Impaired clearance of apoptotic cardiocytes is linked to anti-SSA/Ro and -SSB/La antibodies in the pathogenesis of congenital heart block
Robert M. Clancy, … , Tom P. Gordon, Jill P. Buyon
Robert M. Clancy, … , Tom P. Gordon, Jill P. Buyon
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2413-2422. https://doi.org/10.1172/JCI27803.
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Research Article Cardiology

Impaired clearance of apoptotic cardiocytes is linked to anti-SSA/Ro and -SSB/La antibodies in the pathogenesis of congenital heart block

Abstract

The role of cardiocytes in physiologic removal of apoptotic cells and the subsequent effect of surface binding by anti-SSA/Ro and -SSB/La antibodies was addressed. Initial experiments evaluated induction of apoptosis by extrinsic and intrinsic pathways. Nuclear injury and the translocation of SSA/Ro and SSB/La antigens to the fetal cardiocyte plasma membrane were common downstream events of Fas and TNF receptor ligation, requiring caspase activation. As assessed by phase-contrast and confirmed by confocal microscopy, coculturing of healthy cardiocytes with cardiocytes rendered apoptotic via extrinsic pathways revealed a clearance mechanism that to our knowledge has not previously been described. Cultured fetal cardiocytes expressed phosphatidylserine receptors (PSRs), as did cardiac tissue from a fetus with congenital heart block (CHB) and an age-matched control. Phagocytic uptake was blocked by anti-PSR antibodies and was significantly inhibited following preincubation of apoptotic cardiocytes with chicken and murine anti-SSA/Ro and -SSB/La antibodies, with IgG from an anti-SSA/Ro– and -SSB/La–positive mother of a CHB child, but not with anti–HLA class I antibody. In a murine model, anti-Ro60 bound and inhibited uptake of apoptotic cardiocytes from wild-type but not Ro60-knockout mice. Our results suggest that resident cardiocytes participate in physiologic clearance of apoptotic cardiocytes but that clearance is inhibited by opsonization via maternal autoantibodies, resulting in accumulation of apoptotic cells, promoting inflammation and subsequent scarring.

Authors

Robert M. Clancy, Petra J. Neufing, Ping Zheng, Marguerita O’Mahony, Falk Nimmerjahn, Tom P. Gordon, Jill P. Buyon

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Figure 1

Reactivity and binding activity of monoclonal antibodies ScFv 60.

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Reactivity and binding activity of monoclonal antibodies ScFv 60. and 6...
and 60.4. Apoptotic and intact cardiocytes were single and double stained using monoclonal antibodies (ScFv) to evaluate accessibility of SSA/Ro60. Apoptotic cardiocytes were prepared by plating fetal human cardiocytes on pHEMA plus TNF-α (10 ng/ml, 18 hours, 37°C). Representative FACS data to evaluate the binding of different monoclonal antibodies to cell preparations are shown in A and B (see also Table 1). (A) Apoptotic cardiocytes as well as intact cardiocytes (normal culture conditions) and digitonin-permeabilized and nonpermeabilized fetal cardiocytes were single stained (isotype control [ISO] and 2 anti-Ro60 monoclonal antibodies, m60.1 and m60.4). (B) Intact and apoptotic cardiocytes were double stained using annexin V–FITC and a second-stage PE antibody reporting binding of isotype or anti-Ro60 monoclonal antibody (m60.1, m60.4). FL1 (x axis) and FL2 (y axis) indicate the binding of annexin and ScFv antibody, respectively. (C) Real-time recording of the interaction between SSA Ro60 and the monoclonal antibodies 60.1 (top) and 60.4 (bottom) as assessed by surface plasmon resonance imaging. In each tracing, a time interval was used to measure association (from 125 to 300 seconds) and dissociation (from 330 to 600 seconds) at each concentration of monoclonal antibody. The concentrations used were 31.3 nM, 62.7 nM, 125 nM, 250 nM, and 500 nM (tracings 1–5, respectively). Note that, for the association, saturation was achieved at the 250 nM and 500 nM concentrations. Resp. diff., response difference.