Cardiovascular Research 55 (2002) 150–160
www.elsevier.com / locate / cardiores
Acute stress induces cardiac mast cell activation and histamine release,
effects that are increased in Apolipoprotein E knockout mice
Man Huang, Xinzhu Pang, Richard Letourneau, William Boucher,
Theoharis C. Theoharides*
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
Received 8 November 2001; accepted 25 February 2002
Abstract
Objectives: Cardiac mast cells have recently been found to be activated in atherosclerotic coronary arteries, but no mediator has so far
been documented to be released from them, nor have they been investigated in Apolipoprotein (Apo) E knockout (k / o) mice that develop
atherosclerosis. Psychological stress triggers acute coronary syndrome, while acute restraint stress stimulates rat cardiac mast cells, the
main mediator of which histamine is a coronary constrictor. Here, we investigated the effect of acute stress on the activation of cardiac
mast cells morphologically, as well as the levels of cardiac and serum histamine in normal and genetically deficient mice. Methods: Male,
8–14 week-old ApoE k / o mice and their corresponding control C57BL / 6J mice were used. Significant reduction of cardiac histamine
from 396.7645.6 to 214.6641.5 ng / g was observed over 120 min restraint stress with a corresponding increase in serum histamine from
126.964.0 to 188.4617.3 ng / ml in C57BL mice. Cardiac mast cell activation was observed by light and electron microscopy. Both basal
cardiac and serum histamine in ApoE k / o mice was significantly higher than that in C57BL mice. Although the extent of mast cell
activation in ApoE k / o mice was similar to that of C57BL mice, the number of cardiac mast cells in ApoE k / o mice was 37% higher.
Histamine levels were hardly detectable with or without stress in W/ W v mast cell deficient mice. Conclusions: Acute restraint stress
triggered cardiac histamine release in mice that was clearly derived from mast cells, as it was absent in W/ W v mice. The high basal
cardiac and serum histamine in ApoE k / o mice, along with the high number of cardiac mast cells, suggest possible ongoing cardiac mast
cell activation that may participate in atherosclerosis. These results may possibly help better understand stress-related cardiovascular
pathology.  2002 Elsevier Science B.V. All rights reserved.
Keywords: Atherosclerosis; Coronary disease; Cytokines; Ischemia; Vasoactive agents
This article is referred to in the Editorial by A.H.
Chester ( pages 13 – 15) in this issue.
1. Introduction
Increasing evidence implicates acute psychological
stress in cardiovascular pathology, especially silent
myocardial ischemia (MI). MI occurring without angina on
presentation now appears to be a sizable portion of the MI
population [1–4]. Stress also precipitates or exacerbates
certain neuroinflammatory conditions, many of which
*Corresponding author. Tel.: 11-617-636-6866; fax: 11-617-6362456.
E-mail address: [email protected] (T.C. Theoharides).
involve mast cells [5,6]. Mast cells are critical for allergic
reactions, but they also release numerous vasoactive,
neurosensitizing and inflammatory molecules [7]. Mast
cells are located close to neurons (for review see Ref. [8])
where they can be activated by neuropeptides [9], by
antidromic trigeminal ganglion stimulation [10], as well as
by restraint stress [11,12]. These findings point to the
significance of mast cell–neuron interactions [5] and have
heightened interest in the versatile role of mast cells [13].
There is growing evidence that cardiac mast cells may
participate in the development of atherosclerosis, coronary
inflammation and cardiac ischemia [14–17]. Mast cells are
increased and / or activated in association with MI [15] and
atherosclerosis [16], as well as ischemic cardiomyopathy
Time for primary review 26 days.
0008-6363 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 02 )00336-X
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
[18]. Mast cells were also recently shown to accumulate in
the shoulder region of human coronary atheromas, the
predilection sites of atheromatous erosion / rupture [15,16],
and have also been implicated in coronary arteries during
spasm or plaque rupture [14]. The human mast cell
proteolytic enzyme chymase has been shown to be the
main cardiac source of converting enzyme generating the
coronary constrictor angiotensin II [19]. Cardiac mast cell
derived histamine [20] can constrict the coronaries [21]
and can sensitize nerve endings [22]; such an action is
supported by recent findings showing adventitial mast cells
localized close to nerve endings in atherosclerotic coronary
arteries [23].
Acute restraint stress in rodents has been established as
a useful model to study stress-related events especially
those involving inflammatory processes [24]. It was shown
that acute stress induced rat cardiac mast cells activation
documented morphologically, an effect blocked by the
‘mast cell stabilizer’ disodium cromoglycate (cromolyn)
[12]. However, the source of any mediators released was
not investigated, neither was the possible contribution of
this effect to atherosclerosis.
