Mol. Hum. Reprod. Advance Access originally published online on December 3, 2004
Molecular Human Reproduction 2005 11(1):35-41; doi:10.1093/molehr/gah129
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Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level
1Departments of Clinical Immunology, 2Immunology, 3Oncology and 4Obstetrics and Gynecology, Umeå University, S-90185 Umeå, Sweden
5 To whom correspondence should be addressed. Email: lucia.mincheva-nilsson{at}climi.umu.se
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Abstract |
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The local immune privilege of the fetus is created by the placenta. Fas ligand (FasL) expression in trophoblast has been implied as one of the mechanisms of fetal tolerance. However, the expression of membranal FasL by trophoblast has failed to explain this role of FasL. Two objections can be raised: (1) there have been contradictions considering which trophoblast cells, syncytiotrophoblast (ST) or cytotrophoblast, express FasL; (2) in vivo and in vitro studies have shown that the membranal form of FasL evokes inflammatory response and thus may promote fetal rejection. Using different assays and the FasL-specific antibody G247-4 we demonstrate beyond doubt that in vivo, (1) FasL is produced by and stored in the first trimester human ST only and (2) the human ST lacks surface membranal FasL. Instead, FasL, loaded in microvesicles, is stored in cytoplasmic granules. These results complement the recent in vitro studies of the microvesicular form of FasL secretion by cultured trophoblast cells, and suggest that placental FasL is synthesized by villous ST, stored in microvesicular form and secreted as exosomes. Secretion of the exosome-associated form of FasL may be one mechanism by which the placenta promotes a state of immune privilege. Additionally, FasL expression in Hofbauer cells is also demonstrated.
Key words: cytoplasmic granules/electron microscopy/FasL/microvesicles/trophoblast
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Introduction |
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The survival of the semi-allogeneic fetus during human gestation is still a puzzling issue for immunologists to explain. The local fetal tolerance seems to be maintained by the placenta. In early pregnancy, two layers of epithelial cells form the villous trophoblast of the human haemochorial placenta, an inner layer of cytotrophoblast (CT) adhering to the basal membrane and an outer layer of syncytiotrophoblast (ST) derived from the fusion of CT. The physical contact of ST, preferentially expressing paternal alloantigens, with the maternal blood circulating in the intervillous space of the placenta exposes these cells to the risk of being attacked by maternal immune cells, and to become the target of antibodies and complement activation products with deleterious consequences for the pregnancy and the fetus. The villous trophoblast uses different escape mechanisms to evade immune destruction. These mechanisms of fetal immunotolerance are multiple and overlapping (Thellin et al., 2000
). It has been postulated that the villous trophoblast evades attack by the maternal immune system through the establishment of immune privilege at the feto–maternal interface. The immune privilege has been attributed to the placental expression of Fas ligand (FasL, CD95L) triggering local deletion of activated maternal lymphocytes that recognize placental paternal antigens and express the FasL receptor–Fas (CD95) (Bamberger et al., 1997; Uckan et al., 1997; Mor et al., 1998; Kauma et al., 1999).
FasL, a type II membrane protein, is a member of the tumour necrosis factor superfamily. The intracellular and extracellular domains of FasL are located in the N- and C-terminal regions, respectively (Suda et al., 1993). A proline-rich sequence in the cytoplasmic domain is responsible for sorting FasL to secretory lysosomes in haematopoietic cells (Bossi and Griffiths, 1999). The extracellular domain of FasL can be cleaved by the metalloproteinase, matrilysin, producing a soluble form of FasL (Vargo-Gogola et al., 2002). After trimerization membrane-bound FasL cross-links Fas and induces apoptosis of the Fas-expressing cells (Holler et al., 2003).
FasL is mainly expressed by NK cells and activated T cells and is involved in the regulation of activation-induced cell death as well as in cell-mediated cytotoxicity (Nagata and Golstein, 1995). In recent years, constitutive expression of FasL was shown for non-lymphoid cells within immune privileged sites like the eye, brain, testis, thyroid, uterus and the placenta (Hunt et al., 1997; Uckan et al., 1997; Mor et al., 1998; Kauma et al., 1999; Green and Ferguson, 2001). These observations strongly implicate non-lymphoid FasL in the control of immune responses via elimination of the Fas-bearing immune cells invading these sites. However, although very attractive, this suggestion is still a matter of unresolved controversy (Ferguson et al., 2002).
