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International Immunology Advance Access originally published online on June 18, 2007
International Immunology 2007 19(7):857-865; doi:10.1093/intimm/dxm052
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The efficacy of specific IVIG anti-idiotypic antibodies in antiphospholipid syndrome (APS): trophoblast invasiveness and APS animal model

Miri Blank1,2, Liat Anafi1, Gisele Zandman-Goddard3, Ilan Krause1, Shlomit Goldman4, Eliezer Shalev4, Ricard Cervera5, Josep Font5, Mati Fridkin6, Hans-Jurgen Thiesen7 and Yehuda Shoenfeld1,3,8

1 The Autoimmune Disease Center, Sheba Medical Center
2 Department of Human Microbiology, Sackler Faculty of Medicine, Tel-Aviv University, Israel
3 Department of Medicine B, Sheba Medical Center, Tel-Hashomer and Sackler faculty of Medicine, Tel-Aviv university, Israel
4 Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha’Emek Medical Center, Afula, Israel
5 Department of Autoimmune Diseases, Institute Clinic de Medicina i Dermatologia, Hospital Clinic, Barcelona, Catalonia, Spain
6 Department of Organic Chemistry, The Weizman Institute for Sciences, Rehovot, Israel
7 Department of Immunology, University of Rostock, Schillingallee 70, 18055 Rostock, Germany
8 Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases, Tel-Aviv University, Israel

Correspondence to: Y. Shoenfeld; E-mail: shoenfel{at}post.tau.ac.il


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Objectives: Administration of intravenous Ig (IVIG) is a recognized, safe and efficient mode of immunomodulatory therapy for many autoimmune diseases. Anti-idiotypic antibody binding to pathogenic autoantibodies and hence inhibition of binding to the corresponding antigen is one postulated mechanism of the beneficial effect of IVIG. The aim of this study was to fractionate the anti-beta-2-glycoprotein-I (ß2GPI) anti-idiotypic antibodies from a commercial IVIG preparation and use it as a treatment in an experimental antiphospholipid syndrome (APS) mouse model. Methods: Anti-ß2GPI polyclonal antibodies were purified on a ß2GPI column. The purified antibodies were bound to CN–Br-activated sepharose and employed for purification of IVIG-anti-anti-ß2GPI (anti-idiotypic antibodies), defined as specific intravenous Ig (sIVIG). The idiotype specificities were confirmed by competition assays. The effect of sIVIG in vitro was tested in a trophoblast and choriocarcinoma matrigel/invasion assay (i.e. proliferation and metalloproteinase (MMP)2/MMP9 expression) and in vivo in a fetal loss model of APS. Results: Anti-ß2GPI antibodies inhibited human trophoblast cell invasion in vitro. The function was attributed to the Fab portion of the anti-ß2GPI Igs, since ß2GPI-related synthetic peptides specific for the Fab part of the anti-ß2GPI antibodies neutralized its activity. APS sIVIG fraction reduce human trophoblast invasion in vitro by 560 times more than the whole IVIG compound and improved the MMP2 and MMP9 production by trophoblast cells. sIVIG improved significantly (200 times more) the pregnancy outcome in BALB/c mice passively infused with anti-ß2GPI antibodies, in comparison to treatment with IVIG (P < 0.02). Conclusions: Based on the current results, we propose that APS sIVIG may be considered as potential specific therapeutic safe compound for developing a treatment for APS patient's early fetal loss.

Keywords: antiphospholipid syndrome, autoantibodies, beta-2-glycoprotein-I, experimental model, IVIG


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The classical antiphospholipid syndrome (APS), ‘Hughes syndrome', is characterized by the presence of anti-phospholipid antibodies which bind negatively charged phospholipids, platelets, endothelial cells and neuronal cells via beta-2-glycoprotein-I (ß2GPI), associated with recurrent fetal loss, thromboembolic phenomena and thrombocytopenia (1–4). Recurrent fetal loss is one of the ‘Sapporo’ criteria for APS, encompass unexplained fetal death >10 weeks of pregnancy or three or more unexplained fetal loss prior to 10 weeks (5, 6). Currently, it is well established that anti-cardiolipin-ß2GPI-dependent antibodies are the main cause for APS-related fetal loss and thrombosis (7–11). Passive transfer of these antibodies into naive mice resulted in elevated percentage of fetal loss, impaired embryonic implantation and induction of other findings characteristic of APS (10–14).

