From the Division of Clinical Immunology and Allergy (V.P., I.M., G.M.),
University of Naples Federico II, School of Medicine, Napoli, Italy;
Department of Pathology (E.A., L.V.), University of Pavia, School of Medicine,
Pavia, Italy; and Department of Anesthesiology (B.L.-S., M.A.), Deutsches
Herzzentrum Berlin, Berlin, Germany.
Correspondence to Gianni Marone, MD, Division of Clinical Immunology and Allergy, University of Naples Federico II, Via S Pansini 5, 80131 Napoli, Italy. E-mail marone{at}unina.it
Methods and Results—HHMC density in DCM (18.4±1.6
cells/mm2) and ICM (18.4±1.5 cells/mm2) was
higher than that in control hearts (5.3±0.7 cells/mm2;
P<.01). The histamine and tryptase contents of DCM and
ICM hearts were higher than those of control hearts. The histamine
content of the hearts was correlated with mast cell density
(rs=.91; P<.001). Protein
A/gold staining of heart tissue revealed stem cell factor (SCF), the
principal growth, differentiating, and activating factor of human mast
cells, in HHMC secretory granules. Histamine release from cardiac mast
cells caused by immunological (anti-IgE and rhSCF) and nonimmunological
stimuli (Ca2+ ionophore A23187) was higher in patients with
DCM and ICM compared with control subjects. Immunological activation of
HHMC induced a significantly greater release of tryptase and
LTC4 in patients with DCM and ICM compared with control
subjects.
Conclusions—Histamine and tryptase content and mast cell density
are higher in failing hearts than in control hearts. SCF, present
in secretory granules of HHMC, might represent an autocrine
factor sustaining mast cell hyperplasia in heart tissue in these
patients. The increased local release of fibrogenic factors (eg,
histamine, tryptase, and leukotriene C4) might
contribute to collagen accumulation in the hearts of patients with
cardiomyopathy.
The concentration of histamine and the density of mast cells are
increased in the arteries of cardiac
patients,4 5 9 and coronary arteries from
cardiac patients are hyperresponsive in vitro to
histamine.4 Furthermore, in vivo administration
of histamine and other mast cell-derived mediators (peptide
LTC4) in humans causes significant
cardiovascular effects.10 11 12
Finally, serum IgE levels are increased in patients with
coronary artery disease.13 14 Taken
together, these observations raise the possibility that local
activation of cardiac mast cells might contribute, through the release
of vasoactive mediators, to certain cardiovascular
diseases.15 16
Fibrosis is a hallmark of failing hearts in DCM and
ICM.17 The cells and the mediators responsible
for fibroblast proliferation and collagen accumulation in failing
hearts in DCM and ICM are largely unknown. Mast cells are involved in
many types of inflammation and repair processes and are found in
increased numbers in fibrotic tissues. For example, increased mast cell
density has been documented in the lesional skin of scleroderma
patients18 and in the lung of patients with
fibrotic lung disorders.19 A possible association
between increased mast cell density and
endomyocardial fibrosis was first suggested by
Fernex in 1968.20 More recently, cardiac mast
cells (HHMC) have been implicated in eosinophil
myocarditis21 22 and in DCM secondary to systemic
sclerosis.23 However, the studies in this area
have been limited by the difficulties in isolating and purifying mast
cells from human heart tissue.
We established a technique for the efficient dispersion of mast cells
from human heart tissue and identified the immunological and
nonimmunological stimuli that induced HHMC to release vasoactive and
fibrogenic mediators in vitro.22 24 The
present study was performed to compare the cardiac mast cell
density, concentration of mast cell–derived mediators (histamine and
tryptase), and immunological and nonimmunological release of chemical
mediators from mast cells isolated from failing hearts obtained from
patients with DCM and ICM undergoing heart transplantation and from
control subjects who died in accidents. We also identified
ultrastructural localization of SCF, the principal growth,
differentiating, chemotactic, and activating factor for human mast
cells,25 26 27 28 in cardiac tissue.
