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Regular Article |
a Departments of Microbiology and Immunology,
b Obstetrics and Gynaecology, and
c Pathology, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 4LP, United Kingdom
ABSTRACT
Complete hydatidiform moles are entirely paternally derived and, therefore, represent a complete intrauterine allograft that might be expected to provoke an altered maternal immune response compared with that of normal pregnancy. Uterine decidua contains a large leukocyte population, of which 10%–20% are T lymphocytes. Fas ligand (FasL) expression by placental trophoblast may induce apoptosis of Fas+ lymphocytes, thereby facilitating immune tolerance and survival of the molar trophoblast. Our previous studies have shown an increase in activated CD4+ decidual T cells in molar pregnancy compared with normal pregnancy. This study was designed to characterize and quantitate Fas/FasL expression by decidual leukocytes in complete and partial hydatidiform mole compared with that in normal early pregnancy using single and double immunohistochemical labeling (i.e., avidin-biotin-peroxidase and avidin-biotin-alkaline phosphatase). A significant increase was found in Fas and FasL expression by decidual CD4+ T cells in complete (Fas+, P = 0.0106; FasL+, P = 0.0081) and partial (Fas+, P = 0.0131; FasL+, P = 0.0051) hydatidiform moles, as was a significant decrease in Fas expression by decidual CD8+ T cells in complete (P = 0.0137) and partial (P = 0.0202) hydatidiform mole compared with normal early pregnancy. The implications of altered Fas/FasL status of decidual T-cell subsets in hydatidiform mole are also discussed.
apoptosis, pregnancy, reproductive immunology
INTRODUCTION
Hydatidiform mole is a gestational trophoblastic disease in which placental trophoblast cells proliferate abnormally. The incidence of molar gestation varies geographically, being highest in Asian countries [1]. Complete hydatidiform moles are usually diploid, with a 46,XX karyotype; derive totally from the paternal genome; and are characterized by generalized trophoblastic hyperplasia with no fetus [1–3]. In contrast, partial hydatidiform moles are generally triploid, resulting from fertilization of a normal ovum by two spermatozoa [4]. The characteristic histological features affect only part of the placenta, and a fetus, often with congenital malformation, is present. Both partial and, more frequently, complete hydatidiform moles are associated with persistent trophoblastic disease [1].
During normal pregnancy, approximately 30% of stromal cells in decidualized uterine endometrium are leukocytes [5]. Of these, as many as 70% are endometrial granulated lymphocytes (eGL), approximately 20% are macrophages, and 10%–20% are T lymphocytes, with a CD8:CD4 ratio of approximately 3:1 [5, 6]. These leukocytes are potential sources of cytokines in uteroplacental tissues [6], but their in vivo role in the maternofetal relationship remains unclear. Because its chromosomes are paternal in origin, a complete hydatidiform mole (unlike the normal, semiallogeneic conceptus) forms a complete intrauterine allograft and, therefore, may be expected to provoke an altered maternal immune response. An earlier study noted increased CD4+ decidual T cells in complete hydatidiform mole [7]. Recently, we reported an increase in activated CD4+ T cells in decidua associated with both partial and complete hydatidiform moles compared with normal early pregnancy, suggesting an altered maternal immune response against molar trophoblast [8].
Activated CD4+ and CD8+ T cells characteristically express Fas ligand (FasL; CD95-ligand), which is an inducible, cell surface-associated, trimeric protein belonging to the tumor necrosis factor family that induces apoptosis of Fas (CD95/Apo-1)-bearing cells. Fas is a monomeric molecule that is trimerized when bound to its ligand and sends a signal through its death domains that leads to a cellular suicide cascade [9]. In the immune system, Fas/FasL expression is involved in apoptosis-induced regulation of immune reactions [9].
