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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013102
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BIOLOGY OF REPRODUCTION 69, 718–724 (2003)
DOI: 10.1095/biolreprod.102.013102
© 2003 by the Society for the Study of Reproduction, Inc.

Pregnancy

Regulation of FasL/Fas in Human Trophoblasts: Possible Implications for Chorioamnionitis1

Dhruv R Balkundi2,3, Judy A Ziegler3, Jon F Watchko3, Catherine Craven4, and Massimo Trucco5

Department of Pediatrics,3 Division of Neonatology and Developmental Biology, University of Pittsburgh, Magee Womens Research Institute, Pittsburgh, Pennsylvania 15213 Department of Pathology,4 Magee Womens Hospital, Pittsburgh, Pennsylvania 15213 Department of Pediatrics,5 Division of Immunogenetics (MT), University of Pittsburgh, Pittsburgh, Pennsylvania 15213


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chorioamnionitis is a common cause of premature birth and is associated with significant morbidity and mortality in the mother and infant. Preterm birth shares similarities with rejection of the fetal allograft, which is characterized by increased apoptosis of placental trophoblasts. We hypothesized that there is increased trophoblast apoptosis in chorioamnionitis and that this increased apoptosis is mediated by the Fas ligand (FasL)/Fas pathway. To test our hypothesis, we examined placental villous tissues from patients with chorioamnionitis and used the TUNEL assay to demonstrate enhanced trophoblast apoptosis in patients with chorioamnionitis. When the same samples were stained for Fas, there was increased trophoblast Fas expression in patients with chorioamnionitis. To define the mechanisms responsible for this increase in trophoblast apoptosis, we cultured villous explants from uncomplicated term placentas with proinflammatory cytokines and demonstrated a marked increase in trophoblast apoptosis. By blocking FasL, we reduced tumor necrosis factor {alpha}-induced and interferon {gamma}-induced apoptosis. These data suggest that chorioamnionitis is associated with increased trophoblast apoptosis and enhanced trophoblast Fas expression. As a complement to our in vivo study, we demonstrated that cytokine-induced trophoblast apoptosis is mediated in part by the FasL/Fas pathway, suggesting that cytokines promote sensitivity to Fas-mediated apoptosis. These mechanisms may be important in perpetuating inflammation in the placental microenvironment and may contribute to the pathogenesis of chorioamnionitis.

apoptosis, cytokines, immunology, placenta, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chorioamnionitis is the most common cause of premature delivery in gestations of <30 wk and occurs in approximately 10% of all births [1, 2]. Histologic signs of chorioamnionitis [3] and an increase in inflammatory mediators [4–6] can be detected in >50% of women who deliver prematurely. Intrauterine infection during chorioamnionitis activates placental and decidual cells to produce a number of proinflammatory cytokines at the fetomaternal interface [6–8], including tumor necrosis factor {alpha} (TNF{alpha}), interleukin (IL) 1ß, interferon {gamma} (IFN{gamma}), IL-6, IL-8, and granulocyte colony-stimulating factor (G-CSF) [5, 8, 9]. Recent evidence suggests that proinflammatory cytokines are potentially harmful to pregnancy in mice [10] and humans [11]. For example, excess production of TNF{alpha} and IFN{gamma} is thought to be involved in premature delivery [11, 12]. In addition to influencing the maternal immune response, many of these cytokines affect trophoblast physiology, specifically the proliferation and apoptosis of trophoblast cells [13].

Apoptosis is an important process that maintains the appropriate cell numbers by removing excess cells. The Fas ligand (FasL)/Fas pathway is an important pathway of apoptosis that controls cell proliferation and tissue remodeling [14]. Fas (CD95) is a transmembrane protein of the TNF/nerve growth factor superfamily that is expressed on both immune and nonimmune cell types [15]. Fas when bound by FasL activates a signal transduction pathway that eventually results in apoptosis of the cell. FasL/Fas-mediated apoptosis is essential for immune homeostasis, a process by which unnecessary peripheral T lymphocytes are removed from the active repertoire [16]. FasL also plays an important role in several immune processes, including immune privilege [17, 18]. We and others have demonstrated that FasL present on human trophoblasts may confer immune privilege to the fetus by destroying maternal T cells. [19–23].