For more than three decades, intravenous Ig (IVIG) preparations were reported to be beneficial in patients with a variety of autoimmune disorders (15–21), including a review on IVIG treatment in APS patients (16–18). The main mechanisms known so far to explain its broad activity comprise: (i) provision of anti-idiotypic antibodies and their function as immunomodulators; (ii) interference with the activation of complement and the cytokine network; (iii) modulation of the expression and function of Fc receptors and (iv) differentiation and effector functions of T and B cells, all summarized in (15, 18). Currently, it is well established that commercial IVIG preparations contain anti-idiotypic antibodies against variety of idiotypes such as anti-factor VIII, anti-DNA, anti-intrinsic factor, anti-thyroglobulin, anti-neutrophil cytoplasmic antibodies, anti-microsomal, anti-neuroblastoma, anti-phospholipid, anti-platelet, anti-Sm idiotype (4B4), anti-GM1 and anti-desmoglein-3 antibodies (15–23). In the past specific intravenous Ig (sIVIG) preparations, prepared for anti-Fas, were already studied (24, 25). Previously, we have fractionated IVIG specific for anti-DNA anti-idiotypic antibodies, employing a column composed of anti-dsDNA affinity purified from 55 lupus patients at active stage of the disease. This IVIG fraction showed specific activity for systemic lupus erythematosus patient's idiotypes in vitro and was 200 times more effective than the whole IVIG commercial compound in NZBxW.F1 mice (26).

In this study, we attempted to fractionate IVIG-specific anti-idiotypic antibodies (anti-anti-ß2GPI) from IVIG preparation (named sIVIG) and to study its biological functions in vitro and in an APS mouse model.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell cultures
JAR (HTB 144, American Type Tissue Collection) human choriocarcinoma cell line was established from trophoblast tumor of the placenta. Cells were grown in dodecyl maltoside-199 medium supplemented with 5% FCS and penicillin/streptomycin (Beit-Ha’Emek, Israel).

Human trophoblast cells were obtained from legal abortions (9–12 weeks of gestational age), with the approval of the local ethical committee (in compliance with the Helsinki Declaration) and the consent of the participating patients. Trophoblast cells were isolated as described previously in detail elsewhere (27, 28).

Synthetic peptides
The following ß2GPI-related peptides were used in the study: Peptide A: 58LKTPRV63(12), B: 208KDKATF213(11), C: 133TLRVYK13 (12), D: 274GDKVSFFCKNKEKKC288 (29) and E: 298KEHSSLAFWK317 (30). The D form of the synthetic peptides was used as control peptide. The concentration of each peptide in the used cocktail was 10 mg. The peptides were prepared by conventional solid-phase peptide synthesis, using an ABIMED AMS-422 automated solid-phase multiple peptide synthesizer (Langfeld, Germany). For purity determination, analytical reversed-phase HPLC was performed using a prepacked-100 RP-18 column (Merck, Darmstadt, Germany) (12).

Igs
IVIG—IVIG preparation named OMRIGAM was kindly provided by Omrix Pharmaceutical Ltd, Nes-Ziona, Israel. C-IgG—IgG from one healthy individual was loaded on anti-human-IgG sepharose column (Sigma), the bound IgG was eluted by using 5 M MgCl2 and dialyzed against PBS pH 7.4.

The sIVIG, IVIG, c-IgG or anti-ß2GPI antibodies were biotinylated as described before (12).

F(ab)2 fragments
IVIG was dialyzed against 100 mM Na-acetate buffer, pH 4.0 and digested with pepsin (2% w/w; Sigma chemical Co., St Louis MO, USA), at 37°C for 18 h. Any remaining traces of undigested IgG and Fc fragments were removed by binding to a protein-A column (Pharmacia Biotech, Norden AB Sollentuna, Sweden). The efficiency of the IVIG digestion was confirmed by running on 10% SDS-PAGE.

Fractionation of sIVIG from IVIG reparation
APS sIVIG, anti-anti-ß2GPI fraction of IVIG was prepared in three stages.

Stage I.
Preparation of ß2GPI.
Plasma from a healthy donor were incubated with 60% perchloric acid for protein sedimentation, neutralized with NaHCO3 and dialyzed against PBS. The dialyzed proteins were loaded on a Heparin column. The ß2GPI was eluted with Tris buffers pH 8 0.15 M, 0.35 M and 0.50 M. The fractions were further purified on protein-G column (Pharmacia). The protein concentration was defined by BCRTM protein assay kit (Pierce, Rockford, IL, USA) and tested for purity by western and immunoblot analyses. The purified ß2GPI was used for construction of ß2GPI column using activated CN–Br-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions.

