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Biology of Reproduction 65, 1766-1770 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Monoclonal Anti-Annexin V Antibody Inhibits Trophoblast Gonadotropin Secretion and Induces Syncytiotrophoblast Apoptosis

Nicoletta Di Simonea, Roberta Castellania, Dario Caliandroa, and Alessandro Caruso1,a

a Department Obstetrics and Gynecology, Universita' Cattolica del S. Cuore, 00168 Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathogenic role of anti-annexin V antibodies remains unclear. Anti-annexin V antibodies are frequently associated with higher incidences of intrauterine fetal loss, preeclampsia, and arterial and venous thrombosis. The present study investigated the in vitro ability of anti-annexin V antibody to bind human trophoblast cells, to affect trophoblast gonadotropin secretion and invasiveness, and to induce placental apoptosis. Cytotrophoblast cells were dispersed in Ringer bicarbonate buffer containing trypsin and DNase I, filtered, and layered over a Percoll gradient in Hanks balanced salt solution. In the case of monoclonal anti-annexin V antibody, the highest binding was found when the cells displayed the greatest amount of syncytium formation. Anti-annexin V antibody, but not its negative control, induced trophoblast apoptosis and significantly reduced trophoblast gonadotropin secretion. These findings suggest that recognition by anti-annexin V antibody of adhered annexin V on trophoblast cell structures might represent a potential pathogenic mechanism by which these antibodies can cause defective placentation.

apoptosis, placenta, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antiphospholipid antibodies are clinically associated with thrombocytopenia, recurrent thrombosis, repeated pregnancy loss, and a combination of these events [1]. However, some patients with poor obstetrical outcome and presumed antiphospholipid syndrome have negative results for antiphospholipid antibodies with a sustained anti-annexin V titer [2]. Anti-annexin V antibodies have been detected in patients with systemic autoimmune diseases, especially systemic lupus erythematosus and rheumatoid arthritis, and this observation was associated with higher incidences of intrauterine fetal loss, preeclampsia, and arterial and venous thrombosis [3–6]. These findings suggest that anti-annexin V autoantibodies might be the cause of thrombotic events in such patients, possibly by interfering with the function of annexin V, which is believed to be a potent anticoagulant [7, 8].

Annexin V is a Ca2+-dependent, phospholipid-binding protein that is abundant in cells exposed to blood, such as platelets, trophoblasts, and endothelial cells [9, 10]. The high concentration of annexin V in placental syncytiotrophoblasts has been proposed as a mechanism to maintain blood fluidity on the surfaces of placenta so that maternofetal nutrition exchange and fetal viability are unimpaired [11]. A significant reduction of annexin V on the placental villi in patients with antiphospholipid syndrome has been reported [12], and antiphospholipid antibodies have decreased the level of annexin V on the surfaces of cultured trophoblasts and placental villi [13]. These observations suggest that the reduced expression of annexin V in these placentas leads to a hypercoagulable state in the intervillous space and may be associated with obstetrical complications.

Direct proof for the pathogenicity of anti-annexin V antibodies came from studies in which anti-annexin V antibody infusion caused fetal loss, placental thrombosis, and tissue necrosis in pregnant BALB/c mice [14]. The infused anti-annexin V antibodies might act in a manner similar to that of antiphospholipid antibodies, causing the loss of annexin V in placenta and promoting thrombosis at the maternofetal tissue junction [13], thereby compromising the nutrient-exchanging functions and, consequently, causing fetal death and absorption. The large-scale necrosis and thrombosis formation in the placentas of partially absorbed embryos in the study by Wang et al. [14] are consistent with this idea.

Assuming that several pathogenic mechanisms can be present at the same time, and even in the same patients, another hypothesis is that anti-annexin V antibodies are responsible for the induction of apoptosis in endothelial cells. Immunoglobulin G (IgG) antibodies were isolated from the plasma of patients with lupus erythematosus, and the apoptosis-inducing activity was localized in the annexin V-binding antibodies [15, 16].