A number of studies have demonstrated that FasL is expressed by villous trophoblast of human early and late pregnancy. However, there have been different opinions considering the precise localization of FasL in the villous trophoblast. According to several reports (Bamberger et al., 1997; Uckan et al., 1997; Balkundi et al., 2000; Pongcharoen et al., 2004) FasL is expressed by both ST and CT in early pregnancy. Others, however, could reveal FasL in CT only with no access of the FasL expressing cells to the maternal blood (Huppertz et al., 1998; Hammer and Dohr, 2000). Occasional, spot-like membranal staining of ST was explained as being a site of freshly fused CT cells (Huppertz et al., 1998; Zorzi et al., 1998). The explanation for these conflicting results is still unclear.
Several observations support a role for placental FasL in apoptosis of maternal-activated lymphocytes in vitro (Jiang and Vacchio, 1998; Kauma et al., 1999) and in vivo (Mor et al., 1998; Hammer and Dohr, 1999) suggesting a mechanism of immune suppression at the feto–maternal interface. However, these reports have been questioned as engineered expression of FasL in grafts and tumours (Allison et al., 1997; Kang et al., 1997, 2000) have shown, on the contrary, that membranal FasL promotes an accelerated allograft rejection in association with a neutrophilic inflammatory response (Restifo, 2000). In this context, the latest report of Abraham et al. (2004) is of considerable interest showing that the first trimester trophoblast cells in vitro do not express membranal FasL but a cytoplasmic form, which is probably secreted via microvesicles. Such microvesicle-mediated secretion of FasL suggests a new mechanism by which trophoblast cells might eliminate maternal Fas-expressing immune cells whilst avoiding an inflammatory response to membranal FasL.
Summarizing the results presented above, two important questions should be raised: (1) Which cells express FasL in human first trimester chorionic villi? (2) Which cellular compartments harbour FasL and in what form?
In this study, we confirm that freshly isolated human trophoblast cells constitutively express FasL mRNA. Using the FasL-specific antibody G247-4 and immunoelectron microscopy we show that FasL is exclusively localized in ST of the first trimester chorionic villi. Moreover, we characterize for the first time the ultrastructural localization of FasL in ST, demonstrating an active synthesis of the protein and its presence in microvesicles of cytoplasmic granules. Additionally, we show expression of FasL in Hofbauer cells.
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Samples and donors
Healthy women undergoing elective termination of early pregnancy (8–14 weeks of gestation) donated specimens of chorionic villous tissue after the ethical committee's approval and informed consent. The operative method used for pregnancy termination was vacuum extraction under a mild narcosis. The tissue was collected in Tris–Hanks' solution immediately after extraction and kept on ice until processing. To prevent possible cleavage of membrane-bound FasL by placental metalloproteinases some of the tissue samples were collected in the presence of 1 mM of the metalloproteinase inhibitor 1,10-phenanthroline (Mincheva-Nilsson et al., 2000
).
FasL antibodies
The immunohistochemistry at the light and electron microscopic level was performed with the mouse anti-human FasL monoclonal antibody G247-4 (isotype IgG1, Pharmingen, Germany). As an isotype-specific control antibody, DAK-G01 mouse IgG1 mAb was used (against Aspergillus niger glucose oxidase, DAKO A/S, Denmark).
