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*
Hospital for Joint Diseases, New York University School of Medicine, New York, NY 10003;
Department of Obstetrics and Gynecology, Beth Israel Medical Center, New York, NY 10003; and
The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin and contractility
induced by 1.8 mM calcium. Incubation with 0.5 µM staurosporine or
0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic
morphologic and biochemical changes of apoptosis. The cellular topology
of Ro and La was evaluated with confocal microscopy and determined in
nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence.
In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly
nuclear, and propidium iodide (PI) stained the nucleus. In early
apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained
nucleus was observed, accompanied by Ro/La fluorescence around the cell
periphery. In later stages of apoptosis, nuclear Ro and La staining
became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained
blebs emerged from the cell membrane, a finding observed in
nonpermeabilized cells, supporting an Ab-Ag interaction at the cell
surface. In summary, induction of apoptosis in cultured cardiocytes
results in surface translocation of Ro/La and recognition by Abs.
Although apoptotic cells are programmed to die and do not
characteristically evoke inflammation, binding of maternal Abs and
subsequent influx of leukocytes could damage surrounding healthy fetal
cardiocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The candidate Ags and their cognate Abs have been characterized
extensively at the molecular level. Initial cloning of 60-kDa SSA/Ro
identified a zinc finger and an RNA-binding protein consensus motif
(10, 11), both of which could account for its direct interaction with
small cytoplasmic hY-RNAs (12). It has been suggested recently that
60-kDa SSA/Ro may function as part of a novel quality control or
discard pathway for 5S rRNA production in Xenopus oocytes
(13). Anti-SSB/La Abs recognize a 48-kDa polypeptide that does not
share antigenic determinants with either 52- or 60-kDa SSA/Ro (14, 15).
SSB/La directly binds a spectrum of RNAs and associates at least
transiently with 60-kDa SSA/Ro (16). It facilitates maturation of RNA
polymerase III transcripts (17), and has recently been shown to be
required for 3'-endonucleolytic cleavage that matures tRNA yeast
precursors (18). In addition to the well-characterized 60-kDa SSA/Ro
and 48-kDa SSB/La autoantigens, another target of the autoimmune
response in mothers whose children have CHB is the 52-kDa SSA/Ro
protein (19). The full-length protein, 52, has three distinct
domains: an N-terminal region rich in cysteine/histidine motifs
containing two distinct zinc fingers known as RING finger and B-box; a
central region containing two coiled coils with heptad periodicity, one
being a leucine zipper, both with potential for intermolecular
dimerization; and a C-terminal "rfp-like" domain (20, 21). We have
recently described an alternative 52 mRNA transcript derived from the
splicing of exon 4 encoding amino acids 168–245 inclusive of the
leucine zipper, which results in a smaller protein, 52ß, with a
predicted m.w. of 45 kDa (22). This isoform is maximally expressed in
the human fetal heart between 14 and 16 wk of gestation.
Despite new insights into pathogenic mechanisms of autoantibodies (23, 24, 25), an explanation of how maternal autoantibodies directly interact with intracellular Ags is not apparent, raising the possibility that they are clinical markers and not truly causal. However, in two studies of fatal CHB, culpable footprints were described: in one, maternal IgG bearing anti-SSB/La Ids was demonstrated on the surface of the fetal myocardial fibers (26); in another, anti-SSA/Ro Abs were eluted from the affected fetal heart (27). Apoptosis has been proposed as a means of presenting otherwise sequestered Ags to the immune system (28). Applicability of apoptosis to the pathogenesis of CHB is supported by several observations. It is a selective process of physiologic cell deletion in embryogenesis and normal tissue turnover, plays an important role in shaping morphologic and functional maturity (29, 30), and affects scattered single cells rather than tracts of contiguous cells (31). Therefore, Ab binding to apoptotic cells could trigger an inflammatory response that damages surrounding healthy tissue. Casciola-Rosen et al. have demonstrated the clustering of SSA/Ro and SSB/La in apoptotic blebs on the surface of apoptotic keratinocytes (28).
To investigate the hypothesis that apoptosis facilitates accessibility of SSA/Ro and SSB/La in the heart to circulating maternal autoantibodies, a reliable system for culturing human fetal cardiac myocytes was first established. Apoptosis was then readily induced with either staurosporine or DMNQ, and cellular topology of SSA/Ro and SSB/La was evaluated by indirect immunofluorescence.