To our knowledge, no information is available regarding the effects of anti-annexin V antibodies on the functions of human trophoblast. The purpose of the present study was to investigate the in vitro ability of anti-annexin V monoclonal antibody (mAb) to bind human trophoblast cells and to affect hCG secretion and cellular apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures

Placentas were obtained from healthy women immediately after uncomplicated vaginal delivery at 36 wk of gestation. Informed consent for the use of human tissues in this study was obtained from all patients.

Cytotrophoblast cells were isolated as detailed elsewhere [17]. Briefly, placental tissues were rinsed two or three times in cold Dulbecco modified Eagle medium (DMEM) and 10% (v/v) fetal calf serum (FCS; Gibco BRL, Grand Island, NY). After mincing, the tissues were submitted to repeated enzymatic digestions in Ringer-bicarbonate buffer containing 0.25% trypsin (Gibco BRL) and DNase I (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO) at 37°C in a shaking water bath for 2 h. The supernatants were filtered through a 42-µm mesh filter and centrifuged (200 x g at room temperature for 7 min); the cell suspension was layered over a preformed Percoll (Pharmacia-Biotech, Uppsala, Sweden) gradient in Hanks balanced salt solution (HBSS; Gibco BRL). The gradient was made from 5% to 70% (v/v) Percoll by dilutions of 90% Percoll (nine parts Percoll, one part 10x HBSS) and layered in a 50-ml, conical polystyrene tube. After centrifugation (200 x g at room temperature for 20 min), the middle layer was removed, washed, and then resuspended in DMEM. Cell viability was >90% as determined by trypan blue exclusion.

The purity of the cell preparation was evaluated by immunohistochemical staining for markers of fibroblasts (2%, determined using a polyclonal antivimentin antibody; Labsystems, Helsinki, Finland) and macrophages (3%, determined using a polyclonal anti-{alpha}1-chymotrypsin antibody; Dako, Santa Barbara, CA). The enriched (95%) cytotrophoblast cells (5 x 105 cells/ml) were cultured in DMEM-10% FCS in 96-well plates at 37°C in 5% CO2/95% air. Cell cultures were performed for 24, 48, and 72 h in standard medium. The medium was changed daily [18].

Binding Assay

On Day 1 (24 h), Day 2 (48 h), and Day 3 (72 h) of culture, the medium was removed, and the cells were incubated at room temperature for 1 h with serial protein concentrations of the different antibody preparations [19].

Mouse mAbs of the IgG1 isotype were used. Bindings of the mAb anti-annexin V (0.018–10 µg/ml, clone RUU-WAC2A; Monosan, Uden, The Netherlands) and the mAb anti-ß-actin (0.018–10 µg/ml, clone AC-15; Sigma-Aldrich), as a negative control, were measured in complete medium at a final volume of 100 µl. The mouse mAb anti-FAS (5 µg/ml, clone 11G10; Ylem S.r.L., Avezzano, Italy) was used as a positive control [20].

The plates were then extensively washed to remove unbound antibodies and proteins. A secondary alkaline phosphatase-conjugated anti-mouse IgG antibody (Sigma-Aldrich) was added to the plates. After incubation and washing, p-nitrophenylphosphate substrate (Sigma-Aldrich) was used to measure the IgG binding on trophoblast cells. The optical density values of the samples were read at 405 nm by a microplate photometer (Bio-Rad Platereader Model; Bio-Rad Laboratories S.r.L., Milan, Italy).

The mAb anti-annexin V (5 µg/ml) was incubated with varying concentrations of annexin V (0.01–10 µg/ml; Sigma-Aldrich) at 37°C for 1 h. After incubation, the binding to trophoblast cells (72 h of culture) was measured by ELISA, as described above.