In addition to FasL, the trophoblasts express its receptor Fas. Several other tissues, including mammary gland [14], human ovary [24], and endometrium [25], express both FasL and Fas. Despite expressing both FasL and Fas, villous trophoblasts undergo apoptosis in limited numbers during normal pregnancy [26]. However, trophoblasts exhibit increased apoptosis in pregnancies complicated by fetal growth restriction [26] and preeclampsia [27, 28]. Recent evidence suggests that the expression of FasL and Fas in nonimmune cells is correlated with the presence of a tissue-specific microenvironment [29, 30]. A number of factors in the placental microenvironment, including hormones [31] and cytokines [32], modulate the immune response by regulating the expression of FasL and Fas. Chorioamnionitis is a commonly occurring inflammatory state associated with a marked increase in proinflammatory cytokines in the placental microenvironment. In the present study, we tested the hypothesis that chorioamnionitis is associated with increased villous trophoblast Fas-mediated apoptosis and that proinflammatory cytokines regulate the expression and activity of FasL and Fas at the fetomaternal interface. We demonstrated that trophoblast apoptosis is markedly increased in patients with chorioamnionitis in association with increased Fas expression on trophoblasts. Proinflammatory cytokines also induce trophoblast apoptosis in part via the FasL/Fas apoptosis pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Collection and Processing

Placentas from patients with chorioamnionitis This study was approved by the Institutional Review Board at Magee Womens Hospital. Paraffin-embedded placental villous tissues from patients (n = 5) with grade 3 chorioamnionitis [33] were obtained from an archival collection in the Department of Pathology at Magee Womens Hospital. Placental tissues from gestational age-matched patients without chorioamnionitis were used as controls. The diagnosis of chorioamnionitis was confirmed by a perinatal pathologist, and the severity categorized as grades 1–3 using a modification of the Blanc classification [33]. Grade 3, or severe chorioamnionitis, is characterized by a dense infiltration of neutrophils (>30 neutrophils/high-power field) that extends from the subchorionic space throughout the chorion.

Placentas from uncomplicated pregnancies Human placental tissues were obtained from normal pregnant women at term (39–41 wk gestation, n = 15) after elective cesarean section. Inclusion criteria were 1) a single fetus, 2) no preexisting clinical conditions such as diabetes, hypertension, or autoimmune disease, and 3) no clinical evidence of chorioamnionitis or group B streptococcal infection. Once received, the tissues were processed immediately.

In Situ End Labeling of DNA for Cell Death Detection

Trophoblast apoptosis in placental villous tissues from patients with chorioamnionitis was detected by enzymatic labeling of DNA strand breaks using the TUNEL assay performed using a cell death detection kit (Oncogene Research Products, San Diego, CA). Tissue sections were first treated with 2 mg/ml of proteinase K, followed by quenching with 3% H2O2 and equilibration with TdT equilibration buffer. The sections were then blocked with blocking buffer followed by incubation with the substrate diamimobenzidine (DAB) and counterstained with methyl green. The slides were then examined by one of us (D.R.B.) who was blind to the diagnosis of chorioamnionitis. Ten high-power fields (200x) were examined under a light microscope. Only the fields that showed complete villous structure were included in the study. Villi at the edge of the section were excluded from the study to avoid false-positive results due to an edge artifact. Positive staining was defined as brown nuclei. Apoptotic nuclei were counted in 10 random fields and reported as number of apoptotic nuclei per 10 high-power fields.