Recent studies have demonstrated constitutive expression of FasL by placental trophoblast [10–13] as well as apoptosis of decidual leukocytes [14], possibly T cells [15], suggesting a mechanism for immune tolerance in normal pregnancy mediated by the Fas/FasL system. To date, the Fas/FasL status of cells in uterine decidua associated with molar gestation has not been established. Fas/FasL expression may play a role in maternal immune tolerance to abnormal trophoblast in hydatidiform mole, and the aim of this study was to quantitate and to compare Fas and FasL expression by decidual leukocytes during molar pregnancy with those during normal early pregnancy to serve as a basis for future functional studies of molar trophoblast proliferation and survival.
MATERIALS AND METHODS
Tissues
Formalin-fixed, paraffin-embedded decidual tissues from six normal first-trimester pregnancy terminations and from six partial and five complete hydatidiform moles evacuated during the first trimester were retrieved from the archive files of the Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom. Three-micrometer sections were mounted on 3-aminopropyl-triethoxysilane (Sigma Chemical Co., Poole, UK)-coated slides. Cases with histological evidence of decidual necrosis or inflammation, identified by the presence of neutrophil polymorphs or plasma cells, were not included.
Antibodies
Fas and FasL were detected using rabbit polyclonal antibodies raised against Fas (sc-715; diluted 1:500 v:v) and FasL (sc-834; diluted 1:200 v:v), respectively (Santa Cruz Biotechnology, Santa Cruz, CA). Optimal dilutions, incubation times, and pretreatments were established using similarly fixed and processed, positive-control tonsil tissues. A panel of murine monoclonal antibodies against leukocyte antigens (Table 1) was employed to investigate and quantitate Fas and FasL expression by decidual leukocyte populations in molar and normal pregnancy using double immunohistochemical labeling. All antibodies were diluted in 0.1 M Tris 0.05 M saline (pH 7.6; TBS).
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Single Immunohistochemical Labeling
Fas and FasL expression were examined using an avidin-biotin-peroxidase technique (Vectastain Elite; Vector Laboratories, Peterborough, UK). Briefly, sections were deparaffinized, rehydrated, and incubated for 10 min with 0.5% hydrogen peroxide in methanol to block endogenous peroxidase activity. For anti-Fas, sections were pretreated for 10 min with 2.5% (w/v) trypsin (Difco Laboratories, Detroit, MI) in 2.5% (w/v) calcium chloride (pH 7.8). Anti-FasL did not require pretreatment. The sections were then incubated for 30 min with 1.5% (v/v) normal goat blocking serum (NGS) in TBS before primary antibody was applied for 60 min. After washing in TBS, sections were incubated for 30 min with biotinylated anti-rabbit immunoglobulins, washed again in TBS, and incubated in avidin-biotin-peroxidase for 30 min. After further TBS washing, the reaction was developed with 3,3'-diaminobenzidine (DAB; Sigma Chemical Co.) containing 0.01% (v/v) H2O2 to give a brown reaction product. The sections were lightly counterstained with Mayer's hematoxylin, dehydrated, cleared in xylene, and mounted with DPX synthetic resin (Raymond A. Lamb Ltd., London, UK). Positive controls were performed using normal tonsil tissues. Negative controls were performed by replacing the primary antibody with normal rabbit serum diluted 1:200 v:v in TBS.
To verify that the immunostaining patterns obtained with these polyclonal antibodies on paraffin-embedded samples truly reflected Fas and FasL expression, the same antibodies were used to immunostain acetone-fixed cryostat sections of snap-frozen, normal early pregnancy decidua. In addition, representative paraffin-embedded samples from the study were immunostained with mouse monoclonal antibodies directed against Fas and FasL (NCL-FAS-310 and NCL-FASL; Novocastra Laboratories, Newcastle upon Tyne, UK). For both antibodies, sections were pretreated by pressure cooking in citrate buffer. Antibodies were diluted 1:50 v:v in TBS, and the reaction was detected using a mouse Vectastain Elite kit as described previously [8].
