Circulation. 1998;98:699-710
(Circulation. 1998;98:699-710.)
© 1998 American Heart Association, Inc.
Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-
, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion
Nikolaos G. Frangogiannis, MD;
Merry L. Lindsey, BA;
Lloyd H. Michael, PhD;
Keith A. Youker, PhD;
Robert B. Bressler, MD;
Leonardo H. Mendoza, MS;
Robert N. Spengler, PhD;
C. Wayne Smith, MD;
; Mark L. Entman, MD
From the Section of Cardiovascular Sciences, Department of Medicine, The
Methodist Hospital and the DeBakey Heart Center, the Department of
Microbiology and Immunology, the Speros P. Martel Laboratory, Section of
Leukocyte Biology, Department of Pediatrics and Texas Children's
Hospital, Baylor College of Medicine, Houston, Tex, and the Department of
Pathology (R.N.S.), State University of New York at Buffalo.
Correspondence to Mark L. Entman, MD, Department of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston, TX 77030-3498. E-mail mentman{at}bcm.tmc.edu
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Abstract
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Background—Neutrophil-induced
cardiomyocyte injury requires the expression of myocyte
intercellular adhesion molecule (ICAM)-1 and ICAM-1–CD11b/CD18
adhesion. We have previously demonstrated interleukin (IL)-6 activity
in postischemic cardiac lymph; IL-6 is the primary stimulus
for myocyte ICAM-1 induction. Furthermore, we found that induction of
IL-6 mRNA occurred very early on reperfusion of the infarcted
myocardium. We hypothesized that the release of a preformed
upstream cytokine induced IL-6 in leukocytes infiltrating on
reperfusion.
Methods and Results—Constitutive expression of TNF- and not
IL-1ß was demonstrated in the normal canine myocardium
and was localized predominantly in cardiac mast cells. Mast cell
degranulation in the ischemic myocardium was
documented by demonstration of a rapid release of histamine and TNF-
in the cardiac lymph after myocardial ischemia. Histochemical
studies with FITC-labeled avidin demonstrated degranulating mast cells
only in ischemic samples of canine myocardium.
Immunohistochemistry suggested that degranulating mast cells were the
primary source of TNF- in the ischemic
myocardium. In situ hybridization studies of reperfused
myocardium localized IL-6 mRNA in infiltrating mononuclear
cells and in mononuclear cells appearing in the
postischemic cardiac lymph within the first 15 minutes of
reperfusion. Furthermore, isolated canine mononuclear cells incubated
with postischemic cardiac lymph demonstrated significant
induction of IL-6 mRNA, which was partially blocked with a neutralizing
antibody to TNF-.
Conclusions—Cardiac mast cells degranulate after myocardial
ischemia, releasing preformed mediators, such as histamine and
TNF-. We suggest that mast cell–derived TNF- may be a crucial
factor in upregulating IL-6 in infiltrating leukocytes and initiating
the cytokine cascade responsible for myocyte ICAM-1 induction
and subsequent neutrophil-induced injury.
Key Words: cells • ischemia • reperfusion • myocardial infarction • cytokines
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Introduction
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The association
of inflammation with myocardial infarction has been recognized for more
than a century1 and is properly considered part
of the healing process. In recent years, the potential role of
accelerated inflammation in extension of injury after reperfusion of
the infarcted myocardium has been suggested by a variety of
experimental studies. Strategies designed to deplete
neutrophils,2 inhibit
complement,3 or block critical adhesion molecules
controlling leukocyte trafficking4 5 have been
effective in reducing infarct size.
Our laboratory has concentrated on characterizing the biological basis
for inflammatory injury in a canine model of reperfused myocardial
infarction. Early in vitro experiments suggested that neutrophil
adhesion to cardiac myocytes is dependent on CD18 integrin
activation6 and also on the induction of ICAM-1
on cardiac myocytes.7 Additional studies showed
that Mac-1/ICAM-1–dependent neutrophil adherence to cardiac myocytes
activates the neutrophil respiratory burst accompanied by a
highly compartmented transfer of reactive oxygen and resultant myocyte
oxidative injury.8 Thus, we postulated that
induction of myocyte ICAM-1 was an essential factor for
neutrophil-induced cardiac injury.
