Molecular Human Reproduction, Vol. 10, No. 1, pp. 55-63, 2004
© European Society of Human Reproduction and Embryology 2004
First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis
1Department of Obstetrics and Gynecology, Reproductive Immunology Unit, Yale University, School of Medicine, 333 Cedar Street, FMB 301, New Haven, CT 06520, USA and 2Department of Obstetrics and Gynecology, New York University School of Medicine, New York, NY 10016, USA
3 To whom correspondence should be addressed. e-mail: gil.mor{at}yale.edu
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Since the invading trophoblast represents a semi-allograft, it should be rejected by the mother. It has, therefore, been postulated that during normal pregnancy the trophoblast evades the maternal immune system though the establishment of immune privilege by triggering the death of activated lymphocytes which may be sensitized to paternal alloantigens. Such peripheral tolerance may be directed through the Fas/Fas ligand (FasL) apoptotic pathway and mediated by FasL expressed by the trophoblast. However, in vivo studies show that membrane-associated expression of FasL may instead promote allograft rejection, rather than protection. The aim of this study was to determine if there is a role for FasL in trophoblast immune privilege. In this study, we demonstrate that isolated first trimester trophoblast cells lack membrane-associated FasL, but express a cytoplasmic form in association with a specialized secretory lysosomal pathway. Furthermore, this intracellular FasL is constitutively secreted by trophoblast cells via the release of microvesicles. Following disruption of these microvesicles, the whole 37 kDa secreted FasL is able to induce T-cell death by apoptosis through activation of the Fas pathway. Therefore, we propose that secretion of FasL may be one mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition.
Key words: Key words: FasL/immune privilege/implantation/microvesicle/trophoblast
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Introduction |
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The invading trophoblast represents a semi-allograft which, therefore, should be rejected by the mother (Medawar, 1953). A number of hypotheses exist to explain how the maternal immune system fails to reject the trophoblast, and consequently the fetus, during normal pregnancy. These include the regulation of the complement system at the maternal–fetal interface (Holmes et al., 1990; Hsi et al., 1991), tryptophan catabolism by the enzyme indoleamine 2,3-dioxygenase (Munn et al., 1998) and the expression of non-classical HLA molecules by trophoblast cells (Loke and King, 2000). Recently, it has been postulated that the establishment of immune privilege at the implantation site is a result of the clonal deletion of immune cells that recognize paternal antigens present in the placenta (Tafuri et al., 1995). Such peripheral tolerance is believed to be mediated by the expression of Fas ligand (FasL) on trophoblast cells (Runic et al., 1996; Jiang and Vacchio, 1998). These and other studies postulate the hypothesis that invading trophoblast cells actively control maternal immune responses by triggering the death of activated lymphocytes (Griffith and Ferguson, 1997; Jiang and Vacchio, 1998; Mor et al., 1998).
FasL is a type II transmembrane protein, expressed by NK cells, activated T cells and within immune privileged sites, such as the eye and brain (Bechmann et al., 1999). Membranal FasL (mFasL) is expressed on the surface of cells as a 
37 kDa protein, and is proteolytically cleaved by matrix metalloproteinases (MMPs) to generate its soluble, 26 kDa form (sFasL) (Kayagaki et al., 1995). Both forms can self-associate, and following trimerization, FasL binds to, and activates its receptor, Fas (CD95). However, trimerization and bioactivity of mFasL is more efficient than that of its soluble form (Shudo et al., 2001). Fas is a type I membranal protein that is a member of the tumour necrosis factor (TNF) receptor family and is expressed by a wide variety of cell types. Upon cross-linking by either FasL or an agonistic anti-Fas monoclonal antibody (mAb), Fas intracellularly recruits Fas-associated death domain (FADD) and procaspase-8 forming the death-inducing signalling complex (DISC), after which the caspase pathway can be activated, resulting in apoptosis and cell death (Krammer, 2000).
