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Regular Article |
a Department of Obstetrics and Gynecology and Department of Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) and tumor necrosis factor 
(TNF) and with the anti-inflammatory cytokines interleukin (IL)-6 and IL-10. Sensitivity to Fas-mediated apoptosis was measured using an activating anti-Fas monoclonal antibody. Cell viability was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and CellTiter 96 assay. Fas/FasL mRNA and protein expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Trophoblast cells normally express FasL, but low levels of Fas, and they are resistant to Fas-mediated apoptosis. IFN- and TNF promote Fas expression and sensitivity, whereas IL-6 and IL-10 increase the resistance of trophoblast cells to Fas-mediated apoptosis. Furthermore, IL-10 treatment activates FLICE-like inhibitory protein (FLIP), a downstream inhibitor of Fas apoptotic signaling. Although trophoblast cells express Fas, susceptibility to Fas does not necessarily correlate with its expression. In this study, we demonstrate that Th-2 cytokines increase the resistance of trophoblast cells to Fas-mediated apoptosis either by inhibiting Fas expression or by inducing FLIP activation. This "trophoblast-cytokine-Fas/FasL triad" determines the ability of the Fas/FasL system to regulate trophoblast viability and, consequently, the success or failure of pregnancy.
apoptosis, cytokines, Fas/FasL, placenta, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normal pregnancy is characterized by a lack of maternal cell-mediated immune response against the implanting embryo. Instead, humoral immunity predominates during pregnancy [2, 9]. This switch, from cytolytic to humoral immunity, is the result of maternal changes in the cytokine profile [9]. The balance between T helper (Th)-1, or proinflammatory, cytokines and Th-2, or anti-inflammatory, cytokines is thought to be crucial for determining the success or failure of a pregnancy. It is currently believed that production of proinflammatory cytokines, including interleukin (IL)-2, tumor necrosis factor (TNF), and interferon- (IFN-) is suppressed, whereas production of Th-2 cytokines, such as IL-4, -6, and -10, is enhanced [3, 10].
Placental and decidual tissues isolated from normal pregnancies, however, have been shown to produce both Th-1 and Th-2 cytokines [11–16]. Interestingly, the type of cytokines thought to be potentially harmful to pregnancy in humans as well as in mice are those classified as proinflammatory cytokines. For example, excess production of TNF [17] and IFN- [18] is believed to be involved in preterm delivery. Besides influencing the maternal immune response, many of these cytokines have been shown to affect trophoblast physiology, specifically the proliferation and apoptosis of trophoblast cells [19, 20].
Apoptosis is one of the primary mechanisms in the body that maintains appropriate cell number by removing senescent cells and allowing tissue renewal. The Fas/Fas ligand (FasL) system represents one of the main apoptotic pathways controlling cell proliferation [21] and tissue remodeling [22]. Both Fas and FasL are type I and type II transmembrane proteins, respectively, and are members of the TNF-receptor family. The interaction between Fas and FasL or activating anti-Fas antibodies results in the trimerization of Fas, followed by the assembly of other intracellular proteins to form the death-inducing signal complex (DISC) [21, 23]. Caspase-8 recruitment to the DISC results in its proteolytic activation, which, in turn, activates other members of the caspase family, eventually ending in apoptosis [24].
The Fas/FasL system was originally implicated in the maintenance of lymphocyte homeostasis, during which activated peripheral T cells are removed by a Fas-dependent mechanism [25, 26]. More recently, Fas and FasL have been characterized in several immune processes, including immune privilege [27–30]. Along with other investigators, we have demonstrated the presence of FasL expression in normal trophoblast cells. Moreover, we have shown that FasL is localized to sites where placental tissue is in contact with the maternal immune system [30]. We have suggested that this may represent one of the mechanisms by which the trophoblast escapes maternal immune surveillance [31–34]. However, trophoblast cells also express Fas, which complicates the role of the Fas/FasL system in these cells [35–37].
Recently, we documented that the Fas/FasL system mediates involution of the mammary gland at the end of lactation [22] and is involved in tissue remodeling processes in the human ovary and endometrium [38]. Similarly, French et al. [39] have shown that Fas and FasL are constitutively expressed in tissues characterized by high cell turnover. These and other findings suggest a critical role for Fas-mediated apoptosis in tissue renewal.