We developed a canine model of a chronically cannulated cardiac lymph
duct to obtain cardiac extracellular fluid under conditions in which
the inflammatory mediators associated with acute surgery have
dissipated. After reperfusion of the infarcted myocardium,
we demonstrated the rapid appearance in cardiac lymph of activity
capable of stimulating ICAM-1 expression on isolated cardiac
myocytes.9 10 The observation that an anti–IL-6
antibody neutralized the synthesis of ICAM-1 in cardiac myocytes
stimulated by postischemic cardiac lymph suggested that
myocyte ICAM-1 induction was mediated by IL-6.9
These findings led to in vivo experiments on ischemic and
reperfused myocardium that demonstrated ICAM-1 mRNA
induction in the viable myocytes on the border of infarcted
tissue.10 11 IL-6 mRNA induction was seen (with
an earlier peak) in the myocardium in the same
ischemic segments in which ICAM-1 mRNA is
found.12 The early IL-6 induction and subsequent
ICAM-1 upregulation was dependent on reperfusion of the
ischemic myocardium. Because leukocyte influx is
similarly dependent on reperfusion, we have postulated that leukocytes
participate in the cascade leading to myocyte ICAM-1
induction.12
Recent findings indicate that mast cells can influence biological
responses through the production of cytokines. Gordon
and Galli13 identified mouse mast cells as the
first example of a cell type that contains stores of preformed TNF-.
They suggested a potential role of mast cell activation, in part
through the release of TNF-, in influencing the recruitment and
function of additional effector cells.14
Furthermore, Ito and colleagues15 recently
presented evidence suggesting that porcine cardiac mast cell
degranulation occurs after intracoronary infusion of C5a.
The present study was designed to investigate the role of
resident cardiac mast cells in myocardial ischemia/reperfusion
as a potential source of preformed TNF-. Our data suggest that mast
cell degranulation and TNF- release initiate a cytokine
cascade involving IL-6 induction in infiltrating mononuclear cells and
subsequent ICAM-1 induction in cardiac myocytes.
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Methods
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Ischemia/Reperfusion Protocols
We used a chronic canine model of myocardial ischemia
and reperfusion.10 12 This model includes
placement of a hydraulically activated occluder on the left
circumflex coronary artery and cannulation of the cardiac lymph
duct. After surgery, the animals were allowed to recover for 72 hours
before occlusion. Ischemia/reperfusion protocols were performed
in awake animals as described.10 The
coronary artery was occluded by inflation of the
coronary cuff occluder until mean flow in the coronary
vessel was zero, as determined by the Doppler flow probe. At the
end of 1 hour, the cuff was deflated and the myocardium was
reperfused. Reperfusion intervals ranged from 1 to 3 hours. During the
experiment, the cardiac lymph was collected in 30-minute intervals. The
samples were centrifuged, and the supernatant was collected and
immediately frozen at -80°C. The pelleted cells were fixed in 4%
paraformaldehyde and used for
histological studies. After the reperfusion periods,
hearts were stopped by the rapid intravenous infusion of 30
mEq of KCl and removed from the chest for sectioning from apex to base
into 4 transverse rings 
1 cm thick. The posterior papillary muscle
and the posterior free wall were identified. Tissue samples were
isolated from infarcted or normally perfused myocardium on
the basis of visual inspection. Myocardial segments were fixed for
histological analysis. Duplicate samples were
also processed for blood flow determinations with radiolabeled
microspheres as previously described.10
Samples described as ischemic were all from areas in which
ischemic blood flow was <25%. Samples of control tissues were
taken from the anterior septum and had normal blood flow during
coronary occlusion.