Immunohistochemistry for Fas

Trophoblast Fas expression in paraffin-embedded placental villous tissues from patients with chorioamnionitis was determined by immunohistochemistry using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Tissue sections were deparaffinized and rehydrated through graded alcohols using standard procedures. Endogenous peroxidase activity was quenched by a 30-min incubation with 0.3% H2O2 in water. Nonspecific binding sites were blocked by incubation with 5% goat serum. Samples were incubated with Fas antibody (polyclonal antibody C-20 at 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Sections were then washed twice and incubated with a biotinylated secondary antibody diluted at 1:200 in PBS. Two successive washing steps were followed by incubation with preformed avidin-biotinylated peroxidase complex. Sections were developed with DAB as the chromogen substrate, rinsed, and counterstained with Mayer hematoxylin. Sections incubated with PBS instead of primary antibody served as negative controls.

Culture of Placental Villous Explants with Cytokines and FasL Blocking Antibody

Placental villous tissues (100 mg) obtained from uncomplicated pregnancies were rinsed with warm 0.9% NaCl solution, and explants were established in six-well culture plates. The explants were cultured with different concentrations of IL-1ß, IFN{gamma}, or TNF{alpha} (PeproTech, Rocky Hill, NJ) for 24 h. The culture medium consisted of Dulbecco modified Eagle medium (DMEM; Mediatech, Herndon, VA) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and antibiotics. The explants were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The concentrations of cytokines used were 0.1, 1, 10, and 50 ng/ml. These concentrations are consistent with the amniotic fluid concentrations seen in patients with chorioamnionitis [4]. After 24 h of culture, the explants were fixed in formaldehyde for TUNEL assay.

In experiments using FasL-blocking antibody, villous explants from placentas of uncomplicated pregnancies were processed as above. The explants were initially treated with a specific FasL-blocking protein Fas-Fc (Alexis Corporation, San Diego, CA) for 1 h at a concentration of 2 µg/ml. Pretreatment with Fas-Fc for 1 h completely blocked FasL-induced apoptosis in an ovarian carcinoma cell line [24]. Proinflammatory cytokines (TNF{alpha}, IL-1ß, or IFN{gamma}) were then added to the six-well plates at a concentration of 10 ng/ml and cultured under standard cell culture conditions for a further 24 h. Explants pretreated with Fas-Fc were also cultured in 10% serum without cytokines. At the end of 24 h, the explants were fixed in formaldehyde, and paraffin-embedded sections were prepared. The sections were then stained to reveal apoptotic nuclei using the TUNEL technique.

Isolation of Cytotrophoblasts

Cytotrophoblasts were isolated and purified using a modified version of the protocol established by Kliman et al. [34]. Term placental tissues were obtained after elective cesarean section under sterile conditions and processed immediately. Several cotyledons from a single placenta were removed from the underlying fibrous elements and rinsed thoroughly in 0.9% NaCl and penicillin (50 U/ml)/streptomycin (50 µg/ml) (Sigma Chemical Co., St. Louis, MO). Soft villous material from the fetal surface was cut away from the connective tissues and blood vessels. Approximately 30–40 g of tissue was collected from each placenta. The tissue was coarsely minced and transferred to 150 ml warmed calcium- and magnesium-free Hanks solution (CMF Hanks) containing 25 mM Hepes (Sigma). The tissue was then digested with trypsin (1 mg/ml) and DNase 1 (5 mg/ml; Sigma) at 37°C for 20 min each in a shaking water bath. The flask was then set at an angle, and tissue fragments were allowed to settle for 5 min; the supernatant was then discarded. The tissue fragments were subjected to the enzymatic digestion three more times as described above, and the supernatant was collected each time. The supernatant was layered over 1.5 ml calf serum in a 15-ml polystyrene conical centrifuge tubes and pelleted. The resultant pellets were resuspended in DMEM containing 25 mM Hepes and 25 mM glucose (DMEM-H-G) at room temperature. The final pellet was then resuspended in 3 ml DMEM-H-G and layered on a preformed discontinuous Percoll (Pharmacia, Piscataway, NJ) gradient made up in CMF Hanks solution. The gradient was centrifuged at room temperature for 20 min, and a multilayered preparation of eight bands was obtained. The middle four to six layers from the bottom (25%–35% density area) containing cytotrophoblasts were then washed with DMEM-G, and the cells were counted using a hemocytometer. The cell suspension was then purified by eliminating CD45+ cells following culture with CD45-coated magnetic beads (Polysciences, Warrington, PA). This step is needed to obtain a pure population of cytotrophoblasts. We routinely have been able to isolate 1–1.5 x 106 cells/g of placental tissue with this method. The purity of the cytotrophoblast population (generally >95%) was ascertained using anti-cytokeratin and anti-CD45 antibodies (R&D Systems, Minneapolis, MN). The freshly isolated cells were used for cell culture and Western immunoblotting.