Isolation of trophoblast cells from first trimester chorionic villi
Isolation of trophoblast cells from the first trimester chorionic villous tissue was carried out using a modified procedure previously described by Clover et al. (2000). Briefly, chorion villi from normal first trimester pregnancy were collected, washed in Tris–Hank's solution and cut into small pieces. The minced specimen was then incubated in enzyme solution (5 ml/g tissue) containing protease Type XIV (1 mg/ml), collagenase Type IV (0.5 mg/ml), and DNase Type IV (50 µg/ml) in RPMI 1640 supplemented with antibiotics. The enzymes were purchased from Sigma Chemical Co., St. Louis, MO, USA. The digestion was carried out at 37°C with constant stirring for 30 min, divided into three consecutive digestion steps of 10 min each with one-third of the total incubation volume. The digestion was ‘stopped’ by the addition of an equivalent volume of RPMI supplemented with 10% FCS. The released cells were collected and cleaned of debris and red blood cells by gradient centrifugation with a gradient of 25% and 60% Percoll (Pharmacia Amersham, Sweden) at 700 g for 30 min. The cells, gathered at the interface between the two gradients, were collected and washed twice in RPMI 1640 with 10% FCS and antibiotics. As a final step a negative selection of CD45+ cells was done. In brief, appropriate concentration of immunomagnetic beads (Dynabeads, Dynal A/S Oslo, Norway), loaded with anti-CD45 mAbs were added to the cell suspension and incubated for 30 min at 4°C with end-to-end rotation. The non-bound, CD45-negative cells were collected, washed and counted in Trypan Blue solution to assess viability. The yield was 1.78±0.23 x 106/g tissue (n=6), and the viability was 94.33±0.8%. Absence of CD45+ cells and positivity for cytokeratin 7 (clone OV-TL 12/30, DakoCytomation Norden AB, Denmark) were checked by immunoflow cytometry. More than 98±1.2% (n=6) of the isolated cell population was positive for cytokeratin 7 and negative for CD45 (data not shown). The cells were used for total RNA extraction.
Total RNA extraction, RT–PCR amplification and sequencing of the amplified products
Lysates of isolated trophoblast cells from individual samples (n=10) were used to extract total RNA by the acid guanidinium thiocyanate-phenol-chloroform method as previously described (Mincheva-Nilsson et al., 2000). The isolated RNA samples were analysed by RT–PCR with the following specific primers for FasL: forward primer 5'-GGATTGGGCCTGGGGATGTTTCA-3' and reverse primer 5'-TTGTGGCTCAGGGGCAGGTTGTTG-3'. The primer sequences were located in different exons so that amplification of mRNA could be distinguished by the size of the amplified product (344 bp). ß-Actin served as an RNA quality control. The RNA isolated from decidual 
T cells was used as a positive control (Mincheva-Nilsson et al., 2000). Single-stranded cDNA copies were made from 1 µg of total RNA using random hexamers and murine leukaemia virus reverse transcriptase (AB Applied Biosystems, Forster City, CA). RT was performed at 42°C for 15 min followed by denaturation at 99°C for 5 min. Subsequent PCR was performed in a total volume of 50 µl in a reaction mixture containing 1.0 U of Taq DNA polymerase (Promega) in 1.5 mM MgCl2, 10 mM Tris (pH 8.3), 50 mM KCl, 0.01% gelatin (w/v) supplemented with 0.25 µM each of dATP, dGTP, dCTP, and dTTP and 0.20 µM of each primer. The following thermocycle programme was used: 1 cycle of 95°C for 4 min; 39 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by an extension step of 72°C for 5 min. The amplicons were analysed in 2% SeaKem LE agarose (BioWhittaker Molecular Applications) gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination.
RT–PCR products were subjected to gel electrophoresis in 1.5% SeaKem®LM agarose (FMC BioProducts, Rockland, ME, USA), fragments were cut out and purified with QiaexII® Gel extraction kit (Qiagen, Germany) and then ligated into dT-treated EcoRV pBluescript (SK+). Competent Escherichia coli XL1blueMRF' was transformed with plasmids and grown on plates containing 100 µg/ml ampicillin, 12.5 µg/ml tetracycline, 20 µg/ml X-gal and 40 µg/ml IPTG. Transformants were checked for inserts of the expected size on agarose gel after restriction cleavage of plasmids using XbaI/XhoI (Life Technologies). DNA, 2–3 µg, was used for cycle-sequencing using T7 or Rev primers and Thermo Sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham/Pharmacia Biotech, UK). Cycle-sequenced products were analysed by ALFexpress (Pharmacia Biotech). The PCR product nucleotide sequence showed 100% identity to FasL (NM_000693 [GenBank] ) in the NCBI Entrez database.