In nonimmune tissues, the expression of Fas and FasL correlates with the presence of tissue-specific microenvironment factors and cytokines, which are known to locally modulate immune reactions [40–42]. Therefore, changes in Fas and FasL expression reflect not only a shift in the cytokine profile [41, 43] but also alterations in the hormonal environment of these tissues [22, 44, 45].
In the present study, we examine the hypothesis that decidual and immune cells regulate the expression and activity of Fas/FasL in trophoblast cells via the cytokine composition at the implantation site. The cytokine composition has been studied by numerous investigators and was summarized recently by Hunt et al. [6]. We demonstrate that Th-1 proinflammatory cytokines promote Fas expression, whereas Th-2 anti-inflammatory cytokines inhibit the expression of Fas, thereby decreasing the sensitivity of trophoblast cells to Fas-mediated apoptosis. The enhanced sensitization to Fas results in trophoblast autocrine-induced apoptosis once FasL binds to and activates the Fas receptor. An imbalance in this system may have major relevance in pathologic conditions such as preeclampsia and recurrent abortion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RPMI 1640 and fetal bovine serum were purchased from Life Technologies (Grand Island, NY). Cytokines were obtained from PeproTech, Inc. (Princeton Business Park, NJ), and the anti-human Fas monoclonal antibody was purchased from R&D Systems (Minneapolis, MN). All other chemicals, unless otherwise specified, were obtained from Sigma Chemical Co. (St. Louis, MO).
Clinical Material
First-trimester human placentas (gestational age, 7–12 wk) were obtained from clinically normal pregnancies, which were terminated for nonmedical reasons, at the Yale University School of Medicine, Department of Obstetrics and Gynecology. Each patient completed a signed, written consent form. The Yale University Human Investigation Committee approved the form and the use of the placental tissue. Following vacuum-suction curettage, tissue specimens were collected in cold, sterile PBS and immediately transported to the laboratory for cell culture preparation.
Trophoblast Cell Cultures
Trophoblast cells were prepared from first-trimester placentas as previously described [46, 47]. Briefly, placental material obtained from terminated pregnancies was washed with Dulbecco modified Eagle medium (D-MEM)/F-12 (without phenol red) to remove excess blood. Cells were removed from the membranes by scraping and transferred to digestion buffer solution (D-MEM/F-12 that contained trypsin EDTA). The mixture was stirred with a magnetic stirrer bar for 10 min. An equal volume of D-MEM/F-12 containing 20% calf serum was added to inactivate the trypsin and filtered through two layers of gauze. The filtrate was centrifuged at 430 x g for 5 min. The supernatant was discarded, and the pellet was resuspended in an equivalent volume of Lymphocyte Separation Media (Organon Teknika Corporation, Durham, NC). Following centrifugation at 610 x g for 20 min, the trophoblast cells at the interface were removed and washed with D-MEM/F-12 medium. Finally, the cells were resuspended in D-MEM/F-12 containing 10% fetal calf serum, 2 mM L-glutamine, and 100 U/ml of penicillin/streptomycin (Sigma).
The isolated human trophoblast cells were plated in plastic Petri dishes (diameter, 35 mm) and incubated at 37°C for 40 min to allow the contaminating macrophages to adhere to the plastic. The nonadherent trophoblast cells were transferred to fresh plates and cultured in minimum essential media (MEM) with 10% fetal bovine serum and D-valine substituted for L-valine at a concentration of 1 x 106 cells/ml at 37°C in 95% air and 5% CO2. The purity of the trophoblast cell preparation was approximately 98% as determined by immunohistochemistry, in which anticytokeratine antibody was used and the production of hCG was confirmed [47, 48].
Cell Lines
The trophoblast stable cell line A3 was purchased from ACTT (Gaithersburg, MD), and the trophoblast stable cell line HTR8 was a gift from Dr. Charles Graham (Queen's University, Kingston, ON, Canada). Cells were grown in 35-mm plates containing RPMI media with 10% fetal bovine serum.
