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a Departments of Medical Microbiology and Immunology and
b Obstetrics and Gynecology,
c The University of Alberta Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
d Department of Obstetrics, City Hospital, University of Nottingham, Nottingham, NG7 2UH, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TNFß. Mutational analysis of the cytoplasmic regions of Fas and TNF receptor p55 (TNFRp55) reveals the existence of a homologous 70-amino acid "death" domain required for the transduction of the apoptotic signals that typify these two receptors. In addition to the membrane-anchored form of Fas, three soluble forms are produced by alternate splicing that eliminates the transmembrane domain [5], and a death domain-deficient variant is expressed by tumor cells [6]. Fas is constitutively expressed by a number of tissues such as thymus, liver, heart, and kidney (reviewed by Nagata, [4]). FasL is expressed as an inducible protein on activated T lymphocytes and natural killer cells, where it participates in the programmed elimination of activated T cells after strong immune challenges. FasL is also constitutively expressed by specialized epithelial or endothelial cells associated with sites of T cell selection (thymus epithelium) or peripheral elimination (the eye, testis, brain, and placenta) [7–9]. From these sites of expression, FasL is thought to trigger apoptosis of homing T lymphocytes, thereby enforcing immune privilege [10] and mediating immune tolerance [11].
Placental trophoblasts, epithelial cells of fetal origin, separate all maternal from fetal tissue (reviewed in [12]). Within the villous placenta, maternal blood faces a multinucleated, nonreplicating syncytium (the syncytiotrophoblast), beneath which lie immature, replicating, mononuclear cytotrophoblasts, which are frequent in the first and second trimesters of pregnancy but much less so in the third trimester as the trophoblast thins to facilitate gas, nutrient, and waste flow. The syncytiotrophoblast appears to constantly renew by loss of aged nuclei and cytoplasm in structures called syncytial knots [13] and replenishment through fusion of underlying cytotrophoblasts [14]. A role for programmed death in this process is suggested by electron microscope demonstrations of apoptotic nuclei within the syncytiotrophoblast [15]. Observations of trophoblast apoptosis throughout gestation, with a significantly higher frequency in the third than first trimester, suggest it to be a normal placental aging process [16]. A higher-than-normal incidence of apoptosis in placentas of pregnancies complicated by intrauterine growth restriction (IUGR) suggests that increased trophoblast apoptosis might be a primary pathological event [17].
Both Fas and FasL are expressed in the villous placenta. FasL expression, initially in the decidua and later in the exchange trophoblast and fetal stroma, has been suggested to limit migration, initially of fetal cells into maternal tissue, then later of maternal cells into fetal tissue [18–20]. Fas is expressed in chorionic trophoblasts and selectively in villous stromal and endothelial cells but has not yet been identified in villous trophoblasts [9, 21]. Preliminary studies in our laboratory (see Results) showed Fas expression on pure populations of term villous cytotrophoblasts, and thus the potential for juxtacrine killing via trophoblast-expressed Fas and FasL.
Although Fas expression in chorionic trophoblasts is associated with apoptosis [9], whether villous (or chorionic) trophoblast Fas transduces cytotoxic signals (as does TNFRp55 in trophoblasts [22]) is not known. Apoptosis is not the only possible outcome of Fas expression. The existence of alternative splice forms lacking the death domain or plasma membrane anchoring sequences suggests translation products with possible antagonistic functions [5, 6], and Fas signal transduction is subject to regulation [23–26]. We therefore asked what the form and function of Fas in cytotrophoblasts might be. We found that cytotrophoblasts express full-length, cell-surface Fas. However, Fas death signal transduction is completely blocked by what appears to be a novel mechanism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Powdered Iscove's Modified Dulbecco's Media (IMDM) was purchased from Gibco (Grand Island, NY) and prepared according to the manufacturer's instructions. Fetal calf serum (FCS) was purchased from Hyclone (Logan, UT). Recombinant human TNF was a gift from Hoffman LaRoche (Basel, Switzerland), and recombinant human interferon gamma (IFN
) was purchased from Collaborative Biomedical Products (Bedford, MA). Monoclonal mouse anti-Fas (CH-11) was from Immunotech (Westbrook, ME); monoclonal mouse anti-Fas (clone SM1/23) was from Chemicon International; and control mouse IgM, IgG, and goat anti-mouse horseradish peroxidase (HRP) conjugate were from Southern Biotechnology Associates, Inc. (Birmingham, AL). The nitric oxide synthase (NOS) inhibitors aminoguanidine (AG), N-monomethyl-L-arginine (NMMA), and N-nitro-L-arginine methyl ester hydrochloride (L-NAME) were all from Sigma (Oakville, ON, Canada).