Here, we report that acute stress by restraint triggered
cardiac histamine release, as evidenced by reduced cardiac
histamine content and corresponding increases in serum
histamine levels in C57BL and ApoE k / o mice. Although
the extent of cardiac mast cell activation in ApoE k / o mice
that develop atherosclerosis was similar to that of C57BL
mice, the number of cardiac mast cells in ApoE k / o mice
was higher than that in C57BL mice; moreover, both
cardiac and serum histamine levels were significantly
higher in ApoE k / o mice than in C57BL mice, indicating
higher basal release of histamine possibly from on-going
cardiac mast cell activation in ApoE k / o mice. Both
cardiac and serum histamine levels in W/ W v mice were
very low and did not change with stress, suggesting that
stress-induced cardiac histamine release was derived from
mast cells.
2. Methods
2.1. Restraint stress
Normal (C57BL / 6J), W/ W v (WBB6F1 / J-W/ W v) genetically mast cell deficient mice and their wild type 1 / 1
control (WBB6F1) mice, as well as ApoE k / o mice
(JR2052 C57BL / 6J-Apoe tmlunc) for which C57BL / 6J mice
are considered their wild type controls (Jackson Laboratories, Bar Harbor, ME) were housed in plastic cages
(four mice per cage) with a wire top in a modern animal
facility under the supervision of veterinarians. Mice were
allowed food and water ad libitum and were maintained in
an automatic 14:10 h dark–light cycle. Animals were kept
in the animal facility for at least 1 week before use. Each
mouse was brought into an isolated procedure room,
151
adjacent to the animal holding room inside the animal
facility between 09.00 and 11.00 h (to avoid any effect of
diurnal rhythms) for 30 min every day for 3 days in order
to reduce the stress of handling. During the day of the
experiment, each control animal was allowed to stay in its
cage for the designated period of time on a bench top at
room temperature in the procedure room. At a different
time, the experimental mouse was placed in a clear
restraint chamber with openings for sufficient aeration
(Harvard Apparatus, Cambridge, MA) for 15–120 min.
Mice were stressed inside the animal facility in order to
reduce the degree of mast cell activation noticed in control
animals upon transferring them to the laboratory [12]. No
mouse was ever present or in close proximity, while
another was stressed or dissected.
At the end of the experiment, each animal was anesthetized with a single i.p. injection (0.1 ml) of ketamine
(80 mg / kg) and xylazine (10 mg / kg), killed by asphyxiation under CO 2 vapor and decapitated. This protocol was
approved by the University’s Animal Research Committee
and the investigation conforms with the Guide for the Care
and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23,
revised 1996).
2.2. Corticosterone measurements
Blood was collected from the neck vessels after the
mouse was decapitated. Blood samples were allowed to
clot overnight at 2–8 8C before centrifuging for 20 min at
20003g. The serum was collected and subjected to
corticosterone radioimmunoassay using a Corticosterone
125
I-RIA kit (ICN, Costa Mesa, CA).
2.3. Histamine measurements
For histamine measurements, the heart was rapidly
removed, immediately frozen on dry ice and kept at
280 8C until the histamine assay. The heart was cut into
small pieces (about 1 mm 3 ) and washed with phosphate
buffered saline (PBS, Sigma, St. Louis, MO). Heart tissue
fragments were then disrupted using a Polytron (Brinkmann Instruments, Westbury, NY) at 4 8C in PBS. The
weight of tissue per total volume was recorded for each
sample. The homogenized samples were centrifuged
(15 0003g) at 4 8C for 15 min and the supernatant was
collected. Histamine in both the heart and serum samples
was measured by radioimmunoassay ( 125 I-RIA kit, Immunotech, Westbrook, ME). For serum histamine levels,
blood was collected from the neck vessels after decapitation and there was no significant difference in histamine
levels between serum and plasma samples. Thereafter, only
serum was used for all the assays. Therefore, blood
samples were allowed to clot overnight at 2–8 8C before
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M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
centrifuging for 20 min at 20003g at 4 8C; the serum was
then collected and kept at 280 8C until use.
3. Results
3.1. Effect of acute stress on serum corticosterone levels
2.4. Light microscopy
For light and electron microscopy, the heart was rapidly
removed and fixed en bloc by immersion in 4% paraformaldehyde for 2 h at room temperature and then overnight
at 4 8C. The tissue was frozen using Tissue Freezing
Medium (Triangle Biomedical Sciences, Durham, NC) and
thin sections (7 mm) were cut using a cryostat (Jung CM
3000, Leica, Inc. Deerfield, IL). Cardiac sections were
stained with acidified (pH,2.5) toluidine blue (Sigma, St.
Louis). Mast cells were counted at 4003 in an area of
0.2948 mm 2 using six random cardiac sections from each
mouse by two researchers blinded to the experimental
conditions using a Diaphot inverted Nikon microscope
(Don Santo, Natick, MA).
Results are presented as scattergram of percent activated
mast cells stained with toluidine blue (the number of mice
is shown in parentheses for each condition). Mast cell
activation was defined as the presence of extruded granules
close to the surface of the cell in question or staining of
about half or less of the cell section with toluidine blue. A
scattergram is used to appreciate the individual variation,
while the mean6standard deviation is also provided for
easier comparison. This subjective evaluation clearly records only mast cells that have been maximally stimulated.