Cytokine Treatment
Cells were grown to 80% confluence, at which stage the medium was replaced with reduced-serum, phenol-depleted Opti-MEM for 24 h before cytokine treatment. IFN-, IL-6, leukemia inhibitory factor, IL-10, and TNF were added to the cells at various doses (0.1, 1, and 10 ng/ml) and for different time periods (3, 6, 24, and 48 h of treatment).
Preparation of Total RNA and Protein Samples
Total RNA and protein were isolated from trophoblast cells with the use of Trizol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer's instructions. This method allowed us to simultaneously extract both RNA and protein from the same sample.
Reverse Transcription-Polymerase Chain Reaction
Details of the RT-PCR characterization have been previously described [41, 44]. In short, RT was performed using the RT-PCR kit from Amersham Pharmacia Biotech (Piscataway, NJ) according to the manufacturer's instructions. The cDNA synthesis was performed using 0.2 µg of pd(N)6 and 5 µg of total RNA. The primers used for the human FasL amplification have been previously described [29] and had the following sequence: forward, 5'-ATAGGATCCATGTTTCTGCTCTTCCACCTACAGAAGGA-3'; reverse, 5'-ATAGAATTCTGACCAAGAGAGAGCTCAGATACGTTGAC-3'. Each PCR cycle for human FasL consisted of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, and elongation at 72°C for 1 min for a total of 35 cycles. The primers used for the amplification of human Fas had the following sequence: forward, 5'-AAGGAGTACACAGACAAAGCCC-3'; reverse, 5'-AAGAAGAAGACAAAGCCACCC-3'. Each PCR cycle for human Fas consisted of denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, and elongation at 72°C for 30 sec for a total of 30 cycles. The PCR products were visualized on 1%–2% agarose gels stained with ethidium bromide in 1x Tris-acetate EDTA.
The level of Fas and FasL expression was measured by densitometric analysis and standardized by comparison to the ß-actin control using a digital imaging and analysis system (1D Image Analysis software; Eastman Kodak Company, Scientific Imaging Systems, Rochester, NY). The linearity of the system was determined using a serial dilution of cDNA. The cDNA dilution factor was linear (y = 2881.125x - 785.75), and the correlation coefficient was r = 0.994, as previously described [41, 44].
Western Blot Analysis
Proteins were separated by SDS-PAGE using 10% polyacrylamide gels and transferred to nitrocellulose membranes as previously described [28]. To inhibit nonspecific binding, the membranes were blocked in 5% powdered milk before immunoblotting. The blots were incubated with the following primary antibodies: clone 33 monoclonal antibody (1:1000 [v/v] dilution; Transduction Laboratory, Lexington, KY) for FasL, M-20 polyclonal antibody (1:500 [v/v] dilution; Santa Cruz Biotechnology, Santa Cruz, CA) for Fas, and rabbit polyclonal immunoglobulin (Ig) G (Upstate Biotechnology, Lake Placid, NY) for FLIP, an intracellular component of the Fas apoptotic pathway. After washing with PBS-Tween, the membranes were incubated with either horse anti-mouse gamma globulin or goat anti-rabbit (Vector, Burlingame, CA) peroxidase-labeled secondary antibodies for 1 h. Finally, the blots were developed using either the TMB Peroxidase substrate kit (Vector) or the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). To ascertain the specificity of the clone 33 anti-FasL monoclonal antibody used in the present study, we compared the polyclonal antibody N-20 from Santa Cruz and the clone NOK-1 from Pharmingen (Palo Alto, CA) by Western blot analysis (for details, see [41, 44]).
All experiments were repeated at least 3 times. The intensity of the signal was analyzed using 1D Image Analysis software.
Evaluation of the Effect of Cytokines on Trophoblast Viability
The effect of cytokines on trophoblast cell viability was measured using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) proliferation assay (Sigma) and the CellTiter 96 proliferation assay (Promega, Madison, WI).
