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(Circulation. 1998;97:900-906.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Differential Effects of Anti–ß₂-Glycoprotein I Antibodies on Endothelial Cells and on the Manifestations of Experimental Antiphospholipid Syndrome

Jacob George, MD; Miri Blank, PhD; Yair Levy, MD; Pierluigi Meroni, MD; Maya Damianovich, PhD; Angela Tincani, PhD; ; Yehuda Shoenfeld, MD

From the Research Unit of Autoimmune Diseases (J.G., M.B., Y.L., M.D., Y.S., S.C.), Department of Medicine B Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Israel; IRCCS Policlinico, Milan, Italy (P.M.); and Clinical Immunology Unit (A.T.), Spedali Civili, Brescia, Italy.

Correspondence to Yehuda Shoenfeld, MD, Department of Medicine B, Sheba Medical Center, Tel-Hashomer, 52621, Israel. E-mail shoenfel{at}post.tau.il

	Abstract

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Background—The antiphospholipid syndrome (APS) entails a prothrombotic state associated with the presence of anticardiolipin antibodies (aCL). aCL were shown to promote endothelial cell and platelet activation and to induce an APS-like syndrome in mice when administered intravenously. Recent data suggest that aCL target the plasma cofactor ß₂-glycoprotein I (ß2GPI) rather than negatively charged phospholipids. However, it has not been determined whether different epitope-specific anti-ß2GPI antibodies obtained from one patient possess pathogenic properties.

Methods and Results—Three ß2GPI-binding IgM monoclonal antibodies (mAbs) (ILA-1, ILA-3, and ILA-4) were cloned from a patient with APS. The three antibodies were shown to bind ß2GPI immobilized on irradiated plates, yet only ILA-1 bound ß2GPI coated onto nonirradiated plates. Furthermore, when using the anti-ß2GPI enzyme-linked immunosorbent assay, ILA-1 was the only mAb inhibited by fluid phase ß2GPI. ILA-1 and ILA-3, but not ILA-4, induced adherence of U937 cells to endothelial cells in vitro (reflecting activation of endothelial cells). mAbs ILA-1 and ILA-3 as opposed to ILA-4 induced significant expression of adhesion molecules when preincubated with human umbilical vein endothelial cells. Passive administration of ILA-1 and ILA-3 to pregnant BALB/c mice induced clinical findings consistent with APS (increased fetal resorptions, reduced platelet counts, and prolonged activated partial thromboplastin time), whereas both ILA-4 and the control human IgM did not produce similar effects.

Conclusions—The results of the study demonstrate the differential effects of various populations of anti-ß2GPI antibodies on endothelial cell activation and on experimental APS.

Key Words: endothelium • adhesion molecules • antibodies • immune system

	Introduction

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The APS is characterized by a combination of clinical findings consisting of thromboembolic events, thrombocytopenia, and recurrent fetal loss in association with aCL.¹ The syndrome can accompany additional disorders defining it as secondary or exist as an isolated primary entity.

The clinical manifestations of APS have been considered to result from the occurrence of antibodies against negatively charged PLs. However, various hypotheses have been raised as to the true target of these antibodies. It has been suggested that aCL target either negatively charged PLs, various plasma cofactors, or, alternatively, an epitope exposed on either these molecules (ie, PLs or plasma cofactors) on their mutual interaction.^{2 3 4 5} One of the conceivable targets of aCL appears to be a 50-kD plasma cofactor termed ß2GPI.⁶ This glycoprotein, initially described by Schulze et al in 1961,⁷ is composed of five respective consensus ("sushi") repeats.⁸ ß2GPI binds negatively charged PLs through a lysine-rich locus in the fifth domain^{9 10} and possesses several in vitro properties that define it as an anticoagulant (ie, inhibition of prothrombinase activity, ADP-induced platelet aggregation, or platelet factor IX production).⁸

Several recent studies suggest that ß2GPI alters its density¹¹ or exposes a cryptic epitope on its binding to PLs, which allows subsequent aCL binding.^{12 13} Emerging clinical studies^{14 15} reinforce these observations, thus confirming the specificity of "autoimmune" aCL to ß2GPI to the point that some consider them to be anti-ß2GPI antibodies.