Serum corticosterone levels increased during stress (Fig.
1A–C). Acute restraint stress for 15 min rapidly raised
these levels from 30.3614.8 to 311.1669.6 ng / ml (10fold) in C57BL mice (Fig. 1A, P,0.05), from 24.565.9 to
243.4647.6 ng / ml (10-fold) in W/ W v mast cell deficient
mice (Fig. 1B, P,0.05) and from 42.7626.4 to
338.66114.7 ng / ml (8-fold) in ApoE k / o mice (Fig. 1C,
P,0.05). By 120 min, serum corticosterone levels were
2.5. Electron microscopy
Tissue samples were fixed in modified Karnovsky’s
medium containing 2% paraformaldehyde, 3% glutaraldehyde and 0.5% tannic acid in 0.1 M cacodylate buffer
(pH 7.4) and were processed as previously described [25].
They were examined and photographed using a Phillips300 transmission electron microscope.
2.6. Statistical analysis
For histamine measurements, Student’s t-test was used
to compare the differences between the stressed group and
the control group. For light and electron microscopic
observations, results were compared by non-parametric
analysis using the Mann–Whitney U-test. Comparisons
were done between C57BL control and stressed animals,
while the W/ W v mast cell deficient mice were compared to
their respective wild-type age-matched controls after both
were stressed. The means and standard deviations of the
results are reported in the text only, while the mean is also
shown by a horizontal line in the scattergram. For corticosterone analysis, the P-values were obtained using a onetailed t-test; for all of the other analyses, P-values were
obtained using a two-tailed t-test. A P-value of less than
0.05 was considered to indicate statistical significance.
Fig. 1. Serum corticosterone levels were measured in control (unstressed)
and stressed (A) C57BL (n514); (B) W/ W v mast cell deficient (n58);
and (C) ApoE k / o mice (n58). The mean6S.D. is derived from one
measurement from each animal, the number of which is shown in the
parentheses.
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
equivalent in C57BL mice (456.6666.8 ng / ml, Fig. 1A,
P,0.05), in W/ W v mast cell deficient mice (389.96144.4
ng / ml, Fig. 1B, P,0.05) and in ApoE k / o mice
(453.3666.6 ng / ml, Fig. 1C, P,0.05). It can, therefore,
safely be concluded that the different results in mast cell
activation, as well as cardiac and serum histamine levels
obtained in C57BL, W/ W v mast cell deficient mice and
ApoE k / o mice (see below) were not due to any difference
in their response to stress.
3.2. Effect of acute stress on cardiac histamine levels
Histamine was measured in whole heart from control
and stressed animals. Histamine levels in the hearts from
control C57BL mice (n514) were 354.36160.1 and
445.06196.8 ng / g tissue; these values decreased significantly to 211.1697.2 ng / g (P,0.05) and 175.0681.7
ng / g (P,0.05) after 15 and 30 min of stress, respectively.
The reduction from 390.96130.2 to 257.86151.8 ng / g
after 2 h of stress was not significant (P.0.05) (Fig. 2A).
This result confirms the morphological findings of mast
cell activation (see below) and shows that at least one
mediator is in fact secreted from cardiac mast cells in
response to acute stress.
W/ W v mast cell deficient mice (n58) had very low
histamine levels in the heart either in control mice
(25.869.0, 22.567.1 and 25.069.3 ng / g) or after they
were stressed (22.6610.3, 23.467.1 and 25.0612.0 ng / g)
(Fig. 2B). In contrast, their wild type 1 / 1 control mice
(n58) showed similar results to C57BL mice. The unstressed 1 / 1 mice had 422.56211.9 and 422.56245.3
ng / g of cardiac histamine, which after 15 and 30 min of
stress significantly decreased to 197.5666.1 ng / g (P,
0.05) and 176.3686.8 ng / g (P,0.05), respectively. The
reduction after 2 h of stress from 410.06185.4 to
213.56112.33 ng / g was not significant (P.0.05) (Fig.
2C). These results imply that nearly all of the histamine
secreted under stress comes from mast cells.
The activation of cardiac mast cells in ApoE k / o mice
was also significantly induced by restraint stress. Cardiac
histamine levels in ApoE k / o mice (n58) were
520.06206.2 and 481.36214.5 ng / g; these values decreased significantly after 15 and 30 min of stress to
310.0699.0 ng / g (P,0.05) and 296.76165.6 ng / g (P,
0.05), respectively. The reduction after 2 h of stress from
511.36231.6 to 418.336107.41 ng / g was not significant
(P.0.05) (Fig. 2D). The mean basal cardiac histamine
level of 504.2620.3 ng / g in ApoE k / o mice was significantly higher than that of 396.7645.6 ng / g in C57BL
mice (P,0.05), and so was the level after stress, which
was 341.7666.7 ng / g in ApoE k / o mice and 214.6641.5
ng / g in C57BL mice (P,0.05) (Fig. 2E). These results
indicate that ApoE k / o mice may have increased number
of mast cells that appear to be already activated before
stress in association with atherosclerosis.