Idiopathic DCM
Coronary Artery Disease Group
Normal Donor Heart
Reagents
Buffers
Isolation and Partial Purification of Human Heart Mast
Cells
Measurement of Histamine Content and Mast Cell Density of
Heart Tissues
Histamine Release Assay
RIA of Tryptase and LTC4
Ultrastructural Study
Electron Microscopic Immunocytochemistry Study
Statistical Analysis
Histamine Content of Heart in Idiopathic DCM and ICM
Expression of SCF in Mast Cells of Failing Human Hearts
Mediator Release From Isolated Cardiac Mast Cells Induced by
Immunological Stimuli
Mediator Release From Isolated Cardiac Mast Cells Induced by
Nonimmunological Stimuli
Tryptase Content in the Failing Heart
Tryptase and LTC4 Release From Isolated Cardiac Mast
Cells Induced by Immunological Stimuli
Mast cells are the only cells in connective tissues that possess
high-affinity receptors for IgE and synthesize a variety of vasoactive
and fibrogenic mediators1 and proinflammatory
cytokines.41 42 Mast cells and
their mediators have been implicated in several disorders involving the
human heart. Mast cell density is increased in the coronary
arteries of cardiac patients,3 4 5 6 9 and
coronary arteries from patients with ischemic heart
disease are hyperresponsive in vitro to
histamine.4 Furthermore, in vivo administration
of histamine causes coronary spasm in
Even more suggestive is the role of mast cells in various aspects of
fibrotic lesions of the human heart. Mast cell density is increased in
myocardial fibrosis in Africans20 and in DCM
secondary to systemic sclerosis.23 The mechanism
linking mast cells to collagen accumulation and fibrosis is complex and
largely unknown. However, mast cell–derived mediators (ie, histamine,
tryptase, and LTC4) are mitogens and comitogens
for human fibroblasts39 40 44 45 ; they stimulate
collagen synthesis, and collagen accumulation is a hallmark of ICM and
idiopathic DCM.17 Finally, SCF, a major
product of human fibroblasts,46 is a
principal growth, differentiating, chemotactic, and activating factor
for human mast cells.25 26 27 28 36 These observations
raise the possibility that cardiac mast cells might play a role in the
fibrotic cascade in cardiomyopathy.
The increased cardiac mast cell density in patients with failing hearts
may result from in situ differentiation from mast cell precursors, in
situ replication from preexisting mast cells, and migration from other
sites. Our data do not allow us to distinguish between these
possibilities; however, SCF, also termed "mast cell growth factor,"
c-kit ligand (KL), or steel factor, is the principal growth,
differentiating, chemotactic, and activating factor for human mast
cells.25 26 27 28 36 SCF produced by mesenchymal cells
(fibroblasts, epithelial cells, endothelial cells,
keratinocytes, neurons, and so
on)46 47 48 49 exerts its biological activities via
interaction with c-kit, its cognate
receptor.46 47 The SCF receptor is a
transmembrane tyrosine kinase receptor of fundamental importance for
the normal development, maturation, and functioning of mast
cells.25 28 The SCF receptor is expressed on the
plasma membrane of mast cells.26 37 A novel
finding of our study is the subcellular localization of SCF in
secretory granules of HHMC. The gene for human SCF consists of at least
eight exons. Alternative RNA splicing gives rise to two SCF
transcripts: one that contains sequences representing all
exons (exon 6+) (SCF248)
and a second, from which exon 6 is excluded (exon
6-)
(SCF220).50 SCF translated
from exon 6+ transcripts exists in both
membrane-bound and soluble form. The predominant soluble protein is
produced by proteolytic cleavage of the membrane-bound
precursor.51 SCF translated from exon
6- transcripts lacks the major proteolytic
cleavage site. As a result, SCF220 is
inefficiently cleaved at an alternative site and resides almost
exclusively in the plasma membrane.50 Here, we
demonstrate that SCF is contained in the cytoplasmic secretory granules
of HHMC. The specificity of this observation is supported by results
obtained with two different monoclonal and two polyclonal anti-SCF
antibodies, which presumably recognize different epitopes of human
rhSCF. Thus, it is unlikely that different anti-human SCF monoclonal
and polyclonal antibodies recognize cross-reactive epitopes present
in secretory granules of HHMC.