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Materials and Methods |
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Human fetal hearts are aseptically obtained after elective termination of normal pregnancy by dilatation and evacuation. This is done in accordance with the guidelines of the Institutional Review Board and after obtaining consent from the mothers. No cardiac toxic drugs are administered to the mothers during these procedures. Gestational age is defined by sonographic measurement of biparietal diameter and femur length. The hearts, weighing 3 to 5 g each, are immediately dissected from the thoracic cavity with the great vessels intact and transported to our laboratory within 15 min on iced HBSS minus calcium and magnesium (Life Technologies, Gaithersburg, MD) containing 100 U/ml of heparin.
The aorta is cannulated for continuous perfusion of the coronary arteries with calcium-free Tyrode’s solution (117 mM NaCl, 5.7 mM KCl, 11 mM glucose, 4.4 mM NaHCO3, 1.5 mM KH2PO4, 1.7 mM MgCl2, and 20 mM HEPES, pH 7.4) containing 1 U/ml of Na-Heparin at 37°C, bubbled with 100% O2, as described for the Langendorff preparation (32). After 15 min of washing to clear the blood from the heart, fresh calcium-free Tyrode’s solution containing 1.5 mg/ml collagenase A (type III) is recirculated for approximately 20 min. The heart initially becomes completely pale and flaccid, and subsequently dissociates spontaneously, allowing cells to slowly drip and fall on a petri dish containing 0.25% trypsin, 1 mM EDTA in HBSS. At this point, clumps of cells are gently dissociated with forceps, and the resulting cell suspension is poured over a cell strainer, composed of 70-µm nylon mesh (Fischer, Pittsburgh, PA). Cells are centrifuged to remove the trypsin, and the pellet is resuspended in 20 ml of culture medium (DMEM supplemented with 10% FBS, 50 U/ml penicillin, 50 U/ml streptomycin, 100 mg/ml gentamicin, 1 mM nonessential amino acid (Life Technologies), 0.1 mM essential medium vitamins (Life Technologies), 2 mM glutamine, and 0.1 mM sodium pyruvate). To minimize fibroblast overgrowth of the cardiac cultures, preplating of the cells is done in culture medium for 20 min in 5% CO2 at 37°C. The nonadherent cells are then plated at approximately 3 x 106 cells/25-cm2 culture flask and grown in 5% CO2 at 37°C.
Cardiocyte contractility
To evaluate cell contractility and establish purity of the cardiocyte culture, medium containing 1.8 mM CaCl2 was added to the cultured cells on days 4 and 14. Flasks were maintained on a warmed microscope stage. Morphologic analysis was conducted using a Scientific Imaging Solutions workstation (BDS, Bethesda, MD) consisting of a Macintosh IIfx personal computer equipped with a Pixel Pipeline video acquisition board (Perceptics, Knoxville, TN), a video camera (CCD72; MTI, Fremont, CA), and TCL-image software (Oncre Imaging Systems, Rockville, MD). Backscattered electron images were acquired through the video camera directly from the screen of a high resolution image acquisition system, and projected onto a VCR-TV unit. Using the imaging workstation, the gray-scale images were adjusted for contrast and brightness, and cells beating in culture could be observed and recorded.
Immunocytochemistry
On day 4 of culture, cells were harvested and transferred to
Lab-Tek 4 chamber slides (7 x 105 cells/well). Twenty-four
hours later, cells were washed in PBS containing 0.1 mM
CaCl2 (PBS-C) for 5 min, fixed with 4% paraformaldehyde
for 20 min, and permeabilized with 100% acetone for 5 min at room
temperature. Cells were then washed for 5 min in PBS-C and incubated
with monoclonal anti--actinin (sarcomeric) mouse IgG1 (Sigma,
St. Louis, MO) at a dilution of 1/500 in PBS-C for 1 h. Monoclonal
anti--actinin is specific for -skeletal muscle actinin and
-cardiac muscle actinin. It stains Z lines and dots in stress fibers
of skeletal and cardiac muscle, but not in nonsarcomeric muscle
elements such as connective tissue, epithelium, nerves, or smooth
muscle. Cells were again washed for 5 min with PBS-C and incubated with
anti-mouse IgG (whole molecule) FITC conjugate at 1/500 dilution in
PBS-C for 30 min. After extensive washing for 5 min, the cells were
mounted in Vectashield (Vector Laboratories, Burlingame, CA).