Hormone Secretion

Primary trophoblast cells were cultured in complete medium for 48 h. The medium was then removed, and the cells were incubated in the presence of mAb anti-annexin V (0.05–5 µg/ml) or the mAbs anti-ß-actin and anti-FAS (5 µg/ml). After 48 h of culture, the media were removed and stored at -20°C for hCG determination. The assay was performed with a commercial radioimmunoassay kit (generously provided by Radim, Rome, Italy). The intra- and interassay coefficients of variation were <12% and <8%, respectively.

Measurements of Fragmented DNA by ELISA

Trophoblast cells were labeled with 10 mM 5-bromo-2'-deoxy-uridine for 48 h and then detached by EDTA-trypsin (Gibco BRL) treatment. The cells were collected by centrifugation at 250 x g for 10 min and suspended in a fresh culture medium to make 1 x 105 cells/ml. Next, 100 µl of the cell suspension were transferred to each well of a microculture plate and incubated with the test samples (mAb anti-annexin V or the control mAbs, 5 µg/ml). At 24 and 48 h, 20 µl of lysis buffer were added to the well or to the supernatants obtained by centrifugation; the amounts of fragmented DNA were measured with a cellular DNA fragmentation ELISA kit (Boehringer Mannheim, Mannheim, Germany) [16].

The experiments were done five times on different placentas, with duplicate within each experiment.

DNA Fragmentation Analysis

Cytotrophoblast cells (106 cells/ml) were cultured in complete medium. After a 48-h culture, the cells were treated for 48 h with the mAb anti-annexin V or the control mAbs (5 µg/ml). At the end of the incubation period, cells were washed twice in PBS. Cell pellets were resuspended and incubated in lysis buffer (50 mM Tris-HCl, 100 mM EDTA, and 0.5% SDS) supplemented with proteinase K (0.7 mg/ml; Sigma-Aldrich) and then incubated for 1 h at 55°C. The DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1 [v/v]), followed by absolute ethanol and addition of 70% ethanol. The DNA was dissolved in 10 mM Tris (pH 7.5) and 1 mM EDTA (pH 8) after evaporation of ethanol. The DNA was loaded into wells of a 1.5% agarose gel and electrophoresed at 75 mV using 100 mM Tris, 100 mM boric acid, and 0.2 mM EDTA as running buffer. The DNA was visualized by ethidium bromide staining.

Statistical Analysis

Significant differences were determined using the Student t-test, with P < 0.05 considered to be significant for all experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monoclonal Antibody Binding to Trophoblast Cells

Recently, we showed that in vitro differentiation progresses in trophoblast cultures from 24 h of culture [19]. At the beginning of the cell culture, enriched cytotrophoblasts were single, mononucleated cells with two or three aggregates. After 48 h, an increasing number (mean ± SD) of syncytia (31% ± 3% of 200 counted cells) were detectable, and they became the dominant form at 72 h of culture (81% ± 2% of 200 counted cells). These syncytia had centrally placed nuclei with no visible intervening membranes, and after 72 h of culture, they showed increasing amounts of vacuolated cytoplasm [19].

The trophoblastic differentiation process is characterized by the externalization of phopshatidylserine, and this physiological event may explain why the placenta is sensitive to the effect of anti-annexin V antibody. Exposure of anionic phosphatidylserine can, in turn, offer a substrate for annexin V binding, and the annexin V-membrane phosphatidylserine complex may increase antigen density to a point suitable for anti-annexin V antibody binding.

As shown in Figure 1, the mAb anti-annexin V displayed time-dependent trophoblast binding. The highest binding was seen at 72 h of culture, when the largest differentiation of cytotrophoblast into syncytiotrophoblast was found. Whereas the mAb anti-annexin V retained its trophoblast-binding activity with protein concentrations as low as 0.07 µg/ml, the negative control mAb did not display any significant binding at any tested concentration. Significant binding was observed with the positive control (mAb anti-FAS; data not shown).