The role of aCL in conferring the prothrombotic predisposition in APS has been strengthened by in vitro studies showing their platelet-^{16 17} and EC-^{18 19} activating properties. Additionally, it has been demonstrated that passive transfer²⁰ or active immunization²¹ with aCL of naive mice led to the clinical manifestations of APS. aCL have also been shown to possess thrombogenic properties in mice.^{22 23} However, it has not been established whether antibodies with different epitope specificities (ie, targeting different loci of the ß2GPI) are capable of promoting activation of ECs and induction of an APS-equivalent mouse model.

In the present study, we generated three IgM anti-ß2GPI mAbs from a patient with primary APS. We provide a description of their binding characteristics and show that despite their various epitope specificities, two mAbs had pathogenic properties manifested by in vitro activation of cultured ECs. Infusion of these mAbs to naive mice resulted in the clinical manifestations of APS.

	Methods

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Patient
The patient was a 32-year-old woman with primary APS, diagnosed after the occurrence of a previous episode of deep vein thrombosis and two abortions. The patient's serum was found to be positive for aCL (using the standard aCL ELISA) in high titers and for lupus anticoagulant.

Production of Human mAbs
Single-cell suspension of lymphocytes were derived from peripheral blood cells of a patient with primary APS. After a 5-day exposure to pokeweed mitogen (GIBCO Laboratory, Grand Island Biological Co), the lymphocytes were fused with the GM4672 human lymphoblastoid cell line according to a method described previously.²⁴ After fusion, the cells were seeded onto 96-well tissue culture plates (Nunc-Immunol), with hypoxanthine aminopterin thymidine selection media for 4 to 5 weeks. The clones were screened for binding to ß2GPI with the modified aCL ELISA. All clones were subjected four times to limiting-dilution cloning procedures in a regular medium. The hybridomas were grown in culture supernatant. The culture fluid was collected, and ammonium precipitation (50%) was performed. Dialyzed ammonium precipitate was loaded onto anti-HIgM Sepharose columns (Pharmacia), and the immunoglobulins were eluted using 5 mol/L MgCl₂ and dialyzed against PBS.

The total IgM and IgG from the patient was prepared by combining the IgG and IgM prepared by loading 1 mL of the patient serum onto Sepharose anti-HIgG and anti-HIgM columns.

Detection of aCL
ELISA plates were coated with cardiolipin (Sigma Chemical) at a concentration of 50 µg/mL in ethanol. Plates (Nunc) were blocked with 1% gelatin in PBS, and serial dilutions of the mAbs were added for 2 hours. Bound antibodies were detected using 1:10 000 dilution of goat anti-human IgM conjugated to alkaline phosphatase (Jackson ImmunoResearch) and the addition of its substrate p-nitrophenylphosphate. Absorbance was read at 405 nm in a Titertek ELISA reader (S.L.T. Laboratory Instruments).

Detection of mAb Reactivity by Modified aCL and Anti-ß2GPI ELISA
ß2GPI dependence of the binding of the mAbs to cardiolipin was assessed with a modified aCL ELISA. Cardiolipin coated onto ELISA plates were blocked with 1% gelatin in TBS and washed after 2 hours. Serial concentrations of ß2GPI (0 to 100 µg/mL) with 10 µg/mL purified mAbs were added. The binding was detected as described above (aCL ELISA).

To detect reactivity of the mAbs with human mouse and bovine ß2GPI, the CL-coated plates were blocked with different dilutions of human, mouse, or bovine sera (1% to 10% in TBS), followed by the addition of the mAbs (10 µg/mL), and the assay was performed as described above.