FasL is expressed at the maternal–fetal interface and has been localized to villous cytotrophoblast, extravillous cytotrophoblast and syncytiotrophoblast populations (Runic et al., 1996; Uckan et al., 1997; Mor et al., 1998; Kauma et al., 1999; Ohshima et al., 2001). In addition, apoptotic leucocytes, of which most are T lymphocytes, have been localized to the maternal–fetal interface (Mor et al., 1998; Hammer and Dohr, 1999). These observations support a role for FasL in the elimination of maternal immune cells, which may be sensitized to paternal alloantigens expressed by the trophoblast (Tafuri et al., 1995; Jiang and Vacchio, 1998). Furthermore, in vitro FasL-expressing trophoblasts can induce Fas-mediated apoptosis in activated lymphocytes (Kauma et al., 1999). However, several animal studies which have focused on the role of FasL in immune privilege, have challenged such reports (Allison et al., 1997; Kang et al., 1997). In these studies, it was found that the expression of membranal FasL promoted allograft rejection rather than protection and that this was associated with an intense immune response evidenced by a neutrophilic infiltrate (Allison et al., 1997; Kang et al., 1997, 2000). Whether FasL mediates immune privilege or inflammation at the maternal–fetal interface, therefore, remains to be elucidated.
In this study, we demonstrate for the first time that first trimester trophoblast cells lack cell surface FasL, but instead express high levels of intracellular FasL that is secreted via microvesicles. We demonstrate that the secreted form of FasL, which is distinct from the soluble form, induces Fas-mediated apoptosis in T cells. We propose that the secretion of FasL via microvesicles represents a mechanism by which trophoblast cells may induce apoptosis in Fas-bearing maternal immune cells and thus establish a state of immune privilege for the placenta, while avoiding the inflammatory response associated with membranal FasL.
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Patients and samples
First trimester placentas (n = 14) were obtained from elective terminations of normal pregnancies performed at New York University School of Medicine and at Yale-New Haven Hospital. All patients signed consent forms and the use of patient samples was approved under both New York University’s and Yale University’s Human Investigations Committees.
Reagents and antibodies
Camptothecin and Triton X-100 were purchased from Sigma (St Louis, MO). The pan-caspase inhibitor (Z-VAD-FMK) and the agonistic mouse IgM anti-FasL mAb (clone E0S9.1) were obtained from PharMingen (San Diego, CA). Immunohistochemical staining for FasL was carried out using the rabbit polyclonal antibody (N20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and confirmed using the mouse mAb, G247.4 (PharMingen). Co-localization studies for FasL with Lamp-1 (CD107a) were performed using the anti-FasL polyclonal antibody (N-20) and a mouse anti-Lamp-1 mAb (PharMingen, clone H4A3). Fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse and Texas red-conjugated goat anti-rabbit secondary antibodies were used (Vector Laboratories, Burlingame, CA). FasL was detected by western blot using the mouse mAb, clone 33 (Transduction Laboratories, Lexington, KY) and confirmed using clone G247-4. The mouse anti-caspase-8 mAb was purchased from Oncogene Research Products (San Diego, CA) and the rabbit anti-cleaved caspase-3 (Asp175) polyclonal was obtained from Cell Signaling Technology (Beverly, MA). The mouse mAb for beta-actin was purchased from Sigma. Specific signals were detected using either a peroxidase-conjugated horse anti-mouse, or a peroxidase-conjugated goat anti-rabbit secondary antibody (Vector Laboratories).
Isolation and culture of trophoblast cells from first trimester placenta
Primary trophoblast cells from first trimester placentas (n = 5) were prepared as described previously (Loke et al., 1989) with some modifications (Aschkenazi et al., 2002). Briefly, tissue specimens were washed with cold Hanks’ balanced salt solution (HBSS; Gibco) to remove excess blood. Cells were scraped from the membranes, transferred to trypsin-EDTA (Gibco-Invitrogen, Carlsbad, CA) digestion buffer and incubated at 37°C for 10 min with shaking. An equal volume of D-MEM media (Gibco) containing 10% fetal bovine serum (FBS) was added to inactivate the trypsin. This mixture was vortexed for 20 s, allowed to sediment and the supernatant was collected. This was repeated twice and the collected supernatant was centrifuged at 400 g for 10 min. Contaminating red blood cells were removed by resuspending the cellular pellet with HBSS, layering this over the same volume of lymphocyte separation media (ICN Biomedicals, Inc., Aurora, OH) and centrifuging at 500 g for 25 min. The cellular interface containing the trophoblast cells was collected and resuspended in D-MEM supplemented with 10% normal human serum (Gemini Bio-Products, Woodland, CA) and cultured at 37°C/5% CO2 for no more than three passages. Purity of the trophoblast cells was >98% as determined by immunostaining for cytokeratin-7 (Sigma) (Aschkenazi et al., 2002).