Double Immunohistochemical Labeling
Expression of Fas and FasL by decidual CD45+, CD3+, CD4+, CD8+, CD56+, and CD68+ cells was assessed by double immunohistochemical labeling using an avidin-biotin-peroxidase method (Vectastain Elite) to detect leukocytes, followed by an avidin-biotin-alkaline phosphatase method (Vectastain alkaline phosphatase kit) to localize Fas and FasL expression.
Leukocytes were detected as described in detail previously [8] with appropriate pretreatments and blocking steps (Table 1). This reaction was developed with DAB to give a brown reaction product. After washing in tap water, sections were incubated sequentially with 1.5% (v/v) NGS for 30 min, anti-Fas or anti-FasL for 60 min, biotinylated anti-rabbit immunoglobulins for 30 min, and finally, with avidin-biotin-alkaline phosphatase for 30 min. The reaction was developed with alkaline phosphatase substrate kit III (Vecta Blue; Vector Laboratories) in the dark for 20–30 min until the appropriate blue color had developed. Sections were mounted with Supermount permanent aqueous mounting medium (BioGenex, San Ramon, CA).
Positive controls were performed for all antibodies using normal tonsil or neuroblastoma (anti-CD56) tissues. Negative controls included replacing both primary antibodies with normal serum. In addition, separate negative controls were performed for each level of the double-labeling technique by replacing each primary antibody with the appropriate normal serum in separate sections.
Quantification of Fas+ and FasL+ Decidual Leukocytes in Double-Labeled Sections
Single-positive decidual leukocytes were identified by the presence of membranous brown reactivity, whereas single-labeled Fas+ or FasL+ cells were identified by blue reactivity. Double Fas+ or FasL+ leukocytes showed distinctive membranous, dark blue-brown staining, which could be clearly distinguished from single-labeled (brown) leukocytes. Positive cells were counted in comparable areas of consecutive sections at 400x magnification in at least four fields to give a minimum of 100 cells counted. Double-positive cells were assessed independently by two investigators who were blinded as to the group designation of each case. Anti-CD4 also stained macrophages; therefore, only small, rounded, lymphocytic cells were counted. Similarly, anti-CD3 and, occasionally, anti-CD8 reacted also with eGL, and care was taken to count only small, rounded, agranular CD3+ or CD8+ lymphocytes with scanty cytoplasm. Granulated lymphocytes were clearly distinguished by their reactivity for CD56. The proportion of Fas+ or FasL+ leukocytes was calculated as the percentage of the total of the individual leukocyte populations. Statistical analysis was performed using the nonparametric Mann-Whitney test (two-sided test) with the conventional significance level of P < 0.05.
RESULTS
Single Immunohistochemical Labeling
Single immunohistochemical labeling revealed expression of Fas by cells in decidua from normal and molar pregnancy. These Fas+ cells included all decidualized stromal cells as well as small cells within the decidual stroma that appeared to be leukocytes (Fig. 1A). Small numbers of Fas- cells with a leukocytic morphology were clearly identified within the same fields. FasL+ cells were also demonstrated in decidua associated with normal early pregnancy and hydatidiform mole. These FasL+ cells included all decidualized stromal cells and a proportion of decidual leukocytes (Fig. 1B). FasL- leukocytes, although scanty, were obvious. Negative controls were performed for all samples and showed no reactivity (data not shown).
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Frozen sections immunostained with the polyclonal anti-Fas and anti-FasL antibodies showed similar reactivity to the paraffin-embedded samples. Representative study samples immunostained with monoclonal anti-Fas and FasL antibodies showed identical reactivity to that described for the polyclonal antibodies (data not shown).
Double Immunohistochemical Labeling
Negative controls showed no reactivity if both primary antibodies were replaced with normal serum, and the appropriate single immunolabeling pattern was obtained when one of the primary antibodies was replaced with normal mouse or rabbit serum as appropriate. No nonspecific staining and no spurious double immunolabeling were found (data not shown). In normal early pregnancy and complete and partial hydatidiform moles, the percentage of decidual CD45+ cells coexpressing Fas was similar, accounting for 33.0%–52.0% of the total CD45+ cells (Table 2). Similarly, the percentage of decidual CD3+ T cells that coexpressed Fas did not significantly differ between normal and molar pregnancy and ranged from 42.5%–53.0% of total CD3+ T cells (Table 2).