Internalization of c-kit together with SCF has been
demonstrated recently on human fetal liver–derived mast
cells.52 Moreover, SCF protein has been detected
bound to skin mast cells in lesions of patients with urticaria
pigmentosa.47 Although we have not demonstrated
SCF synthesis by human mast cells, we show for the first time that
mature mast cells express SCF in their secretory granules.
In addition to providing the first ultrastructural localization of
granule-associated cytokine in HHMC, this observation raises
the intriguing possibility that SCFs present in the secretory
granules of HHMC represents an autocrine factor that
contributes to mast cell hyperplasia in failing human hearts. The
increased density of cardiac mast cells and the increased local release
of factors mitogenic for fibroblast (eg, histamine,
tryptase, LTC4, and so on) might explain the
collagen overproduction and accumulation in failing
hearts.17
Several clinically relevant stimuli activate HHMC isolated from
DCM, ICM, and control subjects. Serum IgE levels are increased in
patients with several cardiovascular
diseases,13 14 and anti-IgE induces the release
of preformed (histamine and tryptase) and de novo synthesized
(LTC4) mediators from cardiac mast cells.
Moreover, SCF, a cytokine synthesized by
fibroblasts46 that induces mast cell growth in
vitro25 27 and in vivo,28
activates HHMC. These stimuli caused the same percentage of
mediator release from HHMC isolated from normal and failing hearts.
However, given the higher density of mast cells in DCM and ICM compared
with that in control subjects, the absolute release per gram of wet
tissue is significantly increased in the group of failing hearts.
Enhanced release of cardiac mast cell mediators in DCM and ICM also
occurred when cells were activated by such nonimmunological
stimuli as A23187.
Our finding of increased mediator release in patients with
cardiomyopathy might have clinical relevance.
Histamine is arrhythmogenic10 16 and can cause
coronary vasoconstriction in a significant percentage of
patients with ischemic heart disease and unstable
angina.11 16 In vivo administration of peptide
leukotrienes can increase coronary vascular
resistance in man.12 Thus, the increased density
of cardiac mast cells and the greater release of these mediators in
failing hearts might contribute to certain
cardiovascular derangements in these patients.
In conclusion, our results demonstrate that the histamine and tryptase
content and cardiac mast cell density are markedly increased in
patients with either of two different
cardiomyopathies compared with control subjects. In
addition, the immunological and nonimmunological release of mast
cell–derived mediators from mast cells isolated from failing hearts is
higher than that in the control group. SCF, identified for the first
time in secretory granules of HHMC, might act as an autocrine
factor that sustains mast cell hyperplasia and modulates mast cell
function in heart tissue of patients with
cardiomyopathy. These findings suggest that
increased local release of mast cell–derived fibrogenic factor (eg,
histamine, tryptase, and LTC4) could play a role
in the fibrotic cascade in idiopathic DCM and ICM.
Received July 10, 1997;
revision received October 22, 1997;
accepted October 31, 1997.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Stem Cell Factor in Mast Cells and Increased Mast Cell Density in Idiopathic and Ischemic Cardiomyopathy
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—We compared cardiac mast
cell (HHMC) density and the immunological and nonimmunological release
of mediators from mast cells isolated from heart tissue of patients
with idiopathic dilated (DCM) (n=24) and ischemic
cardiomyopathy (ICM) (n=10) undergoing heart
transplantation and from control subjects (n=10) without
cardiovascular disease.