Cells
Placentas were obtained from normal delivery or elective cesarean section from uncomplicated pregnancies. Villous trophoblasts (> 99.9% pure) were isolated by trypsin/deoxyribonuclease (DNase) digestion of minced chorionic tissue and immunoabsorption on Ig-coated glass beads (Biotex, Edmonton, AB, Canada), cryopreserved in liquid nitrogen, thawed, and cultured at 1 x 105 per microwell as described previously [27, 28]. HeLa cells (American Type Culture Collection [ATCC], Rockville, MD) were cultured in IMDM supplemented with 10% FCS. Cells were detached from culture flasks with 0.05% trypsin, then plated in 96-well plates at 104 cells/microwell and incubated overnight to allow the cells to adhere and become confluent. A1.1 cells (from D. Green [29, 30]) were cultured in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin/streptomycin, and 2-mercaptoethanol. Jurkat cells (ATCC) were cultured in the same medium as A1.1 cells. BeWo cells (ATCC) were cultured in 15% FCS in IMDM in tissue culture flasks (Nunc T80; GIBCO/BRL, Inc., Toronto, ON, Canada) and passaged by detaching with 0.05% trypsin.
Immunohistochemical Staining
After adhering to 96-well plates, cytotrophoblasts were washed twice with PBS, fixed with 50 µl ice-cold methanol:acetone (95:5) or 2% paraformaldehyde (phosphate-buffered, pH 7.2) for 5 min, washed three times with PBS, then blocked for 1 h with 1% BSA-1% FCS-PBS at room temperature. Cells were then incubated overnight at 4°C with 5 µg/ml mouse anti-Fas (CH11) or mouse IgM in 1% BSA-1% FCS-PBS. After being washed 3 times with PBS, cells were incubated at room temperature for 2 h with goat anti-mouse Ig-HRP at 1:200 in 1% BSA-1% FCS-PBS and washed 4 times with PBS; then the color was developed with 3-amino-9-ethylcarbozole (AEC kit; Zymed, San Francisco, CA).
Cellular ELISA
Confluent cytotrophoblast cultures in 96-well plates were blocked for 1 h with 1% BSA-1% normal goat serum-PBS at 4°C and then incubated at 4°C with 5 µg/ml monoclonal mouse anti-human Fas (SM1/23) or IgG2b isotype control in blocking buffer for 2 h. The plates were washed 3 times with cold PBS and incubated at 4°C with goat anti-mouse-HRP conjugate at 1:2000 in blocking buffer for 1 h. After 5 washes with cold PBS, color was developed with ABTS (Sigma, St. Louis, MO) solution (2 mM 2,2'-azino-bis[3-ethylbenzthiazoline–6-sulfonic acid], 45 mM disodium phosphate, 30 mM citric acid, 0.003% H2O2), and optical density (OD) was measured at 405 nm with a reference wavelength of 490 nm as previously described [31].
Anti-CD95 Cytotoxicity
After cytotrophoblast and HeLa cells were seeded in 96-well plates as described, medium was aspirated and replaced with fresh medium containing 1 µg/ml cycloheximide (CHX; Sigma) and either anti-CD95 or normal mouse IgM at varying concentrations, and incubated at 37°C for varying times. In experiments in which cells were pretreated with IFN, medium was aspirated, replaced with fresh medium containing 100 U/ml IFN, and incubated 24 h at 37°C. The medium was aspirated and replaced with fresh medium containing IFN, CHX, and antibodies. Cell viability was determined by (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma) assay [32] as previously described [33].
A1.1-Induced Cytotoxicity
A1.1 cells in log phase were pelleted, resuspended in fresh 10% FCS-RPMI containing 10 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) and 3 µg/ml ionomycin (Sigma) for 2 h to stimulate expression of FasL, then washed and resuspended in IMDM supplemented with 10% FCS. Control (mock-activated) cells were incubated for the same period of time in 10% FCS-RPMI. A1.1 cells were added to confluent trophoblast and HeLa cells in 96-well plates and incubated at 37°C. Cell viability was determined by MTT assay after gentle washing to remove A1.1 cells, and DNA fragmentation was determined.