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In contrast, the percentage of decidual CD4+ T cells that coexpressed Fas increased significantly in both complete (84.0%, P = 0.0106) and partial (74.5%, P = 0.0131) hydatidiform moles compared with normal early pregnancy (51.0%) (Figs. 1, C and D, and 2A, Table 2). Moreover, a significant decrease in the percentage of Fas+ decidual CD8+ T cells was detected in both complete (21.0%, P = 0.0137) and partial (17.5%, P = 0.0202) hydatidiform moles compared with normal early pregnancy (37.0%) (Figs. 1, E–G, and 2B, Table 2). The percentage of Fas+ CD56+ eGLs and Fas+ CD68+ macrophages did not differ significantly in partial and complete hydatidiform moles compared with normal early pregnancy (Table 2).
The percentage of CD4+ T cells that coexpressed FasL was also increased significantly in both complete (76.0%, P = 0.0081) and partial (78.5%, P = 0.0051) hydatidiform moles compared with normal early pregnancy decidua (34.5%) (Figs. 1H and 3, Table 2). For other leukocyte populations, the proportion of FasL+ cells did not differ significantly between molar pregnancy and normal early pregnancy (Table 2).
DISCUSSION
In the present study, Fas and FasL expression by cells within decidua associated with both normal early pregnancy and hydatidiform molar pregnancy has been demonstrated. These Fas+ and FasL+ cells included most decidualized stromal cells and a proportion of decidual leukocytes. Using double immunohistochemical labeling, a significant increase in the proportion of CD4+ T lymphocytes that coexpressed both Fas and FasL was noted in both complete and partial hydatidiform mole. Recently, we demonstrated that decidual CD4+ T-cell numbers are increased in complete and partial hydatidiform mole compared with normal early pregnancy, and that these CD4+ T cells are activated/memory cells expressing CD45RO, both of which are findings that suggest an altered maternal immune response against molar trophoblast compared with normal pregnancy [8]. The present study demonstrates that these CD4+ T cells coexpress both FasL and Fas, suggesting that, despite an altered activated decidual T-cell response, their Fas-mediated apoptosis [9] may ensure the survival of the molar pregnancy allograft. The precise biological significance of placental Fas and FasL expression remains to be established.
In contrast, the decrease in Fas+ CD8+ decidual T cells in hydatidiform mole noted in the present study suggests that CD8+ T cells may not be primarily responsible for immune responses against the molar trophoblast and do not undergo apoptosis following activation. The decrease in Fas+ CD8+ decidual T cells possibly reflects their down-regulation by transforming growth factor (TGF)-ß [16] and interleukin (IL)-15 [17], two cytokines normally present in the fetoplacental unit. Recent in vitro studies have demonstrated that TGF-ß [18] and IL-15 [19] up-regulate the de novo expression of CD94/NKG2A heterodimer killer inhibitory receptor (KIR) complex on CD8+ cytotoxic T cells. This, in turn, results in the inhibition of T-cell receptor-mediated cytotoxicity and cytokine production by CD8+ T cells. The level of TGF-ß and IL-15 and the KIR status of CD8+ decidual T cells in molar pregnancy are unknown.
In support of a role for Fas-mediated apoptosis in survival of the molar pregnancy allograft, recent reports of FasL expression by placental trophoblast and choriocarcinoma cell lines in vitro [10–13] and Fas expression by decidual CD45+ cells [13] that undergo apoptosis [14] raise the possibility that immune tolerance to placental trophoblast in both normal and pathological pregnancy may be mediated by the Fas/FasL pathway. The present studies of normal early pregnancy have confirmed the expression of Fas by decidual leukocytes and further demonstrated that Fas+ cells include CD4+ and CD8+ T cells, eGLs, and macrophages, suggesting that these cells may be regulated by either FasL-expressing leukocytes and/or trophoblast at the maternofetal interface. Recent studies have also demonstrated decreased Fas+ CD56+ cells in the decidua from pre-eclamptic women, but Fas+ CD4+ and Fas+ CD8+ decidual T cells [20] have been demonstrated to be unaffected. These findings suggest an alteration of maternal immune response to inappropriate trophoblast invasion, which may influence Fas-mediated apoptosis of eGLs.