Induction of apoptosis
On the third day after isolation, cardiac myocytes were harvested and transferred to eight-well chamber slides. Twenty-four hours later, various concentrations of staurosporine (Sigma) (0.5, 0.8, 1, 1.2 µM) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ, a gift from Dr. Gerald Cohen, Medical Research Council, Toxicology Unit, Leicester, U.K.) (0.03, 0.3, 3 mM) were added to the culture medium; subsequently, cells were incubated in 5% CO2 at 37°C. The morphology was assessed every hour by phase-contrast light microscopy and fluorescence microscopy (propidium iodide (PI) staining).
Assessment of apoptosis
The morphologic characteristics of apoptosis were assessed by phase-contrast light microscopy, trypan blue exclusion, and fluorescence microscopy (PI staining) (33). For electron microscopy, cells were processed as described (34, 35). DNA from whole cell populations was extracted, processed, and electrophoresed on 1.5% agarose gels (33). Cleavage of poly(ADP-ribose) polymerase (PARP) was detected in immunoblot of cell lysates (36) using a highly specific human antiserum (37).
Cellular topology of SSA/Ro and SSB/La in nonapoptotic and apoptotic cardiocytes
Human sera.
To evaluate the cellular localization of SSA/Ro and SSB/La, cells were
incubated with one of the following antisera at a dilution of 1/100 for
1 h in PBS-C: human autoimmune serum containing 48-kDa
anti-SSB/La Abs alone, as evaluated by ELISA, immunoblot, and
immunoprecipitation (Ze (38)); or anti-SSB/La Abs affinity purified
from serum (Lew); or 52- and 60-kDa SSA/Ro Abs alone (Ge (20) or Ohl);
or serum from a healthy multigravida with no known autoantibodies (Mo).
In experiments in which cells were double labeled with monoclonal
antisarcomeric -actinin and human sera, the second-stage Ab used to
detect SSA/Ro or SSB/La was goat anti-human IgG (whole molecule)
Texas Red conjugate (Accurate Chemical, Westbury, NY).
Immunofluorescence. Apoptotic and control cells were double stained with human antisera, as described above, and with PI (33), to study simultaneously the redistribution of the Ags and the morphologic changes in the nucleus. Staining was performed in living as well as in fixed/permeabilized cells.
Cardiocytes were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 20 min and permeabilized with 100% acetone for 5 min at room temperature before incubating with human serum (diluted 1/100 in PBS-C) for 1 h, followed by goat anti-human IgG (whole molecule) FITC conjugate (Sigma) for 30 min. PI (5 µg/ml) was then added for 5 min. Slides were mounted with Vectashield (Vector Laboratories). To study the surface expression of Ags on living cells, human antisera were added for 60 min at 37°C before fixation in 4% paraformaldehyde, followed by incubation with the fluorescein conjugate and PI.
Conventional immunofluorescence microscopy and photomicrography were conducted using an Axiophot microscope (Carl Zeiss, Thornwood, NY). A scanning laser confocal microscope system (model MultiProbe 20001; Molecular Dynamics, Sunnyvale, CA) was used to determine the cellular localization of the labeled Ags (39).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial attempts to culture human cardiocytes employed mechanical mincing to facilitate enzymatic dispersion of tissue (40); however, the final cell yield was insufficient and extremely variable. Accordingly, a novel approach was developed employing a Langendorff apparatus with constant flow (32, 41). Perfusion of fetal hearts (16–22 wk) with collagenase in Ca2+-free Tyrode’s solution at 37°C resulted in complete loosening of the intercellular matrix and intercellular adhesion forces, leading to spontaneous dissociation of cells. Initial inspection of the dissociated fetal heart cells by phase-contrast light microscopy revealed a homogeneous population of rounded cells. After 24 h in culture on standard tissue culture-grade plasticware, the cells could be observed to adhere, and by day 3 a clear monolayer of 75 to 85% confluent cells was apparent. This method consistently yielded approximately 45 to 60 x 106 cells per 3 to 5 g fetal heart.
The purity of cultures was evaluated by the induction of contractility. On day 4 of culture, CaCl2 (1.8 mM) was added to the medium, and the cardiac myocytes were placed on a warmed stage to maintain the temperature at 37°C. The cells were viewed in an enhanced computer-generated image (x250). In the majority of fields, more than 90% of cells were observed to contract in synchrony at rates of 25 to 75 beats per minute. However, the number of beating cells per field became progressively fewer over time, with only isolated groups of beating cells at day 14.
