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* Department of Rheumatology, Hospital for Joint Diseases, New York University School of Medicine, New York, NY 10003; and
Department of Laboratories, Children’s Hospital and Regional Medical Center, University of Washington, Seattle, WA 98105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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V
3 integrin. Fetal cardiac fibroblasts
exposed to supernatants obtained from macrophages incubated with
opsonized apoptotic cardiocytes (but not nonopsonized) dramatically
increased expression of the myofibroblast marker -smooth muscle
actin (SMAc). The "opsonized" supernatant reversed an inhibitory
effect of the "nonopsonized" supernatant on proliferation of
fibroblasts (120 vs 69%, p < 0.05). Parallel
experiments examined the effects of two cytokines and their
neutralizing Abs on fibroblasts. TGF1 increased SMAc staining but
decreased proliferation. TNF- did not affect either readout.
Addition of anti-TGF1 Abs to the "opsonized" supernatant
blocked SMAc expression but increased proliferation, while
anti-TNF- blocking Abs had no effects. These data suggest that
transdifferentiation of cardiac fibroblasts to a scarring phenotype is
a pathologic process initiated by maternal Abs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Subsequent events initiated by the candidate maternal autoantibodies likely result in scarring, given that fibrosis of the atrioventricular (AV) node is the characteristic histopathologic lesion of CHB. Ho et al. (5) reported replacement of the AV node by fibrosis in seven hearts with CHB in association with maternal SSA/Ro Abs. The response of the fetal cardiac fibroblast may be a critical factor contributing to the ultimate expression of disease. Under physiologic conditions, healing of a wound is a well-orchestrated phenomenon involving a sequence of events in which the ability to lay down granulation tissue depends on a proliferative phase of "repair" cells and a remodeling phase to achieve a maturation of the connective tissue matrix. The transdifferentiation of a fibroblast to a myofibroblast is recognized as a pivotal component of granulation tissue formation (6). However, there is evidence that scarring results from disruption of the healing process due to the continued presence of myofibroblasts. For example, myofibroblasts persist in hypertrophic scars and in fibrotic lesions of many organs such as liver cirrhosis, lung fibrosis, kidney fibrosis (the mesangium of experimental glomerulonephritis), stromal reaction to epithelial tumors, and cardiac fibrosis (6).
Because the signature lesion of CHB is fibrosis of the AV node, the
present study was initiated to determine the relationship between early
Ab-mediated inflammatory events and the final sequelae leading to
fibrosis. It is hypothesized that CHB is a consequence of unresolved
scarring of the AV node secondary to the transdifferentiation of
cardiac fibroblasts to unchecked proliferating myofibroblasts, a
pathologic process initiated by maternal autoantibodies. To address
this hypothesis, we extensively defined the histopathology of a heart
obtained from a neonate who died of CHB (7). In
vitro studies were performed to recapitulate the cascade leading
to fibrosis. Separate cultures of myocytes and fibroblasts isolated
from human fetal hearts were established. The cardiac myocytes were
rendered apoptotic and subsequently treated with IgG fractions from a
healthy donor, a patient with systemic lupus erythematosus absent
anti-SSA/Ro-SSB/La Abs, or a mother with anti-SSA/Ro-SSB/La Abs whose
child had cutaneous neonatal lupus, or with Abs to components of SSA/Ro
affinity-purified from a mother whose child has CHB, and incubated with
macrophages isolated from the PBMC of healthy controls. The
supernatants generated under these varied conditions were tested for
their effects on the cardiac fibroblasts. To confirm activation of the
fibroblasts, evidence was sought for transdifferentiation to a
myofibroblast phenotype (assessed by expression of -smooth muscle
actin (SMAc)) and for proliferation (assessed by incorporation of
tritiated thymidine). Molecular characterization of the effects on
fibroblast phenotype was assessed by the direct addition of two
candidate cytokines, TNF- and TGF, as well as supernatants
(generated by the macrophages) that were preincubated with TNF-- and
TGF-neutralizing mAbs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed paraffin sections were obtained from hearts of an infant diagnosed in utero with CHB and dying at birth (7), a human fetus electively aborted at 24 gestational weeks, and a term newborn dying of noncardiac causes. Sections were immunostained with primary mAbs: anti-SMAc (used at 1/200; DAKO, Carpinteria, CA), which reacts with smooth muscle cells lining blood vessels and myofibroblasts, and anti-CD68 (1/3200) (Accurate Chemical and Scientific, Westbury, NY), which stains macrophages. Sections were visualized using the Vectastain avidin-biotin-peroxidase detection system (Vector Laboratories, Burlingame, CA), and counterstained with hematoxylin before photography.