Immunohistochemistry
Chorionic villous tissue samples were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3 for 2 h, washed in 0.1 M phosphate buffer supplemented with 3.5% sucrose and 1% fish gelatin and snap-frozen in liquid nitrogen. Eight micrometre-thick sections were placed on SuperFrost Plus glasses (Menzel-Gläser, Germany) and air-dried for 15 min. The sections were then rehydrated in phosphate-buffered saline (PBS) and incubated for 20 min in PBS containing 50 mM glycine, followed by a 20 min incubation with a blocking solution containing 1.5% normal rabbit serum, 0.2% bovine serum albumin and 0.05% saponin in PBS (pH 7.3). Mouse anti-human FasL antibodies (clone G247-4) were diluted to an appropriate concentration in the blocking solution and applied to the sections for 1 h at room temperature. For control of specific staining, parallel sections were incubated either with the irrelevant isotype-specific antibody DAK-G01, or with PBS instead of the antibody itself. The sections were then washed twice in PBS and the presence of endogenous biotin was blocked using the biotin blocking system (DAKO, Glostrup, Denmark) according to the manufacturer's instructions. A secondary antibody of biotin-conjugated F(ab')2 fragments of rabbit anti-goat IgG (Jackson ImmunoResearch laboratories Inc., West Grove, PA, USA) was applied for 1 h at room temperature followed by two PBS washings. Endogenous peroxidase activity was blocked by incubation in PBS containing 0.03% H2O2 and 0.02% NaN3 for 1 h at 37°C followed by three PBS washings. Thereafter, the sections were incubated with peroxidase-conjugated streptavidin (DAKO, Glostrup, Denmark) for 1 h and the specific staining was developed using 0.05 M Tris–HCl solution, pH 7.6, containing 0.5 mg/ml of 3,3-diaminobenzidine tetrahydrochloride (DAB) and 0.03% H2O2.
Immunoelectron microscopy
Small pieces of the first trimester chorionic villi were fixed by immersion in 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M PBS (pH 7.3) for 4 h at 4°C. The fixed specimens were washed in the same buffer containing 3.5% sucrose and 0.05% saponin (Merck) at 4°C overnight, snap-frozen and sectioned on a cryostatic microtome. Frozen 16 µm-thick sections were placed on poly-L-lysine (Sigma Chemical Co, St. Louis, MO, USA)-coated Thermanox coverslips (Nunc, Roskilde, Denmark) and air-dried for 3–5 min. After rehydration in PBS, the sections were stained using the indirect immunoperoxidase technique. Briefly, the sections were soaked in PBS containing 2 mM sodium azide (Sigma) and 0.003% H2O2 for 30 min at 37°C to block the endogenous peroxidase activity, followed by 20 min incubation with 0.1 M lysine monohydrochloride (Sigma) in PBS to block free-aldehyde groups. After blocking with 0.3% bovine serum albumin in PBS an appropriate concentration of the anti-FasL mAb G247-4 in blocking solution containing 0.05% saponin was applied and allowed to bind at room temperature for 4 h. The sections were then rinsed in PBS for 1 h. F(ab)'2 fragments of goat anti-mouse IgG labelled with horse-radish peroxidase (Pharmacia Amersham, Buckinghamshire, UK) were added and incubated overnight at 4°C. Peroxidase activity was revealed with DAB (Sigma) using a two-step procedure: the sections were first incubated with 0.05% DAB in 0.05 M Tris–HCl buffer (pH 7.6) for 10 min and then incubated with the same solution containing 0.003% H2O2 for 20 min. The sections were then fixed with 1.33% OsO4 (Sigma) for 1 h, dehydrated in graded acetone and flat embedded in Epon–Araldite mixture (Fluka, Buchs, Switzerland). Semi-thin (5–8 µm) sections of the embedded cryosections were examined at the light microscopic level for morphological orientation. Representative areas were then cut in an LKB ultramicrotome. Ultrathin sections were examined without additional staining in a Zeiss EM 109 electron microscope. Sections incubated either with DAK-G01 mouse IgG1 mAbs or with PBS instead of the first mAb were used as negative controls.