Staining with monoclonal antisarcomeric -actinin was done at day 4
of culture to further assess purity of the cells. At this time,
approximately 90% of the plated cells displayed the expected
striations characteristic of differentiated cardiac myocytes (Fig. 1
). The percentage of cells expressing
sarcomeric -actinin decreased over time, and was only 10% after 14
days in culture. Although dedifferentiated cardiac myoblasts do not
express contractile proteins and have the capacity to proliferate, the
cells were considered to be cardiac fibroblasts because of their
characteristic flat appearance and large nuclei (40).
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Various concentrations of staurosporine (Sigma) (0.5, 0.8, 1, 1.2 µM) or DMNQ (0.03, 0.3, 3 mM) were tested, and it was established that the optimal concentrations required to induce apoptosis were 0.5 µM staurosporine and 0.3 mM DMNQ.
By phase-contrast microscopy, fluorescence microscopy of PI-stained
cells, and electron microscopy, morphologic signs of early apoptosis
were observed in 40% of the cardiocytes after 3 and 4 h of
incubation with 0.5 µM staurosporine or 0.3 mM DMNQ, respectively;
after 7 h, 97% of the cells showed signs of advanced apoptosis.
Trypan blue exclusion indicated membrane integrity. Fluorescence
microscopy of nonapoptotic cardiocytes showed strong nuclear and
diffuse cytoplasmic staining with PI; the nucleus was large and ovoid,
with a diameter of 16 to 24 µm. The cytoplasm was between 75 and 110
µm in length. During the initial stages of apoptosis, chromatin
condensation could be appreciated, followed by shrinkage of the
nucleus, which decreased to 5 to 8 µm before fragmenting. The
cytoplasm also progressively decreased in size as blebs ranging from
1.3 to 5.2 µm emerged from the cell surface. Blebs tended to be
larger in the later stages of apoptosis. Apoptotic cardiocytes were
rounded and measured between 10 and 18 µm (Fig. 2).
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B, 5 h
after incubation with 0.5 µM staurosporine, shrinkage of the nucleus
and formation of condensed chromatin could be seen. In later stages of
apoptosis, the nucleus was highly condensed and the cytoplasm was
fragmented (Fig. 3C). Agarose gel electrophoresis of cell
lysates revealed the presence of a nucleosomal ladder in cells
harvested 8 h after the addition of 0.5 µM staurosporine.
Immunoblotting of cell lysates obtained from cultures incubated for
8 h with 0.5 µM staurosporine demonstrated cleavage of PARP
yielding the characteristic 85-kDa fragment (Fig. 4).
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Cultured human fetal cardiac myocytes were stained with human
antisera and counterstained with PI to assess the nuclear morphology of
nonapoptotic and apoptotic cells. In cultures from fetal hearts aged 16
to 22 wk, affinity-purified anti-SSB/La Ab and two sera (Ze (38)
and Lew), each containing 48-kDa SSB/La Abs, demonstrated a homogeneous
nuclear immunofluorescence pattern with marginally detectable green
staining of the cytoplasm when applied individually to separate
chambers of cardiocytes (Fig. 5A). Sera Ge (20) and Ohl,
each containing anti-SSA/Ro Abs (52 and 60 kDa), also stained the
nuclei, but demonstrated a stronger cytoplasmic signal than that
observed for SSB/La (Fig. 5D). A normal human serum (Mo) did
not stain nonapoptotic cardiocytes (Fig. 5G).
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, B and C).
Similar redistribution of Ags was observed in apoptotic cardiocytes
stained with anti-SSA/Ro (Fig. 5, E and F).
SSA/Ro and SSB/La generally colocalized with PI in the surface blebs;
however, this was not absolute, and occasional blebs appeared to be
stained with PI exclusively or only with anti-SSB/La or SSA/Ro. In
apoptotic cardiocytes stained with normal human serum, no
immunofluorescent signal could be appreciated (Fig. 5, H and
I). In several apoptotic cardiocytes, SSB/La was present in
the cytoplasm and surface blebs, but was only marginally detected in
the nucleus (Fig. 6). When cardiocytes
were incubated for 7 h with 0.3 mM DMNQ, SSA/Ro localized
predominantly in the periphery of the cells and in the apoptotic blebs
(Fig. 7).
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, A and D). In
contrast, both SSB/La and SSA/Ro were clearly present in the periphery
and surface blebs of apoptotic cardiocytes (Fig. 8, B and
C (SSB/La), E and F (SSA/Ro)). Normal
human serum did not stain nonpermeabilized apoptotic cardiocytes,
confirming that apoptosis does not render cells nonspecifically sticky
(Fig. 8, H and I). Optical horizontal sections of
nonpermeabilized apoptotic cardiocytes viewed by confocal microscopy (Z
series) confirmed that anti-SSA/Ro stains the surface blebs, but
not the nucleus or cytoplasm (Fig. 9).