Affinity purification of anti-ß2GPI antibodies.
Human anti-ß2GPI antibodies were affinity purified from 15 patients with APS at an active stage of disease (the blood was taken when the patients experienced episodes of pregnancy loss and/or thrombosis). The plasma was passed on ß2GPI column, elution with Glycin–HCl pH 2.5, neutralization with Tris and dialysis against PBS. The binding of anti-ß2GPI to ß2GPI was confirmed by ELISA and inhibition studies.

Stage II.
Construction of anti-ß2GPI column.
A mixture of affinity-purified anti-ß2GPI antibodies from 15 patients with APS were used since different APS patients’ sera entail diverse anti-ß2GPI anti-idiotypic antibodies. The mixture of anti-ß2GPI antibodies was dialyzed against coupling buffer (0.1 M NaHCO3 pH 8.3 containing 0.5 M NaCl) overnight at 4°C and covalently bound to CN–Br-activated Sepharose 4B. The remaining active NH groups were blocked by incubation with 0.2 M Glycine, pH 8.0 overnight at 4°C. Three cycles of washings with coupling buffer followed by 0.1 M acetate buffer pH 4.0 was used to remove the excess of unbound Ig. The washed column was equilibrated in Tris buffer pH 7.4.

Stage III.
Fractionation of IVIG anti-anti-ß2GPI anti-idiotypic antibodies—sIVIG.
Five hundreds milligrams of the commercial IVIG preparation (Omrix, Nes-Ziona, Israel) were dialyzed against loading buffer (0.05 M Tris containing 0.5 M NaCl, pH 8.0), diluted in 50 ml of loading buffer, filtered through the 0.45-µm filter (Minisart, Sartorius AG, Germany) and passed through the anti-ß2GPI column for 16 h at 4°C. Unbound material was washed out and the bound antibodies were eluted with 0.2 M Glycine–HCl, pH 2.5 and neutralized with 2 M Tris. The IgG containing fractions were pooled, dialyzed against PBS and tested for anti-idiotypic activity.

The anti-idiotypic binding sIVIG was analyzed by direct binding of biotinylated sIVIG or commercial IVIG to anti-ß2GPI bound to goat-anti-human-Fc-coated ELISA plates. The inhibition of anti-ß2GPI binding by sIVIG in comparison to IVIG was analyzed by inhibition assays. sIVIG, IVIG and IgG affinity purified from a single donor (c-IgG) were used as competitors and tested for their ability to inhibit the binding of biotinylated human anti-ß2GPI affinity purified from five patients to ß2GPI-coated ELISA plates.

Matrigel invasion assay
Matrigel invasion assay was performed as previously described in the literature (27, 28), briefly: Matrigel (100 mg ml–1) (BD Biosciences, Beit-Ha’Emek, Israel) in serum-free cell culture media was added to upper chamber of 24-well transwell plate (Corning) and incubated at 37°C 3–4 h for gelling. JAR cells were harvested from tissue culture flasks by Trypsin/EDTA, washed and resuspended in 5% FCS in M-199 medium and added to upper wells at a amount of 105 cells per well in 200 µl medium, while 500 µl medium was added to lower well. First trimester trophoblasts were cultured in upper wells at a density of 2 x 105 cells per well in 100 µl medium. The same density of cells, in the absence or presence of anti-ß2GPI 10 mg ml–1, was seeded in a well without transwell and counted at time of the invasion assay, as reference of total cells. IVIG, sIVIG or c-IgG at different concentrations were added to medium in upper and lower wells. Plates were incubated at 37°C for 48 h for JAR cells and 72 h for trophoblast cells. Then, the non-invaded cells on top of the transwell were scraped off with a cotton swab. The amount of invaded cells in the lower well as a percent of total seeded cells was evaluated with XTT Reagent kit (equivalent to MTT kit). The percent of invasion was calculated as: [absorbance of invaded cells/absorbance of seeded cells] x100 = invasion (%). Inhibition of invasion was calculated as percent of invasion in the presence of Ig from percent of control invasion without Ig.

Proliferation assay
Evaluation of cell proliferation was performed with XTT Reagent kit (XTT, cell proliferation kit, Beit-Ha’Emek, Israel, equivalent to MTT kit) according to the manufacture's protocol.