Immunohistochemistry and Histology
For histological study of cardiac tissue,
sections taken from endocardium to epicardium were fixed in 4%
phosphate-buffered formalin, 2%
paraformaldehyde, and Carnoy's, Mota's, or B*5
fixatives and embedded in paraffin. Sequential 2- to 5-µm sections
were cut by microtomy. Sections were immunostained for
TNF- adjacent to serial sections stained for tryptase with an
enzymatic stain as described by Caughey et al.16
Chymase activity was detected as previously described by
Seppa.17 Immunostaining was
performed with the ELITE rabbit or mouse kit (Vector Laboratories)
according to the manufacturer's instructions. The following primary
antibodies were used: rabbit polyclonal antibody to human TNF-
(Genzyme) known to cross-react with canine
TNF-,18 polyclonal antibody to human IL-1ß
(Genzyme) known to cross-react with canine
species,19 and the neutrophil-specific monoclonal
antibody SG8H6.20 Antibody was detected with a
peroxidase-based system using DAB (Vector Laboratories) as a substrate.
Slides were counterstained with eosin. Appropriate controls were
performed with rabbit or mouse serum substituted for the primary
antibody. Fluorescent labeling of mast cells with FITC-avidin
was performed as previously described by Bergstresser and
colleagues.21 Double-fluorescent staining
was done by fluorescent immunohistochemistry using a
rhodamine-labeled anti-rabbit IgG as a secondary antibody and
counterstaining with FITC-avidin.
Histamine Assay
Histamine in lymphatic drainage was measured by competitive
immunoenzymatic assay22 (AMAC Inc).
TNF- Bioassay
Cardiac lymph samples were assayed for TNF- activity by use
of the WEHI 164 subclone 13 fibroblast cytotoxicity assay, as
previously described.23 The WEHI 164 cells are
very sensitive to the lytic effects of both murine and human TNF-,
detecting as little as 2 pg/mL. The cytotoxic effect of canine rTNF-
on these cells was recently demonstrated.24 WEHI
cells (5x105 /mL) were cultured in 96-well
microtiter plates (Costar) with test samples and 1 µg/mL actinomycin
D (Carbiochem, Boehring Diagnostics). After 20 hours'
incubation at 37°C, 180 µL of supernatant in each well was replaced
with 180 µL of fresh culture medium with 1 µg/mL of actinomycin D.
A 5-mg/mL MTT (20 µL; Sigma) solution was added to all wells, and the
plates were incubated at 37oC. After 4 hours of
incubation, 150 µL of supernatant was removed and discarded from all
wells, and 100 µL of a 0.04N HCl/isopropanol solution was added to
each well to dissolve the crystals characteristic of this particular
assay. Plates were wrapped in aluminum foil and stored overnight in a
dark moist area at room temperature. The level of lysis was determined
with a microELISA Autoreader (550 nm). Units of activity were
calculated according to internal rTNF- standards (Genzyme).
Riboprobe Preparation
Digoxigenin-labeled probes were prepared by in vitro
transcription from a linearized template according to the method used
by Boehringer Mannheim in the Genius RNA probe labeling kit. A
216-bp fragment of canine IL-6 cDNA was obtained from the published
sequence by PCR amplification and was subcloned into the PCR plasmid
(Invitrogen) so that the use of SP6 polymerase would result in the
generation of single-stranded antisense (3'-5') and the use of T7
polymerase would result in the generation of the sense (5'-3') RNA
probe. Before beginning the transcription reaction, we linearized the
DNA templates by digestion with restriction enzymes that cut downstream
of the insert to avoid transcription of undesirable plasmid sequences.