Key Words: cardiomyopathy • fibrosis • leukotrienes • cells, mast
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Mast cells are the
only tissue cells expressing on their surface the high-affinity
receptor for IgE (Fc
RI) and synthesizing vasoactive and fibrogenic
factors (histamine, tryptase, and
LTC4).1 Mast cells are
present in human heart tissue2 3 and in
adventia and intima of coronary arteries of patients with
coronary artery disease.4 5 6 Moreover,
the in vitro immunological activation of human heart tissue with
anti-IgE induces the release of histamine and prostaglandin
D2.7 8
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Patients
The heart tissue used in this study was obtained from
patients undergoing heart transplantation at the Deutsches Herzzentrum
(Berlin) and from individuals not affected by
cardiovascular disease who died in accidents
(control subjects).
Twenty men and 4 women, aged 26 to 64 years (mean age, 50.6±2.3
years), constituted the idiopathic DCM group. The diagnosis of DCM was
based on the World Health Organization criteria29
with the demonstration of dilated and poorly contracting left and/or
right ventricle in the absence of known cause. Patients were excluded
from this group if there was evidence of (1) coronary heart
disease at coronary arteriography, (2) ischemia during
exercise testing, (3) systemic blood pressure of 
150/90 mm Hg,
(4) concomitant systemic or endocrine diseases known to cause left
ventricular impairment, or (5) excessive alcohol
consumption.30
Nine men and 1 woman, aged 38 to 63 years (mean age, 49.6±2.7
years), constituted the ICM group. All patients had congestive heart
failure and a history of acute myocardial infarction. Before
transplantation, both groups of patients had been treated with multiple
drugs (ACE inhibitors, cyclosporin A, digoxin, furosemide,
and heparin).
The control subjects consisted of 9 men and 1 woman, aged 29 to
58 years (mean age, 43.4±3.2 years), who died from
noncardiovascular causes and whose hearts were
designated "normal" on gross examination and histopathology.
We purchased 60% HClO4 from Baker
Chemical. BSA, PIPES, hyaluronidase, collagenase type II,
and synthetic LTC4 were from Sigma Chemical.
Hanks' balanced salt solution and fetal calf serum were from GIBCO.
Deoxyribonuclease I and pronase were from Calbiochem. RPMI 1640 with
25 mmol/L HEPES buffer and Eagle's minimum essential medium were
from Flow Laboratories. Percoll was from Pharmacia Fine Chemicals.
(3H)-LTC4 (39.3 Ci/mmol)
was from New England Nuclear Research Products. Protein A/gold
complex was from British Biocell International. rhSCF was from Genzyme.
Rabbit anti–human-Fc antibody was a generous gift from Drs Teruko
and Kimishige Ishizaka (La Jolla Institute for Allergy and Immunology,
La Jolla, Calif). The anti-peptide LTC4 antiserum
was donated by Dr Edward Kusner (Zeneca Pharmaceuticals, Philadelphia,
Pa).31 The monoclonal anti-rhSCF (7H6) was kindly
donated by Dr Keith Langley (Protein Chemistry Group, Amgen, Thousand
Oaks, Calif); this antibody recognizes the region 79 to 97 of human
SCF (K. Langley, personal comunication). The monoclonal (hkl-12)
and rabbit polyclonal anti-rhSCF antibodies were kindly provided by Dr
Manfred Brockhaus (Hoffman-LaRoche, Basel, Switzerland). The monoclonal
hkl-12 recognizes epitopes in the region 150 to 164 (M. Brockhaus,
personal comunication). The polyclonal sheep anti-human SCF was
obtained from Genzyme. Irrelevant monoclonal mouse anti–E-selectin was
from R&D Systems. Rabbit and sheep polyclonal IgGs from nonimmunized
animals were obtained from Sigma Chemical. The tryptase RIA kit
(Pharmacia Tryptase RIACT 50; Pharmacia Diagnostics AB) was
kindly donated by Kabi Pharmacia SpA (Milan, Italy).