Zymography (substrate-gel electrophoresis)
In order to detect proteolytic activity of matrix metalloproteinases (MMP2, MMP9) in conditioned media (CM) collected after 48–72 h culture, substrate-gel electrophoresis (Zymography) on gels containing gelatin as the substrate were used as was previously described (31): CM were electrophoresed through a 10% polyacrylamide gel containing 0.5% gelatin (50 mg ml–1). Afterward gels were washed twice in 2.5% Triton X-100 for 30 min and incubated for 24 h at 37°C in 0.2 mol l–1 NaCl, 5 mmol l–1 CaCl2, 0.2% Brij 35 and 50 mmol l–1 Tris, pH 7.5. The buffer was decanted and the gels stained with Coomassie Blue followed by distaining. Identification of each gelatinase band was done in accordance to their molecular weight and commercial standards.

APS experimental model treatment with IVIG
Induction of fetal loss by passive transfer of anti-ß2GPI antibodies and treatment.
BALB/c mice female (10–12 weeks old) and male mice (12–14 weeks old) were purchased from Tel-Aviv University and were mated. After confirming vaginal plugs, the mice were infused with anti-ß2GPI (20 mg in 200 ml per mouse) through the tail vein on day 0 of pregnancy in order to induce fetal loss (9–11). On day 16 of pregnancy the mice were analyzed for fecundity, fetal resorptions and number of embryos.

The anti-ß2GPI-infused group was divided in to five subgroups. The first left untreated, the second was treated with high dose (HD) of commercial IVIG (IVIG 400 mg kg–1 12 mg per mouse), the third was treated with low dose (LD) of commercial IVIG (IVIG 2 mg kg–1 60 µg per mouse), the forth was treated with sIVIG (sIVIG 2 mg kg–1 60 µg per mouse) and the fifth was treated with control IgG (400 mg kg–1 12 mg per mouse) through the vein on day 1 from the vaginal plug formation. Each group contained 50 mice.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Fractionation and in vitro characterization of IVIG specific for APS (sIVIG) from a polyclonal preparation of IVIG
The anti-anti-ß2GPI IVIG (sIVIG) was fractionated from a commercial compound of IVIG on an anti-ß2GPI column. The anti-idiotypic activity of the sIVIG was tested by ELISA by introducing biotinylated sIVIG, commercial IVIG to anti-ß2GPI bound to anti-Fc-coated ELISA plates. sIVIG bound significantly (P < 0.001) to anti-ß2GPI antibodies affinity purified from five APS patients in comparison to binding to IgG from healthy donor, [optical density (OD) at 405 nm ranged between 0.845 and 1.242 in comparison to OD 0.142 to 0.118 at 405 nm for IgG from healthy donor]. No significant binding was detected for 5 mg ml commercial IVIG to anti-ß2GPI antibodies (OD 0.109 at 405 nm, in comparison to OD 0.084 binding to IgG from a healthy donor, P > 0.05). When we compared the binding of sIVIG at 5 mg ml–1 to the binding of the commercial IVIG, we could see that the efficacy of sIVIG was 100 times more efficient than the commercial IVIG in binding to anti-ß2GPI antibodies.

Furthermore, sIVIG was more effective in inhibiting the binding of affinity-purified anti-ß2GPI antibodies from one patient to ß2GPI, in comparison to the commercial IVIG, in a dose-dependent manner (Fig. 1a). Similarly, sIVIG significantly inhibited the binding of anti-ß2GPI affinity purified from five different APS patients, P < 0.001, (Fig. 1b). Moreover, sIVIG was significantly more efficient than the commercial IVIG in inhibiting the ß2GPI binding by the preparations of anti-ß2GPI antibodies from five patients, 76% inhibition with sIVIG in comparison to 22% inhibition at 50 mg ml–1 (Fig. 1b).