Culture of cell lines
All cells were maintained at 37°C/5% CO2. Two first trimester trophoblast cytotrophoblast cell lines were used in these studies. The 3A cell line was purchased from ATCC (Manassas, VA) and the HTR8 (referred to from here on as H8) cell line was a kind gift from Dr Charles Graham (Queens University, Kingston, ON, Canada). Jurkat cells, a human T-cell leukaemia line, were obtained from ATCC. All cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Hyclone, South Logan, UT), 10 mmol/l HEPES, 0.1 mmol/l MEM non-essential amino acids, 1 mmol/l sodium pyruvate, 100 nmol/l penicillin/streptomycin (Gibco).
Immunohistochemistry
The cellular localization of FasL expression in trophoblast cells from first trimester placenta was performed as previously described (Gutierrez et al., 1999). In short, placental samples were fixed with 4% paraformaldehyde and then paraffin embedded. Sections of placenta (5 µm) or paraformaldehyde-fixed trophoblast cells, previously adhered to glass slides, were blocked with 10% goat or horse serum in phosphate-buffered saline (PBS) for 1 h at room temperature. Following three washes with PBS, samples were incubated overnight at 4°C with either the rabbit polyclonal N-20 (1:200 dilution) or the mouse G247.4 (2 µg/ml) anti-FasL antibody. Rabbit serum or mouse IgG1 served as isotype controls. After three washes with PBS, specific staining was detected by incubating with either a peroxidase-conjugated goat anti-rabbit or horse anti-mouse antibody (1:1000 dilution) for 1 h followed by a 5 min incubation with DAB substrate (Vector Laboratories). Cells and tissue sections were then counterstained with haematoxylin (Sigma) before dehydration with ethanol and Histosolve (Shandon, Inc., Pittsburg, PA). Slides were then mounted with Permount (Fisher Scientific, Pittsburg, PA) and visualized by light microscopy.
Co-localization of FasL with Lamp-1
Trophoblast cells, previously adhered to glass slides, were fixed with 4% paraformaldehyde and then blocked with 10% goat and 10% horse serum in PBS for 1 h at room temperature. Following three washes with PBS, samples were incubated overnight at 4°C with the rabbit polyclonal anti-FasL antibody (N-20; 1:200 dilution) and the mouse anti-Lamp-1 mAb (H4A3; 10 µg/ml). Rabbit serum and mouse IgG1 served as isotype controls. Following three washes with PBS, cells were incubated with a FITC-conjugated horse anti-mouse antibody and a Texas red-conjugated goat anti-rabbit antibody (both at 1:1000) for 1 h at room temperature in the dark. Following three washes with PBS, slides were mounted and visualized by fluorescent confocal microscopy (Olympus) using Magnafire software (Microsoft).
Isolation of microvesicles
Microvesicles were isolated from trophoblast cell culture supernatants as described previously (Abrahams et al., 2003). Briefly, the cell culture supernatants were collected 72–96 h after cells were plated and centrifuged twice at 500 g for 20 min at 4°C to remove any cellular debris. The cell-free supernatant was then ultra-centrifuged at 125 000 g at 4°C for 3 h. The supernatant was discarded and the microvesicle-containing pellet resuspended in sterile PBS. Following treatment with or without 2% Triton X-100 at 4°C for 30 min, the microvesicle suspension was centrifuged at 16 000 g and 4°C for 1 h. The supernatant fractions were collected and the vesicle fractions resuspended in sterile PBS and both were stored at –40°C until further use.