Culture of Cytotrophoblasts with Cytokines

The cytotrophoblasts were cultured for 24 h in DMEM with 10% FCS in 12-well plates at a cell density of 3 x 106 cells/well. The cells were then washed with sterile PBS, dead (floating) cells were gently aspirated, and the adherent cells were incubated with increasing concentrations (0.1, 1, 10, 50, and 100 ng/ml) of individual cytokines (IL-1ß, TNF{alpha}, or IFN{gamma}). These concentrations were chosen because they represent the range of cytokines in amniotic fluid in women with proven histologic chorioamnionitis [4]. The cell lysates were then subjected to Western blot analysis.

Western Blot Analysis for FasL and Fas Expression

Protein extraction and Western blot analysis of FasL and Fas were performed as previously described [22]. After 24 h of culture with cytokines, 3 x 106 cytotrophoblasts cultured with cytokines were washed once in PBS (1x) and harvested in PBS containing 1 mM PMSF and 10 µg/ml leupeptin (Sigma). The resultant cell pellet was resuspended in 15 µl of Laemmli sample buffer and then sonicated on ice for 15–20 sec. After centrifugation at 14 000 rpm for 15 min, the supernatants were collected, and total protein concentrations determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the protein standard.

Equal amounts of protein extracts were separated by SDS-PAGE using 10% polyacrylamide gels and then transferred to nitrocellulose membranes. After blocking nonspecific binding sites by treatment with 5% milk powder and 0.05% Tween-20 in PBS, the blots were washed and probed with anti-FasL (polyclonal antibody C-178 at 1:1000 dilution; Santa Cruz Biotechnology) or anti-Fas (polyclonal antibody C-20 at 1:200 dilution; Santa Cruz Biotechnology) for 1 h at room temperature. Following incubation with a donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2500 dilution; Amersham), the blots were washed. The protein bands were visualized using an enhanced chemiluminescence kit (NEN Life Science Products, Boston, MA) and exposed on BioMax MR film (Eastman Kodak, Rochester, NY). The films were electronically scanned, and the band densities were quantified using Molecular Analyst/MacIntosh software (Bio-Rad). Equal protein loading was confirmed by Coomassie blue staining of all the gels.

Statistical Analysis

Data were analyzed by chi-square tests and Student t-tests. The differences were considered significant at P < 0.05. All experiments were performed in triplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chorioamnionitis Is Associated with Increased Villous Trophoblast Apoptosis

To test our hypothesis that chorioamnionitis is associated with increased villous trophoblast apoptosis, we studied placental villous tissues from women with chorioamnionitis (n = 5). For the negative control, we chose placentas from otherwise normal pregnancies, and these placentas were matched for gestational age. The placental tissues from patients with chorioamnionitis showed increased apoptosis in the trophoblast layer involving both syncytiotrophoblasts and cytotrophoblasts, whereas the control tissues were generally negative for this increase (Fig. 1A). There was a marked increase in apoptotic nuclei in randomly chosen fields for patients with chorioamnionitis (Fig. 1B). The median number of apoptotic nuclei found in chorioamnionitis was significantly higher than that in the controls (4 vs. 0, P < 0.001; Fig. 1C).