Isolation and culture of cardiac myocytes and fibroblasts from human fetal tissue
Human fetal cardiocytes and human fetal fibroblasts were
cultured as described (8). Briefly, human fetal hearts of
gestational age 16–24 wk were aseptically obtained after elective
termination of normal pregnancy by dilatation and evacuation. This was
done in accordance with the guidelines of the Institutional Review
Board and after obtaining consent from the mothers. The aorta was
cannulated for continuous perfusion of the coronary arteries using a
Langendorff preparation (9). The heart was treated with
collagenase A (type III), which was recirculated for 
20 min. The
heart dissociated spontaneously, allowing cells to slowly drip and fall
on a Petri dish containing 0.25% trypsin, 1 mM EDTA in HBSS. Clumps of
cells were dissociated and the resulting suspension was poured over a
cell strainer. Cells were centrifuged and the pellet was
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, Rockville,
MD), 0.1 mM essential medium vitamins (Life Technologies), 2 mM
glutamine, 0.1 mM Na pyruvate).
The cell isolate contained both cardiac myocytes and fibroblasts.
Separate enriched cultures of each cell type were generated by an
initial adhesion step in which 1.2 x 107
cells were plated per 75-cm2 culture flask in
DMEM plus 20% FCS (20 min, 37°C). The nonadherent cells (cardiac
myocytes) were centrifuged and then plated at 1.2 x
107 cells per 75-cm2
culture flask and grown in 5% CO2 at 37°C.
After 4 days in culture, spontaneous contraction (30–40 beats per min)
was observed under phase-contrast microscopy. Greater than 75% of the
cells were stained by a murine monoclonal anti- actinin
(sarcomeric) Ab which 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 (3).
To obtain cardiac fibroblasts, the primary isolate was plated in flasks (20 min at 37°C). Fibroblasts at passages 3–5 were routinely used in these studies. A fibroblast enrichment in the cell culture was observed (fibroblasts are rapidly proliferating vs myocytes) which was >90%, as assessed using mAb clone IB10 (F-4771; Sigma-Aldrich, St. Louis, MO) which recognizes fibroblasts.
Affinity-purified Abs and serum IgG
Affinity-purified Abs were prepared as described (4). Briefly, affinity-purified Abs against 52 and 60 kDa SSA/Ro proteins were isolated from serum (of a mother whose child has CHB) by affinity column chromatography and the eluted Abs were tested for specificity by ELISA, immunoblot, and immunoprecipitation. Using a Protein A-IgG isolation kit (Pierce, Rockford, IL), human IgG was isolated from three sources: a healthy control; a patient with systemic lupus erythematosus whose serum contains antinuclear Abs but not anti-SSA/Ro-SSB/La Abs; and a mother whose serum contains Abs to all components of the SSA/Ro-SSB/La complex and whose child had cutaneous manifestations of neonatal lupus. Protein concentration of affinity-purified Abs and IgG fractions was assessed by a protein quantification kit (Pierce). Affinity-purified Abs and normal human IgG were endotoxin-free as assessed by the E-toxate (Limulus amebocyte lysate) assay (Sigma-Aldrich). Samples are routinely processed by application to Detoxi-Gel endotoxin removing gel (Pierce) to remove any contaminating LPS. These columns reduce LPS levels to below 1 pg/ml.
Induction of apoptosis
Cardiocytes were treated to induce apoptosis by plating on tissue culture dishes coated with poly (2-) hydroxyethylmethacrylate (polyHEMA) (10) for 18 h at 37°C. Cells were retrieved and apoptosis was assessed by TUNEL staining using a commercial kit as per the recommendation of the manufacturer (no. 1684817; Boehringer Mannheim, Indianapolis, IN).