Western blot analysis
Microvesicles were prepared as described above and then analysed by western blot. For analysis of intracellular proteins, cells were lysed using 1% NP-40 and 0.1% SDS in the presence of protease inhibitors. Protein concentrations were calculated by BCA assay (Pierce Biotechnology, Rockford, IL). Proteins were then diluted with gel loading buffer to 20 µg and boiled for 5 min. Proteins were resolved under reducing conditions on either 10 or 12% SDS–PAGE gels and then transferred onto PVDF paper (Perkin Elmer, Boston, MA). Membranes were blocked at room temperature for 1 h with 5% fat-free powdered milk (FFPM) in PBS/0.05% Tween-20 (PBS-T). Following three washes for 10 min each with PBS-T, membranes were incubated overnight at 4°C with primary antibody in PBS-T/1% FFPM. Following this incubation, membranes were washed three times as before and then incubated at room temperature for 1 h with the appropriate secondary antibody conjugated to peroxidase (Vector Laboratories) in PBS-T/1% FFPM. Following three washes for 10 min each with PBS-T and three washes for 10 min each with distilled water, the peroxidase-conjugated antibody was detected by enhanced chemiluminescence (Perkin Elmer). All experiments were repeated at least three times and the intensity of the signals was analysed using a digital imaging analysis system and 1D Image Analysis Software (Scientific Imaging Kodak Company). Where appropriate, beta-actin was used as internal control, in addition to Ponseau Red, to validate the amount of protein loaded onto the gels.
Assessment of secreted FasL bioactivity
The bioactivity of the secreted FasL was determined using the CellTiter 96 viability assay (Promega, Madison, WI), as previously described (Mor et al., 2000; Neale et al., 2002). Jurkat cells were plated in wells of a 96-well plate at 5 x 104 cells per well in the reduced serum medium, Opti-MEM (Gibco) and cultured overnight at 37°C. Jurkat cells were then incubated with either the secreted FasL medium as a negative control, or an agonistic anti-Fas mAb (500 ng/ml) as a positive control. Cells were incubated at 37°C for 24 h after which the CellTiter substrate, MTS tetrazolium, was added. Following a 1–4 h incubation at 37°C, optical densities were read at 490 nm. All samples were assayed in triplicate and cell viability was presented as a percentage relative to the untreated control.
Flow cytometry studies
Apoptosis was monitored by flow cytometry as described previously (Abrahams et al., 2003). Following treatment, Jurkat cells (1 x 106) were washed twice with cold PBS and centrifuged at 400 g for 5 mins at 4°C. The cell pellet was then resuspended in 1 ml of cold PBS and incubated on ice for 20 min with propridium iodide (Sigma) at 1 µg/ml and Hoechst 33342 dye (Molecular Probes) at 5 µg/ml. Unstained cells served as a negative control. Samples were then analysed using a FACS Vantage (Becton Dickinson) with 488 nm/UV dual excitation. Propridium iodide staining was detected in the FL-2 channel and Hoechst staining was detected in the SSc-W channel. Data were analysed using CellQuest software (Becton Dickinson).
Trophoblast cells were analysed for surface FasL expression by indirect immunofluorescence and flow cytometry as described previously (Abrahams et al., 2000). Trophoblasts were harvested using a rubber policeman. Cells were then washed twice with ice-cold buffer (PBS, 1% BSA, 0.1% sodium azide) and then incubated at 4°C for 45 min with the mouse anti-FasL mAb (NOK-1, Pharmingen) or an isotype-matched control. After washing twice with buffer, the cells were incubated with FITC-conjugated goat F(ab)2 anti-mouse (Dako, Carpinteria, CA) at 4°C for 45 min in the dark. Following two washes with buffer, the cells were fixed with 2% paraformaldehyde in PBS and analysed by flow cytometry using a FACScan (Becton Dickinson). Data were analysed using CellQuest software (Becton Dickinson). Isolated microvesicles were immunostained for FasL expression as described previously (Monleon et al., 2001) using the same antibodies and analyses as described above.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Statistical significance (P < 0.05) was determined using one-way ANOVA with the Bonferonni correction.