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FasL mRNA expression by isolated trophoblast cells from the first trimester chorionic villi
FasL mRNA expression in freshly-isolated trophoblast cells of human early placental samples was investigated using RT–PCR and confirmed by subsequent sequencing of the amplicons obtained. Using intron-spanning primers, a cDNA signal of the expected size (344 bp) was amplified in all the trophoblast cell samples studied (n=10). Figure 1 illustrates FasL transcripts amplified by RT–PCR with specific primers from three representative individual samples. Total RNA from decidual
T cells was used as a positive control (Mincheva-Nilsson et al., 2000). The amplification of ß-actin served as a housekeeping gene control. The 344 bp fragments obtained by RT–PCR were cut out and purified with QiaexII Gel extraction kit (Quiagen, Germany), ligated into dT-treated EcoRV pBluescript (SK+ or KS+) and sequenced. The sequence of the amplicons matched the FasL cDNA sequence available from GenBank under accession No. NM_000693
[GenBank]
. These results confirmed previously published data, based on the analysis of human total placental mRNA, that FasL mRNA is present at the feto–maternal interface (Uckan et al., 1997; Mor et al., 1998). However, in our study we used freshly isolated trophoblast cells which allowed us to exclude possible mRNA contamination from other types of placental cells and to single out the villous trophoblast cells as the source of FasL mRNA expression.
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In the next step of the investigation we had to establish which cells, ST or/and CT were possibly producing and expressing FasL protein.
Immunohistochemical localization of FasL in the first trimester chorionic villi
To study the cellular distribution of FasL, anti-FasL mAb G247-4 was used in immunoperoxidase staining of cryosections of the first trimester placental villi. In all placental samples (n=5) FasL staining was mainly observed in the cytoplasm of ST (Figure 2). CT appeared to be negative for FasL staining. In some sections, stained individual cells could be seen inside the villi (Figure 2). To further examine the intracellular location of FasL, immunoelectron microscopy was applied.
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Immunoelectron microscopic analysis of FasL expression in the first trimester chorionic villi
The electron microscopic localization of FasL supported and extended the observations made at the light microscopy level. In all placental samples examined, both metalloproteinase inhibitor-treated and non-treated, a distinct staining for FasL was noted only within the ST layer (Figure 3A–F). Interestingly, we did not see any staining of the apical surface membrane of ST (Figure 3A,C,D). Instead, ST showed a prominent intracellular staining (Figure 3A). In contrast, CT was always negative for FasL expression (Figure 3A). At low magnification, ST showed a diffuse cytoplasmic staining and a selective immunolabelling of multiple cytoplasmic granules (Figure 3A). The former was undoubtedly due to the positive staining of rough endoplasmic reticulum (RER) cisternae (Figure 3B). When the Golgi complex was in the plane of the section, it was also stained by the reaction product (Figure 3B). The cytoplasmic granules, sized approximately 1.5–2 µm, contained a varying amount of a compact material that was more or less strongly stained by the reaction product masking underlying structures (Figure 3C,D). Often, the granules formed a close contact with the apical surface membrane of ST, and even an opening to the intervillous space (Figure 3D). At higher magnification the cytoplasmic granules had a multivesicular ultrastructure, displaying numerous internal microvesicles of 60–100 nm diameter, which form clusters and were definitely immunolabelled (Figure 3E,F). Thus, we conclude that the first trimester ST actively synthesizes FasL in the RER and the Golgi complex, and transports it as FasL-loaded microvesicles to cytoplasmic granules for further storage and probably secretion. The lack of visible immunolabelling of the apical surface membrane of ST contradicts the existing opinion of cell-surface membranal FasL in human early placenta.
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Additionally, we found that Hofbauer cells in placental villi (Figure 4) also expressed FasL. These cells with different shapes were distributed in the villous core with a predominant localization in close vicinity to the trophoblast basal membrane. Some of the numerous cytoplasmic granules of the Hofbauer cells were stained by the reaction product and contained internal FasL-loaded microvesicles (Figure 4). These results confirm a previous report of FasL expression in Hofbauer cells by Zorzi et al. (1998)
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Negative controls, carried out omitting the anti-FasL antibody, revealed no detectable staining in the first trimester chorionic villi (data not shown).
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Discussion |
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In the introduction of the present report, two important questions were raised to clarify the hypothetical role of FasL in the immune privileged status of placenta during human normal pregnancy.