DNA Fragmentation Assay
After adhering to plastic 96-well plates, cytotrophoblasts and HeLa cells were labeled overnight with 1 µCi tritiated thymidine ([3H]TdR; Dupont NEN, Boston, MA). The cells were washed once with fresh medium and incubated for at least 1 h at 37°C. The medium was then aspirated, and 50 µl 10% FCS-IMDM with 2 µg/ml CHX and 200 U/ml IFN (and 20 µg/ml of antagonistic anti-Fas [SM1/23] or IgG2b control in some wells to show that killing was mediated by Fas-FasL interactions) was added, followed by 50 µl A1.1 cells (105), or 50 µl medium with 20 ng/ml TNF, or medium alone for spontaneous fragmentation and total labeling. Final concentrations were 1 µg/ml CHX, 100 U/ml IFN, and 10 ng/ml TNF. Cytotrophoblasts were incubated for 18–20 h (HeLa for 3–4 h) at 37°C, medium was aspirated, and experimental and control samples were lysed with 100 µl of 1% Triton X-100 (Sigma) in PBS, 2 mM EDTA (Sigma). The lysates were transferred to microfuge tubes and centrifuged at 13 000 x g for 10 min; then 50 µl of the supernatant was transferred to scintillation vials. For total DNA labeling, samples were lysed with 2% SDS in 0.1 N NaOH, and 50 µl was transferred to vials for scintillation counting.
Western Blot Analysis
Cells were harvested in 25 mM Tris-HCl pH 7.5, 0.5% Triton X-100 and then sonicated for 5 sec. PAGE of 10 µl of lysate equivalent to 2 x 104 cells was performed on 12% gels (8.3 cm x 7.4 cm x 0.75 mm) in SDS with a pH 6.8, 3.5% stacking gel (discontinuous SDS-PAGE) at room temperature for 1.5 h at 120 volts on an EC120 Minivertical gel/power source system (E-C Apparatus, Holbrook, NY; Fisher Scientific, Nipean, ON, Canada). Separated prestained standards (Bio-Rad, Mississauga, ON, Canada) and lysate proteins were transferred electrophoretically to a nitrocellulose membrane (0.22-µm pore size; MSI Inc., Westborough, MA) at 15 volts for 2.5 h with an EC140 Miniblot module (EC Apparatus); then the membrane was blocked overnight at 4°C with 5–7% milk solution in 0.1 M Tris-HCl, 0.5 M NaCl (TBS). Gels were routinely stained with Coomassie blue dye to confirm protein transfer. The milk-blocked membrane was reacted first with anti-human Fas mouse monoclonal IgM clone CH11 (Transduction Laboratories, Lexington, KY) at a dilution of 1:1000 in 0.1% Tween in TBS (TBST), then with HRP-conjugated goat AffiniPure goat anti-mouse (Jackson Immunoresearch, Biocan Scientific, Mississauga, ON, Canada) at a dilution of 1:2000 in TBST. Ig-reacted bands were visualized with an enhanced chemiluminescence kit (Amersham Canada Ltd., Oakville, ON, Canada), and autoradiography was carried out with scientific imaging paper (X-OMAT AR; Kodak, Rochester, NY).
Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Assay
First-strand cDNA was synthesized from 10 ng total RNA with 50 U Moloney Murine Leukemia Virus RT in 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 10 U recombinant ribonuclease (RNase) inhibitor, 400 µM nucleotides (dATP, dCTP, dGTP, TTP; Sigma), and 6 µg/ml oligo(dT12–18) primer (Boehringer-Mannheim, Laval, PQ, Canada) in a total reaction volume of 50 µl at 37°C for 60 min; then reverse transcriptase was inactivated by heating at 94°C for 10 min. Resulting cDNA stocks were stored at -20°C until use. Specific sequences were amplified from 1–4 µl of the cDNA stocks by nested PCR in a PTC-200 DNA engine (GRI Ltd., Dunmow, Essex, UK). The first 20-cycle round of PCR for Fas was performed with primers Fas1 and Fas2 (see below) designed with the Primer3 program at Massachusetts Institute of Technology (http://www.genome.wi.mit.edu/cgi-bin/primer) based on the published sequence for Fas [34], and the second 30-cycle round carried out using 0.2 µl of the first-round product with primers Fas II F and Fas II R [5]. Fas primers were prepared by Gibco BRL Life Technologies (Paisley, Scotland). PCRs for glyceraldehyde phosphate dehydrogenase (GAPDH) and ß-actin were carried out for 32 cycles, using primers