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First trimester trophoblast cells express cytoplasmic FasL
FasL expression has been previously detected in trophoblast cells of normal placental tissues (Runic et al., 1996; Bamberger et al., 1997; Mor et al., 1998; Kauma et al., 1999; Balkundi et al., 2000; Hammer and Dohr, 2000; Ohshima et al., 2001). While some studies have described trophoblast FasL expression as membranal (Hammer and Dohr, 2000), others have described intracellular expression in trophoblast cells, and this was thought to be FasL synthesized which was then translocated to, and expressed on, the plasma membrane (Runic et al., 1996; Mor et al., 1998). As shown in Figure 1A, FasL expressed in normal first trimester placental villi appeared to be mainly cytoplasmic, as determined by immunohistochemistry. To further characterize the intracellular localization of FasL expression, an in vitro system consisting of either isolated first trimester trophoblast primary cultures or first trimester trophoblast cell lines were utilized. As shown in Figure 1B, FasL was absent from the cell surface of all first trimester trophoblast cells, however, positive immunoreactivity for FasL was found to be localized to the cytoplasm. Flow cytometric analysis of these cells confirmed the lack of surface-expressed FasL (Figure 1C). Furthermore, the intracellular FasL was distributed within the cytoplasm in a polarized manner, indicative of a protein that is secreted (Figure 1B).
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Intracellular FasL is associated with a secretory pathway
In order to determine whether the cytoplasmic FasL expressed by first trimester trophoblasts had the potential to be secreted, the association of this FasL with secretory organelles was evaluated. For this, cells were immunostained for both FasL (Figure 2, panel 1, red) and the specialized secretory lysosome marker, Lamp-1 (Figure 2, panel 2, green). Fluorescent confocal microscopy revealed that the majority of FasL staining was co-localized with that for Lamp-1 (Figure 2, panel 3, yellow). However, this was not the case for Lamp-1, which could be seen both alone as punctate staining (green), as well as in co-localization with FasL (yellow) (Figure 2, panel 3). This observation suggested that the FasL expressed by first trimester trophoblast cells is associated with a specialized secretory pathway.
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FasL is secreted via microvesicles
To determine whether the lysosomal-associated FasL expressed in first trimester trophoblast cells could be secreted, microvesicles were isolated from the culture supernatants collected from both trophoblast primary cultures and first trimester trophoblast cell lines. The microvesicles were then analysed for FasL expression by western blot. A 37 kDa protein was detected by the anti-FasL mAbs in the microvesicles isolated from the culture supernatants of all the studied trophoblast cells (Figure 3A).
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The FasL-positive microvesicles which were isolated from the culture supernatants of first trimester trophoblasts were then further characterized. For this, FasL expression was analysed by flow cytometry. No positive staining was detected on any of the preparations, indicating that the microvesicles lacked surface FasL (Figure 3B). These results suggested that the FasL detected by western blot analysis was stored inside of the microvesicles. To test this hypothesis, microvesicles were disrupted by treatment with the detergent, Triton X-100. Following treatment with or without Triton, the supernatant fractions were separated from the vesicle fractions by centrifugation and both the vesicle and supernatant fractions were then tested for the presence of FasL by western blot. As shown in Figure 3B, the only fractions where FasL was detectable were either the vesicle fraction from untreated, intact microvesicles, or the supernatant fraction of microvesicles disrupted by treatment with Triton (Figure 3C). Together, these results suggest that first trimester trophoblast cells secrete FasL via microvesicles, within which the protein is contained.
