Hydroxychloroquine reduces binding of antiphospholipid antibodies to syncytiotrophoblasts and restores annexin A5 expression

Abstract

Objective: Antibody-mediated disruption of the annexin A5 anticoagulant shield has been posited to be a thrombogenic mechanism in the antiphospholipid syndrome. We recently showed that the antimalarial drug, hydroxychloroquine, dissociates antiphospholipid immune complexes and restores annexin A5 binding to planar phospholipid bilayer. Using quantitative immunoassays, we demonstrated similar effects on BeWo trophoblasts. We therefore, investigated the effects of the drug on localization of annexin A5 in primary cultures of human placental syncytiotrophoblasts.

Study design: Laser confocal microscopy with computer-based morphometric analysis was used to localize annexin A5 and antiphospholipid antibodies on syncytiotrophoblasts exposed to polyclonal and monoclonal antiphospholipid and control immunoglobulin-Gs.

Results: Hydroxychloroquine reversed the effects of the antiphospholipid antibodies on the syncytiotrophoblasts by markedly reducing immunoglobulin-G binding and restoring annexin A5 expression.

Conclusion: These results provide the first morphologic evidence for this effect of hydroxychloroquine on human placental syncytiotrophoblasts and support the possibility of novel treatments that target antiphospholipid antibody binding.

Conflict of interest statement

DISCLOSURE: None of the authors have conflict of interest

Copyright © 2011 Mosby, Inc. All rights reserved.

Figures

Figure 1. Effects of HCQ on polyclonal aPL IgG binding and AnxA5 expression on cultured SCTs

Representative images of three-channel laser confocal microscopy show that in the absence of HCQ, cells treated with polyclonal control IgG showed A) very little bound-IgG (red fluorescence) and B) normal expression of AnxA5 (green fluorescence). Cells treated with polyclonal aPL IgG showed D) a large amount of bound-IgG (red fluorescence) and E) markedly reduced expression of AnxA5 (green fluorescence). Treatment with 1μg/ml of HCQ G) reduced the amount of bound-aPL IgG and H) increased the expression of AnxA5 on the cells. Addition of HCQ to the polyclonal control IgG-treated cells had no discernible effect (data not shown). C, F and I show merged images of green (FITC), red (rhodamine) and blue (DAPI) fluorescence marking AnxA5, IgG and nuclei, respectively. [bars = 50 μm. Confocal microscopy 3-dimensional projections of Z-axis image stacks, voxel size (μm): width 0.73, height 0.73, and depth 1.50]

Figure 2. Effects of HCQ on monoclonal aPL IgG binding and AnxA5 expression on cultured SCTs

Representative images of three-channel laser confocal microscopy are similar to the results with polyclonal IgGs shown in Figure 1. In the absence of HCQ, cells treated with control mAb showed A) very little binding of IgG (red fluorescence) and B) normal expression of AnxA5 (green fluorescence). Cells treated with aPL mAb showed D) extensive binding of IgG (red fluorescence) and E) markedly reduced expression of AnxA5 (green fluorescence). Treatment with 1 μg/mL HCQ G) reduced the amount of bound aPL-mAb on the cells and H) increased the expression of AnxA5. Addition of HCQ to the control mAb-treated cells had no discernible effect (data not shown). C, F and I show merged of green (FITC), red (rhodamine) and blue (DAPI) fluorescence marking AnxA5, IgG and nuclei, respectively. [bars = 50 μm. Confocal microscopy 3-dimensional projections of Z-axis image stacks, voxel size (μm): width 0.73, height 0.73, and depth 1.50]

Figure 3. Computer-based quantitative measurements of areas with positive immunofluorescence for AnxA5 and IgG antibodies on the SCTs

A) In the absence of HCQ, polyclonal aPL IgG significantly reduced the area of AnxA5 with positive immunofluorescence as compared to polyclonal control IgG. In the presence of HCQ, the drug reversed the aPL IgG-mediated reduction of AnxA5 and restored AnxA5 expression on the SCTs. B) HCQ reversed the binding of aPL IgG to the SCTs. In the absence of HCQ, the cells incubated with polyclonal aPL IgG had a significantly larger area of intense fluorescence for rhodamine-labeled IgG than the cells incubated with polyclonal control IgG. Incubation of the SCTs with aPL IgG together with HCQ markedly reduced the area containing aPL IgG to a level that were much close to the control IgG.

Figure 3. Computer-based quantitative measurements of areas with positive immunofluorescence for AnxA5 and IgG antibodies on the SCTs

A) In the absence of HCQ, polyclonal aPL IgG significantly reduced the area of AnxA5 with positive immunofluorescence as compared to polyclonal control IgG. In the presence of HCQ, the drug reversed the aPL IgG-mediated reduction of AnxA5 and restored AnxA5 expression on the SCTs. B) HCQ reversed the binding of aPL IgG to the SCTs. In the absence of HCQ, the cells incubated with polyclonal aPL IgG had a significantly larger area of intense fluorescence for rhodamine-labeled IgG than the cells incubated with polyclonal control IgG. Incubation of the SCTs with aPL IgG together with HCQ markedly reduced the area containing aPL IgG to a level that were much close to the control IgG.