For the MTT assay, 200 µl containing 5000–10000 cells in media with 10% serum were seeded in a 96-well, flat-bottom microplate (Becton Dickinson and Company, Lincoln Park, NJ) and incubated at 37°C for 48 h. Cells were grown to 80% confluence, at which stage the medium was replaced with reduced-serum, phenol-depleted Opti-MEM for 24 h before cytokine treatment. The medium was then removed, and the cells were treated with the cytokines at 37°C for 24–48 h. Following treatment, 20 µl of the MTT reagent were added to each well and incubated at 37°C for 4 h. The medium was decanted, and 100 µl of acid-isopropyl alcohol were added to solubilize the reactive crystals. Absorbency was measured at a wavelength of 540 nm on an automatic microplate reader (Model 550; Bio-Rad, Hercules, CA).
The effect of cytokines on cell proliferation was also tested with the CellTiter 96 assay. Cells were treated as described above. The CellTiter 96 Aqueous One Solution was then added to the wells, and the absorbency was measured 1–4 h later at a wavelength of 490 nm. As the cells undergo apoptosis, total viability decreases. Thus, the values of the treated cells were compared to the values generated from the untreated control cells and reported as the percentage viability.
Evaluation of Trophoblast Sensitivity to Fas-Mediated Apoptosis
The sensitivity of trophoblast cells to Fas was evaluated with an activating anti-human Fas monoclonal antibody (R&D Systems). Following cytokine treatment as described above, the trophoblast cells were incubated with various doses of mouse monoclonal IgG anti-Fas antibody in 200 µl of medium. Incubation with normal mouse, IgG-isotype control antibody (R&D Systems) was used as a nonspecific control. Cell viability was evaluated using the MTT assay and the CellTiter 96 assay. The presence of apoptotic cells was confirmed by labeling the cells with propidium iodide (PI) and by measuring caspase-3 activity using the CaspACE assay system (Promega) according to the manufacturer's instructions.
Blocking of Apoptosis with Anti-FasL Monoclonal Antibody
Trophoblast cells were treated with different concentrations (20, 100, and 500 ng/ml) of either anti-FasL monoclonal antibody (NOK-mAb; Pharmingen) or mouse IgG1 isotype-matched control monoclonal antibody (Pharmingen) for 24 h before cytokine treatment. Cell viability was determined using the MTT assay and the CellTiter 96 assay. Each assay was performed at least 3 times in triplicate.
PI Staining
The presence of apoptotic cells was determined by PI staining. Trophoblast cells (1 x 105) were incubated in chamber slides and treated as described above. After each treatment, 1 µg/ml of PI was applied to the cells and fixed with 4% paraformaldehyde. The presence of positively stained trophoblast cells was evaluated with the Zeiss fluorescent microscope (Oberkochen, Germany) and Image Analysis software Openlab 2 (Improvision, Lexiton, MA).
Statistical Analysis
The data were analyzed for statistical significance using the Student t-test, Mann-Whitney test, or Kruskal-Wallis test. All experiments were repeated 2 or 3 times with indistinguishable results.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we reported that conditioned media from resting macrophages was beneficial to trophoblast cell growth, whereas that obtained from lipopolysaccharide-activated macrophages had a detrimental effect [41, 47]. Based on these results, we propose that the activation status of the macrophages might determine the type of cytokines (Th-1 vs. Th-2) present at the implantation site. Consequently, this may affect the fate of the trophoblast cells.
To determine whether the type of cytokine has a differential effect on trophoblast cell survival and apoptosis, we tested two different in vitro systems. The first consisted of primary culture trophoblast cells prepared from first-trimester human placentas, and the second consisted of A3 and HTR/8 stable, first-trimester trophoblast cell lines. No major differences were observed between these two systems. Hereafter, we will refer to both the primary culture cells and the cell lines as trophoblast cells, but we will distinguish between the two when differences were detected.
Treatment with 5 ng/ml of IFN- or 10 ng/ml of TNF for 48 h decreased the number of viable trophoblast cells in both the primary cultures (42% decrease) and the cell lines (38% decrease for HTR/8 cells and 41% decrease for A3 cells) (Fig. 1A). Treating the cells with either IL-6 or IL-10 had the opposite effect, and an increase in trophoblast cell number was observed when compared to the nontreated cells (54% increase). Figure 1 shows the effect of these cytokines on H8 cells.