The PIPES buffer used in these experiments was made up of
25 mmol/L PIPES, pH 7.37, 110 mmol/L NaCl, and 5 mmol/L
KCl; the mixture is referred to as P. P2CG contains, in addition to P,
2 mmol/L CaCl2 and 1 g/L
dextrose.32 PGMD contains 0.25 g/L
MgCl2 · 6H2O, 10
mg/L DNase, and 1 g/L gelatin in addition to P, pH 7.37.
The explanted hearts and the hearts from subjects who died in
accidents were immediately immersed in cold (4°C) cardioplegic
solution, shipped by air (4°C), and processed within 5 to 18 hours of
removal. The heart tissue (100 to 600 g) was placed in a beaker
(Nalgene) containing buffer P at 22°C. Hearts were dissected to
separate the left and right ventricles and the septum. Two small
samples (
2 g) representative of all myocardial
layers of each section were obtained for microscopic examination of
tissue mast cells and for measurement of total histamine and tryptase
content. Fat tissue, large vessels, and pericardium were removed. HHMC
were isolated from human heart tissue through the use of a technique
recently described in detail.22 24 HHMC were
partially purified through flotation through a discontinuous Percoll
gradient as detailed elsewhere.22 24 The purity
of these populations ranged from 0.2% to 18%. The viability of mast
cells was routinely evaluated, and it was always >95%, as detected
with Trypan blue exclusion.
Samples of human hearts were separated from fat and large
vessels through blunt dissection. For each heart, samples were obtained
from left and right ventricles and septum. Tissue samples were weighed
and boiled in 8% of HClO4 for 30 minutes. The
mixture was filtered to remove particles, and supernatants were assayed
for histamine.33 Morphometric assessment was
performed with a Nikon lens system by analyzing images obtained from 10
consecutive fields per section of the heart under x250 magnification.
Mast cell numbers obtained from the consecutive fields were added
together, and the density of mast cells expressed as the mean value
per mm2. Mast cells were counted blindly by
two independent investigators.
Cells (3x104 mast cells per tube)
resuspended in 0.3 mL of P2CG were placed in 12x75-mm polyethylene
tubes; 0.2 mL of each prewarmed releasing stimulus was added, and
incubation was continued at 37°C for 45
minutes.24 Each experimental group was performed
in duplicate. Cell-free supernatants were stored at -70°C for
subsequent assay of histamine, tryptase, and
LTC4. The cell-free supernatants were assayed for
histamine with the use of an automated fluorometric
technique.33 To calculate histamine release as a
percentage of total cellular histamine, the "spontaneous" release
of histamine from mast cells (3% to 12% of the total cellular
histamine) was subtracted from both numerator and denominator. All
values are based on means of duplicate or triplicate determinations.
Replicates differed from each other in histamine content by <10%.
Total tryptase content was assessed by lysis induced by
incubation of cells with 100 µL of Triton X-100 (0.1%). Tryptase was
analyzed by a solid-phase RIA (Tryptase RIACT 50;
Pharmacia).24 In some experiments, 200-µL
fractions were taken from the supernatant fluids for the
analysis of LTC4. The samples were stored
at -70°C until analyzed for eicosanoid content. The
LTC4 assay was carried out with a previously
described RIA.32
Samples for ultrastructural study were fixed in Karnovsky
solution (0.5% glutaraldehyde, 2%
paraformaldehyde in Na-cacodylate buffer, pH 7.3, 0.1
mol/L) at 4°C for 2 hours, rinsed in Na-cacodylate buffer, postfixed
with 1% osmium tetroxide in 0.1 mol/L cacodylate buffer for 1 hour at
4°C, dehydrated in ethanol and propylene-oxide, and embedded in
Epon-Araldite. Ultrathin sections were stained with uranyl acetate and
Reynold's lead citrate. The stained sections were examined with a
Zeiss EM10 electron microscope.24 30
Ultrathin sections were deosmicated in aqueous saturated
solution of 5% sodium metaperiodate for 10 minutes, rinsed in 1% OVA
in 0.01 mol/L Tris buffer and 0.5 mol/L NaCl, pH 7.6 (TBS/Triton