Preparation of opsonized and nonopsonized apoptotic cardiocytes
Apoptotic fetal cardiocytes were incubated for 30 min with affinity-purified Abs reactive with 52 and 60 kDa SSA/Ro (1 µg/ml) or IgG (5 µg/ml) from the mother whose child had cutaneous neonatal lupus ("opsonized"), or with purified IgG (5 µg/ml) from a healthy control or from a patient with systemic lupus erythematosus absent anti-SSA/Ro-SSB/La Abs ("nonopsonized").
Isolation and culture of macrophages
Macrophages were derived from PBMCs of healthy donors or from white blood cell concentrate (Leukopak; New York Blood Center, New York, NY) as described (11). Mononuclear cells were isolated by centrifugation on Ficoll-Hypaque gradient. Cells were then suspended in 10 ml of RPMI 1640 + 10% human serum and incubated in Falcon T-75 tissue culture flasks for 1 h at 37°C. Nonadherent cells were removed by washing, leaving an adherent cell population which was retrieved and cultured in Teflon beakers (RPMI 1640 + 10% human serum, 7 days). The purified cell population consisted of >90% monocyte-derived macrophages as measured by phagocytosis of IgG-coated sheep RBC (data not shown).
Macrophages (105/well) were added to polyHEMA
plates in the absence or presence of wells containing apoptotic
cardiocytes (nonopsonized or opsonized). Supernatants (conditioned
media) from the cocultures were collected and analyzed separately. The
macrophages from each condition were retrieved, fixed with
paraformaldehyde, and stained with the following primary Abs (at 1/200
dilution): mouse IgG (isotype control), ligand-induced binding site 1
(generous gift from M. H. Ginsberg, The Scripps Research Institute, La
Jolla, CA),
anti-V3 integrin
(Chemicon International, Temecula, CA). After addition of
anti-mouse IgG FITC, the samples were analyzed by FACS analysis
(FACScan) or by indirect immunofluorescence using phase contrast
microscopy.
Assessment of fibroblast phenotype and proliferation
To evaluate the phenotype of the cultured fibroblasts, cells were plated on glass coverslips and treated with supernatants generated as described above. Cells were fixed with paraformaldehyde, and permeabilized with 100% acetone. Primary Abs at 1/200 dilution reactive with SMAc (Sigma-Aldrich) were added. After addition of anti-mouse IgG FITC (Sigma-Ald-rich), the samples were analyzed by FACS (FACScan) or by indirect immunofluorescence. To evaluate proliferation, cells were assessed via a tritiated thymidine incorporation assay as described (12). Fibroblasts were incubated at 37°C (in 5% CO2) for a total of 48 h in the presence of 1 µCi/well of [3H]thymidine. The cells were harvested onto glass-fiber filter paper, and [3H]thymidine incorporation was determined by liquid scintillation spectroscopy.
In addition, recombinant human TNF- and TGF were added
individually to fibroblasts at 5 ng/ml, incubated for 48 h at
37°C, and evaluated for SMAc expression and thymidine incorporation
as above.
Statistical analysis
The Student t test for unpaired data was used to compare proliferation and FACS mean fluorescent measurements between the different groups. Values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical description and routine postmortem evaluation
of a term male infant, diagnosed with AV block at 19 wk and dying at
birth, have previously been published (7). Available
slides were immunostained with Abs to SMAc (for detection of
myofibroblasts) and CD68 (for detection of macrophages). Representative
photomicrographs are shown in Fig. 1
. As
expected, SMAc-positive smooth muscle cells lined the blood vessels.
Notably, the ventricular tissue contained numerous areas of fibrosis
and microcalcification (Fig. 1, A and B) in
which a predominant SMAc-positive infiltrate, indicative of
myofibroblasts, was readily observed (Fig. 1, C and
D). Small clusters of macrophages could also be appreciated
in areas of scar tissue (Fig. 1, G and H). In
contrast, there were no SMAc-positive cells (other than those lining
the blood vessels) in ventricular tissue from an otherwise
healthy 24-wk abortus (Fig. 1E) or a term neonate dying at
birth of noncardiac causes (Fig. 1F). The CD68 Ab showed no
immunostaining in the subendocardial zones shown (Fig. 1, I
and J). Based on these novel findings, in vitro experiments
were initiated to examine the role of maternal anti-SSA/Ro Abs in
the pathologic cascade leading to fibrosis.