Identical results were obtained for SSB/La (data not shown). The
finding that cardiocytes were not permeable to PI in the absence of
prior fixation, but were stained by antisera containing SSA/Ro and
SSB/La Abs under these identical conditions, further substantiated that
an interaction with the target Ags occurs at the cell surface.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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from scavenger macrophages than
that elicited by nonopsonized apoptotic cells (47, 48). Apoptosis in the context of fetal disease is of interest since it is a selective process of physiologic cell deletion in embryogenesis and normal tissue turnover, and plays an important role in shaping morphologic and functional maturity (29, 30). In the normal adult myocardium, apoptosis has been observed only rarely (49, 50). In contrast, apoptosis does occur during cardiac development, a point relevant to neonatal lupus since the maternal heart is unaffected despite identical circulating autoantibodies (3). In the 1970s, Pexeider extensively characterized the temporal and spatial distribution of cell death in the hearts of chicken, rat, and human embryos (51). Major foci of apoptosis included the AV cushions and their zones of fusion, the bulbar cushions and their zones of fusion, and the aortic and pulmonary valves. Albeit much of the cell death was noted in nonmyocytes, a region of myocyte death was apparent in the muscular interventricular septum as it grew toward the AV cushions in midgestation. More recently, Takeda and colleagues demonstrated apoptosis in midgestational rat hearts using terminal deoxynucleotidyl transferase dUTP nick end labeling (31). It has also been suggested that apoptosis contributes to the postnatal morphogenesis of the sinus node, AV node, and His bundle (52). Although initial clinical detection of bradyarrhythmia is most often between 18 and 24 wk of gestation (6, 7, 53), progression of incomplete blocks after birth has been observed in several cases (53, 54). Moreover, a significant degree of myocyte apoptosis has been noted to occur in the right ventricle and interventricular septum of rats in the immediate postnatal period, which may contribute to right ventricular remodeling during the transition from fetal to adult circulations (55). Intractable cardiomyopathy, despite pacing, has been noted in several infants postpartum (53, 56).
The establishment of a reproducible high yield method for culturing
human fetal cardiac myocytes was a requisite for the studies of
apoptosis described in this work. The availability of cultured
keratinocytes, the other most common target in neonatal lupus, has
facilitated the identification of conditions that induce expression of
SSA/Ro and SSB/La Ags and their translocation to the cell surface. We
have applied the Langendorff technique to isolate cardiac myocytes and
to subsequently maintain cells in culture. The presence of cardiac
myocytes was confirmed by two independent assays. Contractility, after
the addition of physiologic calcium and warming, assured that the cells
were functionally viable cardiac myocytes. Staining with a mAb
recognizing sarcomeric -actinin was used to track the percentage of
cardiac myocytes at different days in culture. Although the Langendorff
technique has been used to obtain mammalian cardiac myocytes for patch
clamping and recording of ion channels (25, 41), to our knowledge all
published methods for the culturing of human fetal heart cells describe
initial mechanical dispersion (minced pieces) of tissue, followed by
incubation with various proteases (40, 57, 58, 59, 60, 61). Little information is
provided on cell yields per heart for use in primary cultures.
Cannulation of the aorta with subsequent perfusion of the heart via the
coronary arteries facilitates greater accessibility of proteolytic
enzyme to the tissue, thereby allowing a high cell yield since the
majority of cells in a heart are dissociated and recovered. The
isolation method we describe provides a constant O2 supply
to all cells, which reduces the time of ischemia, thereby increasing
cardiac myocyte viability. This is particularly important with regard
to studies of apoptosis since ischemia can induce programmed cell death
(49).
The physiologic triggers of apoptosis in fetal tissues are unknown.