buffer), and washed for 1 hour in TBS/Triton/lysine buffer; the
sections were then incubated with 10% heat-inactivated
normal goat serum and subsequently incubated overnight with the
anti-SCF antiserum (7H6) diluted 1:100 in TBS/1% BSA/0.5% sodium
azide buffer. The sections were washed three times in TBS/1% BSA/0.5%
sodium azide buffer for 10 minutes and incubated for 1 hour with
protein A/gold complex diluted 1:30 with TBS/1% BSA/0.5% sodium azide
buffer.34 After a 2-hour wash in TBS/1%
BSA/0.5% sodium azide buffer, the grids were dried and stained for 15
minutes with aqueous uranyl acetate (5%) and for 10 minutes with
Reynold's lead citrate. The stained sections were examined with a
Zeiss EM10 electron microscope. In parallel experiments, ultrathin
sections were incubated with another monoclonal anti-SCF antibody
(hkl-12) or a rabbit or a sheep polyclonal anti-SCF antibody. The
following controls were performed: omission of the antibody layer,
replacement of specific antibody with isotype-matched irrelevant
antibody at the same concentration, and neutralization of specific
antibody with rhSCF (3 µg/mL) (equal amounts were mixed and allowed
to stand for 1 hour at 22°C until used for immunolabeling). The
results of the control procedures excluded nonspecific reactivity.
The results are mean±SEM. The data subjected to linear
regression were calculated with the least-squares method
(y=a+bx), where a is the
y-axis intercept, and b is the slope of the line.
The rank correlations were calculated with the Spearman rank
coefficient (rs). The multiple comparisons
between groups were assessed using the Student's t test
with Bonferroni's correction for multiple
comparisons.35
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Ultrastructural Features and Density of Cardiac Mast Cells in
Cardiomyopathy
Human hearts obtained from patients with DCM (n=24) or ICM (n=10)
undergoing heart transplantation and from subjects who died from
noncardiovascular causes (n=10) were studied by gross
examination, histopathology, and electron microscopy. The hearts from
the control subjects were designated "normal" on gross examination
and histopathology. Microscopic examination of sections stained with
toluidine blue and electron microscopic studies of hearts revealed the
presence of mast cells, mainly around blood vessels and between
myocardial fibers in all preparations from the control, DCM, and ICM
groups. Mast cells were also found in the intima of atherosclerotic
lesions, as reported previously.6 9 Mast cells in
human hearts were pleomorphic with respect to shape. Some mast cells
were large and round or oval (62%) and were almost completely filled
with cytoplasmic granules; other mast cells were slender and elongated
(38%) (Fig 1
). Cytoplasmic contents were
dominated by numerous, membrane-bound heterogeneous
(scrolls, homogeneously dense and crystals) granules and
non–membrane-bound lipid bodies. In situ examination of mast cells
from DCM and ICM revealed a small percentage (5%) of
activated (ie, partially degranulated mast cells), as reported
previously.2 24 The mast cell density of heart
tissue (left ventricle) from patients with DMC (18.4±1.6 mast
cells/mm2) and ICM (18.4±1.5 mast
cells/mm2) was higher than that from control
subjects (5.3±0.7 mast cells/mm2)
(P<.01) (Fig 2). Similar
results were obtained when the right ventricles or septum were examined
(data not shown).
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Figure 1. Electron micrograph of a mast cell in heart tissue
of a patient with DCM. Note the elongated shape and cytoplasm filled
with numerous granules. The mast cell is surrounded by collagen and is
in close proximity to a blood vessel (V) and myocyte (My). (Uranyl
acetate and lead citrate stained; original magnification,
x11 000.)
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Figure 2. Mast cell density of human heart from control
subjects ( 
) and patients with DCM (
) and ICM (
). Each point
represents the value from a single donor. The hatched area
represents the mean±SEM.
