrane, 8-µm pore; Corning Cornstar Corp., Cambridge, MA) were coated with 100 µl of a 1 in 10 dilution of reconstituted ECM (Growth Factor Reduced Matrigel; Collaborative Biomedical Products, Bedford, MA) in serum-free RPMI (Gibco-BRL). The Matrigel was allowed to dry overnight and was subsequently rehydrated with 100 µl of serum-free RPMI (Gibco-BRL). A total of 750 µl of medium was placed in the compartment below the Transwell chamber. After removing the rehydration medium, 5 x 104 cells suspended in 100 µl of FBS-supplemented RPMI (Gibco-BRL) were overlaid on the surface of the polycarbonate membrane of the Transwells, with or without TNF (1 and 0.04 ng/ml; Sigma) or macrophages at various ratios (trophoblast:macrophage = 8:1, 4:1, or 2:1), and left to incubate for 24 h.
After incubation, a cotton swab was used to remove cells and excess Matrigel from the upper surface of the polycarbonate membrane of the Transwell. Cold methanol (750 µl) was added to the Transwell chamber and left to incubate for 7–10 min with the Transwell reinserted. Methanol was then removed and replaced with 0.3% H2O2 (750 µl; Fisher Scientific, Fair Lawn, NJ) in methanol, which remained in the chamber for 30 min. Following several washes with PBS, the membranes were removed using a scalpel and transferred with the upper surface facing down to a 24-well plate.
Immunocytochemistry
Membranes were blocked for 30 min with 10% normal horse serum (NHS; Gibco-BRL) in PBS. Afterward, membranes were treated with Pan-cytokeratin antibody (1 in 50 dilution; DAKO Corp., Carpinteria, CA), which is specific for epithelial cells, and thus will not detect macrophages. Subsequently, membranes were treated with a biotinylated anti-mouse immunoglobulin G (IgG; 1 in 200 dilution; Vector Laboratories, Burlingame, CA). Finally, to complete the avidin-biotin complex reaction, membranes were treated with Vectastain (Vector) and diaminobenzidine (Sigma). All the trophoblast cells that invaded throughout the membrane were counted with the aid of a microscope.
Zymographic Analysis
To determine cell-associated and secreted uPA levels, HTR-8/SVneo cells were incubated in 24-well plates in the presence of macrophages, TNF (0.04 ng/ml), anti-TNFRI antibody (1 ng/ml; R&D Systems, Minneapolis, MN), or a combination of these. HTR-8/SVneo cells (150 000 in number) were plated with various ratios of macrophages (trophoblast:macrophage = 8:1, 4:1, and 2:1). Two hundred microliters of cell suspensions were plated per well. For controls, culture medium, HTR-8/SVneo cells, or macrophages were each plated alone in a 24-well plate. After incubation, media were collected and centrifuged (300 x g for 10 min), and cells were lysed in 2% SDS, 10 mM Tris HCl (pH 7.5), and 0.15 mM NaCl. The lysates were subjected to DNA shearing (ejected 10 times into a 25
-gauge needle), and then centrifuged (15 min at 14 000 x g). Both media and cell supernatants were stored at –80°C. Samples were combined with SDS sample buffer, loaded onto gels containing 0.8 mg/ml casein (with or without plasminogen; 50 µg in 10 ml of acrylamide), and subjected to electrophoresis at 165 V for about 90 min. After incubating overnight in 2 mM Tris and 15 mM CaCl2 (with or without 100 mM of amiloride, a selective inhibitor of uPA) for catalytic activation of proteolytic enzymes, gels were stained with 0.4% Coomassie brilliant blue and dried to reveal caseinolytic activity.
Determination of PAI1 Secretion
To determine the levels of PAI1 secreted, 1.5 x 105 HTR-8/SVneo cells suspended in 200 µl were incubated in 24-well plates with activated or nonactivated macrophages, or with various concentrations of TNF (0.04–10 ng/ml). Activated macrophages were also incubated with or without 10 µg/ml of a neutralizing antibody against TNF (anti-TNF; Sigma). After 24 h, media were collected and centrifuged at 300 x g for 10 min. Subsequently, the amount of PAI1 secreted in the various treatment groups was determined by ELISA (American Diagnostics, Greenwich, CT).
