β2-glycoprotein I and protection from anti-SSA/Ro60-associated cardiac manifestations of neonatal lupus

Abstract

One mechanism to molecularly explain the strong association of maternal anti-Ro60 Abs with cardiac disease in neonatal lupus (NL) is that these Abs initiate injury by binding to apoptotic cardiomyocytes in the fetal heart. Previous studies have demonstrated that β(2)-glycoprotein I (β(2)GPI) interacts with Ro60 on the surface of apoptotic Jurkat cells and prevents binding of anti-Ro60 IgG. Accordingly, the current study was initiated to test two complementary hypotheses, as follows: 1) competition between β(2)GPI and maternal anti-Ro60 Abs for binding apoptotic induced surface-translocated Ro60 occurs on human fetal cardiomyocytes; and 2) circulating levels of β(2)GPI influence injury in anti-Ro60-exposed fetuses. Initial flow cytometry experiments conducted on apoptotic human fetal cardiomyocytes demonstrated dose-dependent binding of β(2)GPI. In competitive inhibition experiments, β(2)GPI prevented opsonization of apoptotic cardiomyocytes by maternal anti-Ro60 IgG. ELISA was used to quantify β(2)GPI in umbilical cord blood from 97 neonates exposed to anti-Ro60 Abs, 53 with cardiac NL and 44 with no cardiac disease. β(2)GPI levels were significantly lower in neonates with cardiac NL. Plasmin-mediated cleavage of β(2)GPI prevented binding to Ro60 and promoted the formation of pathogenic anti-Ro60 IgG-apoptotic cardiomyocyte complexes. In aggregate these data suggest that intact β(2)GPI in the fetal circulation may be a novel cardioprotective factor in anti-Ro60-exposed pregnancies.

Figures

FIGURE 1

Purified native human β2GPI binds to apoptotic human fetal cardiomyocytes and inhibits binding of maternal anti-Ro60 IgG. (A) Apoptotic cardiomyocyte populations were gated based on Annexin V and propidium iodide (PI) staining. Annexin V-positive, PI-negative cells (Quadrant, Q4) were termed early apoptotic and Annexin V/PI-positive cells (Q2) were late apoptotic. (B) β2GPI bound both early and late populations in a dose-dependent and saturable manner. (C) Increasing concentrations of β2GPI inhibited the binding of anti-Ro60 IgG to both early and apoptotic cardiomyocyte populations. (D) The inhibition of anti-Ro60 IgG binding to apoptotic cardiomyocytes is specific for β2GPI as GAS6 and MFG-E8 do not alter antibody binding.

FIGURE 2

β2GPI levels are lower in anti-Ro60 exposed neonates with cardiac NL compared to non-cardiac NL. (A) β2GPI concentration, measured by ELISA, in umbilical cord blood from children affected by cardiac NL (n=53) and non-cardiac NL controls (n=44). Solid horizontal lines represent mean β2GPI concentration. Dashed lines connect β2GPI levels in umbilical cord blood from twins discordant for cardiac NL. (B) Immunoblot analysis of a panel of 9 umbilical cord blood samples (4 non-cardiac NL and 5 cardiac NL) probed with anti-β2GPI antibody were quantitatively consistent with those measured by ELISA.

FIGURE 3

The Ro60 binding site on β2GPI is within the plasmin cleavage site, Lys317-Thr318. (A) The β2GPI binding site on Ro60 was measured by ELISA using recombinant maltose binding protein (MBP) expressing various regions of Ro60. Ro60 fragments encompassing amino acids (aa) 82–244, aa 82–192, and aa 82–146 bound to purified native human β2GPI compared to MBP-Ro60 aa 149–244, aa 193–236, and MBP control. Values are the mean ± standard deviation (SD) of triplicate determinations (n=3). (B) Molecular surface models of Ro60 and β2GPI with the β2GPI fifth domain oriented towards a cleft in Ro60 formed by the mapped aa 82–146 region. Surface colours are calculated using a coulombic algorithm for electrostatic potential where blue is positive charge, white is neutral, and red is negatively charged. (C) The binding of β2GPI to apoptotic cardiomyocytes is abrogated by plasmin cleavage. (D) Plasmin reverses the β2GPI-mediated inhibition of anti-Ro60 binding to apoptotic cells. Representative flow cytometry dot plots depicting the binding of anti-Ro60 IgG (green) to late apoptotic cells (n=5).