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  		FIG. 1. Binding of human anti-annexin V mAb to primary trophoblast cells. Serial protein concentrations of anti-annexin V or negative control antibody were evaluated after 24 (A), 48 (B), and 72 (C) h of culture. Dose- and time-dependent binding was seen with the human anti-annexin V mAb but not with the control antibody. Values are the mean ± SD of five experiments. O.D., Optical density. aP < 0.05, bP < 0.01, anti-annexin V mAb versus control mAb

As shown in Figure 2, when the mAb anti-annexin V (5 µg/ml) was incubated with varying concentrations of annexin V before addition to trophoblast cells, the binding was inhibited.



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  		FIG. 2. Inhibition of the anti-annexin V antibody (5 µg/ml) binding. After preincubation with varying concentrations of annexin V, the antibody binding was measured by ELISA. Results are the mean ± SD of three independent experiments. Significant differences from anti-annexin V mAb binding in the absence of annexin V are indicated (aP < 0.02)

Effect of Anti-Annexin V Antibody on Trophoblast hCG Secretion

We evaluated the levels of hCG secreted into the culture medium by trophoblast cells in the presence of anti-annexin V antibody or its negative control. The mAbs were added at 48 h of culture, when a significant differentiation of cytotrophoblast into syncytiotrophoblast was found [19].

After 48 h, anti-annexin V antibody (from 0.05 µg/ml; P < 0.02) significantly inhibited hCG secretion, but the negative control did not change basal hCG secretion (Table 1). The positive control, mAb anti-FAS, did not display any significant effect at the tested concentration (5 µg/ml; data not shown).


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  		TABLE 1. Effect of human anti-annexin V and negative control monoclonal antibodies (mAbs) on basal hCG production.a

Anti-Annexin V Antibody Induces Apoptosis in Trophoblast Cells

The mAb anti-annexin V exhibited apoptosis-inducing activity, as determined by the DNA fragmentation of trophoblast cells. The time-course of apoptosis was monitored by the fragmented DNA derived from all cellular components. Incubation with anti-annexin V antibody produced significant amounts of fragmented DNA in a time-dependent manner, whereas the values for the negative control were similar to those of the untreated cells (Fig. 3). Incubation with 5 µg/ml of the mAb anti-FAS did not induce DNA fragmentation (data not shown).



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  		FIG. 3. Time-course of apoptosis-inducing activities of human anti-annexin V or negative control antibodies on primary trophoblast cells. 5-Bromo-2'-deoxyuridine-labeled cells were incubated for 24 or 48 h in the presence of mAbs, and DNA fragments were measured by ELISA on nonadherent (A) or adherent (B) cells. Results are the mean ± SD of five independent experiments. aP < 0.03, bP < 0.01, anti-annexin V antibody versus negative control antibody or untreated cells

Apoptosis was verified by electrophoretic observations (Fig. 4). The experiments were repeated five times on different placentas.



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  		FIG. 4. Gel electrophoresis of fragmented DNA. Primary trophoblast cells were exposed to 5 µg/ml of human anti-annexin V mAb (lane 3) or anti-ß-actin mAb (negative control, lane 2). Untreated cells are also shown (lane 1). The DNA was extracted and electrophoresed on a 2% agarose gel. M, DNA size markers in base pairs


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Annexin V has been localized in many tissues, and its expression has been described as a function of cell growth [21]. This protein has a high affinity for anionic phospholipids, which it binds to in a Ca2+-dependent manner, inhibiting procoagulant reactions [22]. It has a diverse range of in vitro functions and is probably involved in multiple basic cellular functions, such as transmembrane Ca2+-channel activity [23], inhibition of phospholipase A2 [24] and protein kinase C [25], cell-matrix interactions [26], and regulation of membrane integrity [27].