from Stratagene Ltd. (La Jolla, CA). All amplification products spanned intron-exon junctions to control for genomic DNA contamination. The product amplified by Fas1 and Fas2 spans the transmembrane domain region of the Fas mRNA; thus these primers detect both full-length Fas and the transcript for the soluble FasTMDel. Primer sequences were as follows: Fas1 (5'-CTCTGGTTCTTACGTCTGTTGCTA), Fas2 (5'-GCTTTGGATTTCATTTCTGAAGTT), Fas II F (5'-CATGGCTTAGAAGTGGAAAT), Fas II R (5'-ATTTATTGCCACTGTTTCAGG), GAPDH1 (5'-CCACCCATGGCAAATTCCATGGCA), and GAPDH2 (5'-TCTAGACGGCAGGTCAGGTCCACC). Amplimer sizes are 338 base pairs (bp) (Fas), 275 bp (FasTMDel), 661 bp (ß-actin), and 600 bp (GAPDH). PCR reactions were carried out in the presence of 0.75 U Taq polymerase, 20 mM Tris HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, and 1 mM of each oligonucleotide in a total reaction volume of 30 µl. PCR was performed with temperatures of 94°C for 3 min and 60°C for 3 min followed by a varying number of cycles (see above) at 72°C for 90 sec, 94°C for 45 sec, and 60°C for 45 sec. Final extension was for 10 min at 72°C. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining and UV fluorescence. PCR products were confirmed by restriction digestion of amplifiers extracted from low-melting point agarose gels (results not shown).
Statistical Analysis
Statistical differences were evaluated by analysis with Student's t-test (Excel; Microsoft, Redmond, WA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fas mRNA was detected in both term and first-trimester villous placental tissue and in highly purified (> 99.9% [27, 28]), freshly isolated term cytotrophoblasts by nested RT-PCR of extracted RNA using primers that amplify a sequence encoding extracellular, transmembrane, and cytoplasmic domains [5]. The overwhelmingly major product from both first-trimester and term placentas (Fig. 1A) and purified term cytotrophoblasts (Fig. 1B) was a 300- to 400-bp amplimer of size identical to products from Jurkat cells, a T lymphocyte line known to express transmembrane Fas [35], and BeWo cells, a choriocarcinoma (of trophoblast origin) line [36] (Fig. 1A). Since the predicted size of the amplicon for transmembrane Fas is 338 bp and that for the transmembrane deletant is 275 bp, we conclude that the dominant form of the Fas transcript in villous cytotrophoblasts encodes a transmembrane protein. Restriction analysis of the amplified product supported this conclusion (data not shown). Both whole placenta and cytotrophoblast mRNA generated traces of the smaller amplimer when the gels in Figure 1 were overexposed (data not shown). Western analysis of cellular proteins extracted from freshly isolated and purified term cytotrophoblasts revealed a major band of approximately 45 kDa running at the same molecular mass as the full-length expression product extracted from Jurkat cells (Fig. 2).
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In order to determine the fraction of primary cytotrophoblasts that expressed Fas protein, cultured cells were methanol-acetone-fixed and immunohistochemically stained (Fig. 3, A and B). This analysis showed that all cells expressed Fas, albeit at widely differing levels. Intense perinuclear staining was observed in a significant subpopulation of the cells. Fas protein staining on paraformaldehyde-fixed cells (data not shown) suggested cell-surface expression. This was confirmed by carrying out an enzyme-linked antibody assay of Fas antigen on live cells in culture (a cellular ELISA). The 4-fold difference in color development between specific anti-Fas antibody and the IgG2b control (Fig. 3C) showed strong cell-surface expression on live cells. Surface expressions on cultured HeLa cells (which readily undergo Fas-mediated apoptosis, see below and reference [37]) and cytotrophoblasts were not significantly different (HeLa: 0.44 ± 0.06 OD U/µg DNA; cytotrophoblasts: 0.30 ± 0.15 OD U/µg DNA, n = 3, p > 0.2).