153
3.3. Effect of acute stress on serum histamine levels
Acute stress significantly increased serum histamine
levels (Fig. 3A) in C57BL mice from a basal level of
124.2617.9, 125.0617.5 and 131.5632.3 ng / ml at 15
min, 30 min and 2 h to 193.4648.4 (P,0.05), 169.1633.8
(P,0.05) and 202.5627.5 ng / ml (P,0.05), respectively
after stress.
W/ W v mast cell deficient mice showed almost undetectable serum histamine levels that did not change with stress
(Fig. 3B), whereas 1 / 1 mice showed increased serum
histamine levels similar to those of C57BL mice after
different periods of stress (Fig. 3C). Therefore, any
histamine increased in the serum of the 1 / 1 controls,
could not have derived from any other source than mast
cells.
Serum histamine in ApoE k / o mice was also increased
by stress over different periods of time (Fig. 3D). Moreover, the average serum histamine in control unstressed
ApoE k / o mice was significantly higher (161.164.0 ng /
ml) than that of C57BL mice (126.964.0 ng / ml) (P,
0.05) (Fig. 3E) from which they derive, further indicating
increased basal histamine secretion in these mice. Overall,
the average (results from all time points combined) serum
histamine level of 188.4617.3 ng / ml in stressed C57BL
mice was significantly higher than that of 126.964.0
ng / ml in unstressed ones (P,0.05) (Fig. 3E). Whereas
ApoE k / o mice already show significantly higher serum
histamine at rest (161.164.0 ng / ml) than C57BL mice
(P,0.05) (Fig. 3E), the average serum histamine level
after stress (201.7619.9 ng / ml), although significantly
higher than its own controls (Fig. 3E), was similar to that
of C57BL mice (188.4617.3 ng / ml) (P.0.05).
3.4. Effect of acute stress on cardiac mast cell
activation
Most cardiac mast cells were located close to blood
vessels (Fig. 4A). Mast cells in control C57BL mice
showed minimal activation as evidenced by light microscopy (Fig. 4A). Instead, those from stressed mice were
activated to varying degrees, ranging from involvement of
part of the cell (Fig. 4B,C) to almost complete loss of
staining, indicating maximal activation (Fig. 4D). Ultrastructural studies showed that mast cells from control
animals contained mostly intact, round, homogeneous
electron dense secretory granules (Fig. 5A). In contrast,
mast cells from stressed animals were activated to various
degrees ranging from mild secretory granule changes (Fig.
5B), to extensive granule swelling (Fig. 6A,B). Some mast
cells showed complete dissolution of the electron dense
secretory granule content, although frank exocytosis was
not apparent (Fig. 6C). Some of the milder changes
observed with electron microscopy or those mast cells
showing complete loss of staining (e.g. mast cell in Fig.
4D) may not be easily apparent at the light microscopic
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M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
Fig. 2. Heart histamine levels were measured in control (unstressed) and stressed (A) C57BL (n514); (B) W/ W v mast cell deficient mice (n58); (C) 1 / 1
mice (n58); (D) ApoE k / o mice (n58); and (E) composite comparison of the mean cardiac histamine levels from all time points of control and stressed
mice. The mean6S.D. is derived from one measurement from each animal, the number of which is shown in the parentheses.
level; such cells were often excluded from the final counts
leading to underestimation.
The extent of mast cell activation was estimated by
averaging the counts taken at the light microscope by two
different investigators. Acute restraint stress for 30 min
clearly increased the extent of mast cell activation in the
heart in C57BL mice (Fig. 7) from 25.661.8 to
40.962.2% (P,0.05). The extent of activation was also
assessed in ApoE k / o animals that develop coronary
atherosclerosis. Mast cell activation in the heart of unstressed ApoE k / o mice was 26.764.5%, while 30 min
stress increased it to 37.967.3% (P,0.05), this degree of
activation was similar to that of C57BL mice. However,
the number of cardiac mast cells counted from six random
cardiac sections of ApoE k / o mice was 37% higher than
that of C57BL mice. These findings suggest that some of
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
155
Fig. 3. Serum histamine levels were measured in control (unstressed) and stressed (A) C57BL (n514); (B) W/ W v mast cell deficient mice (n58); (C)
1 / 1 mice (n58); (D) ApoE k / o mice (n58); and (E) composite comparison of the mean serum histamine levels from all time points of control and
stressed mice. The mean6S.D. is derived from one measurement from each animal, the number of which is shown in the parentheses.
the cardiac mast cells in ApoE k / o mice may already be
maximally activated and not recognizable by light microscopy.
We also investigated the effect of stress on the wild type
1 / 1 mice of the W/ W v genetically mast cell deficient
mice. Control, unstressed 1 / 1 animals showed
27.464.1% mast cell activation in the heart which was
increased to 46.769.7% (P,0.05) by 30 min stress (Fig.
