Trophoblast expression of the minor histocompatibility antigen HA-1 is regulated by oxygen and is increased in placentas from preeclamptic women

Abstract

Introduction: Maternal T-cells reactive towards paternally inherited fetal minor histocompatibility antigens are expanded during pregnancy. Placental trophoblast cells express at least four fetal antigens, including human minor histocompatibility antigen 1 (HA-1). We investigated oxygen as a potential regulator of HA-1 and whether HA-1 expression is altered in preeclamptic placentas.

Methods: Expression and regulation of HA-1 mRNA and protein were examined by qRT-PCR and immunohistochemistry, using first, second, and third trimester placentas, first trimester placental explant cultures, and term purified cytotrophoblast cells. Low oxygen conditions were achieved by varying ambient oxygen, and were mimicked using cobalt chloride. HA-1 mRNA and protein expression levels were evaluated in preeclamptic and control placentas.

Results: HA-1 protein expression was higher in the syncytiotrophoblast of first trimester as compared to second trimester and term placentas (P<0.01). HA-1 mRNA was increased in cobalt chloride-treated placental explants and purified cytotrophoblast cells (P = 0.04 and P<0.01, respectively) and in purified cytotrophoblast cells cultured under 2% as compared to 8% and 21% oxygen (P<0.01). HA-1 mRNA expression in preeclamptic vs. control placentas was increased 3.3-fold (P = 0.015). HA-1 protein expression was increased in syncytial nuclear aggregates and the syncytiotrophoblast of preeclamptic vs. control placentas (P = 0.02 and 0.03, respectively).

Discussion: Placental HA-1 expression is regulated by oxygen and is increased in the syncytial nuclear aggregates and syncytiotrophoblast of preeclamptic as compared to control placentas. Increased HA-1 expression, combined with increased preeclamptic syncytiotrophoblast deportation, provides a novel potential mechanism for exposure of the maternal immune system to increased fetal antigenic load during preeclampsia.

Keywords: HA-1; Immune tolerance; Minor histocompatibility antigen; Oxygen; Placenta; Preeclampsia; Pregnancy; Trophoblast.

Figure 1. Expression of HA-1 in the human placenta across gestation

HA-1 expression in the syncytiotrophoblast (small arrows) and Hoffbauer cells (large arrows) of first trimester (n=5), second trimester (n=4) and term placental sections (n=5) was examined by immunohistochemistry and quantified using the H-score method. Expression of HA-1 in the syncytiotrophoblast was significantly higher in first trimester placentas as compared to second trimester (P<0.01) and third trimester placentas (P<0.01). There was no difference in HA-1 protein expression between the second and third trimester placentas (P=0.277). * = P<0.01, ns=no significance.

Figure 2. Cobalt chloride and low oxygen increase HA-1 mRNA expression in culture

First trimester explants (n=6) and purified term trophoblast cells (n=5) were cultured in the presence of cobalt chloride, a hypoxia mimetic. Purified term trophoblast cells from normal, C-section deliveries (n=6) were cultured in 2%, 8% or 21% oxygen for twenty-four hours. HA-1 mRNA expression, determined using qRT-PCR, was normalized to beta-actin and fold change relative to the untreated controls or 21% oxygen condition, as appropriate, was calculated using the ddCt method. HA-1 mRNA expression was elevated in the first trimester placental explants and purified term cytotrophoblast cells treated with 100μM cobalt chloride as compared to the untreated controls (P=0.04 and P<0.01, respectively). HA-1 mRNA expression was significantly higher in the 8% and 2% oxygen conditions as compared to 21% oxygen (P<0.01). HA-1 mRNA expression was also significantly higher in the 2% oxygen condition as compared to the 8% oxygen condition (P<0.01). *=P<0.01.

Figure 3. HA-1 expression is increased in preeclamptic placentas as compared to controls

Whole placental lysate and placental sections were obtained from preeclamptic and gestational-age and mode of delivery matched controls. (A) HA-1 mRNA expression was determined using qRT-PCR normalized to beta-actin. HA-1 mRNA expression (fold change) was significantly higher in the preeclamptic placentas as compared to the control placentas (n=9, P=0.015). (B–D) HA-1 protein expression in the syncytiotrophoblast and syncytial nuclear aggregates was significantly elevated in the preeclamptic placentas as compared to the controls (n=8, p=0.03 and p=0.02, respectively), whereas there was no difference in HA-1 expression in the Hofbauer cells of preeclamptic placentas as compared to controls (n=8, p=0.69). (E–G) HA-1 protein expression in the syncytiotrophoblast layer (black arrows), syncytial nuclear aggregates (red arrows) and Hofbauer cells of preeclamptic and control placentas was determined by a blinded reviewer using the H-score method. PE = Preeclampsia.