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  		FIG. 1. In situ localization of apoptotic nuclei in placental villi from uncomplicated (A) and chorioamnionitis (B) pregnancies. Representative sections were stained using the TUNEL technique. Apoptotic nuclei were stained brown (arrow). Original magnification x200. The total number of apoptotic nuclei per 10 high-power fields was determined in the placental villi in uncomplicated and chorioamnionitis pregnancies (C). The red bar represents the median number of apoptotic nuclei. Significant difference (P < 0.001) between uncomplicated and chorioamnionitis pregnancies

Chorioamnionitis Is Associated with Increased Villous Trophoblast Fas Expression

To determine whether the increased trophoblast apoptosis in chorioamnionitis is associated with increased Fas expression, we performed immunohistochemistry on placental villous tissues from women with chorioamnionitis and compared these tissues with gestational age-matched controls. There was a marked increase in Fas expression in the trophoblasts (both syncytio- and cytotrophoblasts) of the chorioamnionitis placentas (Fig. 2) compared with low levels of constitutive Fas expression in placentas from uncomplicated pregnancies (Fig. 2B). In contrast, there was a marked increase in Fas expression in placental villous trophoblasts in patients with chorioamnionitis (Fig. 2B).



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  		FIG. 2. Immunolocalization of Fas-expressing trophoblasts in placental villi from uncomplicated (A) and chorioamnionitis (B) pregnancies. Representative sections were stained for Fas expression. Fas expression was increased in cytotrophoblasts and syncytiotrophoblasts in patients with chorioamnionitis. Original magnification x200

Proinflammatory Cytokines Induce Trophoblast Apoptosis

To understand the mechanisms for trophoblast apoptosis in chorioamnionitis, we performed in vitro studies using villous tissues from uncomplicated term pregnancies. Villous explants were established and cultured with proinflammatory cytokines TNF{alpha}, IL-1ß, or IFN{gamma} individually. Trophoblast apoptosis was determined by TUNEL assay. There was marked apoptosis noted in both syncytio- and cytotrophoblast layers (Fig. 3, C, E, and G). Figure 3 demonstrates the trophoblast apoptosis at a cytokine concentration of 10 ng/ml, although apoptosis was noted at lower concentrations of individual cytokines (data not shown). The villous explants were then cultured with a combination of all three cytokines, resulting in marked trophoblast apoptosis (data not shown). These results suggest that the proinflammatory cytokines induce trophoblast apoptosis,; however, they do not elucidate the mechanism of apoptosis.



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  		FIG. 3. FasL-blocking protein Fas-Fc reduces TNF{alpha}- and IFN{gamma}-induced apoptosis. Placental villi from uncomplicated term pregnancies were initially treated with 2 ng/ml of Fas-Fc for 1 h and cultured with TNF{alpha}, IFN{gamma}, or IL-1ß for 24 h. Fas-Fc does not reduce the spontaneous apoptosis when villous explants are cultured for 24 h (A and B). Fas-Fc reduces TNF{alpha}-induced (C and D) and IFN{gamma}-induced (E and F) but not IL-1ß-induced (G and H) apoptosis. Original magnification x200

TNF{alpha} and IFN{gamma} but Not IL-1ß Induce Fas-Mediated Apoptosis of Trophoblasts

To determine whether the apoptosis induced by the proinflammatory cytokines was induced by FasL, we cultured the placental villous explants with the specific FasL-blocking protein Fas-Fc for 1 h prior to culture with TNF{alpha}, IL-1ß, or IFN-{gamma}. Following 24 h of culture with Fas-Fc and cytokines, the placental explants were subjected to TUNEL assay. We and others have noted that placental villous trophoblasts undergo spontaneous apoptosis under culture conditions [35] (Fig. 3A). However, this apoptosis was not reduced by blocking FasL (Fig. 3B). In contrast, Fas-Fc reduced trophoblast apoptosis in explants treated with TNF{alpha} (Fig. 3D) and IFN{gamma} (Fig. 3F). Fas-Fc had no effect on the villous explants treated with IL-1ß (Fig. 3H) or a combination of all three cytokines. These findings suggest that TNF{alpha} and IFN{gamma} induce apoptosis by ligation of FasL with Fas, whereas IL-1ß uses a pathway independent of Fas. Contrary to the studies by others, our results suggests that trophoblast FasL and Fas are biologically active and that this activity is dependent on the specific microenvironment in which proinflammatory cytokines are increased.