Figure 1
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  		Fig. 1. Inhibition of anti-ß2GPI antibodies binding to ß2GPI with sIVIG. (a) sIVIG, commercial IVIG and control IgG (c-IgG purified from a healthy donor) were added at different concentrations to anti-ß2GPI affinity purified from one patient with APS at 50% binding to ß2GPI. Following an overnight incubation, the mixture was added to ß2GPI-coated ELISA plates. Anti-ß2GPI binding was detected by anti-human IgG-alkaline-phosphatase and appropriate substrate. The binding was presented in OD at 405 nm and the percent of inhibition was calculated as described in Methods. Data are presented as percent of inhibition mean ± SD of three separate experiments. (b) sIVIG, commercial IVIG and control IgG purified from a healthy donor (c-IgG) were pre-incubated with anti-ß2GPI affinity purified from five representative APS patients separately. Following an overnight incubation, the mixture was added to ß2GPI-coated ELISA plates. The percent of inhibition was calculated as described in Methods. Data are presented as percent of inhibition mean ± SD of three separate experiments.

 
The effect of anti-anti-ß2GPI-sIVIG on reproductive failure in naive mice
Pregnancy impairment was induced in BALB/c mice by passive transfer of a mix of anti-ß2GPI antibodies, affinity purified from five patients with APS at day 0 of pregnancy and treated with sIVIG or commercial IVIG. Fetal loss and the fecundity were followed in all groups of mice. The anti-ß2GPI-infused mice showed reduced pregnancy outcome than the control BALB/c as shown in Table 1: 26.5% and 53.6%, respectively, (P < 0.02). Administration of sIVIG increased the fecundity to 46.43% in the anti-ß2GPI-infused mice, while commercial IVIG at low concentration as the sIVIG did not have any significant effect (P > 0.05). Similar to the effect of sIVIG on mouse fecundity, improvement in fetal loss was observed as well in anti-ß2GPI-infused mice following therapy with LD sIVIG (2 mg kg–1) and HD IVIG (400 mg kg–1), (P < 0.02). Therefore, we can conclude that sIVIG was 200 times more effective in repairing the fecundity of the BALB/c anti-b2GPI-infused mice. LD commercial IVIG did not have any significant effect on fetal loss, P > 0.05.


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  		Table 1. The effect of sIVIG on pregnancy outcome induced by anti-ß2GPI antibodies

 
The effect of sIVIG on in vitro model of implantation
Based on the low pregnancy outcome following passive transfer of anti-ß2GPI in BALB/c matted mice and the improved fecundity following treatment with sIVIG, the possible effect of IVIG on the trophoblast implantation process was assessed. The in vitro double-chamber invasion assay gave us a tool for studying a model of implantation. We used the JAR choriocarcinoma cells and confirmed the results using human 8–9 weeks old trophoblast cells.

Employing the matrigel system found that anti-ß2GPI inhibited significantly the in vitro choriocarcinoma cell invasion in a dose-dependent manner (Fig. 2). For example, 9.5 ± 0.4% of invasion was documented with 50 mg ml–1 of anti-ß2GPI in comparison to 37.3 ± 2.5% of invasion in the presence of IgG originated from a healthy individual at the same concentration (P < 0.03) 39% inhibition of invasion (Fig. 2). The biological function of the anti-ß2GPI antibodies is related to the ß2GPI-binding site of the Ig since a mixture of ß2GPI-related synthetic peptides, previously shown to neutralize anti-ß2GPI activity (11), prevented the anti-trophoblast invasive properties of the antibodies in a dose-dependent manner (Fig. 3). For example, 10 mg ml–1 ß2GPI-related synthetic peptides reduced the percent invasion of choriocarcinoma from 36.6 ± 4.1 to 14.2 ± 1.2%, equivalent to 38.8% inhibition of invasion (Fig. 3).


Figure 2
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  		Fig. 2. Inhibition of choriocarcinoma cell invasion by anti-ß2GPI antibodies. Anti-ß2GPI affinity purified from five patients with APS was administered to choriocarcinoma cell invasion model in a matrigel system at different concentrations. The percentage of invasion was calculated as described in Methods. Data are presented as mean ± SD of three separate experiments.

 

Figure 3
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  		Fig. 3. Prevention of anti-ß2GPI antibody anti-invasive properties by ß2GPI-related synthetic peptides, using human trophoblast cells. To prove which part of the anti-ß2GPI is responsible for the anti-invasive activity, we neutralized the antibody ß2GPI-binding site. The Fab portions of the anti-ß2GPI polyclonal affinity-purified antibodies were neutralized by pre-incubation of the antibodies with a cocktail of ß2GPI-related synthetic peptides at different concentrations (detailed in Methods). The D form of the synthetic peptides was used as controls. The ability of the anti-ß2GPI ± synthetic peptides to inhibit trophoblast invasiveness was studied. The percentage of invasion was calculated as described in Methods. Data are presented as mean ± SD of three separate experiments.