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Three different culturing conditions (plates coated with type I collagen, vitronectin, or polyHEMA) were used to address adhesion (crystal violet assay) and serum-induced proliferation ([3H]thymidine incorporation) of the primary fetal cardiocytes. Cardiac myocytes attached to collagen-coated (110% proliferation vs plastic alone) and vitronectin-coated surfaces (127%), but not polyHEMA-coated surfaces (2%). The latter occurred, in part, because serum protein is unable to adsorb to the polyHEMA surface (10). After 48 h, the thymidine incorporation by cardiac myocytes plated on collagen was equivalent to that by myocytes plated on vitronectin (8430 ± 2010 cpm vs 7200 ± 180 cpm; p = NS). In contrast, polyHEMA did not support proliferation of the cardiocytes. This effect reflects a viability requirement involving anchorage signals. At 18 h, >90% of the polyHEMA-plated cardiocytes, which rounded up in aggregates, were TUNEL-positive, supporting that the cells were apoptotic (not shown).
Subsequent experiments confirmed that SSA/Ro proteins were expressed on
the surface of fetal cardiac myocytes rendered apoptotic by culture on
polyHEMA-coated plates, and were thus accessible to maternal
autoantibodies. As assessed by indirect immunofluorescence with
affinity-purified Abs, there was no surface staining of normally
proliferating cardiocytes (Fig. 2A). In contrast, surface
staining was readily demonstrated on polyHEMA-apoptotic cardiocytes
incubated with the same affinity-purified Abs (Fig. 2B). As
shown by flow cytometry, the polyHEMA-apoptotic cells were not rendered
nonspecifically sticky, because there was no staining with an IgG
fraction from a normal healthy donor (Fig. 2C), but only
with affinity-purified Abs (Fig. 2D).
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The next set of experiments addressed whether the opsonized
apoptotic cardiocytes would induce macrophage activation. Macrophages
were isolated from PBMC of several healthy donors and cocultured for
18 h with opsonized polyHEMA-apoptotic cardiocytes (incubated with
affinity-purified Abs to 52 and 60 kDa SSA/Ro) or with nonopsonized
polyHEMA-apoptotic cardiocytes (incubated with normal serum IgG).
Supernatants (conditioned media) from the cocultures were collected and
analyzed separately for their effects on cardiac fibroblasts (see
below). The macrophages from each condition were fixed in 4%
paraformaldehyde and evaluated for surface expression of the activation
epitope of V3 using
the mAb LIBS1.
Coculture of macrophages for 18 h with opsonized apoptotic
cardiocytes resulted in a three-fold increase in the expression of
active V3 over that
observed on the macrophages alone (p < 0.01,
Fig. 3). In contrast, expression of total
V3 was similar
between macrophages incubated with nonopsonized apoptotic cardiocytes
and macrophages incubated with opsonized apoptotic cardiocytes
(p = NS).
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Subsequent experiments addressed whether fibrosis might be a
consequence of macrophage activation following phagocytosis of
opsonized apoptotic cardiocytes. Primary cultures of fibroblasts were
incubated in the presence or absence of supernatants from cocultures of
apoptotic cardiocytes and macrophages, fixed in 4% paraformaldehyde,
stained using anti-SMAc or monoclonal antihuman fibroblast surface
protein (clone 1B10), and analyzed by indirect immunofluorescence.
Under basal conditions the cultured fibroblasts did not express SMAc
(Fig. 4, A and E).