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Effect of Cytokines on Fas-Mediated Apoptosis
After finding that the proinflammatory (IFN- and TNF) and anti-inflammatory (IL-10 and IL-6) cytokines had different effects on trophoblast survival, we further evaluated whether this variation correlated with changes in sensitivity to Fas-mediated apoptosis. Cells were treated with either IFN-, TNF, IL-10, or IL-6 as described above, then incubated with an anti-Fas monoclonal antibody, which is known to activate the Fas receptor and to induce apoptosis [49]. As shown in Figure 2, trophoblast cells are normally resistant to Fas-mediated apoptosis. However, a statistically significant increase in Fas sensitivity was observed following treatment with IFN- or TNF for 24 h. No effect was observed when the cells were treated with a nonspecific IgG monoclonal antibody under similar conditions.
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Contrary to the effect of IFN- and TNF, treatment with IL-6 increased trophoblast resistance to Fas-induced apoptosis and even stimulated cell proliferation (Fig. 2). Furthermore, IL-10 treatment not only conferred resistance to Fas-mediated apoptosis but also inhibited the apoptotic effect of IFN- and anti-Fas monoclonal antibody (Fig. 3).
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Presence of Apoptotic Cells Following Proinflammatory Cytokine Treatment
To determine whether the decreased percentage of viable cells following IFN- or TNF treatment was due to apoptosis, we stained trophoblast cells (both cell lines and primary-culture cells) with PI. As shown in Figure 4A, treatment with IFN- was associated with an increase in the number of PI-positive cells (Fig. 4A-2). An additional increase in the number of PI-positive cells was observed following treatment with both IFN- and anti-Fas monoclonal antibody (Fig. 4A-3). In contrast, very few PI-positive cells were found in the untreated control group (Fig. 4A-1). Further confirmation of apoptosis was obtained by evaluating caspase-3 activation. Treatment with IFN- and TNF induced a 35% and 120% increase, respectively, in the level of active caspase-3. In addition, an increase in caspase-3 activity was observed in the group of cells incubated with both IFN- and anti-Fas monoclonal antibody (114%) as well as in those incubated with TNF and anti-Fas monoclonal antibody (180%) (Fig. 4B). This was not the case for the untreated control cells or for the cells treated with IL-6 or IL-10 (data not shown).
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Effect of Proinflammatory Cytokines on Fas and FasL Expression in Trophoblast Cells
Because treatment with proinflammatory cytokines and anti-Fas monoclonal antibody increased the number of apoptotic cells, we studied the effects of IFN- and TNF on Fas and FasL expression. Both primary-culture human trophoblast cells and trophoblast cell lines generally express similar amounts of Fas and FasL mRNA and protein. When trophoblast cells were treated with IFN- (5 ng/ml), however, Fas protein expression increased after 12 h and remained higher than control levels even after 24 h of treatment (Fig. 5A). Similarly, TNF (10 ng/ml) increased Fas expression in a time-dependent manner (Fig. 5B). In contrast, neither IFN- nor TNF treatment had an effect on FasL expression in trophoblast cells (Fig. 5).
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Effect of Anti-Inflammatory Cytokines on Fas and FasL Expression in Trophoblast Cells
After demonstrating that IL-6 stimulates proliferation of trophoblast cells, we sought to determine whether IL-6 affected Fas and FasL protein expression in these cells. Treating primary trophoblast cell cultures with IL-6 inhibited Fas protein expression in a dose-dependent manner (from 0.1 to 10 ng/ml). In contrast, HTR/8 and A3 trophoblast cells did not show any change in Fas expression following IL-6 treatment. In addition, IL-6 had a weak stimulatory effect on FasL expression in the primary-culture cells but not in the trophoblast cell lines (data not shown).
Contrary to IL-6, treatment with IL-10 (10 ng/ml) increased Fas protein expression in trophoblast cells but did not affect Fas mRNA levels (Fig. 6A and data not shown, respectively). This stimulatory effect on Fas protein levels exhibited a differential dose response following IL-10 treatment. Thus, lower doses of IL-10 had a more significant effect on Fas protein levels in trophoblast cells. In addition, treating trophoblast cells with IL-10 increased FasL expression at the protein level in both a dose-dependent (Fig. 6A) and a time-dependent (Fig. 6C) manner.