 
Addressing the effect of sIVIG on the anti-ß2GPI-induced inhibition of trophoblast cell invasion, we observed the following data as described in Fig. 4: pre-incubation of affinity-purified anti-ß2GPI 10 mg ml–1 with IVIG or F(ab)2 portion of IVIG (28 mg ml–1), improved the trophoblast cell invasion (P < 0.02). Fc fraction of IVIG did not affect the anti-invasive properties of anti-ß2GPI (P > 0.05). Neutralizing the anti-ß2GPI-binding site by ß2GPI-related synthetic peptides (directed to the Fab of the Ig) prevented significantly the IVIG effect on the anti-invasive activity of anti-ß2GPI antibodies (P < 0.01), (Fig. 4). Interestingly, when the trophoblastic cells were pre-incubated with the ß2GPI-related synthetic peptides alone, we could see a decrease in the anti-invasive properties (P < 0.001).


Figure 4
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  		Fig. 4. IVIG F(ab)2 and Fc fragments effect on in vitro trophoblast cell invasion. Trophoblast invasiveness was analyzed in the presence of IVIG or its F(ab)2 and Fc derivatives and anti-ß2GPI antibodies. The data are presented as mean ± SD of three different experiments.

 
The effect of sIVIG on trophoblast invasive properties is described in Fig. 5. Anti-ß2GPI antibodies inhibited significantly (P < 0.02) the invasion of human trophoblastic cells (Fig. 5a). Pre-incubation of anti-ß2GPI with sIVIG at 50 mg ml–1 or HD of commercial IVIG (28 mg ml–1) neutralized the anti-ß2GPI activity and improved significantly (P < 0.02) the invasive properties of the human trophoblastic cells. LD IVIG at equal concentration as sIVIG did not affect the anti-ß2GPI-mediated inhibitory effect (P > 0.05). HD IVIG by itself as well as sIVIG did not affect directly trophoblast invasion at the studied concentrations (P > 0.05).


Figure 5
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  		Fig. 5. (a)The effect of IVIG, sIVIG on in vitro human trophoblastic cells invasion. Inhibition of trophoblastic cell invasion in a matrigel invasion system was induced by anti-ß2GPI antibodies. sIVIG (10 mg ml–1) was compared with commercial IVIG LD (10 mg ml–1) and HD (28 mg ml–1) for neutralization of anti-ß2GPI effect.(b) The efficacy of sIVIG, IVIG on in vitro human trophoblastic cells invasion dose response. Dose-dependent effect of sIVIG and IVIG on the anti-ß2GPI-induced trophoblast anti-invasive properties. C-IgG, a control IgG from one individual had no effect on trophoblastic cell invasion, was used as a negative control, (upper dashed line). The anti-ß2GPI anti-invasive effect is shown in the lower dashed line. The data are presented as mean ± SD of three different experiments.

 
As described in Fig. 5(b), subjection of sIVIG or IVIG in concert with anti-ß2GPI to throphoblast invasion assay resulted in a dose-dependent inhibitory effect on anti-ß2GPI-mediated defective trophoblast invasiveness. sIVIG was 560 times more effective than native form of IVIG (P < 0.001) (Fig. 5b). LD of sIVIG (50 mg ml–1) had similar effect as HD IVIG (28 mg ml–1).

sIVIG effect on MMP2 and MMP9 secretion by trophoblast in the presence of anti-ß2GPI antibodies in a trophoblast invasiveness assay
Analysis of the culture fluid of all the trophoblast invasion assays for the presence of MMP2 and MMP9 revealed that anti-ß2GPI inhibited the secretion of MMP2 and MMP9 by human trophoblast cells as shown in a representative Fig. 6 (lane A: MMP secretion by trophoblast cells with no treatment, lane B: MMP secretion upon exposure of trophoblast cells to anti-ß2GPI). sIVIG and HD IVIG neutralized the anti-ß2GPI-induced inhibition of MMP2 and MMP9 secretion as shown in lanes C and D, respectively. LD of IVIG, equivalent to the sIVIG dose, had no effect on anti-ß2GPI-induced inhibitory effect on MMP2 and MMP9 secretion, lane E. HD of IgG from healthy individual (lane F), HD of IVIG lane G and LD IVIG lane H did not have any direct effect on the trophoblast MMP2 and MMP9 secretion.


