Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness by binding directly and through adhered beta2-glycoprotein I

Abstract

Objective: To investigate the in vitro ability of antiphospholipid antibodies (aPL) to bind human trophoblast cells and to affect gonadotropin secretion and invasiveness.

Methods: Antiphospholipid antibody IgG from women with recurrent miscarriages, beta2-glycoprotein I (beta2GPI)-independent IgG aPL human monoclonal antibody (mAb) (519), and IgM anti-beta2GPI human mAb (TMIG2) were investigated for their binding to trophoblasts cultured for various amounts of time, their ability to affect invasiveness of Matrigel-coated filters, and their release of human chorionic gonadotropin (hCG).

Results: Polyclonal IgG aPL, as well as mAb 519 and TMIG2, bound to trophoblasts, the highest binding being found when cells displayed the greatest amount of syncytium formation. TM1G2 binding was found to be betaGPI dependent. Both polyclonal and monoclonal aPL, but not the controls, significantly reduced hCG release and Matrigel invasiveness.

Conclusion: These findings suggest that aPL recognition of both anionic PL and adhered beta2GPI on trophoblast cell structures might represent a potential pathogenetic mechanism for defective placentation in women with the antiphospholipid syndrome.

Figure 1

Percoll gradient-purified human cytotrophoblasts. Cells were stained after 24 hours (A), 48 hours (B), and 72 hours (C) of culture. (Hematoxylin and eosin stained; original magnification × 400.)

Figure 2

Immunohistochemical demonstration of human chorionic gonadotropin in Percoll gradient-purified human cytotrophoblasts at 72 hours of culture (original magnification × 600).

Figure 3

IgG binding to primary cytotrophoblast cells. Serial protein concentrations of IgG from patients with the antiphospholipid syndrome (APS) (▲,▼) or from normal human serum (NHS) (●, ▪) were evaluated after 24 hours (a), 48 hours (b), and 72 hours (c) of culture. Dose- and time-dependent binding was found with APS IgG, but not with NHS IgG. Values are the mean ± SD. * = P < 0.05; § = P < 0.01, APS IgG versus NHS IgG. O.D. = optical density.

Figure 4

Binding of human anti–β₂-glycoprotein I (anti-β₂GPI) monoclonal antibody (mAb) to primary cytotrophoblast cells. Serial protein concentrations of anti-β₂GPI mAb TM1G2 (▪) or control mAb TM1B9 (●) were evaluated after 24 hours (a), 48 hours (b), and 72 hours (c) of culture. Dose- and time-dependent binding was seen with TM1G2, but not with TM1B9. Values are the mean ± SD. * = P < 0.05; § = P < 0.01, TM1G2 versus TM1B9. O.D. = optical density.

Figure 5

Binding of human IgG anticardiolipin (aCL) mAb to primary cytotrophoblast cells in serum-free medium cultures. Serial protein concentrations of mAb 519 (▪) or control mAb 57 (●) were evaluated after 24 hours (a), 48 hours (b), and 72 hours (c) of culture. Dose- and time-dependent binding was seen with 519, but not with 57. Values are the mean ± SD. * = P < 0.05; § = P < 0.01, 519 versus 57. See Figure 4 for other definitions.

Figure 6

Binding of human IgG anticardiolipin mAb 519 (▪) or control mAb 57 (●), in the presence of serial concentrations of human β₂-glycoprotein I (Beta 2GPI) (a) or fetal calf serum (FCS) (b) to trophoblast cells cultured for 72 hours. Values are the mean ± SD. See Figure 4 for other definitions.

Figure 7

Matrigel invasion assay in the presence of a, IgG fractions from normal human serum (NHS) or from patients with the antiphospholipid syndrome (APS) at serial protein concentrations ( = 3 µg/ml; = 30 µg/ml; = 60 µg/ml; = 125 µg/ml), b, human anti–β₂-glycoprotein I (anti-β2GPI) monoclonal antibody (mAb) at serial protein concentrations ( = 3 µg/ml; = 12 µg/ml; = 50 µg/ml) (the effect of mAb TM1G2 was investigated in cultures performed in medium with and without fetal calf serum [FCS] or in serum-free medium with 5 µg/ml of β2GPI added), and c, human anticardiolipin mAb 519 and control mAb 57 at serial protein concentrations ( = 3 µg/ml; = 12 µg/ml; = 50 µg/ml). Values are the mean and SD of triplicate experiments. * = P < 0.05; § = P < 0.01, APS IgG versus NHS IgG, TM1G2 versus TM1B9, and 519 versus 57.