However, in general, all agents that induce apoptosis proceed through a
common final pathway leading to identical characteristic morphologic
and biochemical changes (28, 30). Available literature focuses on the
role of cardiac apoptosis in myocardial infarction, with only scant
data to provide guidelines for the study of apoptosis in the developing
fetal heart. Krown has recently demonstrated that physiologically
relevant levels of TNF- and the endogenous second messenger,
sphingosine, induce apoptosis in cultured adult rat ventricular
myocardial cells (62). Neonatal rat ventricular cardiocytes are also
driven into apoptosis when incubated with sphingosine. Curiously,
TNF- did not produce detectable apoptosis in the neonatal cells,
most likely due to the absence of detectable levels of TNFR1. The
authors conclude that both developing and terminally differentiated
cardiac myocytes are susceptible to apoptosis, although possibly to
different degrees and in response to different triggers. Other studies
have demonstrated the induction of apoptosis in primary cultures of
neonatal rat cardiocytes subjected to prolonged periods of hypoxia (63)
and deprivation of glucose and serum (64). Tanaka et al. have
demonstrated that hypoxia induces apoptosis with enhanced expression of
Fas Ag mRNA in cultured neonatal rat cardiocytes (63). Kajstura et al.
have reported that coronary artery occlusion of rat hearts resulted in
increased expression of Fas (55). The rationale for use of two distinct
approaches was that this work represented a first-time look at
apoptosis of human fetal cardiocytes in culture, and it was not known
whether susceptibility to apoptosis differed depending on the mode of
induction. For example, HL-60 cells exhibit extreme sensitivity to UV
irradiation, while K562 cells exhibit a high degree of resistance to UV
irradiation (65). Initial experiments revealed that the
serine/threonine protein kinase inhibitor, staurosporine, and the redox
cycling quinone, DMNQ, both known to cause apoptosis in various cell
types (66, 67, 68), were readily effective in cultured human fetal
cardiocytes. In preliminary experiments by our group, no Fas or Fas
ligand expression could be demonstrated by immunofluorescence on the
surface of cultured human fetal cardiac myocytes, although Fas mRNA has
been demonstrated previously in the murine heart (69).
Based on indirect immunofluorescence using human antisera, SSA/Ro and SSB/La Ags translocate from an intracellular location to the periphery and to blebs of apoptotic cultured human fetal cardiac myocytes. Our findings are comparable with those of Casciola-Rosen et al., who demonstrated that SSA/Ro and SSB/La ribonucleoproteins are clustered in surface blebs of keratinocytes that have undergone apoptosis in response to UV irradiation (28). These autoantigens are accessible to extracellular autoantibodies since specific human antisera stained the surface of nonfixed, nonpermeabilized apoptotic cardiocytes. No staining with sera containing anti-SSA/Ro or SSB/La was observed in control, nonapoptotic living cells. In permeabilized nonapoptotic human fetal cardiac myocytes, SSB/La is confined to the cell nucleus, while SSA/Ro is predominantly nuclear with minor cytoplasmic localization. The topology observed for SSA/Ro cannot be ascribed uniquely to 52- or 60-kDa SSA/Ro, given the human sera used for detection. The results for SSB/La are equivalent to those reported in other cell types (70, 71). The cellular localization of SSA/Ro has been discrepant in different studies, due in part to differences in fixation technique, human substrate, and the specificity of the Abs used for immunostaining. Most investigators have demonstrated predominantly nuclear and minor cytoplasmic localization for both the 52- and 60-kDa SSA/Ro Ags (19, 28, 72, 73, 74). One study found predominantly cytoplasmic localization of 60 and nuclear of 52 in HeLa and HEp-2 cells (75). In a recent study by Yell and colleagues using affinity-purified antisera, distinct differences were found between 52- and 60-kDa SSA/Ro, the former predominantly cytoplasmic and the latter nuclear (76). More recent work using mAbs reveals that both 52-kDa Ro and 60-kDa Ro are localized predominantly in the nucleus with some cytoplasmic staining, dependent on the cell type and fixation method (77, 78).
The findings reported herein apply to a mixed cardiocyte population, the majority of which are atrial and ventricular myocytes. Acknowledging that damage specific to the conducting system of the human fetal heart is characteristically associated with maternal anti-SSA/Ro-SSB/La Abs, it is anticipated that this novel culture system can be adapted for isolated SA and AV nodal cells. Several explanations, albeit each speculative, could account for the preferential vulnerability of the AV node. There may be a higher rate of apoptosis in this region, as suggested by the observations of Pexeider (51). The timing of transplacental passage of maternal autoantibodies may coincide with the period of maximal remodeling and apoptosis in the AV node. Phagocytosis of apoptotic cardiocytes by adjacent healthy cells or macrophages in the AV node could occur at a decreased rate relative to other areas of the heart. Perhaps there is a lower regenerative capacity of the AV node compared with the working myocardium. It has been described that fetal and neonatal, but not adult myocytes retain the capacity to divide, although there are no reports addressing this issue in conduction system or pacemaker cells.