The histamine content of left ventricles of control subjects
(1.07±0.23 µg/g wet tissue) was significantly lower than those of
patients with DCM and ICM (5.8±0.7 and 5.9±0.5 µg/g wet tissue,
respectively; P<.05) (Fig 3).
There was a significant correlation between the histamine content of
human hearts and the cardiac mast cell density
(rs=.91; P<.001) (Fig 4), suggesting that mast cells are the
main, if not the only, source of histamine in human heart tissue in
control subjects and patients with
cardiomyopathies. Similar findings were obtained
with the right ventricle and septum (data not shown).
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Figure 3. Histamine content of left ventricle of heart
tissue from control subjects ( ) and from patients with DCM () or
ICM (). Each point represents the mean of duplicate
determinations from a single donor. The hatched area represents
the mean±SEM.
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Figure 4. Correlation between the histamine content of left
ventricle and mast cell density of heart tissue from control subjects
( ) and patients with DCM () or ICM ().
In an attempt to explain the increased mast cell density in
patients with DCM and ICM, we evaluated the presence of SCF in human
heart tissue because this cytokine is the main growth and
differentiating factor of human mast
cells.25 26 27 28 36 We used a monoclonal antibody
against region 79 to 97 of human SCF to detect SCF in HHMC. After
protein A/gold staining of human heart tissue, gold particles were
present throughout all the secretory granules of HHMC and not in
the perigranular cytoplasm (Fig 5).
Similar results were obtained with a rabbit or sheep polyclonal
antibody against multiple epitopes of SCF and another monoclonal
antibody (hkl-12) against region 150 to 164 of SCF (data not shown).
Granules of HHMC incubated with a murine myeloma against an irrelevant
antigen or an IgG from nonimmunized rabbit or from nonimmunized sheep
at concentrations similar to those of the specific antibody lacked
particles. Similar results were obtained when the specific antibodies
anti-SCF were neutralized with rhSCF (3 µg/mL) (data not shown).
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Figure 5. A, Immunogold staining for SCF of a human heart
mast cell from a patient with DCM. Sections were stained for SCF
(monoclonal antibody 7H6 anti-human SCF) as described in "Methods."
Gold particles that locate SCF are present over all secretory
granules of the mast cell (see inset) (uranyl acetate and lead citrate
stained). Bar scale, 1.1 µm. B, On higher magnification of the
inset, the subcellular localization of SCF of secretory granules is
seen. Gold particles are absent in the perigranular cytoplasm (uranyl
acetate and lead citrate stained). Bar scale, 0.4 µm.
SCF exerts its biological activities via interaction with
c-kit, its cognate receptor present on human
skin,26 lung,37 and heart
mast cells3 24 and induces histamine release from
these cells.3 24 26 37 We evaluated the effect of
rhSCF (3x10-1 to 30 ng/mL) on histamine release
from HHMC isolated from control subjects and DCM and ICM patients.
rhSCF concentration-dependently induced more histamine release from
HHMC obtained from failing heart than from control subjects (Fig 6A). The percentages of histamine release
induced by rhSCF from HHMC isolated from control subjects and DCM and
ICM patients were comparable (Fig 6B). Serum levels of IgE antibodies
are increased in the circulation of patients with
cardiovascular diseases.13 14 A
polyclonal antibody against the Fc portion of IgE
cased a concentration-related
(3x10-1 to 5 µg/mL) histamine release from
HHMC from control subjects and DCM and ICM patients. In these
experiments, the percent histamine did not vary among three groups
examined; however, when the absolute level of release was correlated
per gram of wet tissue, the difference was highly significant (Fig 7).
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Figure 6. A, Effect of increasing concentrations of rhSCF on
histamine secretion from HHMC obtained from control subjects
( 
) or DCM (
) and ICM
(
) patients. Each bar represents the
mean±SEM. *P<.05 compared with the corresponding group
of control subjects. B, Effect of increasing concentrations of rhSCF on
percent histamine secretion from HHMC obtained from control subjects
() or DCM () and ICM
() patients. Each bar represents the
mean±SEM.
