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.

Figure 1. Reactivity and binding activity of monoclonal antibodies ScFv 60.

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.

Figure 2. Human fetal cardiac cells remove autologous apoptotic cells.

Cardiac cells were incubated with or without autologous apoptotic cells (5 hours or 18 hours, 37°C). (A) Cells were fixed, permeabilized, and stained using the TUNEL-peroxidase assay. (B) Confocal analysis of a single cell double-labeled with TUNEL-FITC (green) and anti–α-sarcomeric actinin (red) to identify the phagocytosing cell as a cardiocyte.

Figure 3. Evidence for expression of PSR and CD32 in human fetal cardiac cells and tissues.

(A) Expression of PSR and CD32 (FcγRIIa, FcγRIIc) was assessed by RT-PCR in cDNA (prepared from mRNA) isolated from human fetal cardiac cells or from macrophages (left panels). In addition, cells were stained with a specific antibody (solid line) and isotype control (dashed line) for FACS analysis (right panels). (B) Shown are longitudinal sections of septal tissue from a 22-week CHB fetus and from a normal fetus electively terminated at 24 weeks, stained with anti-PSR, with anti-CD32, and with isotype control. For all immunostains, stain was visualized with alkaline phosphatase and counterstained with hematoxylin (magnification, ×40).

Figure 4. Binding of antibody from IgG fractions of CHB mothers to apoptotic cardiocytes inversely correlates with cardiocyte activity as measured in a functional assay that evaluates the clearance of apoptotic cells by neighboring cells in the presence of these IgG fractions.

Apoptotic cardiocytes were treated with healthy control IgG (nonopsonized) or with IgG from an anti-SSA/Ro– and -SSB/La–positive CHB mother (CHB-1; opsonized) as well as 6 additional IgG fractions (from CHB mothers). Each apoptotic cell preparation was subjected to FACS analysis to examine the percentage of human IgG binding. In addition, monolayer cardiocytes were incubated alone, with opsonized apoptotic cells, and with nonopsonized apoptotic cells. After 5 hours, cells were fixed and stained using TUNEL-peroxidase.

Figure 5. Use of annexin V to assess the persistence of opsonized and nonopsonized apoptotic cardiocytes after incubation with proliferating cardiocytes (5 hours).

Apoptotic cardiocytes were treated with normal control IgG (nonopsonized) or with IgG from an anti-SSA/Ro– and -SSB/La–positive CHB mother (CHB-1; opsonized). Monolayer proliferating cardiocytes were incubated alone, with opsonized apoptotic cells (CHB-1 IgG–apoptotic cells), and with nonopsonized apoptotic cells (normal IgG–apoptotic cells). After 5 hours, cells were scraped from the culture dish and stained using annexin V. M1 and M2 indicate gates of FACS analysis.

Figure 6. Reactivity of affinity-purified human anti-Ro60 with wild-type and Ro60-knockout murine cells and effect on apoptotic uptake.

Splenocytes and cardiocytes were isolated from wild-type and Ro60-knockout C57BL/6 mice, as described in Methods. Splenocytes and cardiocytes were rendered apoptotic by plating on pHEMA plus TNF-α (10 ng/ml, 18 hours, 37°C). FACS analyses of digitonin-permeabilized nonapoptotic splenocytes, after incubation with affinity-purified anti-Ro60 antibodies, are shown in A (upper left: wild-type; lower left: Ro60-knockout). Results of FACS analyses of similarly incubated apoptotic cells are also shown (upper right: wild-type; lower right: Ro60-knockout). (B) Uptake of apopotic Ro60-knockout cardiocytes by neighboring healthy wild-type cardiocytes in the absence (N/A) and presence of: IgG from healthy human donor; affinity-purified anti-La48 (AP48); affinity-purified anti-Ro52; and affinity-purified anti-Ro60.