Importantly, as more cases of autoimmune-associated CHB are reported and autopsies reviewed, it is clear that damage is not confined exclusively to the AV node. The working myocardium is also a target of injury. Mononuclear cell infiltration has been demonstrated in the myocardium of a fetus dying in utero at 18 wk of gestation (8), and patchy lymphoid aggregates were observed throughout the myocardium of an infant delivered at 30 wk and dying in the immediate postnatal period (79). Moreover, on postmortem analysis of neonates with CHB, immunofluorescence studies have demonstrated deposition of IgG and complement (including C1q, C4, C3, C6, and C9), not only in the conducting system (nodal tissue, bundle of His, and Purkinje’s fibers), but also in the working myocardium (79, 80, 81). Horsfall and colleagues showed deposition of anti-SSB/La Abs on the surface of myocardial fibers in fetal cardiac tissue of a stillborn (30 wk) with CHB, using polyclonal anti-idiotypic Abs (26). Extensive review of cases in the Research Registry for Neonatal Lupus (established in October 1994 and supported by funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases) confirms that myocardial dysfunction independent of bradycardia can be part of the spectrum of disease (53).
Although apoptosis could explain accessibility of intracellular Ags to
circulating autoantibodies, the reasons for the selective vulnerability
of specific fetal organs (heart, skin, liver), the low rate (1–2%) of
disease in offspring of mothers with anti-SSA/Ro-SSB/La Abs, and
discordance in monozygotic twins are not readily apparent. Undoubtedly,
other fetal organs undergo apoptosis, but the timing relative to
transplacental transport of maternal Abs and the degree have not been
extensively studied. Alternatively, there may be subclinical injury to
other organs, but repair occurs without sequelae. In utero injury
during the mid-second trimester could well be more frequent than
clinically apparent, but some fetuses might heal without permanent
consequence or suffer lesser degrees of scarring. Autoimmune-associated
CHB can be first, second, or third degree (53), and may or may not be
progressive after detection in utero or postnatally (53, 54).
Furthermore, individual differences in the immune mechanisms involved
in Ab-mediated inflammatory responses, such as Fc
R polymorphisms or
differences in the timing of NK cell, neutrophil, or macrophage
maturation, could account for this.
In summary, the evidence reported supports the hypothesis that apoptosis of human fetal cardiocytes may be a means by which the intracellular SSA/Ro and SSB/La target Ags become accessible to circulating maternal autoantibodies. This work was accomplished by the development of a novel method for culturing human fetal cardiac myocytes. Although apoptosis does not readily account for all of the clues observed at the bedside, it may be one fetal factor among several that contribute to the pathogenesis of autoimmune-associated CHB.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jill P. Buyon, Department of Rheumatology, Room 1608, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003.
3 Abbreviations used in this paper: CHB, congenital heart block; DMNQ, 2,3-dimethoxy-1,4-naphthoquinone; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide.
Received for publication March 23, 1998. Accepted for publication June 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced apoptosis in cardiocyte myocytes: involvement of the sphingolipid signalling cascade in cardiac cell death. J. Clin. Invest. 98:2854.[Medline]
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 Y. Qu, G. Baroudi, Y. Yue, and M. Boutjdir Novel Molecular Mechanism Involving {alpha}1D (Cav1.3) L-Type Calcium Channel in Autoimmune-Associated Sinus Bradycardia Circulation, June 14, 2005; 111(23): 3034 - 3041. [Abstract] [Full Text] [PDF]
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J P Buyon, A Rupel, and R M Clancy Neonatal lupus syndromes Lupus, September 1, 2004; 13(9): 705 - 712. [Abstract] [PDF]
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 K. Hu, Y. Qu, Y. Yue, and M. Boutjdir Functional Basis of Sinus Bradycardia in Congenital Heart Block Circ. Res., March 5, 2004; 94 (4): e32 - e38. [Abstract] [Full Text] [PDF]