Annexin V was localized to trophoblast cells in the first-trimester placenta, with most reactivity found in the syncytiotrophoblast [28]. Apical syncytiotrophoblast staining may reflect an anticoagulant function, exposure of phosphatidylserine [29], or placental apoptosis [30], which occurs throughout normal gestation. Clearly, the syncytiotrophoblast layer must present a nonthrombogenic surface to circulating maternal blood in the intervillous space. Annexin V is critical in the maintenance of placental integrity [14], and a reduction of its placental villous expression has been demonstrated in women with antiphospholipid syndrome [12], preeclampsia, and recurrent spontaneous abortion [31].

In the present study, we found that anti-annexin V antibody binds to trophoblast in vitro. The highest degree of binding was found after 72 h of culture, when the cells displayed the largest syncytial groups [19]. Our results are consistent with the demonstration that phosphatidylserine is exposed on the external cell surface during intertrophoblastic fusion, with this exposure being maximal after 72 h of culture. Exposure of anionic phospholipids can, in turn, offer a substrate for cationic annexin V binding. This binding is supposed to "neutralize" the potentially procoagulant anionic phospholipid; on the other hand, it might increase the antigen density to a point that is suitable for anti-annexin V antibodies.

Clinical characteristics in patients with anti-annexin V antibodies match the clinical features of antiphospholipid syndrome, such as arterial or venous thrombosis and intrauterine fetal loss. Kaburaki et al. [32] reported patients with thrombosis who did not have antiphospholipid antibodies but who did have anti-annexin V antibodies, suggesting an important role for these antibodies in thrombotic events. In the study by Matsuda et al. [31], anti-annexin V antibodies were detected in 36% of patients with habitual fetal loss and in 20% of patients with preeclampsia, suggesting a close relationship between anti-annexin V antibodies and pregnancy-associated complications.

The high coagulation status and the high rate of pregnancy loss found among patients with autoimmune diseases might be due to the decrease of annexin V caused by anti-annexin V antibodies, acting either alone or together with antiphospholipid antibodies. In BALB/c mice, infusion of anti-annexin V antibodies decreased the availability of annexin V to bind to the trophoblast surfaces and caused placental thrombosis, necrosis, and fetal loss [14]. Therefore, the pathologic properties of anti-annexin V antibodies might explain the high thrombotic status and recurrent intrauterine fetal loss seen among patients who have autoantibodies against proteins associated with coagulation. However, multiple pathological mechanisms, rather than a single one, may trigger trophoblast in patients with these antibodies.

In our primary trophoblast cultures, the mAb (5 µg/ml) reacting with annexin V induced syncytiotrophoblast apoptosis. Consequently, it decreased placental hCG secretion to approximately 60% of control levels. Human trophoblast cells are presumed to be exposed to this apoptosis-inducing level of effector antibodies, because the anti-annexin V antibody fraction constitutes a very small percentage of the total IgG fraction, of which the serum concentration is 10–20 mg/ml.

To our knowledge, the present study is the first demonstration of an effect of anti-annexin V antibody on trophoblast apoptosis and on placental hormone secretion. Recently, Nakamura et al. [15, 16] presented evidence for the possible involvement of annexin V in lupus anticoagulant-induced apoptosis, showing that the annexin V-binding IgG antibodies induced endothelial cell apoptosis. That anti-annexin V antibody induces trophoblast and endothelial cell apoptosis could provide new information regarding the role of these autoantibody subpopulations.

In conclusion, our data suggest that the antibody binding, syncytiotrophoblast apoptosis, and consequently, inhibition of trophoblast gonadotropin secretion may represent key mechanisms by which anti-annexin V antibodies, once bound, can affect embryo implantation and pregnancy outcome.


   FOOTNOTES
 
First decision: 28 February 2001.

1 Correspondence: Alessandro Caruso, Department Obstetrics and Gynecology, Universita' Cattolica del S. Cuore, Largo Gemelli 8, 00168 Rome, Italy. FAX: 39 6 35510031; acaruso{at}katamail.com Back

Accepted: July 31, 2001.

Received: January 25, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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