For the purpose of determining the binding to human ß2GPI alone, high binding (-irradiated; Nunc Maxisorp) or nonirradiated (Nunc Polysorp) plates were coated with purified human ß2GPI (10 µg/mL), the mAbs were added in different concentrations, and the ELISA was performed as described previously.¹⁴

Inhibition of Binding of Anti-ß2GPI mAbs by ß2GPI
The concentration was determined of mAbs giving 50% maximal binding to ß2GPI-coated plates. Different concentrations of ß2GPI were preincubated with each of the mAbs for 2 hours and placed on precoated high-binding ß2GPI-coated plates (Nunc) overnight at 4°C. The assay was continued as described in the anti-ß2GPI ELISA.

Detection of Anti–Endothelial Cell Antibodies
HUVECs were isolated as described previously and cultured under standard conditions.²⁵ Cells were used at passage 1 to 2 and plated onto gelatin-coated 96-well plates. Cyto-ELISA was performed as detailed previously.²⁶

Biotinylation of mAbs
The mAbs were dialyzed (100 µg/100 µL against 0.1 mol/L NaHCO₃) overnight at 4°C. NHS (biotinyamidocaproate n-hydroxy succinimide ester in 1 mL dimethylformamide; Sigma) biotin solution (2.5 µL/100 µL mAb) was added for 2 hours at room temperature and dialyzed against PBS.

Determination of ß2GPI Epitopes Recognized by Different mAbs
To clarify whether the mAbs recognize similar epitopes on ß2GPI, 50% of the binding of each of the mAbs onto ß2GPI-coated plates was determined. Subsequently, each mAb and total immunoglobulins from the APS patient sera were biotinylated in a set of separate experiments. The purpose of the first experiment was to assess whether all the mAbs accounted for the total anti-ß2GPI reactivity of the APS patient sera. Thus, a mixture of the nonbiotinylated mAbs (ILA-1 plus ILA-2 plus ILA-4; final concentration, 100 µg/mL) were used for competition with biotinylated total IgG. Subsequently, each of the nonbiotinylated mAbs (or control IgM) was used as competitors for binding in different concentrations (0 to 100 µg/mL) to the single biotinylated mAb (at concentration giving 50% maximal binding) in the anti-ß2GPI ELISA. The binding was probed by streptavidin alkaline phosphatase (Jackson) and appropriate substrate. The percentage of inhibition was calculated as follows: % inhibition=absorbance control-absorbance with inhibitor/absorbance controlx100.

Adherence of U937 Cells to Endothelial Cells in the Presence of mAbs
The assay was performed as described previously.²⁷ Briefly, U937 (a monocyte/macrophage–like cell line) cells were pretreated with mouse anti-human anti–MHC class I mAb (PharMingen) for 30 minutes at 37°C (to block Fc receptor binding without affecting the activation of the ECs) and labeled with 0.5 µCi/mL [³H]thymidine (Amersham International) for 24 hours. Adhesion assays were performed on HUVEC monolayers that were preincubated with ß2GPI, and mAbs were added overnight. The EC monolayers were washed extensively, and radiolabeled U937 cells were added to each well, in RPMI 1640 medium containing 0.2% BSA for 30 minutes at 37°C. The nonadherent cells were removed by washing, and the cells were lysed with formic acid. Radioactivity associated with adherence was quantified by ß-scintillation spectroscopy. The results were expressed as percentage of added U937 cells that adhered and are presented as mean±SD from three to five replicate wells.

ELISA for Detecting Expression of Adhesion Molecule
HUVECs were grown onto 96-well plates, preincubated with mAbs (100 µg/mL), washed and fixed with 0.1% glutaraldehyde, and treated with PBS containing 0.2% Triton X-100 to permeabilize the cell membrane.²⁸ Plates were blocked with 3% BSA and incubated with biotinylated mouse anti-human E-selectin, anti-human ICAM-1, or anti-human VCAM-1 (PharMingen) (1 µg/mL) for 1 hour. Cells were then exposed to streptavidin alkaline phosphatase (1:5000 dilution) and the appropriate substrate.

