DECREASED LEVELS OF FOLATE RECEPTOR-β AND REDUCED NUMBERS OF FETAL MACROPHAGES (HOFBAUER CELLS) IN PLACENTAS FROM PREGNANCIES WITH SEVERE PREECLAMPSIA (PE)^*

Patient groups and tissue collection

We studied placental samples from 50 women enrolled in the following groups: severe PE (n=10); intrauterine growth restriction (IUGR) without PE (n=7); PE+IUGR (n=7); sPTB in the absence of documented intra-amniotic inflammation (IAI) (n=11), and sPTB+IAI (n=9). A group of healthy term women with appropriately grown singleton fetuses undergoing cesarean delivery were also included as control (TC, n=6). Placentas were collected under protocols approved by the Human Investigation Committee of Yale University. Written informed consent was obtained from all participants, prior to enrollment. Gestational age was established based on menstrual date confirmed by sonographic examination prior to 20 weeks’ gestation. Characteristics of the study population are shown in Table 1.

Only patients with severe PE (not mild PE) were included in the study. This definition was based on the American College of Obstetricians and Gynecologists (ACOG) criteria. All patients met the diagnosis of PE (gestational proteinuric hypertension) after >20 weeks’ gestation, and had features of severe disease such as: (i) systolic blood pressure of ≥160 mm Hg and/or diastolic blood pressure of ≥110 mm Hg on 2 occasions at least 6 hours apart, (ii) proteinuria ≥5 g in a 24-hour urine specimen or ≥3+ on dipstick testing of 2 random urine samples collected at least 4 hours apart,²³ (iii) symptoms of cerebral or visual disturbances (headache, visual changes) or epigastric or right upper-quadrant pain, (iv) pulmonary edema or cyanosis, (v) oliguria (urinary output < 500 mL/24 h), (vi) elevated liver enzymes (≥ twice normal values), or (vii) thrombocytopenia (< 100,000 cells/μl). IUGR was defined as birth weight <10th percentile for gestational age at birth.

The sPTB group consisted of pregnancies with spontaneous preterm labor and/or preterm premature rupture of membranes (PPROM) without evidence of clinical chorioamnionitis (CAM). Clinical CAM was diagnosed by the presence of maternal fever (≥37.8°C), uterine tenderness, foul smelling amniotic fluid or visualization of pus at the time of the speculum examination, and maternal (>100 beats per minute) or fetal (>160 beats per minute) tachycardia.^{24, 25} All but one of the patients in the sPTB+IAI group had a clinically-indicated amniocentesis to rule out or confirm microbial invasion of the amniotic cavity. The remaining case presented with clinical CAM for which delivery was indicated. Histological examination of the placentas was performed by a certified perinatal pathologist, unaware of the clinical presentation or outcome. From each placenta, sections of chorionic plate, fetal membranes, and umbilical cord were examined for inflammation. Three histologic stages of CAM (stage I: intervillositis, stage II: chorionic inflammation, and stage III: full-thickness inflammation of both chorion and amnion) were assigned based on a histologic grading system.²⁶ The majority of patients in the preterm groups were administered antenatal corticosteroids in compliance with previous clinical recommendations.²⁷

Following delivery, placentas were brought immediately to the laboratory and the decidua basalis layer from a central cotyledon, noted to be free of macroscopic pathology (i.e. fibrin deposition or infarcts), was dissected and discarded. Approximately 1 g of underlying villous tissue was collected, frozen in liquid nitrogen, and maintained at −80°C for use in gene array, qRTPCR, and Western blot studies. In addition, full thickness placental tissue specimens from both PE (n=10) and sPTB groups (n=11) were fixed in formalin for immunohistochemical analysis as described below.

DNA microarray analysis

RNA was extracted from snap frozen placental samples from pregnancies with both PE and IUGR as well as sPTB (n=3 different placentas for each experimental group) using Trizol reagent (Sigma-Aldrich, St. Louis, MO). RNA was then purified using the RNeasy Mini Kit (Qiagen, Germantown, MD), and 20 μg of purified RNA was submitted to the Keck Facility at Yale University School of Medicine for analysis of gene expression using the full human genome chip (HG_U133 Plus 2.0, Affymetrix, Santa Clara, CA) containing approximately 54K transcripts. Gene expression was analyzed using GCOS 1.4 GeneSpring (Affymetrix) and GX 7.3.1 (Agilent, San Jose, CA) software. A 2-fold cut-off was used to establish an increase in gene expression as we have previously described.¹⁷

Quantitative reverse transcription PCR (qRTPCR)