7). Meanwhile, the number of cardiac mast cells counted
from cardiac sections of 1 / 1 mice was similar to that
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M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
Fig. 4. Light photomicrographs of heart sections from C57BL mice stained with toluidine blue to show cardiac mast cells (A) in control (unstressed) (B, C
and D) stressed mice. Empty granules stain pink (arrowhead) as compared to the dark blue color of intact granules (solid arrows); note almost totally
activated mast cell in (D). Bar510 mm.
from C57BL mice. The W/ W v mast cell deficient mice had
no detectable mast cells in the heart to estimate mast cell
activation either before or after stress.
4. Discussion
The present study demonstrated that acute restraint
stress triggered mouse cardiac histamine release evidenced
by decrease in heart histamine by almost 46% and increase
in serum histamine by about 48%. Mast cell activation was
also documented by light and electron microscopy. Acute
restraint stress in rodents is widely used to study stressrelated events and was also shown to activate mast cells in
the skin [24] and the dura [11]. The stress response, as
indicated by the serum levels of corticosterone, was
equivalent in the C57BL, W/ W v and ApoE k / o mice that
develop atherosclerosis [26]. Consequently, any observed
difference in histamine release and mast cell activation
could not be due to any difference in the activation of the
hypothalamic–pituitary–adrenal axis. Cardiac mast cell
activation was present in control unstressed mice (25.6%
activated, 126.9 ng / ml serum histamine). This could be a
functional response to the stress of handling the mice even
though experiments were carried out inside the animal
facility that had been shown to reduce basal activity from
the higher levels seen in the laboratory [12]. Histamine
measured by RIA includes both bound and unbound amine
in the heart tissue. The serum histamine is mostly unbound
as measurements in plasma did not differ from those in
serum. Blood histamine could, theoretically, come from
any tissue containing mast cells. Our results showed that
significant increase of serum histamine was evident at 15
min, a time possibly too short for histamine from other
sources to reach the systemic circulation. Evidence that
mast cell derived histamine from other tissues, such as the
skin, is not likely to contribute to blood levels significantly
comes from a report showing that serum histamine levels
are not high in systemic mastocytosis patients [27]. The
heart histamine levels at 120 min were not significantly
different from controls. This finding may be due to a
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
157
Fig. 5. Transmission electron micrographs of cardiac mast cells from (A) control, unstressed and (B) stressed C57BL mice showing numerous secretory
granules that have released their contents (arrowheads); note tissue from both control and stressed mice have numerous intact granules (solid arrow). Bar51
mm.
feedback inhibition of serum histamine on further cardiac
histamine release through the activation of auto-inhibitory
H-3 receptors [28]; alternatively, or additionally, cardiac
histamine stores may be replenished over time.
Here, we included ApoE k / o mice in order to investigate whether acute stress would have any different effect
on cardiac histamine release and serum histamine levels in
these mice, since mast cells have been reported to be
associated with atherosclerosis and MI [17]. Our results
showed that both the basal cardiac and serum histamine
levels were significantly higher in ApoE k / o mice than
those in C57BL mice. Although the extent of cardiac mast
cell activation before and after stress was similar between
C57BL mice and ApoE k / o mice, the number of cardiac
mast cells counted in ApoE k / o mice was higher than that
from C57BL mice. We believe that these increases in
histamine levels and number of cardiac mast cells may be
mostly due to mast cell recruitment in relation to mast cell
growth factor C-kit ligand (stem cell factor) that was
increased in association with coronary mastocytosis in
reperfusion ischemia [29]. Alternatively, RANTES (regulated upon activation, normal T cell expressed and
presumably secreted) may be involved as it has been
shown to be particularly chemotactic for mast cells [30].
These findings suggest that an increased number of activated mast cells in the heart and coronary arteries in ApoE
k / o mice may be involved in the pathology of coronary
atherosclerosis. Stress-induced cardiac histamine release
may help explain stress-related myocardial ischemia that
could progress to MI when the underlying coronary
pathology is more severe; this may have been true in the
case of double k / o (ApoE and low-density lipoprotein
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M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
Fig. 7. Scattergram of percent of cardiac mast cell activation in C5
control (unstressed) and S5stressed (for 30 min). C57BL, 1 / 1 and
ApoE k / o mice.
Fig. 6. Transmission electron micrographs of cardiac mast cells from
C57BL stressed mice to show the varying degrees of intragranular
changes, without exocytotic degranulation. (A) partial activation with
some intact granules (solid arrow) still apparent; (B) considerable
activation with most granules altered (arrowhead); (C) complete activation. Bar52 mm.
receptor deficient) mice, in which acute stress induced MI
[31].
Histamine released primarily from the heart [20,32]
during the early phase of acute stress could influence
coronary artery disease by: (a) causing direct constriction
[21]; (b) potentiating other constrictors in atherosclerotic
coronary arteries [33]; (c) increasing the thickness of the
intima [34]; and (d) inducing pro-inflammatory cytokine
production from human coronary endothelial cells [35].