Which cells express FasL in the first trimester chorionic villi? Our initial RT–PCR approach confirmed that the FasL gene is functionally active in isolated first trimester villous trophoblast cells. Using the FasL specific antibody G247-4, immunohistochemistry and immunoelectron microscopy we demonstrate for the first time that in the villous trophoblast FasL is exclusively located in the cytoplasm of ST. In contrast, CT was completely devoid of FasL. Previous immunohistochemical studies reported that in normal human early pregnancy FasL was expressed by both ST and CT (Kauma et al., 1999; Balkundi et al., 2000; Pongcharoen et al., 2004) or in CT only (Mor et al., 1998, Hammer and Dohr, 1999). These contradictory results may be due to technical reasons. Some broadly used commercial monoclonal and polyclonal antibodies lack specificity for human FasL (reviewed in Chen et al., 2004). Similar discrepancies in FasL expression observed in other tissues impelled a critical study of 12 antibodies, where the staining results of tissue sections were compared to in situ hybridization (Strater et al., 2001). The only antibody that matched the staining pattern of FasL and the in situ hybridization signal was the monoclonal antibody G247-4. Thus, we would like to emphasize that in every attempt to evaluate FasL expression serious attention should be paid to the choice of anti-FasL antibodies.
The second question was to elucidate the subcellular distribution of FasL in the villous trophoblast. Immunoelectron microscopy enabled us to visualize for the first time the precise cellular compartments containing FasL and provided evidence that the protein is absent from the apical plasma membrane of ST, but is indeed present in the ST cytoplasm. One possible explanation for this lack of cell surface expression could be a rapid cleavage of membranal FasL by placental metalloproteinases, generating its soluble form (Vargo-Gogola et al. 2002). However, this is highly unlikely since the treatment of placental samples with 1, 1-10-phenanthroline did not result in cell surface staining for FasL. Recently, Abrahams et al. (2004) also noted the absence of FasL from the plasma membrane of the first trimester trophoblast cells in vitro and did not detect the soluble form of FasL in culture supernatants. Alternatively, the amount of membranal form of FasL in the apical domain of ST is either below the electron microscopy detection limit or this form of FasL is not expressed by ST at all. Thus, it is no longer sufficient to demonstrate that FasL is expressed by a cell at mRNA or/and protein level to imply that it is present and functions at the cell surface. Rather, it is important to know where in the cell FasL is found.
Careful examination of the cytoplasmic localization of FasL in ST indicated that the protein was present in three compartments: the RER, the Golgi complex and the cytoplasmic granules. The immunolabelling of the RER and the Golgi complex provides, for the first time, a definitive visible proof for the permanent synthesis of FasL. However, a major fraction of the intracellular FasL was concentrated in numerous large cytoplasmic granules. Within these granules FasL was found on clusters of microvesicles of 60–100 nm diameter. These granules seemed to be secretory lysosomes, judging from their multivesicular body-like ultrastructure and the size of the internal microvesicles (Blott and Griffiths, 2002). It is now known that some cells use secretory lysosomes for temporary storage rather than for degradation of secretory proteins. According to Abrahams et al. (2003), cytoplasmic FasL-containing structures of the first trimester trophoblast cells simultaneously expressed Lamp-1, a secretory lysosome marker protein (Blott and Griffiths, 2002). Moreover, since the antibody G247-4 specifically recognizes the extracellular domain of human FasL (Orlinick et al., 1997), we were able to exclusively identify the newly synthesized protein coming directly from the synthetic pathway. If FasL had been expressed on the cell surface prior to the lysosomal compartment then its extracellular domain would have been cleaved by metalloproteinases and we would have failed to stain it in the cytoplasmic granules (Bossi and Griffiths, 1999). Therefore, our results indicate that FasL detected in the cytoplasmic granules is newly synthesized and stored as FasL-loaded microvesicles. Previously, preformed microvesicle-associated FasL was first reported in the cytolytic granules/secretory lysosomes of haematopoietic cells (Mincheva-Nilsson et al., 2000; Monleon et al., 2001). Recently, secretory lysosomes packed with FasL-bearing microvesicles were found in melanoma cells (Andreola et al., 2002). On the basis of these data it is reasonable to suggest that ST may share with haematopoietic cells and with some epithelial tumours a similar intracellular pathway for targeting and storing newly synthesized FasL into secretory lysosomes via the prolin-rich domain in its cytoplasmic tail (Blott et al., 2001).