Figure 6
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  		Fig. 6. The effect of sIVIg on MM2, MMP9 secretion by human trophoblastic cells. Gelatinase activity detected in the trophoblast invasion assay culture fluid. MMP2 and MMP9 activity: lane A: MMP secretion by trophoblast cells with no treatment, lane B: MMP secretion upon exposure trophoblast cells to anti-ß2GPI. Lanes C and D, respectively, sIVIG and HD IVIG. Lane E: LD of IVIG. Lane F, HD of IVIG. Lane G: sIVIG and lane H did not have any direct effect on the trophoblast MMP2 and MMP9 secretion.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results show for the first time the beneficial activity of fractionated specific IVIG, namely anti-anti- ß2GPI specific IVIG (sIVIG), for anti-phospholipid related reproductive failure. This fraction was affinity purified from an IVIG preparation, on a column composed of anti- ß2GPI antibodies affinity purified from 15 APS patients. Infusion of the polyclonal anti- ß2GPI antibodies into naive mice reduced the fecundity of the mice and enhanced the percentage of fetal loss. Administration of APS specific IVIG to anti-ß2GPI -induced-APS mice, was 200 times more efficient in repairing the fecundity and fetal loss than the original IVIG.

The proof that IVIG encompass anti-anti-cardiolipin-ß2GPI-dependent anti-idiotypic antibodies is based on the following studies: (i) IVIG, specifically the F(ab)2, ameliorate the Lupus anti coagulant (LAC) and anti-phospholipid antibodies activities (32, 33); (ii) IVIG reduced in vivo induction of APS experimental model by passive transfer of human anti-ß2GPI idiotype (33). Moreover, administration of monoclonal anti-anti-cardiolipin-ß2GPI-dependent anti-idotypic antibodies into experimental APS mice induced by anti-ß2GPI human mAb abrogated the generation of mouse anti-ß2GPI antibodies as well as fetal loss and other APS clinical features (34).

In order to analyze the mechanisms by which sIVIG is superior to the whole IVIG compound, we employed the in vitro trophoblast invasiveness assay resembling the in vivo implantation process and analyzed the mechanism by which the beneficial activity of sIVIG may regulate the implantation process. Previously, it was reported that anti-ß2GPI antibodies inhibit trophoblast invasiveness in vitro (35–37). Several mechanisms were proposed to explain the effect of anti-ß2GPI on trophoblast such as inhibition of gonadotropin secretion (35, 36) and reduction in the Bcl-2/Bax ratio, without any clear apoptotic effect on the level of protein and mRNA (37). We add in the current study also the ability of anti-ß2GPI to inhibit MM2/MM9 secretion by trophoblast cells, which are crucial for successful implantation, as an additional mechanism.

APS-specific sIVIG in an in vitro trophoblast invasion assay was significantly more competent in preventing trophoblast inhibition of invasiveness induced by anti-ß2GPI antibodies in vitro as defined by proliferation assays. We demonstrated herein that anti-ß2GPI antibodies affect the trophoblast function in vitro via its Fab portion of the molecule since ß2GPI-related synthetic peptides directed to the antigen-binding site of the antibody molecule neutralized its binding to trophooblast and abrogated trophoblast invasiveness. However, incubation of trophoblast cells with a cocktail of ß2GPI-related synthetic peptides inhibited trophoblast invasiveness. This fact could be attributed to the peptide 274GDKVSFFCKNKEKKC288 which has a dual function; on one hand it is a target molecule for subpopulation of anti-ß2GPI antibodies and on the other hand it is the phospholipid-binding site which binds the trophoblast (i.e rich in phosphatidylserine) (29, 38). Thus, the binding of this fifth domain peptide to the trophoblast may prevent the ß2GPI binding. As was demonstrated before in ß2GPI knockout mice lacking ß2GPI, the ß2GPI molecule is required for successful implantation and placental morphogenesis (39). The ability of ß2GPI-related peptides to neutralize the Fab of the anti-ß2GPI, thus reversing the anti-ß2GPI-mediated inhibition of trophoblast invasiveness, support the importance of anti-ß2GPI neutralization by IVIG for normal trophoblast invasion. Indeed, APS-specific IVIG—sIVIG had 560 times more beneficial effect on trophoblast invasiveness properties. Furthermore, we propose herein that one of the mechanisms affected by anti-ß2GPI is the down-regulation of the MMP2 and MMP9 secretion by trophoblast cells, which could be enhanced by sIVIG treatment or by HD IVIG. MMPs, a family of endopeptidases with the ability to degrade extracellular matrices (ECM) proteins, play a fundamental role in inflammation, tissue remodeling, angiogenesis, wound healing, tumor invasion and metastatic progression (31). Among the MMPs, MMP2 and MMP9 are key enzymes synthesized as latent proenzymes, which must be activated in order to show their proteolytic activities and degrade various components of the ECM including type IV, V, VII and X collagens, fibronectin and gelatin (40, 41). MMP2 and MMP9 have been extensively investigated because of their recognized roles in early pregnancy (31–37). It has been demonstrated that MMP2 and MMP9 play key roles in ECM degradation and trophoblast invasion during early pregnancy and are highly expressed during implantation and early stage of pregnancy (42, 43). We have shown herein for the first time that anti-ß2GPI affect trophoblastic invasiveness by inhibiting MMP9 and MMP2 secretion. HD IVIG and LD sIVIG were able to elevate the MMP2 and MMP9 secretion by trophoblast cells by neutralizing the anti-ß2GPI inhibitory activity on trophoblast MMP's secretion. IVIG and sIVIG had no direct effect on trophoblast MMP's secretion.