Levels of mRNA were determined with an ABI 7500 RealTime PCR System (Applied Biosystems, Foster City, CA) using TaqMan Gene Expression Assays for FR-β (Cat# 00265255), CD163 (Cat# 01016661), CD68 (Cat# 00154355), CD206 (Cat# 00267207), CD209 (Cat# 01588349), Flt-1(Cat# 0152937), endoglin (Cat# 00164438), leptin (Cat# 00174977), and 18S RNA (Cat# 99999901). PCR reactions were performed in duplicate in a 20 μl volume of TaqMan Universal PCR Master Mix containing 1 μl of reverse transcription cDNA and 1 μl of assay primer-probe mix. Mean C_t values were analyzed using the 7500 System SDS software 1.3.1. Gene expression was normalized to the housekeeping gene 18S using the formula 2^−ΔΔCt. Results are expressed as relative expression compared to an endogenous control in each experiment as we have previously described.²⁸

Immunohistochemistry

Five μm placental villous tissue sections were deparaffinized at 58°C overnight, washed 3 times with xylene, and progressively rehydrated to 70% ethanol and PBS. Antigen retrieval was achieved by microwaving for 20 min in 10 mM sodium citrate buffer, pH 6.0. Sections were blocked with10% donkey serum and were incubated overnight at 4°C with sheep anti-FR-β (Cat# AF5697, R&D Systems, Minneapolis, MN), mouse anti-CD163 (Cat# MCA1853, AbD Serotec, Raleigh NC), and mouse anti-CD68 (Cat# M0814, Dako, Carpinteria, CA) primary antibodies, all at 1:100 dilution. Mouse IgG (Cat# X0931, Dako) and Sheep IgG (Cat# 013000003, Jackson ImmunoResearch, West Grove, PA) served as negative controls. The following day, endogenous peroxidase was quenched with 3% hydrogen peroxide in 50% methanol for 12 min, and after washing in PBS, biotinylated donkey anti-mouse (Cat# 715065151, Jackson ImmunoResearch) or anti-sheep (Cat# 713065003, Jackson ImmunoResearch) secondary antibodies were added for 30 min at a dilution of 1:1000. Staining was developed using the Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) and diaminobenzidine substrate. Slides were counterstained with hematoxylin, dehydrated to 100% ethanol and then xylene, and were mounted with CytoSeal 60. Visualization and photography was conducted with an IX71 inverted microscope (Olympus, Melville, NY). The number of cells positive for CD163, FR-β, and CD68 in serial sections were quantitated from pictures of 20 random, well-spaced 20× fields in 11 sPTB and 10 PE group placentas. Cells were counted from captured images using Image J software and results are presented as total number of positive cells per 20 fields. H-score analysis was carried out to analyze FR-β intensity semi-quantitatively in 5 sPTB and 6 PE group placentas using AxioVision and the corresponding digital image processing software (Carl Zeiss Microimaging, Inc, Thornwood, New York) according to the following scale: 0+ (no staining), 1+ (weak but detectable staining), 2+ (moderate staining), or 3+ (intense staining). For each slide, a computer-generated Histologic score (H-score) value was determined as previously described^{29, 30} by calculating the sum of the number of cells that stain at each intensity scale and multiplying that value by the weighted intensity scale using the following formula: H-score = Σπ (i+1), where “i” is the intensity scale and the “π” is the percentage of cells staining at the scale. For each slide, 5 fields and at least 100 cells per area were evaluated under a light microscope using 400× magnification. Results are reported as mean + standard error (SE) from a minimum of 5 separate readings from 3 separate tissue sections.

Western blotting

Protein was extracted from frozen placental tissue by homogenization in ProteoJet cell lysis buffer (Fermentas Life Sciences, Hanover, MD) supplemented with Compleat protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) using a ratio of 0.1 g tissue/1 ml of lysis buffer. Protein was quantitated by DC Protein Assay (BioRad Laboratories, Hercules, CA). Protein was run on a Tris-HCl 4–15% SDS PAGE gel (BioRad) then transferred to a nitrocellulose membrane. Following transfer, the membrane was incubated for 1 h at room temperature in Odyssey blocking buffer, and then overnight at 4°C with primary antibody in PBS containing 0.1%Tween (TPBS) with 5% powdered milk. Sheep anti-FR-β (see above) and goat anti-HSP90 (housekeeping protein, Cat# AF3286, R&D Systems) primary antibodies were all used at 1:2,000 dilution. IR-Dye800CW conjugated donkey anti-sheep (Cat# 613731168, Rockland, Gilbertsville, PA), and donkey anti-goat (Cat# 605731125, Rockland) secondary antibodies were all used at a 1:10,000 dilution. Target and housekeeping proteins were detected simultaneously on the same membrane. The membrane was washed in TPBS, and secondary antibodies were added for 45 min at room temperature. Following washing fluorophores were visualized and quantitated with an Odyssey Infrared Imager (LI-COR, Lincoln, NE) as we have previously described.³¹