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Figure 7. Effect of increasing concentrations of anti-IgE on
histamine secretion from HHMC obtained from control subjects
( ) or DCM (-) and ICM
() patients. Each bar represents the
mean±SEM. *P<.05 compared with the corresponding group
of control subjects.
The Ca2+ ionophore A23187 induces histamine
release from mast cells presumably by increasing the intracellular
concentration of Ca2+.24
A23187 caused a significantly higher release of histamine from HHMC
from DCM and ICM patients than from control subjects. As previously
noted for immunological stimuli, the percent release of histamine was
similar in DCM and ICM patients (data not shown).
Tryptase, a neutral protease stored in the cytoplasmic granules of
all human mast cells,24 32 38 can be released
from human mast cells24 31 and is a potent
mitogen for human fibroblasts.39 The tryptase
content of heart tissue from patients with DCM (32.3±5.9 µg/g wet
tissue) and ICM (33.7±4.3 µg/g wet tissue) was higher than that of
control subjects (6.9±2.1 µg/g wet tissue) (P<.05).
Peptide LTC4 is the principal
5-lipoxygenase metabolite synthesized by
HHMC,24 and it induces fibroblast
proliferation.40 We therefore examined the
immunological release of LTC4 and tryptase from
HHMC obtained from patients with DCM (n=4) or ICM (n=4) and in control
subjects (n=4). In these experiments, IgE cross-linking induced
significantly more release of tryptase (Fig 8) and LTC4 (Fig 9) from HHMC obtained from DCM and ICM
patients compared with that from control subjects.
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Figure 8. Effect of increasing concentrations of anti-IgE on
tryptase secretion from HHMC obtained from control subjects
( ) or DCM () and ICM
() patients. Each bar represents the
mean±SEM. *P<.05 compared with the corresponding group
of control subjects.
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[in a new window]
Figure 9. Effect of increasing concentrations of anti-IgE on
LTC4 release from HHMC obtained from control subjects
( ) or DCM () and ICM
() patients. Each bar represents the
mean±SEM. *P<.01 compared with the corresponding group
of control subjects.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
We demonstrate that the density of HHMC is markedly increased in
patients with idiopathic DCM and ICM. Moreover, the release of
vasoactive and proinflammatory mediators caused by immunological,
cytokine, and nonimmunological stimuli is higher in DCM and ICM
than in control subjects. In addition, we provide the first evidence
that the secretory granules of cardiac mast cells store SCF, which is
the principal cytokine inducing growth, differentiation,
chemotaxis, and activation of human mast
cells.25 26 27 28 36 30% of patient with
unstable angina.11 The presence of mast cells
around4 5 and within6 9
coronary blood vessels suggests that local activation of
cardiac mast cells might contribute, through the release of vasoactive
mediators, to the pathophysiology of ischemic heart
disease.43
Selected Abbreviations and Acronyms
BSA
=
bovine serum albumin
DCM
=
dilated cardiomyopathy
ICM
=
ischemic cardiomyopathy
LTC
=
leukotriene
OVA
=
ovalbumin
PIPES
=
piperazine-N,N'-bis(2-ethanesulfonic
acid)
rhSCF
=
recombinant human stem cell factor
RIA
=
radioimmunoassay
SCF
=
stem cell factor
Acknowledgments
This study was supported by grants from CNR (Target Project
Biotechnology) and MURST (Rome, Italy) and by grant 030RFM87/1-IRCCS to
Policlinico S. Matteo (Pavia, Italy) from the Ministry of Health (Rome,
Italy). The authors thank Oreste Marino and Monica Concardi for their
excellent technical assistance.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
, which induces endothelial leukocyte adhesion
molecule 1. Proc Natl Acad Sci U S A. 1991;88:4220–4224.
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