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Activation of Fas with Antibody
Cell-surface Fas can be activated to induce rapid apoptosis by treatment with the IgM monoclonal antibody CH11 [2], an effect enhanced by the protein synthesis inhibitor CHX [38]. CHX at 1 µg/ml increased TNF/IFN-induced loss of cytotrophoblast viability after 24-h treatment from 1 ± 2% to 10.6 ± 3.8% (p < 0.05; determined by MTT analysis, data not shown), showing that even low CHX concentrations effectively enhance cytokine-induced trophoblast cytotoxicity. A 24-h culture treatment with CHX at 1 µg/ml and CH11 at 100 ng/ml reduced the viability of the epithelial cell line HeLa by 40%, but cytotrophoblasts showed no appreciable loss in viability (Fig. 4A). CH11 was tested over a concentration range of 25–200 ng/ml and had no effect on cytotrophoblast viability at any concentration but reached maximum cytotoxicity to HeLa cells at 100 ng/ml (data not shown). Cytotrophoblasts require between 72 and 96 h to lose viability after treatment with TNF [33]; thus they may also require longer activation with anti-Fas. However, 72-h treatment with CH11 antibody in the presence of CHX had no effect on cytotrophoblast viability even though culture of the same cells with TNF significantly reduced viability (Fig. 4B). Coculture of cytotrophoblasts with IFN increases TNF-induced death [33]. However, cotreatment with anti-Fas antibody and IFN at 100 U/ml did not induce cytotrophoblast death (data not shown). CH11 antibody stimulation of the choriocarcinoma cell line BeWo, which expressed approximately the same amount of Fas protein as HeLa cells, also did not induce apoptosis with or without cotreatment with CHX or IFN (data not shown).
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FasL-Expressing Cells Did Not Stimulate Trophoblast Death
Antibody treatment mimics the effect of FasL in activating Fas receptor, presumably by clustering cell-surface receptor molecules. In order to determine whether cytotrophoblast Fas receptor could be activated by functional cell-surface FasL, cytotrophoblasts were incubated with the T lymphocyte cell line A1.1, which up-regulates FasL after activation with PMA and ionomycin [29]. Activated A1.1 cells reduced the viability of CHX-treated HeLa cells in a FasL-dependent manner (blocked by agonistic anti-Fas mAb SM1/23, data not shown) but not of identically treated cytotrophoblasts (Fig. 5A). As expected, nonactivated A1.1 cells killed neither HeLa cells nor cytotrophoblasts, but TNF efficiently killed the cytotrophoblasts in the same experiment. In order to determine whether activated A1.1 cells nonetheless induce cytotrophoblast apoptosis, the extent of trophoblast DNA fragmentation was determined as a function of A1.1 cell activation. Cytotrophoblast DNA was labeled with [3H]TdR, and cytoplasmic label was monitored as a fraction of total cellular label (Fig. 5B). The results show that neither activated nor control A1.1 cells increased DNA fragmentation of underlying cytotrophoblasts beyond spontaneous levels for this experiment but that the combination of TNF and IFN significantly increased fragmentation. We found that cultured first-trimester cytotrophoblasts [39] and term epidermal growth factor-differentiated syncytiotrophoblasts [40] were also resistant to activated A1.1-stimulated apoptosis (data not shown).
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NOS Inhibitors Did Not Up-Regulate FasL-Induced Apoptosis in Cytotrophoblasts
Nitric oxide (NO) inhibits human B lymphocyte apoptosis [41] and blocks signal transduction through the Fas receptor [26]. Since villous syncytiotrophoblasts [42] and cytotrophoblasts [43] express the NOS isozyme eNOS and sufficient NO is produced to react with endogenous reactive oxygen intermediates to effect considerable cellular nitrotyrosine formation (unpublished results), we asked whether the NOS inhibitors AG, L-NMMA, or L-NAME (reviewed in [44]) up-regulate FasL-stimulated DNA fragmentation in cultured cytotrophoblasts. Over a concentration range of 0.02–0.2 mM, the NOS inhibitors did not increase activated A1.1 cell fragmentation of cytotrophoblast DNA (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TNFRp55 is expressed at high levels on approximately 50% of cultured cytotrophoblasts [22]. We therefore compared the relative capacities of Fas and p55 receptors to mediate apoptosis under optimal and identical activation conditions: TNF-stimulated apoptosis in cytotrophoblasts and HeLa cells is enhanced by concomitant treatment with IFN or the protein synthesis inhibitor CHX ([33, 45, 46], this paper), and Fas-mediated apoptosis of HeLa cells requires pretreatment with IFN [37] or is enhanced by CHX [38]. Under these conditions, neither Fas antibody nor FasL-expressing cells induced cytotrophoblast apoptosis. This unresponsiveness was not due to low Fas expression levels: Fas-mediated apoptosis appears to be mediated most efficiently by low- to medium-density expression [47], and we see similar cell-surface expression on Fas-responsive HeLa cells and unresponsive cytotrophoblasts. Thus, our results demonstrate specific inhibition of Fas-mediated killing in placental trophoblasts and suggest that any possible deleterious consequences of simultaneous expression of Fas and FasL in the villous trophoblast layer may be circumvented by blocked Fas signal transduction.