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IL-10 Regulates Cellular FLIP in Trophoblast Cells
Because the effect of IL-10 treatment on Fas expression was contradictory to the protective effect observed in the proliferative assays, we tested the hypothesis that IL-10 may regulate a downstream component of the Fas-apoptotic pathway. Previous reports have suggested that cellular FLIP (c-FLIP) can interfere with Fas-triggered activation of caspase-8. Therefore, we investigated the possible role of c-FLIP in the IL-10-induced resistance of trophoblast cells to Fas-mediated apoptosis. Under normal culture conditions (10% serum), trophoblast cells express both the long, inactive form (FLIPL) and the active, cleaved form (FLIPC) of FLIP. Whereas removal of serum normally decreases the amount of active FLIP, treating the trophoblast cells with IL-10 increased the expression of the active as well as the inactive form in a dose-dependent manner (Fig. 6B). Interestingly, an increase in both FLIPL and FLIPC was detected as early as 3 h, but the level of FLIPL decreased after 24 h of IL-10 treatment (10 ng/ml) (Fig. 6D).
IFN--Induced Apoptosis in Trophoblast Cells Is Blocked by an Anti-FasL Monoclonal Antibody
To confirm that the Fas/FasL system is associated with the IFN--induced apoptosis of trophoblast cells, we incubated the cells with a blocking anti-FasL antibody before IFN- treatment. As shown in Figures 1 and 7, cell viability decreased by 50%–60% following treatment with IFN-. However, this effect was completely abolished as the cells were treated with increasing concentrations of the blocking anti-FasL antibody (Fig. 7). As a control, trophoblast cells were incubated with a nonspecific-isotype IgG antibody, and inhibition of apoptosis was not observed (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both IFN- and TNF, which are Th-1 cytokines, up-regulate Fas expression and increase the sensitivity of trophoblast cells to Fas-mediated apoptosis. Conversely, IL-6 and IL-10, which are Th-2 cytokines, prevent apoptosis by inducing changes in the sensitivity of the cells to Fas, albeit by different mechanisms.
One of the most studied aspects in the immunology of pregnancy is the shift from a Th-1 to a Th-2 cytokine profile [1]. However, previous studies investigating the role of cytokines in implantation generally have focused on the effect of cytokines on the type of activated immune cells and the type of immune response that was elicited [51]. In the present study, we demonstrate a nonimmune effect on trophoblast physiology resulting from a shift in cytokine profile. Moreover, we show a differential effect of proinflammatory and anti-inflammatory cytokines on the survival and apoptosis of trophoblast cells via the Fas/FasL system.
Characterization of the Fas/FasL pathway has evolved dramatically. Since its original description as the mechanism controlling the turnover of peripheral activated mature T cells [21, 52, 53], it has become evident that the Fas/FasL system plays a major role in continuous tissue homeostasis [22, 38]. Furthermore, understanding Fas/FasL interactions has changed the paradigm regarding the establishment and maintenance of immune-privilege sites. Once believed to depend on physical barriers and anatomical isolation, immune privilege is now viewed as an active process by which infiltrating, activated lymphoid cells are eliminated by the Fas/FasL apoptotic pathway [28, 54].
Along with other investigators, we have suggested a mechanism by which placental expression of FasL serves as the key component for establishing immune privilege at the maternal-fetal interface [30, 33, 55]. Contrary to the old concept of a physical barrier, the trophoblast is now believed to function as a dynamic obstacle, averting activated and potentially detrimental T cells. Once they recognize placental alloantigens, activated T cells express Fas, which interacts with FasL on placental trophoblast cells. Consequently, the activated T cells undergo Fas-mediated apoptosis [30, 32, 34].