Effect of Proinflammatory Cytokines on FasL and Fas Expression in Cytotrophoblasts

To determine the effects of cytokines on trophoblast FasL and Fas expression, we cultured cytotrophoblasts with individual cytokines. Because cytotrophoblasts are the stem cells for the different populations of trophoblasts, they serve as good models to determine the effects of cytokines on FasL and Fas expression. Cytotrophoblasts isolated from placentas obtained from uncomplicated term pregnancies were cultured with different concentrations of cytokines for 24 h, and then Western blot analysis was performed. FasL and Fas expression was determined using FasL and Fas antibodies, respectively. Cytotrophoblasts expressed FasL and Fas constitutively (Fig. 4). TNF{alpha}, IFN{gamma}, and IL-1ß had no effect on the expression of FasL. In contrast, all three cytokines increased Fas expression at concentrations consistent with amniotic fluid concentrations in patients with chorioamnionitis. However, this increase in Fas expression was not significant (P = 0.06).



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  		FIG. 4. Western blot analysis demonstrating the effect of TNF{alpha}, IFN{gamma}, and IL-1ß on cytotrophoblast FasL and Fas expression. Cytotrophoblasts (3 x 106) were cultured with different concentrations of individual cytokines for 24 h (0, 0.1, 1, 10, and 50 ng/ml). TNF{alpha} (A), IFN{gamma} (B), and IL-1ß (C) increased Fas expression, but none of the cytokines had an effect on FasL expression. The intensity of the signal was analyzed using a digital imaging analysis system. Bars represent the mean ± SEM. The figures are representative of three independent experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study indicate that chorioamnionitis is associated with increased trophoblast Fas expression and apoptosis. When placental villous explants from uncomplicated pregnancy were cultured with proinflammatory cytokines TNF{alpha} or IFN{gamma}, trophoblast apoptosis occurred by the FasL/Fas pathway. These findings suggest that FasL expressed on the trophoblasts and activated maternal lymphocytes can induce trophoblast Fas-mediated apoptosis by autocrine or paracrine interactions. In addition, the increased Fas expression on trophoblasts may make them more sensitive to Fas-mediated apoptosis.

The increase in trophoblast apoptosis associated with chorioamnionitis provides support for our hypothesis that immune cells in placenta regulate trophoblast apoptosis via cytokines at the fetomaternal interface. One possible reason for the increase in trophoblast apoptosis associated with chorioamnionitis could be the changes taking place in cytokine composition in the placental microenvironment. Chorioamnionitis is characterized by the activation of the placental and decidual immune cells, resulting in the release of inflammatory mediators, specifically cytokines and chemokines, into the placental microenvironment [5]. This release of inflammatory mediators results in dense neutrophil and lymphocytic infiltration in the chorion, amnion, and placental villi, which is the hallmark of chorioamnionitis [3]. In addition to the immune cells, trophoblasts also produce cytokines. The proinflammatory cytokines whose increases are associated with chorioamnionitis are cytotoxic to trophoblasts, resulting in their apoptosis [13, 36].

A possible mechanism for the increased trophoblast apoptosis associated with chorioamnionitis could be the activation of the FasL/Fas pathway of apoptosis. Cells undergo apoptosis by either ligation of cell surface death receptors by their respective ligands or by the mitochondrial pathway [37]. The Fas L/Fas system has been recognized as an important pathway of apoptosis [16, 18, 37]. However, the role of FasL and Fas in trophoblast survival is perhaps more complex than originally anticipated. We confirmed the presence of Fas in normal trophoblasts (Figs. 2 and 4), as has been previously reported [38–40]. FasL and Fas interaction may occur between neighboring trophoblasts or on the same cell by paracrine and autocrine mechanisms.