In the past, several potential mechanisms were proposed to explain these APS-related obstetric complications: (i) the requirement for complement activation in vivo for anti-phospholipase-induced fetal loss was documented (13, 44). Inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene/protein (Crry)-Ig, blocked fetal loss and growth retardation, a phenomenon currently shown also in complement C3-deficient mice (13); (ii) Anti-ß2GPI-mediated endothelial activation leading to induction of adhesion molecule expression including E-selectin, ICAM-I and vascular cell adhesion molecule-I, associated with elevated expression of NFkB, MyD88 and involvement of p38 MAP-kinase in the up-regulation of tissue factor on endothelial cells (12, 45–47); (iii) Anti-ß2GPI antibodies mediated inhibition of HCG secretion in an ex vivo placental model, as well as by in vitro trophoblast invasiveness model (34, 35) and (iv) Anti-ß2GPI inhibit placental differentiation (48). Anti-ß2GPI may impair placentation by increasing apoptosis and attenuating mitosis of trophoblast cells (49). IVIG is known to have immunomodulatory effect on apoptotic process. On one hand it may exert anti-apoptotic properties or pro-surviving function like in pemphigus vulgaris IgGs induced acantholysis, in which IVIG protect target cells from apoptosis by up-regulating endogenous caspase and calpain inhibitors (50). Alternatively, IVIG is known to cause anti-glycoprotein-IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia or may induce apoptotic processes like in human B cells (51, 52). Other group showed that Fc-gamma receptor-independent mechanism is involved in an inhibitory effect of IVIG on the thrombogenic effects of anti-phospholipid in ex vivo model of thrombosis and endothelial cells (53). The trophoblast Fc receptor is Fc-n (neonatal Fc-receptor) and is not involved in the current set of in vitro experiments since control IgG had no effect on the trophoblast function.

In summary, the results of this study support the idea that similar to what has been shown in other humoral-mediated autoimmune diseases, the beneficial effect of IVIG in APS—particularly in pregnancy loss—is due to the presence of specific anti-idiotypic antibodies. Here, we purified specific anti-idiotypic antibodies and demonstrated superior efficiency in a mouse model of APS in comparison to total IVIG. We propose that LD sIVIG and HD IVIG abrogate trophoblast invasion inhibition mediated by anti-ß2GPI antibodies, involving neutralization of the anti-ß2GPI activity and elevation of MMP9 and MMP2 secretion. Our study underscores the possibility to fractionate many specific anti-idiotypic Igs from the same batch of IVIG that may then be utilized in various autoimmune conditions.

We have to keep in mind that different preparation of IVIG may contain different amounts of anti-idiotypic antibodies due to the differences in the population that IVIG was derived from.


   Acknowledgements
 
This work was supported by AUTOROME European Community grant no. LSHM-CT-2004-005264.


   Abbreviations
 
APS, antiphospholipid syndrome
ß2GPI, beta-2-glycoprotein-I
CM, conditioned media
ECM, extracellular matrices
HD, high dose
IVIG, intravenous immunoglobulin
LD, low dose
MMP, metalloproteinase
OD, optical density
sIVIG, specific intravenous Ig

   Notes
 
Transmitting editor: I. Pecht

Received 27 September 2006, accepted 17 April 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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