The role of Fas and FasL in trophoblast physiology, however, is presumably more complex than originally anticipated. Along with other investigators, we have described the presence of Fas in the normal trophoblast during the different stages of pregnancy. Furthermore, Fas is known to be widely expressed in numerous tissues and cell lines. Despite the presence of Fas and FasL, however, the normal first-trimester trophoblast is resistant to Fas-mediated apoptosis. Because susceptibility to Fas does not necessarily correlate with its trophoblast cell expression, cellular inhibitors must exist in the Fas-induced signaling pathway to prevent apoptosis. In the present study, IL-10 treatment increased Fas expression, but it did not alter the sensitivity of trophoblast cells to Fas-mediated apoptosis. Rather, IL-10 treatment increased trophoblast resistance to apoptosis, suggesting a mechanism of action other than regulation of Fas expression. Fas signaling can be inhibited by FLIP, an intracellular component of the Fas apoptotic pathway, by interfering with the activation of caspase-8 at the DISC [56]. FLIP is structurally similar to caspase-8 in that it contains two death-effector domains and a caspase-like domain. Unlike caspase-8, however, FLIP is catalytically inactive. Once FLIP binds to the FADD (Fas associated death domain), procaspase-8 directly interacts with FLIP, thereby preventing the cleavage of procaspase-8 into its active form. Instead, FLIP is cleaved into its active p43 form [57]. Because FLIP expression has been shown to increase tumor resistance to Fas-mediated apoptosis [58], we evaluated whether IL-10 may have an effect on FLIP expression in trophoblast cells. Indeed, IL-10 treatment increased both the expression and the activation of FLIP in trophoblast cells. Thus, the presence of IL-10 in the microenvironment may regulate trophoblast sensitivity to Fas-mediated apoptosis by regulating FLIP expression and activation.
In this context, an important question to consider is which cells are regulating the cytokines at the implantation site? One of the main candidates is the macrophage. As a major cell type in both the maternal and fetal compartments of the uteroplacental unit [59], macrophages constitute an important source of cytokines and growth factors [19, 60]. During the first few weeks of human implantation, macrophages are found in high numbers in the maternal decidua and in tissues proximal to the placenta [61]. Similarly, macrophages accumulate at or near the implantation site in rodents [62]. The dense macrophage infiltration at the maternal-fetal interface suggests that these cells not only perform their normal immune tasks but are also involved in specific pregnancy-associated functions. Hunt [19] proposed that maternal macrophages assist in the tissue remodeling necessary to accommodate expansion of extraembryonic tissue. However, macrophages are not merely scavengers of dying cells; they also actively orchestrate the apoptosis of unwanted cells during tissue remodeling [63]. Similarly, uterine epithelial cells surrounding the blastocyst undergo apoptosis and may form an anti-inflammatory environment by increasing Th-2 cytokines during embryo implantation. This may explain the unexpected cohabitation of macrophages and trophoblast cells at the implantation site. In this view, the uptake of apoptotic cells would actively suppress activated macrophages from secreting proinflammatory cytokines such as TNF and IFN- [64] and promote the release of Th-2 anti-inflammatory and immunosuppressive cytokines [64].
According to our model, the anti-inflammatory action of apoptotic cell phagocytosis may be perturbed in disease processes. For example, antiphospholipid antibodies in apoptotic cells may bind to the Fc receptors in macrophages, resulting in secretion of TNF, a Th-1 proinflammatory cytokine [65]. If this occurs during pregnancy, cytokine production by macrophages and other cells at the maternal-fetal interface may be drastically altered [66]. Our results are in concert with this concept, and they indicate that enhanced levels of proinflammatory macrophage products increase Fas expression and enhance trophoblast sensitivity to Fas-mediated apoptosis.
Two recent studies have described increased trophoblast cell apoptosis in pregnancies complicated by preeclampsia in comparison to normal controls [67, 68]. Concomitant with this increase in apoptosis, those authors found elevated trophoblast Fas expression in the women with preeclampsia [68]. Interestingly, high levels of neutrophil activation have also been described in patients with preeclampsia [69–71]. It is possible that a dense neutrophil infiltration may alter the surrounding environment at the maternal-fetal interface, thereby promoting the up-regulation of Fas expression in nonimmune cells such as trophoblast and vascular endothelium and allowing FasL-induced inflammation and apoptosis [72]. In the same vein, natural killer cells, another important immune component of the implantation site, may help to maintain the adequate balance between pro- and anti-inflammatory cytokines [7].