It is well known that secretory lysosomes can fuse with the plasma membrane resulting in the release of internal microvesicles into the extracellular milieu. The released vesicles are then termed exosomes (Farsad, 2002). Various cell types have unexpectedly been described to secrete exosomes, including epithelial and tumour cells. Depending on their cell type origin, exosomes can play a role in different physiological and pathological processes serving as a novel form of intercellular communication (Thery et al., 2002). Unfortunately, immunoelectron microscopy is not a suitable method to study dynamic processes and, thus, for the demonstration of secretory behaviour of the cytoplasmic granules in ST. We can only suggest that a release of FasL containing internal microvesicles in the intervillous space is taking place. However, the recent study by Abrahams et al. (2004) of cultured first trimester trophoblast cells strongly supports this suggestion and shows that full-length biologically active FasL is secreted in a microvesicular form. Furthermore, there are other indirect observations that indicate the presence of morphologically uncharacterized FasL-containing material in the maternal circulation during pregnancy (Gercel-Taylor et al., 2002; Strand et al., 2004). The FasL-bearing exosomes should be distinguished from similar microvesicles exfoliated from the plasma membrane (Albanese et al., 1998; Jodo et al., 2001). For this purpose the expression of tetraspanines, a hallmark of exosomes, has to be further characterized (Thery et al., 2002). Our preliminary results (unpublished data) suggest that tetraspanin CD63 is preferentially expressed in internal vesicles of the ST cytoplasmic granules implying that the internal vesicles are precursors of FasL-bearing exosomes. In general, the association with exosomes seems to be a general feature of secreted bioactive FasL (Blott and Griffiths, 2002) and has been shown in activated human T cells (Martinez-Lorenzo et al., 1999), cytotoxic T cells and NK cells (Denzer et al., 2000) as well as in ovarian tumour (Abrahams et al., 2003) and melanoma cells (Andreola et al., 2002; Martinez-Lorenzo et al., 2004). Thus, from the data presented above, it is clear that FasL in ST is permanently synthesized, temporarily stored and secreted in the microvesicular/exosomal form.
Although the fate and physiological role of secreted exosome-associated form of FasL in vivo is far from being unravelled, one may, a priori, predict the benefit of this form in induction of apoptosis, in general, and maintenance of placental immune privilege, in particular. One clear general benefit is preservation of the membranal form of FasL, which is the crucial form in apoptosis induction (Jodo et al., 2001). Another is the potentially greater surface area for microvesicular FasL expression compared with the membranal FasL (Martinez-Lorenzo et al., 1999). Moreover, FasL mediated killing via exosomes does not depend on cell–cell contact and de novo protein synthesis. In addition, exosome-associated FasL has a lower mobility compared with its soluble form, thereby achieving a high local concentration (Martinez-Lorenzo et al., 1999). In pregnancy, storage of preformed microvesicle-associated FasL in secretory lysosomes of ST and its secretion into the intervillous space may protect the placenta from potential inflammatory response that appears to occur when membranal FasL is expressed on the cell surface (Hohlbaum et al., 2000; Shudo et al., 2001; Gregory et al., 2002). Moreover, exosome-associated secretion of FasL may provide a mechanism by which the placenta, in a paracrine manner, induces apoptosis of fetus-sensitized Fas-expressing effector cells distant from the organ, facilitating its escape from immunosurveillance (Jiang and Vacchio, 1998).
In conclusion, our study shows that the human first trimester ST actively synthesizes and stores FasL in cytoplasmic granules as FasL-loaded microvesicles. In all examined placental samples FasL is absent from the apical plasma membrane of ST. Our present results complement the recent data of Abrahams et al. (2004) showing that the first trimester trophoblast cells in vitro secrete bioactive FasL via FasL-bearing microvesicles. The secretion of microvesicle-associated form of FasL may be one mechanism by which the placenta promotes a state of immune privilege.
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Acknowledgements |
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This work was supported by grants from The Swedish Cancer Research Foundation Cancerfonden (4565-B03-03XAC) and The Northern Cancer Research Foundation (AMP 03-350).
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References |
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Submitted on September 6, 2004; resubmitted on October 12, 2004; accepted on October 17, 2004.
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