Apoptosis appears to be normal feature in reproductive organs, including the placenta, and may play a role in normal placental development and aging [21]. Apoptosis of placental trophoblast has been reported to be increased during ectopic pregnancy [22] and spontaneous abortion [23]. Furthermore, activated T cells in the decidua also express FasL, which can induce apoptosis of Fas+ trophoblast [24, 25]. Although apoptosis has been demonstrated mainly in the villous syncytiotrophoblast of normal human first-trimester placentas [26, 27], the incidence of apoptosis has been reported to be higher in the villous cytotrophoblast of complete hydatidiform mole compared with that of normal placenta [28], suggesting that an abnormal maternal immune response may have occurred that leads to apoptosis of the molar cytotrophoblast. The precise levels of apoptosis in complete and partial molar pregnancy should be addressed. Using immunohistochemistry, reactivity for Fas has been detected on villous and extravillous trophoblast in both partial and complete hydatidiform moles (unpublished results). We have also found that all placental trophoblast subpopulations in complete hydatidiform mole strongly express FasL, unlike normal early pregnancy, in which different trophoblast subpopulations have different levels of FasL expression (unpublished results). The present observation of a significant increase in the proportion of FasL+ CD4+ T cells in decidua associated with hydatidiform mole suggests the possibility of Fas-mediated apoptosis of Fas+ molar trophoblast. Nevertheless, other studies have shown that Fas expressed by human placental cytotrophoblast does not necessarily mediate apoptosis [29], possibly reflecting inhibition of tumor necrosis factor-
/interferon-
-induced villous apoptosis by epidermal growth factor [30] or down-regulation of Fas expression by soluble FasL [31, 32].
Research into the immunopathology of hydatidiform molar pregnancy may enhance our understanding of the maternal immune response in normal pregnancy, because complete hydatidiform mole is a complete intrauterine allograft. The findings of increased activated decidual CD4 T cells in hydatidiform mole [8] and the present findings of increased Fas and FasL expression by these cells indicate that maternal immune responses may occur in the decidua when maternal cells encounter trophoblast with an abnormal proliferative capacity. In fact, recent investigations regarding the parental origin of triploidy in spontaneous abortion have indicated that paternally derived conceptuses predominate among "typical" spontaneous abortions: maternally derived cases associated with either early embryonic demise or relatively late demise with a well-formed fetus [33]. Paternal rather than maternal triploidy is associated with development of partial hydatidiform moles, although paternal triploidies often are nonmolar [33]. These findings suggest that an additional paternal chromosomal component may result in rejection of the intrauterine allograft, and a maternal immune mechanism for this rejection cannot be ruled out.
Whether FasL expression by decidual CD4+ T cells mediates the apoptosis of Fas+ molar trophoblast, or whether FasL expression by molar trophoblast regulates maternally derived Fas+ decidual cells, remains to be evaluated. Studies are now required to clarify the regulatory mechanisms that may be integral parts of immunoregulation at the maternofetal interface in both normal and molar pregnancy.
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ACKNOWLEDGMENTS
The authors acknowledge Mrs. Barbara Innes and Miss Claire Gilfillan for their technical assistance.
FOOTNOTES
First decision: 3 August 2000.
1 Correspondence: J.N. Bulmer, Department of Pathology, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK. FAX: 0191 222 8100; j.n.bulmer{at}ncl.ac.uk 
Accepted: October 3, 2000.
Received: July 10, 2000.
REFERENCES
/IFN--induced villous trophoblast apoptosis. Placenta 1999; 20:423–430[CrossRef][Medline]
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