Histamine actually provoked acute coronary spasm in
patients with non-exertional chest pain [36] and plasma
histamine elevations correlated with the onset of acute MI
in patients [37]. Anaphylaxis has also been associated with
increased incidence of MI in two retrospective population
studies [32,38]. Human mast cells also secrete chymase,
which can generate the vasoconstrictive angiotensin II
[19,39,40], as well as histamine-releasing peptides [41],
thus propagating mast cell activation. Histamine would
also sensitize local neurons [22], potentially generating a
pro-arrhythmogenic effect since mast cells are found close
to the sinoatrial node [42], and have been shown to have
functional associations with nerve endings [43]. In fact,
adventitial mast cells in atherosclerotic coronary arteries
were recently found localized close to sensory nerve
endings [23]. Mast cells could serve as a link between the
immune and nervous systems [5,44] since neuropeptides
can augment hypersensitivity reactions [9,45]. For instance, neurotensin is found in the heart [46] and stimulates histamine release from the isolated perfused heart, as
well as from mast cells [47].
Mast cells are necessary for allergic reactions, but they
also release many pro-inflammatory cytokines [7]. Inflammation, especially the involvement of interleukin-6 (IL-6),
is now considered a key factor in cardiovascular pathology
[48,49]. Cardiac mast cells may, therefore, release IL-6 in
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
addition to histamine. Our preliminary results actually
showed that acute restraint stress induced serum increase
in IL-6 that was 4-fold higher in ApoE k / o than in C57BL
mice after 120 min of restraint stress [50]. Activated mast
cells may also be involved in atherosclerotic process since
previous studies showed that mast cell granules were
associated with uptake of low-density lipoprotein by
macrophages [51] that leads to the formation of foam
cells—a characteristic pathological change in early atherosclerosis.
Mast cells are known to be present in the heart and have
been shown to have different characteristics from those in
the skin [18]. The possible role of mast cells in coronary
artery disease (CAD) was reviewed recently [52]. The
notion that mast cells may be involved in the pathophysiology of atherosclerosis [15] is supported by their
increased presence [16,53] and / or activation in atheromatous plaques [54], especially in relation to coronary spasm
[14] and coronary plaque rupture [55]. We showed that
ApoE k / o had increased number of cardiac mast cells,
many of which were degranulated under stress in the
vicinity of lipid congested coronary arteries. Increased
numbers of degranulated mast cells were also observed in
the adventitia of ruptured atherosclerotic coronary artery
plaques in association to MI in humans [17]. Our ultrastructural observations indicated the presence of maximally
activated mast cells that would be missed by light microscopy. Such mast cells have previously been termed
‘phantom’ mast cells [56] and were described in
scleroderma [56], fatal cases of which have been associated with myocardial mast cell infiltration [57]. Moreover,
cardiac mast cells were reported to be increased in
ischemic cardiomyopathy [58] and in reperfusion ischemia
[29].
Mast cell–neuron interactions [8] that are increasingly
implicated in neuroinflammatory disorders [5], have led to
the conclusion that mast cells have a more versatile role
[13]. Acute stress can worsen or precipitate [1–4] cardiac
ischemia especially in patients with CAD [4,59]. The
present findings suggest that cardiac mast cell activation by
acute stress may contribute to this pathology through the
release of histamine or proinflammatory mediators.
Acknowledgements
Thanks are due to Yahsin Tien for her word processing
skills and to Dr. Subimal Basu for his comments and input.
This work was funded by a grant from Kos Pharmaceuticals, Inc. (Miami, FL) to T.C. Theoharides.
References
[1] Deedwania PC. Mental stress, pain perception and risk of silent
ischemia. J Am Coll Cardiol 1995;25:1504–1506.
159
[2] Freeman LJ, Nixon PGF, Sallabank P, Reaveley D. Psychological
stress and silent myocardial ischemia. Am Heart J 1987;114:477–
482.
[3] Deanfield JE, Shea M, Kensett M et al. Silent myocardial ischaemia
due to mental stress. Lancet 1984;2:1001–1005.
[4] Rozanski A, Bairey CN, Krantz DS et al. Mental stress and the
induction of silent myocardial ischemia in patients with coronary
artery disease. N Engl J Med 1988;318:1005–1012.
[5] Theoharides TC. Mast cell: a neuroimmunoendocrine master player.
Int J Tissue React 1996;18:1–21.
[6] Marshall JS, Waserman S. Mast cells and the nerves—potential
interactions in the context of chronic disease. Clin Exp Allergy
1995;25:102–110.
[7] Galli SJ. New concepts about the mast cell. N Engl J Med
1993;328:257–265.
[8] Williams RM, Bienenstock J, Stead RH. Mast cells: the neuroimmune connection. Chem Immunol 1995;61:208–235.
[9] Foreman JC. Neuropeptides and the pathogenesis of allergy. Allergy
1987;42:1–11.
[10] Dimitriadou V, Buzzi MG, Moskowitz MA, Theoharides TC.