Secreted FasL is bioactive
We then sought to determine whether this secreted form of FasL was functional. The Jurkat T-cell line, which is an established and widely used model of Fas-mediated apoptosis (Alderson et al., 1995), was used as a target for the microvesicle-associated FasL. Cell viability was determined by means of the CellTiter 96 assay. To ensure that any responses observed were due to the secreted FasL, all vesicle and supernatant fractions were tested for bio activity. The supernatant from Triton-treated vesicles (sup/triton), which contained the released secreted FasL, significantly reduced Jurkat cell viability to 13.5 ± 1.1% (P < 0.001) when compared with the untreated control (Figure 4A). In addition, this effect was dose-dependent (Figure 4B). As a positive control, Jurkat cells were treated with the anti-Fas mAb which reduced Jurkat cell viability to 17.3 ± 1.1% (P < 0.001) (Figure 4A). The vesicle (ves/PBS) and supernatant (sup/PBS) fractions from the untreated microvesicles failed to induce Jurkat cell death. However, the vesicle fraction from the Triton-treated vesicles (ves/triton) did display some inhibitory effect on Jurkat cell viability, reducing it to 85.9 ± 1.8% (P < 0.001). Medium spiked with Triton X-100 had no effect on Jurkat cell viability (data not shown), thus confirming the effects observed were not Triton mediated.
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Secreted FasL induces apoptosis
The next objective was to confirm that the decrease in cell viability induced by the secreted form of FasL was a result of Fas-mediated apoptosis. Thus, Jurkat cells were incubated with the supernatant from Triton-treated microvesicles which contained the secreted FasL. Following incubation, the cells were double stained with propridium iodide and Hoechst 33342 dye and analysed by flow cytometry. Treatment with the secreted FasL resulted in a 50.53% increase in the number of apoptotic Jurkat cells compared with the untreated control. As a positive control, Jurkat cells were treated with the anti-Fas mAb which increased the number of apoptotic cells by 48.84% (Figure 5A).
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Secreted FasL activates the Fas apoptotic pathway
To demonstrate that the secreted form of FasL was able to induce apoptosis through the Fas pathway, caspase activation was evaluated by western blot analysis. Untreated Jurkat cells expressed only the pro-form of caspase-8 (55 kDa) and failed to express the active cleavage products of caspase-3 (Figure 5B). However, treatment with either the secreted FasL, or the agonistic anti-Fas mAb resulted in activation of the caspase pathway, evidenced by the presence of caspase-8 and caspase-3 cleavage products (43/41 and 19/17 kDa respectively) (Figure 5B). In addition, both secreted FasL and the anti-Fas mAb had the ability to induce caspase activation in freshly isolated peripheral blood lymphocytes, while no such activation was observed in the untreated lymphocytes (data not shown).
In order to confirm these results, Jurkat cells were treated with either the secreted FasL or the anti-Fas mAb in the presence or absence of a pan-caspase inhibitor (Z-VAD-FMK). The presence of the pan-caspase inhibitor blocked the caspase-8 and caspase-3 activation that was induced by the secreted form of FasL and the agonistic anti-Fas mAb (Figure 5C).
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Discussion |
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This report shows for the first time that first trimester trophoblast cells do not express membranal FasL, but a cytoplasmic form which is actively secreted in microvesicles. How the genetically distinct fetus avoids rejection by the mother’s immune system represents an immunological puzzle, particularly since the maternal immune system is not entirely ignorant of the implanted semi-allograft (Mor and Abrahams, 2002). What has become clear is that maternal immune recognition and subsequent immune tolerance is directed not at the fetus itself, but rather against the trophoblast, a population of fetally derived cells that surround the fetus (Colbern and Main, 1991). The invading trophoblast is in direct contact with both maternal blood and tissues and it appears to have developed a number of mechanisms by which it can deceive the maternal immune system into accepting itself and, therefore, the fetus (Erlebacher, 2001). One such method may be clonal deletion of activated immune cells through the Fas/FasL apoptotic pathway. However, recently the area of FasL-mediated immune protection has been challenged by in vivo data showing that cell surface, membranal FasL may actually promote graft and tumour rejection, rather than survival (Allison et al., 1997; Arai et al., 1997; Kang et al., 1997; Chen et al., 1998; Shudo et al., 2001; Gregory et al., 2002). In this study, we demonstrate that first trimester trophoblast cells may utilize a novel tactic of promoting immune privilege through the Fas/FasL system. We report, for the first time, the secretion of whole, biologically active FasL by first trimester trophoblast cells via the release of microvesicles. Such release of a secreted form of FasL suggests a mechanism by which the trophoblast might eliminate activated maternal immune cells, whilst avoiding the inflammation observed with membranal FasL expression (Kang et al., 1997; Chen et al., 1998; Shudo et al., 2001; Gregory et al., 2002).
