Role of IgM and angiotensin II Type I receptor autoantibodies in local complement activation in placental ischemia-induced hypertension in the rat

Abstract

Preeclampsia is characterized by development of hypertension during pregnancy and reduced placental perfusion. Previous studies in a rat model of placental ischemia-induced hypertension demonstrated that inhibiting complement activation attenuated increased maternal blood pressure with C3a and C5a identified as the important products of complement activation. Given that in other forms of ischemia both natural IgM and antigen antibody complexes initiate complement activation, we hypothesized that placental ischemia exposes neoepitopes recognized by IgM to cause local complement activation and hypertension. Alternatively, we postulated that autoantibody to angiotensin II Type 1 receptor (AT1-AA) interacts with AT1 receptors to cause complement activation. Since complement activation occurs in kidney and placenta in preeclampsia, we used immunohistochemistry to determine IgM deposition and local complement activation in each organ (C3 deposition), and quantitative real-time polymerase chain reaction (qRT-PCR) to quantitate mRNA for endogenous regulators of complement activation CD55, CD59 and Complement receptor 1-related gene/protein y (Crry). On gestation day (GD)14.5, timed pregnant Sprague Dawley rats underwent Sham surgery or placement of clips on inferior abdominal aorta and ovarian arteries to create placental ischemia using the reduced utero-placental perfusion pressure (RUPP) model. As previously reported, RUPP surgery increased mean arterial pressure and circulating C3a on GD19.5. In placenta, IgM and C3 deposition increased, whereas mRNA for complement regulators Crry and CD59 decreased along with Crry protein in RUPP compared to Sham treated animals. In kidney, IgM deposition increased in animals subjected to RUPP vs Sham surgery without a significant change in C3 deposition and coincident with an increase in mRNA for CD55 and CD59. The AT1 receptor antagonist losartan prevents placental ischemia-induced hypertension as well as AT1-AA interaction with AT1 receptors. However, losartan did not attenuate complement activation as measured by circulating C3a or placental C3 deposition. Importantly, our studies indicate that following placental ischemia, complement activation is not due to AT1-AA but is associated with IgM deposition. These studies suggest a role for natural antibodies interacting with placental ischemia-induced neoepitopes to activate complement and contribute to hypertension.

Keywords: Autoantibody; C3 deposition; Complement; Natural antibody; Preeclampsia; Pregnancy-induced hypertension.

Figure 1

C3a but not sC5b-9 is significantly increased in RUPP compared to Sham animals. RUPP or Sham surgery was conducted on GD14.5 and outcomes measured on GD19.5. A. Mean arterial pressure (MAP); B. Average fetal weight; C. Circulating C3a in serum, units of C3a are relative to a standard pool of yeast activated rat serum as described in Materials and Methods; D. Circulating sC5b-9 expressed as mAu/ml in plasma as determined by ELISA. For Figures 1A–D, values represent the mean ± SE of determinations from 11–14 animals. *p<0.05 vs Sham. E. Representative near infrared images from 2 different Western blots using serum collected from 3 different RUPP and 2 different Sham animals applied to the gel at both a 1/20 and 1/40 dilution.

Figure 2

C3 deposition is significantly increased in placenta (A) but not kidney (B) in RUPP compared to Sham animals. Immunohistochemistry was conducted as described in Materials and Methods, and staining graded by a blinded observer from 0–3, negative to strongly positive. Representative images at 200X magnification are provided. Values for staining intensity represent the mean ± SE of scores obtained from placenta and kidney from 9–11 animals. *p<0.05 vs Sham

Figure 3

Complement regulators Crry, CD55 and CD59 change in placenta and kidney cortex in RUPP compared to Sham animals. A. mRNA was isolated from placenta and kidney cortex and Delta-delta Ct method of relative quantification was used to determine fold change in mRNA expression compared to actin with the change in Sham animals defined as 1. Values represent the mean ± SE of fold change obtained from 4–7 animals for placenta and 7–9 animals for kidney. *p<0.05 vs Sham. B. Immunohistochemistry was conducted as described in Materials and Methods, and staining graded by a blinded observer from 0–3, negative to strongly positive. Representative images at 200X magnification are provided. Values for staining intensity represent the mean ± SE of scores obtained from placenta from 4–5 animals. *p<0.05 vs Sham

Figure 4

IgM deposition is significantly increased in placenta (A) and kidney (B) in RUPP compared to Sham animals. Immunohistochemistry was conducted as described in Materials and Methods, and staining graded by a blinded observer from 0–3, negative to strongly positive. Representative images at 200X magnification are provided. Values for staining intensity represent the mean ± SE of scores obtained from placenta and kidney from 9–11 animals. *p<0.05 vs Sham

Figure 5

Losartan significantly inhibits placental ischemia induced increase in mean arterial pressure (MAP) without affecting increased circulating C3a or placental C3 deposition. Values represent the mean ± SE on gestation day 19.5. *p<0.05 for the indicated comparisons. A. The increase in MAP in RUPP Water (n=10) compared to Sham Water (n=7) was significantly inhibited by administration of 0.3 mg/ml losartan in drinking water (RUPP Losartan, n=9). MAP was also significantly inhibited comparing Sham Losartan (n=7) to Sham Water. B. The increase in serum C3a in RUPP water (n=10) compared to Sham water rats (n=7) was not inhibited by administration of 0.3 mg/ml losartan in the drinking water (RUPP losartan, n=9). Losartan did not affect C3a in Sham losartan rats (n=7) compared to Sham water. Units of C3a are relative to a standard pool of yeast activated rat serum as described in Materials and Methods. C. The increase in placental C3 in RUPP water (n=11) compared to Sham water rats (n=13) was not inhibited by administration of 0.3 mg/ml losartan in the drinking water (RUPP losartan, n=4). Losartan did not affect C3 deposition in Sham losartan rats (n=4) compared to Sham water. Representative images at 200X magnification are provided. Immunohistochemistry was conducted as described in Materials and Methods, and staining graded by a blinded observer from 0–3, negative to strongly positive.