TNF Secretion by Macrophages
Secretion of TNF by LPS-activated and nonactivated macrophages was determined using an ELISA assay from Abazyme (Needham, MA). Following isolation, 2 x 107 macrophages were incubated in 10 ml of DMEM in the presence or absence of LPS (100 ng/ml) for 18 h at 37°C in 5% CO2. The culture medium was then centrifuged at 300 x g for 10 min, aliquoted, and stored at –80°C before TNF ELISA analysis, which was performed according to the manufacturer's instructions. This TNF ELISA kit has a detection limit of 4 pg/ml.
Determination of Apoptosis
To determine the effect of TNF on HTR-8/SVneo apoptosis, 1 x 105 HTR-8/SVneo cells suspended in 100 µl were incubated in culture slides with or without 1 or 10 ng/ml of TNF for 24 h. Relative quantities of apoptotic cells were determined using a terminal deoxynucleotidyl transferase (TdT) FragEL kit (Calbiochem, San Diego, CA) according to the manufacturer's instructions.
Statistical Analysis
All data were normalized using values from control cultures incubated without activated macrophages or TNF. This normalization was performed to facilitate comparisons between experiments. Experiments were repeated at least three times. All data are presented as the normalized means ± SD. Statistical analyses were 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the role of activated macrophages on cytotrophoblast invasion, HTR-8/SVneo cells were cocultured with various ratios of LPS-activated macrophages or nonactivated macrophages isolated from the peripheral blood of five healthy donors. There were no noticeable differences in the results obtained using leukocytes collected from the various donors. The total number of immortalized cytotrophoblast cells that successfully invaded through the reconstituted ECM after 24 h was counted, and the results are presented in Figure 1. The results indicate that the invasiveness of HTR-8/SVneo cells was significantly decreased by approximately 50% (P < 0.05) when 5 x 104 HTR-8/ SVneo cells were cocultured with 2.5 x 104 macrophages (2:1 ratio). This inhibitory effect on the invasiveness of HTR-8/SVneo cells required macrophage activation, because macrophages that were not pre-incubated with LPS were unable to inhibit invasion (Fig. 1). Likewise, similar numbers of HTR-8/SVneo trophoblast cells were incubated with or without the presence of 1 ng/ml or 0.04 ng/ml of TNF. The results show that 1 ng/ml of TNF significantly decreased HTR-8/SVneo invasiveness by about 50% (P < 0.05; Fig. 2). Results of the TNF ELISA revealed that 2 x 106 activated macrophages secreted 1–2 ng/ml of TNF after 20 h of adherence on plastic, whereas TNF levels in cultures of nonactivated macrophages were undetected in four independent determinations (P < 0.01).
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When the invasion assays were performed in a hyperoxic atmosphere (20% O2), the invasiveness of the HTR-8/ SVneo cells was 60%–70% lower than in the more physiologic low O2 atmosphere (P < 0.05). In the hyperoxic conditions, activated macrophages were unable to further inhibit invasiveness (data not shown).
Activated Macrophages and TNF Abrogate the Secretion of a 55-kDa Caseinolytic Band
To further investigate the anti-invasive effect of activated macrophages, the levels of the invasion-promoting enzyme uPA were measured by gel zymography using cell extracts and culture medium supernatants. Results revealed that compared with culture in the absence of activated macrophages, a caseinolytic band of approximately 55 kDa was absent in both extracts (not shown) and conditioned media (Fig. 3) from cells incubated with activated macrophages. Similar results were obtained when HTR-8/SVneo cells were incubated with media conditioned by LPS-activated macrophages, but not with media conditioned by nonactivated macrophages (not shown). Likewise, zymographic analysis was also used to examine the effects of TNF on uPA secretion. As in the case of the cocultures with activated macrophages, the secretion of the 55-kDa caseinolytic band was abolished in cultures incubated for 24 h in the presence of 0.04 ng/ml of TNF. Moreover, inclusion of the anti-TNFRI antibody (1 ng/ml) prevented this TNF-mediated inhibition (Fig. 4). In all zymograms, caseinolytic activity was abolished when plasminogen was not present in the gels or when the gels were incubated with amiloride (not shown).