Apoptotic leucocytes, of which most are T lymphocytes, have been localized to the maternal–fetal interface, suggesting that a mechanism of immune suppression exists at the site of implantation (Mor et al., 1998; Hammer and Dohr, 1999). The Fas/FasL system has been proposed to play a role in such immune privilege during pregnancy (Runic et al., 1996; Uckan et al., 1997; Ohshima et al., 2001), however, the precise role of FasL in immune privilege has yet to be defined and remains controversial. In vitro studies have shown membranal FasL to promote T-cell apoptosis (Shiraki et al., 1997; Rabinowich et al., 1998), thus supporting the hypothesis that cells within immune privilege organs such as the brain, eye or testis express surface FasL and are able to induce apoptosis in Fas-bearing activated immune cells. However, Allison et al. found that FasL-expressing islet beta cells transplanted into allogeneic animals were rejected rather than being protected, and that this expression of FasL promoted a neutrophilic infiltrate (Allison et al., 1997). These observations were subsequently supported by other in vivo studies using tumour cells and islet beta cells engineered to express FasL (Arai et al., 1997; Kang et al., 1997; Chen et al., 1998). As a consequence, the role of FasL in mediating immune privilege has been questioned.
A number of studies have reported the expression of FasL by trophoblast cell populations within normal first trimester villi and term placentas (Runic et al., 1996; Uckan et al., 1997; Mor et al., 1998; Kauma et al., 1999; Ohshima et al., 2001). Some studies have described this FasL expression as membranal (Hammer and Dohr, 2000), while others have reported cytoplasmic expression (Runic et al., 1996; Mor et al., 1998). When intracellular FasL expression has been observed, it has been assumed that the FasL is then translocated to the cell surface where it would bind to Fas expressed in a nearby cell (Runic et al., 1996; Mor et al., 1998). In support of this hypothesis, an in vitro study by Kauma et al. (1999) demonstrated that FasL-expressing trophoblasts can induce Fas-mediated apoptosis in activated lymphocytes. However, FasL expression by these cells was only demonstrated by western blot. Therefore, the exact cellular distribution of trophoblast FasL was not determined.
In this study, while expressed by all first trimester trophoblast cells, FasL was absent from the plasma membrane. One possible explanation for this lack of surface expression could be that the membranal FasL had been proteolytically cleaved from this site by MMPs, generating the 26 kDa soluble form (Kayagaki et al., 1995). However, this soluble form of FasL was undetectable in all culture supernatants. Furthermore, the distribution of the intracellular FasL followed a polarized manner suggesting that this protein might be secreted.
Originally, secretion of whole 37 kDa FasL from the cytoplasm of cells was not thought possible since FasL lacks conventional secretory sequences. However, it has recently been demonstrated that in cytolytic T cells and NK cells, intracellular FasL is sorted into specialized secretory lysosomes as a result of proline-rich domains within the cytoplasmic tail of FasL (Bossi and Griffiths, 1999; Blott et al., 2001). Further investigation of FasL in immune cells revealed that intracellularly expressed 37 kDa FasL could indeed be secreted and that this was through the release of microvesicles (Martinez-Lorenzo et al., 1999; Monleon et al., 2001). This concept was further supported by the report that interleukin (IL)-1ß, which also lacks conventional secretory sequences, could be secreted by monocytes via microvesicles (MacKenzie et al., 2001). Moreover, we have recently described the secretion of lysosomal-associated FasL via microvesicles from epithelial ovarian cancer cells (Abrahams et al., 2003). The cytoplasmic FasL expressed by first trimester trophoblast cells was co-localized with the lysosomal marker, Lamp-1, thus confirming that trophoblast FasL is associated with the specialized secretory lysosomal pathway and, therefore, has the potential to be secreted under a highly regulated system (Blott and Griffiths, 2002). Furthermore, we demonstrated that in the absence of any stimulus, first trimester trophoblast cells constitutively secrete 37 kDa FasL in association with microvesicles.