Contrary to studies by other investigators [38], we demonstrated that Fas on trophoblasts is biologically active. By specifically blocking FasL, we reduced TNF{alpha}- and IFN{gamma}-induced apoptosis (Fig. 3). This finding suggests that Fas is biologically active during inflammation, when proinflammatory cytokines are increased in the placental microenvironment. A possible explanation for the resistance to Fas-mediated apoptosis described by other investigators could be the low levels of trophoblast Fas expression constitutively (Fig. 2A). Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase [41]. Our data demonstrating increased Fas expression may therefore explain the increased sensitivity to Fas-mediated apoptosis in trophoblasts. Another explanation for resistance to Fas-mediated apoptosis is the presence of Th-2 cytokines or inhibitors downstream of Fas, such as FLICE-inhibitory protein [32] and Bcl-x [42].

Although our data (Western blots) did not demonstrate an increase in cytotrophoblast FasL expression following culture with cytokines, an increase in Fas expression may predispose cells to apoptosis. The absence of a detectable increase in FasL expression could be explained by the cleavage of membranous FasL and its release as the soluble form (sFasL), which is chemotactic and proinflammatory [43, 44]. In the presence of increased Fas expression, sFasL has a synergistic effect, making the cells more susceptible to Fas-mediated apoptosis [41]. Thus, sFasL secreted into the placental microenvironment together with the increased Fas expression may furthur amplify the inflammatory process. The increased Fas concentration also may downregulate FasL expression by a negative feedback mechanism. These questions merit future study.

Generally when cells undergo apoptosis there is no inflammatory response; however, when peritoneal macrophages [45] and dendritic cells [46] undergo Fas-mediated apoptosis, cytokines and neutrophil chemotactic factors are released and inflammation is perpetuated. Because trophoblasts can themselves produce cytokines, a similar phenomenon may occur in the placental microenvironment in patients with chorioamnionitis. We propose the following mechanism for inflammation in chorioamnionitis: bacterial infection activates placental and decidual immune cells to produce cytokines, which in turn induce Fas-mediated apoptosis of trophoblasts, and more cytokines and chemotactic factors are released, which amplifies the inflammatory process. The Fas signaling pathway in trophoblasts could activate cytoplasmic mediators that regulate chemokine production and release and activate caspases [47, 48]. Therefore, trophoblasts could undergo apoptosis and release cytokines, functions that are independent of each other [49, 50].

The increased trophoblast apoptosis associated with chorioamnionitis implies that the number of cells necessary to perform the immune privilege function is diminished, possibly contributing to fetal allograft rejection, i.e., premature delivery. Critical events during normal pregnancy, such as immunoprotection, are regulated by cytokines produced locally at the mother-fetus interface [51]. The locally produced cytokines may exert their effect via the FasL/Fas pathway. Our results shed light on the delicate balance between immunoprotection and fratricidal trophoblast death. These data have implications for understanding the pathogenesis of chorioamnionitis and for the development of future therapeutic approaches to this disease.


   ACKNOWLEDGMENTS
 
The authors thank Terry O'Day for help with statistical analysis and preparation of figures, Dr Jonathan Wispe for critical review of the manuscript, and Robert Szmyd and Karen Bellisario for assistance in the procurement of normal term placentas.


   FOOTNOTES
 
1 Supported by grants from the Magee Womens Health Foundation, the Mario-Lemieux Centers for Patient Care and Research, and the 25 Club of Magee Womens Hospital. Back

2 Correspondence: Dhruv Balkundi, Department of Pediatrics, Magee Womens Hospital, 300 Halket St., Pittsburgh, PA 15213. FAX: 412 641 5313; dbalkundi{at}mail.magee.edu Back

Received: 14 November 2002.

First decision: 12 December 2002.

Accepted: 1 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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