A separate set of experiments was designed to test the specificity of the effect of the mAbs on the expression of adhesion molecules. Accordingly, a cocktail of anti–ICAM-1, VCAM-1, and E-selectin mAbs (PharMingen) (20 µg/mL each) was added to the HUVECs after preincubation with each of the mAbs (25 µg/mL), and the assay was continued as described in the adhesion assays.

Induction of Murine Experimental APS
Mice were infused intravenously with 100 µg of each of the mAbs at day 0 (the day at which a vaginal plug was observed after mating) and day 7 of pregnancy.²⁰ The mice were bled and killed on day 15 of pregnancy. Fetal resorptions, aPTT, and platelet counts (markers of the APS-equivalent in mice) were determined as described previously.^{20 21}

Statistical Analysis
In the solid phase assays and competition and inhibition experiments, the Student's t test was used for comparison between the mAbs. The ANOVA test was used for comparison between the clinical parameters in the experimental groups.

	Results

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Binding of Human mAbs to ß2GPI and Cardiolipin
Three mAbs were generated (ILA-1, ILA-3, and ILA-4), all of which were of the IgM isotype. The three mAbs bound ß2GPI added to CL in a dose-dependent manner, yet no binding was evident to cardiolipin alone (Fig 1A). Binding to CL in the presence of ß2GPI (10 µg/mL) was as follows: ILA-3 absorbance, 1.302±0.104; ILA-1 absorbance, 1.008±0.098; ILA-4 absorbance, 1.005±0.118; and HIgM absorbance,0.145±0.113. Binding of mAbs to sample blanks (the assay devoid of CL) was negligible (mean absorbance: ILA-1, 0.091; ILA-3, 0.084; and ILA-4, 0.095).

View larger version (27K): [in this window] [in a new window]   Figure 1. Binding of the mAbs to ß2GPI/aCL using the modified aCL ELISA. Binding of the three mAbs (10 µg/mL each) was assayed on cardiolipin-coated plates. After blocking with 1% gelatin/TBS, concentrations are shown of human ß2GPI (A) and human (B), bovine (C), and mouse (D) sera.

Higher binding of the mAbs to CL was evident in the presence of increasing concentrations of human, mouse, and bovine serum (Fig 1B, 1C, and 1D, respectively).

Binding of all three mAbs to ß2GPI-coated high-binding plates was observed in a dose-dependent manner compared with control human IgM (Fig 2A). However, when the antibodies were assayed on nonirradiated plates, dose-dependent binding to ß2GPI was noted only with ILA-1 (Fig 2B).

View larger version (14K): [in this window] [in a new window]   Figure 2. Binding of the mAbs onto ß2GPI-coated plates. -irradiated (A) and nonirradiated (B) ELISA plates were coated with human ß2GPI (10 µg/mL), and different concentrations of mAbs or control human IgM were added after blocking with BSA. Binding was assayed as described for the anti-ß2GPI ELISA.

Inhibitory Effect of Fluid Phase ß2GPI on Binding of mAbs in Anti-ß2GPI ELISA
Dose-dependent inhibition of binding of ILA-1, but not of ILA-3, ILA-4, or IgM, by fluid phase human ß2GPI was evident in three separate experiment using high-binding plates (Fig 3). The K_d value of ILA-1 was estimated as -2x10^-7.

View larger version (16K): [in this window] [in a new window]   Figure 3. Inhibition of binding of anti-ß2GPI mAbs to ß2GPI. Each of the mAbs at a concentration giving 50% binding to ß2GPI was preincubated with different concentrations of fluid phase human ß2GPI (0 to 500 µg/mL). Binding was probed as described for the anti-ß2GPI ELISA.

Epitope Recognition by mAbs and Total Immunoglobulins for Binding to ß2GPI/CL
The mixture of the three mAbs obtained from the APS patient partially inhibited the binding of biotinylated total immunoglobulins from the APS patient through the use of the anti-ß2GPI ELISA (Fig 4).