Fibroblasts incubated with supernatants from isolated macrophage
cultures alone marginally expressed SMAc (Fig. 4, B and
F), which was completely abrogated with medium from
cocultures of macrophages and nonopsonized apoptotic cardiocytes (Fig. 4, C and G). In contrast, fibroblasts incubated
with supernatant derived from macrophages plus opsonized apoptotic
cells expressed markedly increased SMAc (Fig. 4, D and
H). In all four conditions (basal, supernatant from
macrophages alone, supernatant from macrophages cocultured with
nonopsonized apoptotic cardiocytes, and supernatant from macrophages
cocultured with opsonized apoptotic cardiocytes), total reactivity by
antihuman fibroblast surface protein expression was equivalent (not
shown). Additional nonopsonized and opsonized conditions were used. The
former included an IgG fraction isolated from the serum of a patient
with systemic lupus erythematosus who had antinuclear Abs but no
anti-SSA/Ro-SSB/La Abs. Supernatants generated from macrophages
cocultured with these apoptotic cardiocytes were added to the
fibroblasts and no SMAc staining was observed (data not shown). The
latter included an IgG fraction isolated from the serum of a mother
with Abs to all components of the SSA/Ro-SSB/La complex and whose child
had cutaneous manifestations of neonatal lupus. Equivalent to the
results obtained with affinity-purified anti-52/60 SSA/Ro Abs,
cocultures with these apoptotic cardiocytes markedly increased SMAc
staining of the fibroblasts (data not shown).
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and TGF in the
transdifferentiation of cardiac fibroblasts by the opsonized
supernatant, the effects of these two cytokines and their neutralizing
Abs were evaluated. The direct addition of TNF- to the fibroblast
cultures did not result in SMAc expression (Fig. 5A). In contrast, addition of
TGF alone markedly increased expression of SMAc (Fig. 5B). As might be predicted by these results,
TNF--neutralizing Ab had no effect when added to the macrophage
opsonized supernatant (Fig. 5D). However, incubation of the
same macrophage supernatant with a neutralizing anti-TGF Ab
resulted in complete attenuation of the elicited SMAc expression (Fig. 5E).
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and TGF in the stimulated proliferation of
cardiac fibroblasts by supernatants, both cytokines and their
respective blocking Abs were added as was done for evaluation of SMAc
expression. TNF- did not affect basal fibroblast proliferation,
paralleling the results obtained for expression of SMAc. However,
contrary to its effect on SMAc, TGF significantly inhibited
proliferation (4,378 cpm ± 360 SE vs 1,857 cpm ± 297 SE;
p < 0.001) (Fig. 6).
Correspondingly, TGF-neutralizing Ab reversed this effect and
further increased the proliferative response observed when fibroblasts
were exposed to the macrophage-opsonized supernatant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies to define the pathogenesis of Ab-mediated insult have focused on apoptosis, which may account for the problem of accessibility in the fetal heart and the absence of injury in the maternal heart. Indeed, Tran et al. (14) have recently identified physiologic apoptosis and translocation of SSB/La in the developing murine heart. In this study, we demonstrate that cardiac myocytes readily undergo apoptosis when plated on polyHEMA, which reflects their enhanced sensitivity to apoptotic stimuli. The apoptotic myocytes expressed SSA/Ro and SSB/La on the surface and were opsonized by the cognate maternal Abs. This confirms earlier studies in which staurosporine and 2,3-dimethoxy-1,4-naphthoquinone resulted in apoptosis of cardiac myocytes and binding by anti-SSA/Ro-SSB/La Abs (3, 4). However, it is readily acknowledged that the specificity of these particular Abs for cardiac damage remains to be accounted for. Perhaps other autoantigens (such as Sm or lamin B) which do translocate to the apoptotic blebs (15, 16) are not truly accessible on the surface.
Inadvertent opsonization of the apoptotic cardiac myocyte could
pathologically modify the scavenging macrophages with regard to their
activation state, signaling pathways and release of soluble mediators.