The template (1 µg) was incubated in 20 µL of 1xNTP mixture
(1 mmol/L ATP, 1 mmol/L GTP, 1 mmol/L CTP, 0.35
mmol/L digoxigenin-UTP, and 0.65 mmol/L UTP), T7 or T3 polymerase
(2 U/µL), and DEPC-treated water for 2 hours at 37°C. Both RNA
probes were precipitated with glycogen and sodium acetate, washed with
70% ethanol, and resuspended in DEPC-treated water. Both probes were
verified by hybridization and detection on a Southern blot (both
positive) and a Northern blot (antisense positive) on nylon
membrane.
In Situ Hybridization in Tissue Sections
Paraffin-embedded samples fixed with 2%
paraformaldehyde were sectioned and deparaffinized by
standard protocols and probed with the IL-6–specific
digoxigenin-labeled riboprobes. Immunological detection used an
alkaline phosphatase–labeled anti-digoxigenin antibody
(Boehringer Mannheim) and nitro blue tetrazolium staining of
the alkaline phosphatase reaction as previously
described.11 In additional experiments, in situ
hybridization was followed by immunostaining with the
mouse monoclonal antibody SG8H6, which specifically stains
neutrophils.20 The antibody was detected by use
of a peroxidase-based system with 3-amino-9-ethylcarbazole as a
substrate.
Mononuclear Cell Isolation and Stimulation
Canine mononuclear cells were isolated by use of a
Ficoll/hypaque gradient and resuspended in PBS without calcium and
magnesium. For incubation experiments with cardiac lymph, aliquots of
lymph were obtained before coronary occlusion and during
reperfusion. Mononuclear cells were incubated for 2 hours at 37°C in
the presence or absence of recombinant human TNF- (200 U/mL)
(Genzyme). The postischemic cardiac lymph used for these
experiments was collected during the first 2 hours of reperfusion.
Blocking studies were performed with the addition of the polyclonal
neutralizing antibody to human TNF- IP-300 (7 µL/mL) (Genzyme),
which is known to cross-react with canine species. After incubation, a
500-µL aliquot of cells from each tube was removed and fixed in 50%
ethanol for 15 to 30 minutes, then resuspended in 75% ethanol and
stored at -20°C. The stored cells were subsequently used for in situ
hybridization studies. Analysis of IL-6 induction in isolated
canine mononuclear cells was performed as follows. Random fields from
slides stained for IL-6 were examined with a light microscope at x400.
Five hundred cells from each slide were counted, and the percentage of
IL-6 positive cells was calculated. Subsequently, numbers of
IL-6–positive cells from different incubation conditions were
normalized on the basis of the percentage of positive cells seen in the
control sample (medium=1). Eight consecutive experiments were used for
quantitative analysis, and ANOVA was used to assess the
statistical significance of the findings, followed by Student's
t test with Bonferroni correction for multiple
comparisons.
Statistical Analysis and Sampling
The statistical significance of rising levels of histamine and
TNF- in cardiac lymph was assessed by ANOVA. This was followed by a
Student's t test, corrected for multiple comparisons
(Bonferroni). In histological studies, each experiment
and time point was analyzed as a function of time of
reperfusion after 1 hour of occlusion. The findings described all
occurred in at least 5 consecutive experiments, and degranulation was
observed only in ischemic segments. Induction of IL-6 in
mononuclear cells was analyzed as described above.
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Results
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Characterization of Canine Cardiac Mast Cells
Samples from control canine heart were stained with toluidine blue
(Figure 1a
) and FITC-avidin (Figure 1b),
demonstrating a sizable canine cardiac mast cell population, located
primarily along vessels. Two populations of mast cells were apparent: 1
resembled "typical" or connective-tissue–type mast cells, which
stained with toluidine blue regardless of fixation, and the other
population resembled "atypical" or mucosal-type mast cells and
exhibited metachromasia only after fixation in Carnoy's or Mota's
fixative.25 Histochemical techniques were used to
identify tryptase and chymase activity and demonstrated the presence of
tryptase and chymase in most canine cardiac mast cells (Figure 1c and 1d).