Trigeminal sensory fiber stimulation induces morphologic changes
reflecting secretion in rat dura mast cells. Neuroscience 1991;44:97–
112.
[11] Theoharides TC, Spanos CP, Pang X et al. Stress-induced intracranial mast cell degranulation. A corticotropin releasing hormonemediated effect. Endocrinology 1995;136:5745–5750.
[12] Pang X, Alexacos N, Letourneau R et al. A neurotensin receptor
antagonist inhibits acute immobilization stress-induced cardiac mast
cell degranulation, a corticotropin-releasing hormone-dependent
process. J Pharm Exp Ther 1998;287:307–314.
[13] Gurish MF, Austen KF. The diverse roles of mast cells. J Exp Med
2001;194:1–6.
[14] Forman MB, Oates JA, Robertson D et al. Increased adventitial mast
cells in a patient with coronary spasm. N Engl J Med
1985;313:1138–1141.
[15] Constantinides P. Infiltrates of activated mast cells at the site of
coronary atheromatous erosion or rupture in myocardial infarction.
Circulation 1995;92:1083–1088.
[16] Kaartinen M, Penttila¨ A, Kovanen PT. Accumulation of activated
mast cells in the shoulder region of human coronary atheroma, the
predilection site of atheromatous rupture. Circulation 1994;90:1669–
1678.
[17] Laine P, Kaartinen M, Penttila¨ A et al. Association between
myocardial infarction and the mast cells in the adventitia of the
infarct-related coronary artery. Circulation 1999;99:361–369.
[18] Patella V, de Crescenzo G, Ciccarelli A et al. Human heart mast
cells: a definitive case of mast cell heterogeneity. Int Arch Allergy
Immunol 1995;106:386–393.
[19] Jenne DE, Tschopp J. Angiotensin II-forming heart chymase is a
mast-cell-specific enzyme. Biochem J 1991;276:567.
[20] Gristwood RW, Lincoln JC, Owen DA, Smith IR. Histamine release
from human right atrium. Br J Pharmacol 1981;74:7–9.
[21] Ginsburg R, Bristow MR, Davis K. Receptor mechanisms in the
human epicardial coronary artery. Heterogenous pharmacological
response to histamine and carbachol. Circ Res 1984;55:416–421.
[22] Christian EP, Undem BJ, Weinreich D. Endogenous histamine
excites neurones in the guinea-pig superior cervical ganglion in
vitro. J Physiol 1989;409:297–312.
[23] Laine P, Naukkarinen A, Heikkila L, Penttila¨ A, Kovanen PT.
Adventitial mast cells connect with sensory nerve fibers in atherosclerotic coronary arteries. Circulation 2000;101:1665–1669.
[24] Singh LK, Pang X, Alexacos N, Letourneau R, Theoharides TC.
Acute immobilization stress triggers skin mast cell degranulation via
corticotropin releasing hormone, neurotensin and substance P: A
link to neurogenic skin disorders. Brain Behav Immun
1999;13:225–239.
[25] Letourneau R, Pang X, Sant GR, Theoharides TC. Intragranular
160
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
M. Huang et al. / Cardiovascular Research 55 (2002) 150 – 160
activation of bladder mast cells and their association with nerve
processes in interstitial cystitis. Br J Urol 1996;77:41–54.
Peidrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N.
Generation of mice carrying a mutant apolipoprotein E gene
inactivated by gene targeting in embryonic stem cells. Proc Natl
Acad Sci USA 1992;89:4471–4475.
¨
Granerus G, Lonnqvist
B, Roupe G. No relationship between
histamine release measured as metabolite excretion in the urine, and
serum levels of mast cell specific tryptase in mastocytosis. Agents
Actions 1994;41:C127–C128.
Levi R, Smith NCE. Histamine H 3 -receptors: a new frontier in
myocaridal ischemia. J Pharmacol Exp Ther 2000;292:825–830.
Frangogiannis NG, Perrard JL, Mendoza LH et al. Stem cell factor
induction is associated with mast cell accumulation after canine
myocardial ischemia and reperfusion. Circulation 1998;98:687–698.
Conti P, Reale M, Barbacane RC, Letourneau R, Theoharides TC.
Intramuscular injection of hrRANTES causes mast cell recruitment
and increased transcription of histidine decarboxylase: lack of
effects in genetically mast cell-deficient W/ W v mice. FASEB J
1998;12:1693–1700.
Caligiuri G, Levy B, Pernow J, Thoren P, Hansson GK. Myocardial
infarction mediated by endothelin receptor signaling in hypercholesterolemic mice. Proc Natl Acad Sci USA 1999;96:6920–6924.
Levi R, Burke JA. Cardiac anaphylaxis: SRS-A potentiates and
extends the effects of released histamine. Eur J Pharmacol
1980;62:41–49.
Ginsburg R, Bristow MR, Davis K, Dibiase A, Billingham ME.
Quantitative pharmacologic responses of normal and atherosclerotic
isolated human epicardial coronary arteries. Circulation
1984;69:430–440.