View larger version (15K): [in this window] [in a new window]   Figure 4. Competition assays using the three mAbs and total immunoglobulins in the anti-ß2GPI ELISA. The total immunoglobulins (Ig) from the APS patient at a concentration giving 50% binding (5 µg/mL) were biotinylated (as described in "Methods"), and competition was performed using either the total patient Ig (IgG+IgM), a mixture of the mAbs, or an irrelevant control (IgG+IgM) using the anti-ß2GPI ELISA.

To assess whether the obtained mAbs were directed against different epitopes on ß2GPI, we biotinylated each mAb and performed competition assays with either of the three nonbiotinylated mAbs (ILA-1, ILA-3, and ILA-4). Binding of ILA-1 to ß2GPI was inhibited only by ILA-1, whereas no competition was achieved with ILA-3 and ILA-4 (ILA-1: 89±7% compared with 5±1% for ILA-3 and 7±1% for ILA-4, at ILA-1 concentrations of 30 µg/mL). Binding of biotinylated ILA-3 was inhibited only by nonbiotinylated ILA-3 and not by ILA-1 or ILA-4 (ILA-3: 79±6%, 8±2% for ILA-1 and 6±1% for ILA-4, at biotinylated ILA-3 concentrations of 30 µg/mL), and binding of biotinylated ILA-4 was inhibited only by nonbiotinylated ILA-4 in comparison to ILA-1 and ILA-3 (ILA-4: 90±11%, 9±4% for ILA-1 and 7±2% for ILA-3, at ILA-4 concentration of 30 µg/mL), thus suggesting that the three mAbs target different non-cross-reactive epitopes on the ß2GPI.

Anticoagulant Activity of mAbs
The anticoagulant activity of each of the anti-ß2GPI mAbs was measured by the aPTT as described previously^{20 21} and is shown in Fig 5.

View larger version (17K): [in this window] [in a new window]   Figure 5. Anticoagulant activity of anti-ß2GPI mAbs. aPTT was measured (in seconds) when mAbs (5 µg/mL) were added to a normal plasma using a mixing test with an equivalent dose of cephalin and incubation for 2 minutes at 37°C. Each point represent the mean±SD of three separate experiments.

Binding of mAbs to HUVECs by Cyto-ELISA
All three mAbs bound HUVECs through the use of a cyto-ELISA (Fig 6). No significant differences were noted between the mAbs with respect to the binding to HUVECs. No binding to HUVECs was evident when the ECs were incubated in a serum-free medium (devoid of ß2GPI) (Fig 6).

View larger version (26K): [in this window] [in a new window]   Figure 6. Binding of the mAbs to HUVECs. Cyto-ELISA using HUVECs was performed as described in "Methods." The binding to HUVECs was assessed in the absence and presence of ß2GPI (10 µg/mL).

Effect of mAbs on Adhesion of U937 Cells to HUVECs
The adhesion of monocytes to EC is considered a marker of EC activation. The percentage of adhesion expresses the portion of added U937 cells adhering the HUVECs (thus reflecting the percentage of EC coverage by the moncytic cells). The most significant adhesion of U937 cells to EC was accomplished by preincubation of the U937 cells with ILA-1 and then subjecting them to HUVECs. Adhesion to EC was dependent on the dose of the ILA-1 mAb achieving a maximal 79±6% (at ILA-1 concentration of 50 µg/mL) adhesion compared with control HIgM (3±2%) (P<.001) (Fig 7A). A similar, although less pronounced, dose-dependent effect on adhesion was evident by preincubation with ILA-3 (at a concentration of 50 µg/mL) reaching a 43±4% adhesion compared with control HIgM (3±2%) (P<.003). ILA-4 was not potent in affecting U937 adhesion, giving a negligible 13±2% compared with the control IgM inducing 3±2% adhesion (P<.5). Prior incubation with antibodies to ICAM-1, VCAM-1, and E-selectin using the same assays significantly decreased the effect of the ILA-3 but only partially that of ILA-1 on U937 adherence to ECs (Fig 7B).