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Figure 1. Canine heart contains sizable mast cell
population. a, Control canine heart stained with toluidine
blue and counterstained with fast green (x400). A significant number
of metachromatic cells are seen. b, Control canine heart stained with
FITC-avidin to identify resident mast cells (arrows). c, Histochemical
staining for tryptase (x400). d, Staining for chymase (x400). Note
that mast cells are frequently perivascular.
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TNF- and Not IL-1ß Is Constitutively Expressed in Control
Canine Heart and Localized Predominantly in Mast Cells
TNF- immunoreactivity was present in many resident cells in
the canine myocardium (Figures 2 and 3).
The positively labeled cells were predominantly perivascular and
resembled metachromatically granulated mast cells in number and
morphology. Comparison of adjacent 5-µm sections from control canine
heart revealed that TNF- was localized predominantly to mast cells
(Figure 2A, 2B, 2C, and 2D), identified by their unique granule content
of the neutral protease tryptase (Figure 2C and 2D). Double-labeling
studies combining fluorescent immunocytochemistry for TNF-
and FITC-avidin counterstaining showed granular cytoplasmic TNF-
immunoreactivity to be confined almost exclusively to
FITC-avidin–labeled mast cells (Figure 3). At the antibody
concentration used in these studies (Figures 2 and 3), no TNF-
staining was detected in any other cell type found in the
myocardium. Similar immunohistochemical experiments using
an antibody to IL-1ß demonstrated no IL-1ß immunoreactivity in the
control canine heart (Figure 4).
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Figure 4. IL-1ß is not constitutively expressed in canine
heart. A, Peroxidase-based immunohistochemistry shows no
IL-1ß immunoreactivity in control canine heart. B, Section from
spleen of an endotoxin-stimulated animal was used as a positive
control.
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Histamine Release in the Postischemic Cardiac Lymph
During Reperfusion
Histamine concentrations in the cardiac lymph were measured in
samples collected from 8 ischemia/reperfusion experiments
(Figure 5). Histamine concentration in
the preischemic cardiac lymph was 2257±239 pmol/L.
Significant elevations of histamine levels in the
postischemic cardiac lymph were noted in 7 of 8 experiments
of coronary occlusion (0 to 30 minutes: 2.15±0.31-fold
increase; P<0.05, n=8; histamine concentration 4646±824
pmol/L; range, 1184 to 9986 pmol/L).
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Figure 5. Kinetics of histamine release in
postischemic cardiac lymph. Histamine concentration in
cardiac lymph was measured in 8 consecutive experiments of experimental
canine myocardial infarction. Concentrations from each experiment were
normalized (pre=1). A significant early increase in histamine
concentration was noted (0 to 30 minutes, 2.15±0.3-fold increase;
*P<0.05 vs preischemic lymph). Number of
experiments of ischemia/reperfusion: n=8 for pre, 0 to 30
minutes, and 30 to 60 minutes; and n=6 for 60 to 120 minutes and 120 to
180 minutes.
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Histochemical and Immunohistochemical Evidence of Mast Cell
Degranulation
Sections from ischemic and reperfused
myocardium were stained with FITC-labeled avidin. As
demonstrated in Figure 6A, mast cells in
the ischemic and reperfused myocardium showed
significant degranulation. In contrast, mast cells in normally perfused
segments appeared to be fully granulated (Figure 6B). Degranulating
mast cells were absent in the control canine heart.
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Figure 6. Mast cell degranulation in ischemic
myocardium. Samples obtained from animal exposed to 1 hour
of coronary occlusion and 3 hours of reperfusion. Sections were
stained with FITC-labeled avidin to identify mast cells (x1000).
Degranulating mast cells were found in ischemic myocardial
segments (A), whereas mast cells in control segments appeared fully
granulated (B).