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TNF Increases PAI1 Secretion by HTR-8/SVneo Cells
To assess the effect of TNF on the secretion of PAI1, HTR-8/SVneo cells were incubated for 24 h either alone or with various concentrations of TNF ranging from 0.04 to 10 ng/ml. As indicated in Figure 5, compared with controls (no TNF added), 1 ng/ml and 10 ng/ml of TNF significantly increased PAI1 secretion by 50% and 100% respectively (P < 0.01).
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Activated Macrophages Increase PAI1 Secretion by HTR-8/SVneo Cells
To assess the effect of macrophage activation on the secretion of PAI1 by trophoblast cells, HTR-8/SVneo cells were incubated for 24 h in the presence of LPS-activated or nonactivated macrophages. As indicated in Figure 6, trophoblast cells cocultured with activated macrophages at a 4:1 ratio (trophoblast:macrophage) secreted 60% higher levels of PAI1 than trophoblast cells cocultured with nonactivated macrophages (P < 0.01). Activated macrophages alone did not secrete a detectable level of PAI1. The addition of a neutralizing antibody against TNF (anti-TNF; 10 µg/ml) significantly inhibited (–70%) the macrophage-mediated increase in PAI1 secretion by cytotrophoblasts cocultured with activated macrophages at a 4:1 ratio (P < 0.05).
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Low Concentrations of TNF Do Not Induce Apoptosis in HTR-8/SVneo Cells
The effect of TNF on cytotrophoblast apoptosis was determined following a 24-h incubation of HTR-8/SVneo cells with 1 and 10 ng/ml of TNF. As presented in Figure 7, TNF did not have any proapoptotic effect relative to the control group incubated without TNF. In fact, compared with cultures incubated without TNF, the number of TUNEL-positive cells was significantly reduced by about 50% in cultures incubated with 1 ng/ml TNF (P < 0.05; Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The impaired ability of trophoblast cells to invade and remodel the spiral arterioles is a factor in the development of pre-eclampsia [3]. In addition, previous reports indicate that pre-eclamptic pregnancies are associated with an increased infiltration of macrophages around the uterine spiral arterioles [10]. The infiltrating macrophages could affect trophoblast function in various ways. They may induce apoptosis [31], may alter differentiation or proliferation [32], and may modify the secretion of hormones [18, 19]. The results of the present study suggest that the abnormal increase in macrophage infiltration around the spiral arterioles during pre-eclampsia may also hamper trophoblast invasion and vascular remodeling through a nonapoptotic mechanism.
Trophoblast invasion and remodeling of the spiral arterioles are essential for adequate uteroplacental blood flow in normal pregnancy. This invasion occurs early in pregnancy and continues until about the middle of the second trimester [33]. Because active remodeling of the uterine vasculature occurs in the first trimester of pregnancy, a time when O2 levels in the placenta are relatively low [30, 34], experiments in the present study were conducted in a low O2 environment.
Cellular invasion through the ECM is dependent on the secretion and activity of proteolytic enzymes. Of particular importance are the matrix metalloproteinases (MMPs or matrixins) and the plasminogen activation system. The latter comprises the enzymes tissue-type plasminogen activator (tPA) and uPA. It has been shown that uPA plays a major role in stimulating invasiveness of various cell types, including trophoblast cells [15, 35, 36]. Previous work in our laboratory demonstrated that the expression of the cell surface PLAUR is increased in HTR-8/SVneo cells as well as in various tumor cell types incubated in 1% O2 versus 20% O2 [36]. In the present study we observed that the secretion of a 55-kDa caseinolytic enzyme by HTR-8/ SVneo cells was inhibited by the presence of activated macrophages or medium conditioned by activated macrophages. This observation suggests that one or more soluble factors produced by activated macrophages are responsible for the invasion-inhibitory effect of macrophages. We conclude that the 55-kDa caseinolytic band represents a member of the plasminogen activator enzymes because all caseinolytic activity in the gel was absent when plasminogen was not included in the gel. In addition, the fact that incubation of the gels in the presence of amiloride also resulted in inhibition of all caseinolytic activity indicates that the 55-kDa band represents a form of uPA. Amiloride has been shown to inhibit uPA but not tPA activity [37]. Finally, because of its close association with the 52-kDa (two-chain) active uPA, it is likely that the 55-kDa band represents latent pro-uPA. Of interest, the present findings did not reveal a detectable decrease in the levels of the 52-kDa form of uPA. It is possible that small, undetectable changes in the levels of active uPA are responsible for the observed inhibition of the in vitro invasiveness. In addition, the decrease in the levels of active uPA may occur later in a cascade of events that includes decreased pro-uPA secretion and increased PAI1 secretion.