View larger version (14K): [in this window] [in a new window]   Figure 7. Adhesion of U937 cells to HUVECs preincubated with different mAbs. Adhesion of U937 cells to HUVECs was assayed as described in "Methods" in the presence of increasing concentrations of ILA-1, ILA-3, ILA-4, and control human IgM (A). The adhesion assays were repeated after incubation of the mAb-treated HUVECs (25 µg/mL) with (black bars) or without (hatched bars) a cocktail of anti-human VCAM-1, ICAM-1, and E-selectin (20 µg/mL) (B). Each point represents the mean±SD of two experiments.

Preincubation with heat-aggregated IgG to block Fc receptor interactions (expressed by U937 cells) ruled out the possibility that the effects on adhesion resulted from nonspecific Fc interactions of the mAbs with the ECs or leukocytes.

Effect of mAbs on Expression of Adhesion Molecules by HUVECs
The most pronounced effect on E-selectin expression by HUVECs was evident after their preincubation with ILA-3 (absorbance, 1.674±0.112) (Fig 8). Preincubation of the mAb ILA-1 with HUVECs also resulted in significant E-selectin expression (absorbance, 1.232±0.212), whereas ILA-4 and human IgM did not produce marked effects on E-selectin expression (absorbance, 0.457±0.078 for ILA-4 and 0.112±0.058 for human IgM; Fig 8). Expression of ICAM-1 was most impressive with ILA-3 (absorbance, 1.365±0.01) compared with ILA-1 (absorbance, 0.856±0.075), ILA-4 (absorbance, 0.507±0.075), and control IgM (absorbance, 0.111±0.068).

View larger version (26K): [in this window] [in a new window]   Figure 8. Expression of adhesion molecules after exposure of HUVECs to mAbs. The expression of ICAM-1, VCAM-1, and E-Selectin was assayed after preincubation with 100 µg/mL of each of the mAbs. A, ILA-1; B-, ILA-3; C-, ILA-4; D-, control human IgM (all at a concentration of 25 µg/mL).

A similar pattern was noted with VCAM-1 expression, obtaining the most significant effect with ILA-3 (absorbance, 0.989±0.100) in comparison with ILA-1 (absorbance, 0.779±0.091), ILA-4 (0.377±0.068), and control IgM (0.092±0.012).

Induction of APS Manifestations in Naive Pregnant Mice by mAbs
Significant fetal loss was induced after passive intravenous administration of two of the three mAbs. The fetal resorption percentage was mostly pronounced with ILA-3 compared with ILA-1, ILA-4, and normal IgM (Table). Significantly reduced platelet counts were noted in the mice passively infused with ILA-1 and ILA-3 compared with ILA-4 and control human IgM (Table). ILA-1 and ILA-3 prolonged aPTT significantly compared with ILA-4 and human IgM.

View this table: [in this window] [in a new window]   Table 1. Clinical Parameters in Pregnant Mice Infused With Different mAbs

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we cloned three IgM mAbs that bound ß2GPI attached to solid phase coated CL. The antibodies were obtained from a single primary APS patient with well-documented thrombotic episodes. The competition assays using the different mAbs show that a mixture of the IgM (containing all three mAbs) was responsible for only a fraction of anti-ß2GPI activity in the serum of the patient. Thus, not all ß2GPI-reactive antibodies from the patient serum were recovered. The three mAbs were found to target different domains on human ß2GPI, evident by the competition assays. All mAbs bound ß2GPI immobilized on high binding plates, yet only ILA-1 bound ß2GPI coated onto nonirradiated plates. ILA-1 also was the only mAb inhibited by fluid phase human ß2GPI. Thus, it appears that ILA-1 is directed to an epitope present on the native structure of ß2GPI, whereas ILA-3 and ILA-4 recognize targets that are exposed on ß2GPI after conformational changes (exposed by its binding to -irradiated plates).

Two of the three mAbs (ILA-1 and ILA-3) induced activation of HUVECs manifested by enhanced adhesion of U937 cells, which was associated with an elevated expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin). It should be mentioned in this respect that the activating effect on the EC was not directly correlated with the binding to HUVECs as measured by the ELISA, thus excluding the possibility that mere binding of the mAbs to ECs is sufficient to elicit monocyte adhesion.