Miyazawa N, Watanabe S, Matsuda A et al. Role of histamine H1
and H2 receptor antagonists in the prevention of intimal thickening.
Eur J Pharmacol 1998;362:53–59.
Li Y, Chi L, Stechschulte DJ, Dileepan KN. Histamine-induced
production of interleukin-6 and interleukin-8 by human coronary
artery endothelial cells is enhanced by endotoxin and tumor necrosis
factor-alpha. Microvasc Res 2001;61:253–262.
Ginsburg R, Bristow MR, Kantrowitz N, Baim DS, Harrison DC.
Histamine provocation of clinical coronary artery spasm: implications concerning pathogenesis of variant angina pectoris. Am Heart J
1981;102:819–822.
Zaca F, Benassi M-S, Ghinelli M et al. Myocardial infarction and
histamine release. Agents Actions 1986;18:258–261.
Schwartz HJ, Yunginger JW, Schwartz LB. Is unrecognized
anaphylaxis a cause of sudden unexpected death? Clin Exp Allergy
1995;25:866–870.
Urata H, Ganten D. Cardiac angiotensin II formation: the angiotensin-I converting enzyme and human chymase. Eur Heart J
1993;14:177–182.
Takai S, Jin D, Sakaguchi M, Miyazaki M. Chymase-dependent
angiotensin II formation in human vascular tissue. Circulation
1999;100:654–658.
Cochrane DE, Carraway RE, Feldberg RS, Boucher W, Gelfand JM.
Stimulated rat mast cells generate histamine-releasing peptide from
albumin. Peptides 1993;14:117–123.
[42] Hellstrom B, Holmgren H. Numerical distribution of mast cells in
the human skin and heart. Acta Anat 1995;10:81–107.
[43] Rozniecki JJ, Dimitriadou V, Lambracht-Hall M, Pang X,
Theoharides TC. Morphological and functional demonstration of rat
dura mast cell–neuron interactions in vitro and in vivo. Brain Res
1999;849:1–15.
[44] Theoharides TC. Mast cells: the immune gate to the brain. Life Sci
1990;46:607–617.
[45] Goetzl EJ, Cheng PPJ, Hassner A et al. Neuropeptides, mast cells
and allergy: novel mechanisms and therapeutic possibilities. Clin
Exp Allergy 1990;20:3–7.
[46] Reinecke M, Weihe E, Carraway RE, Leeman SE, Forssmann WG.
Localization of neurotensin immunoreactive nerve fibers in the
guinea-pig heart: evidence derived by immunohistochemistry,
radioimmunoassay
and
chromatography.
Neuroscience
1982;7:1785–1795.
[47] Carraway R, Cochrane DE, Lansman JB et al. Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates
plasma histamine levels. J Physiol 1982;323:403–414.
[48] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med
1999;340:115–126.
[49] Yudkin JS, Kumari M, Humphries SE, Mohamed-Ali V. Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the
link? Atherosclerosis 2000;148:209–214.
[50] Huang M, Basu S, Pang X et al. Stress-induced interleukin-6 release
in mice is mast cell-dependent and also involves cardiomyocytes
stimulated by urocortin. FASEB J 2002;16:A182.
[51] Kovanen PT. Mast cell granule-mediated uptake of low density
lipoproteins by macrophages: a novel carrier mechanism leading to
the formation of foam cells. Ann Med 1991;23:551–559.
[52] Kelley JL, Chi DS, Abou-Auda W, Smith JK, Krishnaswamy G. The
molecular role of mast cells in atherosclerotic cardiovascular
disease. Mol Med Today 2000;6:304–308.
[53] Bankl HC, Radaszkiewicz T, Klappacher GW et al. Increase and
redistribution of cardiac mast cells in auricular thrombosis: possible
role of kit ligand. Circulation 1995;91:275–283.
[54] Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast
cells at the site of coronary atheromatous erosion or rupture in
myocardial infarction. Circulation 1995;92:1084–1088.
[55] Kaartinen M, Van Der Wai AC, Van Der Loos CM et al. Mast cell
infiltration in acute coronary syndromes: implications for plaque
rupture. J Am Coll Cardiol 1998;32:606–612.
[56] Claman HN. On scleroderma: mast cells, endothelial cells and
fibroblasts. J Am Med Assoc 1989;262:1206–1209.
[57] Lichtbroun AS, Sandhaus LM, Giorno RC, Kim H, Seibold JR.
Myocardial mast cells in systemic sclerosis: a report of three fatal
cases. Am J Med 1990;89:372–376.
[58] Patella VP, Marino I, Arbustini E et al. Stem cell factor in mast cells
and increased mast cell density in idiopathic and ischemic cardiomyopathy. Circulation 1998;97:971–978.
[59] Jain D, Burg M, Soufer R, Zaret BL. Prognostic implications of
mental stress-induced silent left ventricular dysfunction in patients
with stable angina pectoris. Am J Cardiol 1995;76:31–35.