Precedent for early involvement of macrophages is supported by the
autopsy findings of a 19-wk fetus with CHB. Histologic studies
disclosed dense lymphohistiocytic infiltrates and myocyte degeneration
in the cardiac conduction system including the AV node and bundle of
His (7). In the present study, coincubation with opsonized
apoptotic cardiocytes resulted in a phenotypic change of the macrophage
as evidenced by increased expression of an activation epitope of
V3. Relative to the
influence on signaling pathways, phosphorylation and nuclear
translocation of p44/p42 MAP kinase (extracellular-regulated kinase has
recently been demonstrated in extracts of macrophages cocultured with
apoptotic cardiocytes preincubated with affinity-purified Abs or IgG
fractions reactive with 48 kDa SSB/La, and 52 and 60 kDa SSA/Ro, but
not in macrophages cocultured with apoptotic cardiocytes preincubated
with normal human IgG.4
The effect of macrophage activation on the
cardiac fibroblast may be the critical component leading to fibrosis of
the AV node (and in some cases extending to other components of the
conduction system and even the working myocardium). Cross-talk between
macrophages and fibroblasts, and consequent tissue fibrosis, has been
emphasized in other organs. In experimental unilateral ureteral
obstruction, the clearance of apoptotic tubular cells by macrophages
resulted in the appearance of proliferating SMAc-positive
myofibroblasts (17). In models of canine hepatic and renal
injury, macrophages are present in less extensive areas of fibrosis,
and myofibroblasts dominate in the more advanced grades of fibrosis
(18). In a murine model of granulomatous experimental
autoimmune thyroiditis, fibrosis was considered secondary to
macrophages which produced TGF1 (19). Interestingly,
however, the irreversible fibrotic replacement of normal conducting
tissue may be unique to autoantibody-associated CHB. Other inflammatory
stimuli, e.g., those that occur with Lyme disease, generally induce
transient AV nodal block (20, 21). Although this may
reflect a more rapid resolution of the inflammatory process than occurs
with CHB, it suggests that fibroblast transdifferentiation is not
merely a final common pathway of inflammation.
Dedifferentiation of fibroblasts is associated with scar formation.
Fibrosis is due to a persistent myofibroblast, a phenotype associated
with wounding. The transient appearance of a myofibroblast is
recognized as a pivotal component of granulation tissue formation
(6). Proliferation of myofibroblasts is accompanied by the
expression of SMAc and the embryonal isoform of smooth muscle myosin H
chain, and by release of types I and III collagen. Thus, the
myofibroblast functionally resembles features of both smooth muscle
cells and fibroblasts (22). A recent study compared the
phenotype of myofibroblasts and normal fibroblasts from a patient with
a recurrent abdominal incision wound herniation (23);
myofibroblasts were distinguished from fibroblasts by their increased
expression of SMAc and of
V3 integrin. This
phenotype may contribute to unresolved scarring because the binding of
collagen to V3
integrin interferes with lattice contraction and also prevents the
organization of matrix (an 2 integrin-collagen
interaction). Unresolved scarring occurs in the heart following diverse
modes of injury, e.g., pressure-overloaded left ventricle hypertrophy
and a mouse model of autoimmune myocarditis (immunized with cardiac
myosin) (24).
The precise composition of the supernatants (inclusive of cytokines,
superoxide radicals, etc.) obtained from macrophages cocultured with
opsonized cardiocytes has yet to be determined. Based on our previously
reported finding of TNF- release (4), and the
association of TGF with clearance of apoptotic cells (25, 26), initial experiments evaluated these two cytokines and their
neutralizing Abs. The present study is the first to demonstrate
increased SMAc expression in human fetal cardiac fibroblasts after
incubation with TGF, and is supported by a previous report in which
s.c. injection of rats with TGF, but not TNF-, resulted in the
formation of granulation tissue with an abundance of SMAc-expressing
cells (27). Although our data support a role for TGF in
the transdifferentiation to a myofibroblast, this cytokine clearly
inhibited proliferation. In contrast, TNF- did not induce any
measurable changes in the cultured fibroblasts. Whether activation of
the cytokines or their respective receptors on the fibroblasts is
relevant remains to be evaluated. Moreover, the ratio of various
cytokines in the opsonized supernatants may be another determining
factor. In contrast, macrophages cocultured with nonopsonized apoptotic
cells secrete factors that do not induce a myofibroblast phenotype and
actually down-regulate proliferation. Importantly, this latter scenario
likely represents the physiologic process of remodeling of the human
heart during fetal development, in which apoptotic cells are rapidly
cleared with no inflammatory sequelae.
Histopathologic studies support that extensive fibrosis occurs in autoantibody-associated CHB. For example, the spectrum of CHB in the clinically affected fetus includes AV nodal replacement by fibrosis or fatty tissue (5), fibrous structures containing microscopic crystalline structures in the conduction system (28), and altered contractility of the working myocardium secondary to endocardial fibroelastosis (29). The immunostaining of the heart described in this study is the first to demonstrate the presence of myofibroblasts in the region of scarring. The selective vulnerability of the heart (in addition to the skin and less commonly the liver) may relate to the susceptibility of the cardiac fibroblast to transdifferentiation. Of interest, pulmonary abnormalities have not been described as part of the spectrum of neonatal lupus. In this study, although the transdifferentiation of cardiac fibroblasts was readily demonstrated, the same supernatants were incapable of transdifferentiating fetal lung fibroblasts and did not cause their proliferation (R. M. Clancy, A. D. Askanase, R. P. Kapur, E. Chiopelas, N. Azar, M. E. Miranda-Carus, and J. P. Buyon, unpublished observation).