Interestingly, the FasL-expressing microvesicles isolated from first trimester trophoblast cells, were functional only after the integrity of the vesicle membrane was disrupted, indicating that the FasL was encapsulated by the microvesicle. This is analogous to that reported for microvesicle-associated IL-1ß (MacKenzie et al., 2001) and FasL secreted from epithelial ovarian cancer cells (Abrahams et al., 2003). Our findings indicate that treatment of Jurkat T cells and peripheral blood lymphocytes with the secreted FasL, once liberated from the microvesicle, induced Fas-mediated apoptosis through activation of the caspase pathway. Other groups have reported the expression of FasL on the surface of microvesicles and found that these intact vesicles were able to induce Fas-mediated apoptosis (Martinez-Lorenzo et al., 1999; Jodo et al., 2000; Andreola et al., 2002). A possible explanation for this discrepancy may be the different cell types used for microvesicle preparation. Alternatively, these studies may have isolated FasL-expressing membrane fragments rather than intact microvesicles.
The data presented in this current study suggest the following mechanism of FasL secretion. Cytoplasmic FasL is contained within microvesicles, further encapsulated by specialized secretory lysosomes. Following fusion of the secretory lysosome with the trophoblast plasma membrane, the FasL-containing microvesicle is released extracellularly. This is in agreement with the finding of Mincheva-Nilsson’s group, who demonstrated intracellular FasL localized to microvesicles, further contained within the cytolytic granules of NK cells (Mincheva-Nilsson et al., 2000). How microvesicle disruption occurs in vivo is presently unclear. However, we postulate that there is a specific protein–vesicle interaction that occurs at the surface of a Fas-bearing immune cell, resulting in either vesicle ‘lysis’ or fusion of the microvesicle onto the surface of the activated immune cell. Through either mechanism, the secreted FasL is delivered, exposed and free to ligate the Fas receptor expressed by activated immune cells. In support of this hypothesis, Gercel-Taylor et al. (2002) have reported the presence of FasL-expressing placental-derived membrane fragments in the maternal circulation during normal pregnancy. These FasL-bearing membrane fragments are able to down-regulate CD3-zeta chain and induce Fas-mediated apoptosis in Jurkat T cells.
To our knowledge, this represents the first report of the constitutive secretion of fully active, 37 kDa, FasL by first trimester trophoblast cells. FasL secretion via microvesicles may provide an active process whereby trophoblasts can establish maternal immune protection by enhancing their survival through the elimination of alloantigen-sensitized immune cells. Such secretion of FasL avoids the cell–cell contact normally associated with Fas-mediated apoptosis, and the inflammatory response that appears to occur when membranal FasL is expressed on the cell surface (Chen et al., 1998). In agreement with studies using non-cleavable mutants of FasL (Hohlbaum et al., 2000; Kang et al., 2000), we propose that the membranal form of FasL in trophoblast cells is fully capable of inducing a neutrophilic response. On the other hand, the secreted form, following release via microvesicles, may initiate cell death in apoptosis-sensitive immune cells. The mechanism by which FasL secretion by trophoblast cells is induced and regulated has yet to be elucidated. We hypothesize that soluble factors, such as cytokines or hormones at the implantation site may play a role by regulating FasL expression, secretion or localization. Therefore, changes in the micro-environment may significantly alter FasL function.
In summary, microvesicle-mediated secretion of FasL may provide a mechanism by which trophoblast cells eliminate hazardous maternal immune cells, during implantation and pregnancy by inducing apoptosis in Fas-bearing immune cells, thus preventing maternal immune recognition. Alterations in either the secretion or the cellular localization of FasL may contribute to the onset of labour or complications of pregnancy such as pre-eclampsia or intrauterine growth retardation.
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Acknowledgements |
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The authors would like to thank Thomas Taylor for his assistance with the FACS Vantage flow cytometry studies. This work was supported by the National Institute of Health grants RO1HD37137-01A2 and RO1CA92435-01.
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Submitted on July 23, 2003; resubmitted on September 8, 2003; accepted on September 12, 2003.
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