In summary, important reproductive events, including implantation, trophoblast invasion, placental development, and immune protection, are regulated by cytokines produced at the maternal-fetal interface [1]. The Fas/FasL system represents one of the major mechanisms through which locally acting cytokines may influence these crucial processes. The present study provides new insight toward the understanding of implantation and maternal-fetal immune interactions. Moreover, the present results shed light on the dual role and delicate balance of the Fas/FasL system in trophoblast immune protection and self-induced apoptosis.
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FOOTNOTES |
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1 Supported by grants from the National Institutes of Health (RO1 HD37137-01A2 and R01 CA92435-01) to G.M. 
2 Correspondence: Gil Mor, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., FMB 202, New Haven, CT 06520. FAX: 203 785 4883; gil.mor{at}yale.edu
Accepted: January 11, 2002.
Received: August 14, 2001.
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V. M. Abrahams, T. M. Schaefer, J. V. Fahey, I. Visintin, J. A. Wright, P. B. Aldo, R. Romero, C. R. Wira, and G. Mor Expression and secretion of antiviral factors by trophoblast cells following stimulation by the TLR-3 agonist, Poly(I : C) Hum. Reprod., September 1, 2006; 21(9): 2432 - 2439. [Abstract] [Full Text] [PDF]
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 S. L. Straszewski-Chavez, V. M. Abrahams, and G. Mor The Role of Apoptosis in the Regulation of Trophoblast Survival and Differentiation during Pregnancy Endocr. Rev., December 1, 2005; 26(7): 877 - 897. [Abstract] [Full Text] [PDF]
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 M. Kimatrai, O. Blanco, R. Munoz-Fernandez, I. Tirado, F. Martin, A. C. Abadia-Molina, and E. G. Olivares Contractile Activity of Human Decidual Stromal Cells. II. Effect of Interleukin-10 J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6126 - 6130. [Abstract] [Full Text] [PDF]
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 G. Jaita, M. Candolfi, V. Zaldivar, S. Zarate, L. Ferrari, D. Pisera, M. G. Castro, and A. Seilicovich Estrogens Up-Regulate the Fas/FasL Apoptotic Pathway in Lactotropes Endocrinology, November 1, 2005; 146(11): 4737 - 4744. [Abstract] [Full Text] [PDF]
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 V. M. Abrahams, P. Bole-Aldo, Y. M. Kim, S. L. Straszewski-Chavez, T. Chaiworapongsa, R. Romero, and G. Mor Divergent Trophoblast Responses to Bacterial Products Mediated by TLRs J. Immunol., October 1, 2004; 173(7): 4286 - 4296. [Abstract] [Full Text] [PDF]
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 B. Huppertz and J. C. P. Kingdom Apoptosis in the Trophoblast--Role of Apoptosis in Placental Morphogenesis Reproductive Sciences, September 1, 2004; 11(6): 353 - 362. [Abstract] [PDF]
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R. M. Gill and J. S. Hunt Soluble Receptor (DcR3) and Cellular Inhibitor of Apoptosis-2 (cIAP-2) Protect Human Cytotrophoblast Cells Against LIGHT-Mediated Apoptosis Am. J. Pathol., July 1, 2004; 165(1): 309 - 317. [Abstract] [Full Text] [PDF]
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S. L. Straszewski-Chavez, V. M. Abrahams, E. F. Funai, and G. Mor X-linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis Mol. Hum. Reprod., January 1, 2004; 10(1): 33 - 41. [Abstract] [Full Text] [PDF]
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V. M. Abrahams, S. L. Straszewski-Chavez, S. Guller, and G. Mor First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis Mol. Hum. Reprod., January 1, 2004; 10(1): 55 - 63. [Abstract] [Full Text] [PDF]
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D. R Balkundi, J. A Ziegler, J. F Watchko, C. Craven, and M. Trucco Regulation of FasL/Fas in Human Trophoblasts: Possible Implications for Chorioamnionitis Biol Reprod, August 1, 2003; 69(2): 718 - 724. [Abstract] [Full Text] [PDF]
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