The studies described represent a novel view of the cardiac fibroblast as a fetal factor in autoantibody-associated CHB. Taken together, these in vivo and in vitro data support the speculation that CHB results from unresolved wound healing subsequent to the transdifferentiation of cardiac fibroblasts into proliferating myofibroblasts, a pathologic process initiated by specific maternal Abs. AV nodal cells and even the working myocardium may be particularly vulnerable to the myofibroblast, ultimately leading to fibrosis. It is acknowledged that this in vitro study may be an exaggerated model only applicable to the more severely affected cases, such as the neonate described in this study whose tissue was available for immunohistology. Thus, a reasonable prediction is that there are both susceptibility and regulatory factors, such as fetal polymorphisms of FcR and cytokines, each of which could influence the extent of the proposed pathologic cascade to result in permanent third degree heart block. Dissecting the individual components should provide insights into the pathogenesis of Ab-associated CHB and the rarity of irreversible injury.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert M. Clancy, Department of Rheumatology, Hospital for Joint Diseases, Room 1608, 301 East 17th Street, New York, NY 10003. E-mail address: clancr01{at}med.nyu.edu
3 Abbreviations used in this paper: CHB, congenital heart block; AV, atrioventricular; SMAc, -smooth muscle actin; polyHEMA, poly (2-) hydroxyethylmethacrylate.
4 M. E. Miranda-Carus, N. Azar, S. Chandrashekhar, R. M. Clancy, A. D. Askanase, E. K. L. Chan, and J. P. Buyon. A role for extracellular-regulated kinase (ERK) activation by opsonized apoptotic cardiocytes in the pathogenesis of congenital heart block. Submitted for publication.
Received for publication March 28, 2002. Accepted for publication June 13, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by macrophages. J. Immunol. 165:5345.1. Mol. Genet. Metab. 71:418.[Medline]
, PGE2 and PAF. J. Clin. Invest. 101:890.[Medline]
in macrophages that have ingested apoptotic cells. J. Immunol. 163:6164.1 induces -smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122:103.This article has been cited by other articles:
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  C. LLANOS, E. K.L. CHAN, S. LI, G. X. ABADAL, P. IZMIRLY, C. BYRNE, R. M. CLANCY, and J. P. BUYON Antibody Reactivity to {alpha}-Enolase in Mothers of Children with Congenital Heart Block J Rheumatol, March 1, 2009; 36(3): 565 - 569. [Abstract] [Full Text] [PDF]
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 J.P. Buyon and R.M. Clancy Dying right to live longer: positing apoptosis as a link between maternal autoantibodies and congenital heart block Lupus, February 1, 2008; 17(2): 86 - 90. [Abstract] [PDF]
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N Costedoat-Chalumeau, S Georgin-Lavialle, Z Amoura, and J-C Piette Anti-SSA/Ro and anti-SSB/La antibody-mediated congenital heart block Lupus, September 1, 2005; 14(9): 660 - 664. [Abstract] [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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 R. M. Clancy and J. P. Buyon Clearance of apoptotic cells: TGF-{beta} in the balance between inflammation and fibrosis J. Leukoc. Biol., December 1, 2003; 74(6): 959 - 960. [Full Text]
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J. P Buyon, A. Rupel, and R. M Clancy Congenital heart block: do fetal factors fuel the fire from inflammation to fibrosis? Lupus, October 1, 2003; 12(10): 731 - 734. [PDF]
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V Fesslova, S Mannarino, P Salice, C Boschetto, L Trespidi, B Acaia, F Mosca, R Cimaz, and P L Meroni Neonatal lupus: fetal myocarditis progressing to atrioventricular block in triplets Lupus, October 1, 2003; 12(10): 775 - 778. [Abstract] [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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