Several studies investigated the EC binding and activating properties of aCL.^{18 19 29} In a very recent work, Simantov et al¹⁹ used purified IgG aCL from patients with APS and demonstrated that these antibodies activated cultured HUVECs, as reflected by enhanced monocyte adherence to ECs and expression of adhesion molecules. The activation properties were found to depend on the presence of ß2GPI in the medium. However, it was not possible to conclude whether the EC-activating effect is contributed by a single monospecific population of antibodies or, alternatively, induced by different subgroups of aCL. More recently, Del-Papa et al¹⁸ showed that anti-ß2GPI antibodies from APS patients activated ECs provided ß2GPI was present in the medium (assayed by the expression of adhesion molecules, IL-6, and 6-keto-prostaglandin₁). However, the correlation of these interesting in vitro findings with the procoagulant state induced in animal models of APS has not been investigated. In this study, we used three mAbs produced from the same patient that had different binding targets on ß2GPI. Despite the differences in epitope binding, mAbs ILA-1 and ILA-3 were potent in promoting EC activation when added to a culture containing ß2GPI. The EC-activating properties of aCL were previously presumed to result from attachment of the antibodies to ß2GPI deposited on the EC surface after its preactivation and resultant exposure of negatively charged phosphatidylserine. This mechanism, however, remains largely speculative in view of the scarce evidence on loss of membrane asymmetry in EC. Regardless of the mechanism, our observations also show that all mAbs bound cardiolipin in the presence of mouse and bovine sera (presumably native ß2GPI), a finding that could support the role of endogenous ß2GPI-mediated activation of the EC in vivo.

Previously, we have shown that passive administration of monoclonal aCL as well as active immunization of naive BALB/c with these antibodies was capable of inducing experimental APS.^{20 21} The aCL-infused mice were found to develop thrombocytopenia, prolonged aPTT, and increased fetal resorptions after mating. These clinical parameters aided in establishing the primary pathogenicity of the antibodies. Moreover, antibodies produced by immunization with human ß2GPI were found to induce APS in naive BALB/c mice,³⁰ thus supporting an important role of aCL in the pathogenesis of the syndrome.

In the present study, we used these parameters to assess the pathogenic potential of the three mAbs to sort out whether it could be attributed to a monospecific antibody. Herein, we show that two of the three mAbs produced from the same patient harbored pathogenic in vivo characteristics evidenced by the induction of a syndrome resembling human APS. The correlation between the pathogenic effects in the passive transfer experiments and the in vitro adherence assays was not complete. These findings could be explained by the multiple effects of aCL on hemostasis, including activation of platelets,¹⁶ induction of tissue factor,³¹ and so on. Furthermore, the effect of the mAbs on the EC seems to be multifactorial and probably is not restricted merely to expression of the three adhesion molecules that were evaluated. This observation is supported by the partial inhibitory effect of prior incubation with antibodies to adhesion molecules on the ILA-1–mediated EC-activation compared with the near- complete inhibition of the ILA-3 effect.

The results of the present study show for the first time that different anti-ß2GPI antibodies possess both EC-activating properties and are capable of inducing a murine APS.

	Selected Abbreviations and Acronyms

 

aCL = anti-cardiolipin antibodies APS = antiphospholipid syndrome aPTT = activated partial thromboplastin time EC = endothelial cell ELISA = enzyme-linked immunosorbent assay ß2GPI = ß2-glycoprotein I HUVEC = human umbilical vein endothelial cell ICAM-1 = intracellular adhesion molecule 1 mAb = monoclonal antibody PL = phospholipid VCAM-1 = vascular cell adhesion molecule 1

	Acknowledgments

 
The work was supported by Israel-Japan Binational Ministry of Sciences Grant 6113 and Israeli Research Grant 6740. We thank Ludmila Rachlin for excellent technical assistance.

	Footnotes

 
Dr George and Dr Blank contributed equally to this work.

Received May 22, 1997; revision received October 2, 1997; accepted October 13, 1997.

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