Reagents

The research protocol was approved by the institutional review board of Montefiore Medical Center, which granted permission for the use of excess plasmas from APS patients that had been obtained from clinical assays or plasmapheresate discards and were anonymized. Human polyclonal antibody immunoglobulin G (IgG) fractions were isolated from citrated plasma of a patient with severe APS and a normal control subject with a protein G column, as described by Sammaritano et al. The patient had severe primary APS, manifested by recurrent spontaneous pregnancy losses, deep vein thrombosis, pulmonary embolism, stroke, high titers of anticardiolipin (aCL) IgG (25.3-30.6 GPL) and antiphosphatidylserine IgG (78.0-92.5 GPS), and positive lupus anticoagulant tests by standard dilute Russell viper venom time assays performed with mixing and confirmatory steps. The preparation of aPL antibodies from the patient was compared with IgG isolated from control plasma.

The findings were validated with a previously characterized human aPL monoclonal antibody (mAb) IgG, designated IS4, that was generated from a cell line generously provided by Dr Pojen P. Chen (Department of Medicine, Division of Rheumatology, University of California at Los Angeles, Los Angeles, CA) from the peripheral blood mononuclear cells of a patient with APS and was purified by affinity columns, as previously described. The aPL mAb does not have lupus anticoagulant activity by dilute Russell viper venom time (dRVVT) or kaolin clotting time. A commercially available nonimmune human IgG derived from patients with monoclonal gammopathies (Sigma-Aldrich, St. Louis, MO) was used as a control. A stock solution of HCQ (gift from Dr Kirk Sperber of New York Medical College, New York, NY) was prepared with HEPES-buffered saline (HBS; 0.01 M HEPES, 0.14 M NaCl, pH 7.5) at 200 mg/mL and stored at 4°C.

Isolation and syncytialization of placental cytotrophoblasts

To obtain human SCTs, cytotrophoblasts were isolated from placentas from women undergoing elective cesarean sections at term, using the method described by Kliman et al with modification, that robustly yields syncytialized trophoblast. In this well-established model of trophoblast differentiation, syncytialization was confirmed based on morphologic assessment of cell fusion, as well as biochemical criteria, including the synthesis of progesterone, estradiol, hCG, and hPL. Briefly, placental villous tissue was dissected free of membranes, minced, and rinsed with calcium and magnesium free Dulbecco’s phosphate buffered salt solution (Mediatech, Inc, Manassas, VA), which were subjected to sequential enzymatic digestion in a solution containing 0.25% trypsin (Invitrogen, Carlsbad, CA), 0.2% DNase I (Roche, Indianapolis, IN), 25 mM HEPES, 2 mM CaCl2, and 0.8 mM MgSO4 in Hanks’ Balanced Salt Solution (Invitrogen). The first digestion was carried out for 15 minutes in 100 mL digestion solution, and the following 2 sequential digestions were carried out for 30 minutes in 150 mL digestion solution. Cells were pelleted from the second and third digestion by centrifugation at 1500 × g for 10 minutes. The cells were resuspended in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12 (DMEM/F12; Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Gemini Bioproducts, Sacramento, CA) and purified on a discontinuous gradient of Percoll (50%, 45%, 35%, and 30%) (GE Healthcare, Waukesha, WI) by centrifugation for 20 minutes without brake at 1000 × g. The cells that migrated to the 45% Percoll layer were recovered and immunopurified using mouse antihuman CD9 (R&D Systems, Minneapolis, MN), mouse antihuman CD45 (GeneTex, Irvine, CA) antibody, and goat antimouse IgG conjugated DynaBeads (Invitrogen). For immunopurification, the cells were incubated with anti-CD9 and anti-CD45 antibody at a ratio of 1 μg antibody per 10 ⁷ cells for 15 minutes at 4°C. The cells were then incubated with 50 μL antimouse IgG conjugated DynaBeads per 10 ⁷ cells for 30 minutes at 4°C, recentrifuged, and washed using DMEM/F12 containing 10% FBS. Dynabeads and the attached cells were removed by placing the cells under a magnetic force for 5 minutes. The supernatant containing immunopurified cytotrophoblasts was then plated in 4-well culture slides (BD Falcon, Franklin Lakes, NJ) at a concentration of 10 ⁶ cells per well in the DMEM/F12/FBS medium and maintained at 37°C in humidified atmosphere containing 5% carbon dioxide and 95% air. After 72 hours of culture, SCTs were obtained after spontaneous differentiation of cytotrophoblasts and were used for the studies described below.