Activation of macrophages with LPS resulted in increased secretion of TNF as determined by ELISA. Furthermore, incubation with TNF resulted in a decrease in HTR-8/SVneo trophoblast invasiveness. This finding supports the results of Bauer et al., who reported that TNF inhibited the migration of extravillous trophoblast cells but had no effect on anchorage and proliferation [12]. Other studies have shown that TNF influences the secretion and activation of proteases involved in cell migration and invasion. For example, it has been reported that TNF is able to stimulate the secretion of MMPs, specifically MMP9 [12, 20]. In the present study, TNF abolished the secretion of pro-uPA, in addition to inhibiting cytotrophoblast invasion in vitro. Furthermore, interference of TNF binding to the TNFRI with a function-blocking anti-TNFRI antibody prevented the inhibitory effect of TNF on pro-uPA secretion.
The present study also revealed that TNF induced the secretion of PAI1 in HTR-8/SVneo cells in a dose-dependent manner. This result is consistent with the findings of others using endothelial cells and explant cultures of chorionic villi [12, 35]. In addition, activated macrophages, but not nonactivated macrophages, were able to significantly induce PAI1 secretion by trophoblast cells, and the addition of a neutralizing antibody against TNF decreased this response. Therefore, a possible mechanism by which activated macrophages block pro-uPA secretion and decrease trophoblast invasiveness may involve a proinflammatory (e.g., TNF)-mediated increase in PAI1 secretion.
Trophoblast cells are reported to undergo apoptosis when incubated with TNF (10 ng/ml) and that this apoptosis may contribute to decreased cytotrophoblast invasion and remodeling of the spiral arterioles [11]. However, other studies have shown that apoptosis occurs only at relatively high concentrations, and that at lower concentrations similar to those used in this study, trophoblast viability and metabolism remain unaffected [11, 19]. Bauer et al. found only very low levels of apoptosis in differentiated first trimester primary extravillous trophoblasts, and these levels did not increase with the addition of TNF [12]. In the present study, incubation of HTR-8/SVneo cells in the presence of TNF resulted in a decrease in the number of TUNEL-positive cells. While TNF is capable of promoting death in various cell types by inducing the activation of caspases, it has also been demonstrated that this cytokine can promote survival via recruitment of distinct sets of adaptor proteins and subsequent activation of nuclear factor-
B [17]. The HTR-8/ SVneo cells were immortalized following transfection with a plasmid that encodes the large T antigen of Simian Virus 40 [24]. Thus, the apoptotic machinery in these cells is likely compromised. Studies have shown that PAI1 inhibits the activation of caspase 3 and results in decreased apoptosis in various cell types [38–40]. Therefore, the increased secretion of PAI1 could be an additional mechanism by which TNF inhibits trophoblast apoptosis. Regardless of its antiapoptotic effect, low concentrations of TNF effectively inhibited trophoblast invasion.
While the present study revealed that the macrophage-derived molecule TNF inhibits the invasiveness of immortalized trophoblast cells, it is also likely that other macrophage-derived factors control trophoblast invasiveness. This conclusion is based on the fact that the addition of a TNF neutralizing antibody to trophoblast cells cocultured with activated macrophages did not fully abrogate the increase in PAI1 secretion. Therefore, other proinflammatory cytokines may be involved in the regulation of trophoblast invasion. In addition, it has been demonstrated that indolamine 2,3 dioxygenase, a tryptophan-depleting enzyme secreted by activated macrophages, can induce cytotrophoblast apoptosis [11]. Also, our laboratory has shown that the in vitro invasiveness of immortalized trophoblasts may be inhibited by nitric oxide [41]. Therefore, to fully elucidate the role of activated macrophages in the etiology of pre-eclampsia it is important to characterize the effect of multiple macrophage-derived molecules on trophoblast invasiveness.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Charles H. Graham, Department of Anatomy and Cell Biology, Botterell Hall Room 859, Queen's University, Kingston, ON, Canada K7L 3N6. FAX: 613 533 2566; grahamc{at}post.queensu.ca
Received: 15 November 2004.
First decision: 21 December 2004.
Accepted: 25 March 2005.
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