These studies contribute to a growing understanding of programmed cell death in the placenta, particularly in the villous trophoblast. The general distribution of apoptotic nuclei in the villous syncytiotrophoblast suggests regulated tissue turnover, a characteristic of most epithelial tissues [16]. The placental apoptotic frequency is higher in conditions such as IUGR [17], in which the outcome (a small fetus) is thought to be caused by dysfunctional placentas [48, 49]. The maternal pregnancy disorder pre-eclampsia is also frequently associated with placental damage [50]. The observation that abnormal villous cytotrophoblast death and proliferation [51, 52] occur together in placentas from pregnancies complicated by pre-eclampsia suggests the existence of homeostatic mechanisms that limit excessive accumulation of cytotrophoblasts in the third trimester of pregnancy, when the villous trophoblast remodels [12]. The simultaneous presence of Fas and FasL in the villous cytotrophoblast layer (FasL in villous cytotrophoblasts and syncytiotrophoblasts [18] and Fas in freshly isolated, homogenous cytotrophoblasts [this paper]) suggests a very private (juxtacrine) regulation of cytotrophoblast density. However, the specific block of Fas-mediated apoptosis in cytotrophoblasts argues that if Fas-mediated elimination occurs, Fas signaling must be subject to up-regulation downstream of the receptor. Some tumor cells also express both Fas and its ligand [53, 54], suggesting similar mechanisms of avoiding autocrine death and immune cell surveillance.
Fas signal transduction is transiently down-modulated in T cells activated to express FasL, presumably to prevent auto-apoptosis within an immunologically functional time frame after antigen-dependent activation (reviewed in [4]). A recently documented mechanism of Fas signal inactivation in T cells involves blocking the death signal pathway by inhibition of caspase 8 (FLICE) activity [23–25]. FLICE inhibitors (FLIP/I-FLICE/FLAME-1) bind the Fas-associated death domain (FADD) and may block the binding of FLICE to FADD, thereby preventing the assembly of the death-signaling complex. FLIP is transiently expressed in activated T lymphocytes and disappears when cells become susceptible to Fas-mediated apoptosis. This inhibitor is constitutively expressed in the placenta [23]; thus it is a candidate for mediating the observed inhibition of placental cytotrophoblast Fas. However, FADD and caspase 8 are common to the apoptotic pathways of both TNFRp55 and Fas [55], and the inhibitors (FLIP/I-FLICE/FLAME-1) block both TNF- and Fas-mediated apoptosis [24, 25]. We found that Fas-stimulated but not TNFRp55-stimulated apoptosis is blocked in cytotrophoblasts; thus, a role for FLICE inhibitors is not indicated. NO, produced by villous trophoblast NOS (eNOS) [42], could also inhibit Fas response as it does in a variety of B and T cell lines [26]. However, we found that cultured cytotrophoblasts release very low levels of NO and that treatment with NOS inhibitors AG, L-NMMA, and L-NAME did not up-regulate Fas-induced apoptosis. Therefore, the mechanism by which Fas-induced apoptotic signals in cytotrophoblasts are blocked appears to be novel.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Larry J. Guilbert, Dept. Medical Microbiology and Immunology, 1–41 Medical Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2H7. FAX: 403 492 9828; larry.guilbert{at}ualberta.ca
3 S. Payne and S. Smith contributed equally to this publication and are co-first authors.
Accepted: December 11, 1998.
Received: October 1, 1998.
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A. Gruslin, Q. Qiu, and B. K. Tsang X-Linked Inhibitor of Apoptosis Protein Expression and the Regulation of Apoptosis During Human Placental Development Biol Reprod, April 1, 2001; 64(4): 1264 - 1272. [Abstract] [Full Text]
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S. Wongweragiat, R. F. Searle, and J. N. Bulmer Expression of Fas/Fas Ligand by Decidual Leukocytes in Hydatidiform Mole Biol Reprod, March 1, 2001; 64(3): 784 - 789. [Abstract] [Full Text]
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 M. I. Garcia-Lloret, B. Winkler-Lowen, and L. J. Guilbert Monocytes adhering by LFA-1 to placental syncytiotrophoblasts induce local apoptosis via release of TNF-{alpha}. A model for hematogenous initiation of placental inflammations J. Leukoc. Biol., December 1, 2000; 68(6): 903 - 908. [Abstract] [Full Text]
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