Incubation with aPL antibodies and HCQ

To determine the effects of aPL antibodies on AnxA5 and whether HCQ might alter the effect, as previously described in other systems, aPL or control antibodies (polyclonal antibody at 0.2 mg/mL and mAb at 0.1 mg/mL), together with either HCQ (1 μg/mL in HBS) or buffer control (HBS) in the DMEM/F12/FBS medium, were added to the SCTSs and incubated in humidified atmosphere for 24 hours. HCQ was used at a concentration of 1 μg/mL, because that is in the therapeutic range of serum concentrations in patients who are administered the drug for systemic lupus erythematosus (SLE), and it was shown in previous studies not to be toxic to cultured cells. Syncytialized trophoblasts exposed to the same concentration of HCQ for 24 hours were assessed for function by measuring hCG levels in the culture media. Levels of hCG were measured using an Immulite 1000 analyzer (Siemens, Munich, Germany); this assay measures hCG using solid phase, 2-site chemiluminescent immunometric technology and has a reportable range from 1.1 to 5000 mIU/mL. The culture media of cells incubated for 24 hours in culture medium containing HCQ (1 μg/mL) had 1.2 mIU/μg cell protein, which was exactly the same concentration as cells incubated in control culture medium.

The cells were then washed with HBS containing 1.25 mM CaCl2, fixed with 5% formalin containing 1.25 mM CaCl ₂ for 4 minutes at room temperature, and washed 3 times with the calcium-containing HBS. To visualize the cell-bound AnxA5, the SCTs were incubated with rabbit antihuman AnxA5 (2 μg/mL) for 1 hour at room temperature, washed 3 times with the calcium-containing buffer, followed by incubation for 1 hour with fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (1:100 dilution in HBS-CaCl2 buffer) (Sigma-Aldrich). The cell-bound IgG was visualized by incubating the SCTs for 1 hour with rhodamine-conjugated goat antihuman IgG (1:100 dilution in HBS-CaCl2 buffer) (Sigma-Aldrich). For the experiments with polyclonal aPL and control IgG antibodies done with and without HCQ, 3 experiments were performed for each condition, with consistent results. For the confirmatory experiments with monoclonal IgGs, 1 experiment performed in duplicate for each condition and these showed consistent results. The immunostained SCTs were then mounted with medium containing DAPI (Vector Laboratories, Inc, Burlingame, CA). The slides were viewed in Analytical Imaging Facility, Albert Einstein College of Medicine. To confirm findings, the experiment described above was carried out 3 times using the SCTs that were isolated from 2 term placentas.

Laser confocal microscopy

The SCT cells, treated as described previously, were observed and images of random areas were taken using a Leica TCS SP2 AOBS confocal microscope (Mannheim, Germany) equipped with Argon lasers (set at 488 nm for excitation of FITC), diode lasers (set at 561 nm and 405 nm for excitations of rhodamine and DAPI, respectively), and objective lens HCX PL APO CS 40.0 × 1.25 OIL ultraviolet. To display 3-dimensional (3D) images, a series of images in the z -axis were taken at every 1.5 μm voxel. Line-by-line sequential scanning was used to eliminate crosstalk between channels. Conditions for imaging were set with the cells that produced the strongest fluorescent signals, and all of the settings were kept constant during the imaging sessions.

Quantitative analysis of the immunofluorescent distributions of anti-AnxA5 and antihuman IgGs was performed using the ImageJ software (available at: http://rsb.info.nih.gov.easyaccess1.lib.cuhk.edu.hk/ij/ ). Z-stack image was prepared from the z- axis slices, and z-projection images with average intensity were then created from the original Z-stacks. The areas covered by SCTs were contoured and the total cellular areas were determined, along with the areas that were positive for fluorescence in each image; positivity was defined objectively as having fluorescence intensity band of 155-255. The results were expressed as percentage of total cellular area with positive fluorescence. The data obtained from 6 culture wells for polyclonal antibody treatment and 2 wells for mAb treatment, without and with HCQ added, were then statistically analyzed using unpaired t test using GraphPAd InStat software ( www.graphpad.com ; GraphPad Software, San Diego CA).