Statistical analysis

Data sets were subjected to normality testing using the Kolmogorov-Smirnov method as previously carried out by our group.¹⁷ Demographic, clinical, and laboratory characteristics are reported as a mean ± SE. Most comparisons between groups were performed using one-way analysis of variance (ANOVA). Kruskal-Wallis ANOVA was carried out for data that were not normally distributed and are presented as a median with quartiles. Proportions were compared with Chi square tests. Differences in placental mRNA and protein expression between patient groups measured by qRTPCR and Western blotting were analyzed by Kruskal-Wallis ANOVA, Mann-Whitney rank sum, and Student’s t test. SigmaStat software was used for statistical analyses. A P < 0.05 was considered significant in all studies.

Decreased levels of macrophage-associated mRNA and protein in placentas from pregnancies with severe preterm PE

The characteristics of the six groups in the study population are shown in Table 1. Gestational age at delivery was significantly earlier in the severe PE (n=10), IUGR (n=7), PE+IUGR (n=7), and sPTB+IAI (n=9) groups compared to term control (TC, n=6, P <0.001), but not significantly different than that of the sPTB group (n=11). Initial studies using gene array analysis revealed that pregnancies complicated by preterm PE+IUGR were associated with a 2.6-fold reduction in levels of placental FR-β mRNA compared to the sPTB group (n=3 per group, P<0.05). Since FR-β is a major folate transporter in macrophages,²¹ we then used qRTPCR to compare expression of mRNA for several macrophage proteins in placentas from PE (n=10) and sPTB groups (n=11) (Fig. 1A). We observed that expression of FR-β and CD163 were significantly reduced 73% and 79% respectively in the PE group compared to the sPTB group. Levels of CD68 expression were reduced 59% in the PE group, but this effect did not reach statistical significance. In addition, mRNA levels of macrophage markers CD206 and CD209 were similarly down-regulated 1.8- and 4.3-fold in PE group placentas (not shown). These results indicate coordinate down-regulation of macrophage gene expression in PE placentas. Conversely, and consistent with prior publications,^{14, 32, 33} levels of endoglin, Flt-1, and leptin mRNA were up-regulated by 2.4-, 5.4-, and 310-fold, respectively in PE group placentas (Fig. 1B).

Of note, qRTPCR revealed that levels of FR-β mRNA was significantly down-regulated 85% in pregnancies complicated by both PE+IUGR (n=7) compared to sPTB (n=11), whereas levels of these placental mRNAs were not significantly different in IUGR (n=7), sPTB+IAI (n=9), and term (n=6) groups compared to the sPTB group (Fig. 2). These results indicated that macrophage gene expression was selectively down-regulated in pregnancies with severe PE. Western blotting revealed that FR-β protein levels were significantly down-regulated by 65% in the PE group (Fig. 3), but not in pregnancies complicated by IUGR or sPTB+IAI (Fig. 4).

Decreased numbers of HBCs in placental villi in pregnancies with PE

Immunohistochemistry localized CD163, FR-β, and CD68 to select cells within the villus core (i.e. the same location of HBCs in placenta) (Fig. 5, Panels A to H). Staining for CD68 staining was relatively low in the stroma, but present in syncytiotrophoblast and endothelial cells (Fig. 5, Panels I to L). Non-macrophage staining in the placenta has been noted by other groups using several different CD68 antibodies.^{34, 35} No staining was observed when isotype-matched control antibodies were substituted for primary antibodies (Fig. 5, Panels M and N). Quantitation of immunohistochemistry results as described revealed significant 28%, 34%, and 58% decreases in the numbers of FR-β⁺, CD163⁺, and CD68⁺ cells in the placental stroma of the PE group compared to the sPTB group (Fig. 6A). H-score analysis, which combines measurement of cell number and intensity of staining, revealed a significant 45% decrease in levels of FR-β⁺ cells in the villous stroma in PE group placentas (Fig. 6B). Taken together, these results indicate that severe PE is associated with a decrease in placental levels of macrophage marker mRNA and protein as well as a reduction in the number of HBCs.