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R. Caricchio, L. McPhie, and P. L. Cohen Ultraviolet B Radiation-Induced Cell Death: Critical Role of Ultraviolet Dose in Inflammation and Lupus Autoantigen Redistribution J. Immunol., December 1, 2003; 171(11): 5778 - 5786. [Abstract] [Full Text] [PDF]
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P. K. A. Mongini, A. E. Jackson, S. Tolani, R. J. Fattah, and J. K. Inman Role of Complement-Binding CD21/CD19/CD81 in Enhancing Human B Cell Protection from Fas-Mediated Apoptosis J. Immunol., November 15, 2003; 171(10): 5244 - 5254. [Abstract] [Full Text] [PDF]
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R. M. Clancy, C. B. Backer, X. Yin, R. P. Kapur, Y. Molad, and J. P. Buyon Cytokine Polymorphisms and Histologic Expression in Autopsy Studies: Contribution of TNF-{alpha} and TGF-{beta}1 to the Pathogenesis of Autoimmune-Associated Congenital Heart Block J. Immunol., September 15, 2003; 171(6): 3253 - 3261. [Abstract] [Full Text] [PDF]
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A Brucato, A Jonzon, D Friedman, L D Allan, G Vignati, M Gasparini, J I Stein, S Montella, M Michaelsson, and J Buyon Proposal for a new definition of congenital complete atrioventricular block Lupus, June 1, 2003; 12(6): 427 - 435. [Abstract] [PDF]
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 A. Shiratsuchi, T. Mori, Y. Takahashi, K. Sakai, and Y. Nakanishi A Presumed Human Nuclear Autoantigen That Translocates to Plasma Membrane Blebs during Apoptosis J. Biochem., February 1, 2003; 133(2): 211 - 218. [Abstract] [Full Text] [PDF]
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R. M. Clancy, A. D. Askanase, R. P. Kapur, E. Chiopelas, N. Azar, M. E. Miranda-Carus, and J. P. Buyon Transdifferentiation of Cardiac Fibroblasts, a Fetal Factor in Anti-SSA/Ro-SSB/La Antibody-Mediated Congenital Heart Block J. Immunol., August 15, 2002; 169(4): 2156 - 2163. [Abstract] [Full Text] [PDF]
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A D Askanase, D M Friedman, J Copel, M R Dische, A Dubin, T J Starc, M C Katholi, and J P Buyon Spectrum and progression of conduction abnormalities in infants born to mothers with anti-SSA/Ro-SSB/La antibodies Lupus, March 1, 2002; 11(3): 145 - 151. [Abstract] [PDF]
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 J. P. Buyon, D. Nugent, E. Mellins, and C. Sandborg Maternal Immunologic Diseases and Neonatal Disorders NeoReviews, January 1, 2002; 3(1): e3 - 10. [Full Text] [PDF]
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 G.-Q. XIAO, Y. QU, K. HU, and M. BOUTJDIR Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits FASEB J, July 1, 2001; 15(9): 1539 - 1545. [Abstract] [Full Text] [PDF]
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G.-Q. Xiao, K. Hu, and M. Boutjdir Direct Inhibition of Expressed Cardiac L- and T-Type Calcium Channels by IgG From Mothers Whose Children Have Congenital Heart Block Circulation, March 20, 2001; 103(11): 1599 - 1604. [Abstract] [Full Text] [PDF]
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J. S. Navratil, S. C. Watkins, J. J. Wisnieski, and J. M. Ahearn The Globular Heads of C1q Specifically Recognize Surface Blebs of Apoptotic Vascular Endothelial Cells J. Immunol., March 1, 2001; 166(5): 3231 - 3239. [Abstract] [Full Text] [PDF]
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M.-E. Miranda-Carus, A. D. Askanase, R. M. Clancy, F. Di Donato, T.-M. Chou, M. R. Libera, E. K. L. Chan, and J. P. Buyon Anti-SSA/Ro and Anti-SSB/La Autoantibodies Bind the Surface of Apoptotic Fetal Cardiocytes and Promote Secretion of TNF-{alpha} by Macrophages J. Immunol., November 1, 2000; 165(9): 5345 - 5351. [Abstract] [Full Text] [PDF]
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 Y. Xiao, J. He, R. D. Gilbert, and L. Zhang Cocaine Induces Apoptosis in Fetal Myocardial Cells through a Mitochondria-Dependent Pathway J. Pharmacol. Exp. Ther., January 1, 2000; 292(1): 8 - 14. [Abstract] [Full Text]
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 K. Ayukawa, S.'i. Taniguchi, J. Masumoto, S. Hashimoto, H. Sarvotham, A. Hara, T. Aoyama, and J. Sagara La Autoantigen Is Cleaved in the COOH Terminus and Loses the Nuclear Localization Signal during Apoptosis J. Biol. Chem., October 27, 2000; 275(44): 34465 - 34470. [Abstract] [Full Text] [PDF]
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