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Immunostaining for TNF- in ischemic sections
after 1 hour of coronary occlusion and 3 hours of reperfusion
showed that TNF- immunoreactivity was localized predominantly in
mast cells. No significant IL-1ß expression was noted in the
ischemic heart in early reperfusion. Some of the
TNF-–positive cells in the ischemic segments showed
evidence of degranulation, whereas all the mast cells in the normally
perfused tissue samples appeared to be fully granulated. Figure 7 shows a transmyocardial section
spanning the region from the endothelial surface to
400 µm into the wall. The mast cells in the subendocardial area
show evidence of TNF- egress (degranulation), whereas those from the
midmyocardium (well outside the infarct zone) are not
degranulated. In the area of degranulation only, leukocyte and
endothelial staining is seen (Figure 8); the potential significance of the
latter staining will be discussed below.
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Figure 7. TNF- immunoreactivity in ischemic and
reperfused myocardium. Transmural 300-µm segment from 1
hour ischemia and 3 hours reperfusion, spanning from injured
subendocardial area to normal epicardial region (top, x100). Note that
significant mast cell degranulation is found only in cells located in
injured subendocardial area (B) (bottom, x400). Mast cells in
epicardial region appear fully granulated (A) (bottom, x400). Staining
of endothelium and infiltrating cells is also noted in
area of mast cell degranulation.
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Presence of TNF- Activity in the Cardiac Lymph After
Ischemia/Reperfusion
TNF- bioactivity was measured in lymph samples collected during
the ischemia/reperfusion experiments, and the values were
expressed as a percentage of the baseline value (Figure 9). TNF- bioactivity in the
preischemic cardiac lymph was 2.6±1.17 pg/mL. Release of
TNF- bioactivity in the cardiac lymph was noted in all experiments
and peaked in the first 30 minutes of reperfusion (0 to 30 minutes,
7.94±3.11-fold increase; P<0.05, n=8; range, 0.26 to 31.5
pg/mL). A second peak in the third hour after reperfusion was noted in
2 experiments.
Localization of IL-6 mRNA in Mononuclear Cells Infiltrating the
Ischemic and Reperfused Myocardium
In situ hybridization for IL-6 mRNA was performed in sections of
canine myocardium obtained after occlusion and reperfusion
experiments (Figure 10). By examining
samples obtained after 1 hour of occlusion and 1 hour of reperfusion,
we demonstrated that IL-6 mRNA was localized predominantly in
infiltrating mononuclear leukocytes. Minimal staining was observed in
endothelium, smooth muscle cells, and myocytes. There
was no staining in normally perfused samples. Serial sections incubated
with the sense riboprobe demonstrated no staining.
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Figure 10. Cellular origin of IL-6 in ischemic and
reperfused myocardium (1 hour occlusion and 3 hours
reperfusion). Ischemic myocardial segments demonstrated
induction of IL-6 mRNA localized predominantly in infiltrating
leukocytes identified as mononuclear cells (arrowheads). These cells
did not stain with neutrophil-specific antibody SG8H6 (x400). Several
neutrophils are also identified (arrows) and do not stain for
IL-6.
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Samples of cardiac lymph taken in the first 15 minutes of reperfusion
were studied by in situ hybridization, and the isolated leukocytes
demonstrated IL-6 mRNA expression (Figure 11). No staining was seen in cardiac
lymph–derived leukocytes obtained before coronary occlusion.
In both Figures 10
and 11, the IL-6–positive cells showed
morphological characteristics of mononuclear cells. This was confirmed
by the absence of staining for the neutrophil-specific antibody SG8H6
(Figures 10 and 11) in the leukocytes staining for IL-6 mRNA in both
tissue and lymph.
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Figure 11. Early appearance of IL-6 mRNA in cardiac
lymph–derived leukocytes. In situ hybridization with antisense probe
for canine IL-6 (blue) followed by immunohistochemistry with
neutrophil-specific antibody SG8H6 developed with DAB (black).
Leukocytes obtained during first 15 minutes of reperfusion
consistently demonstrated IL-6 mRNA induction and were
identified morphologically as mononuclear cells (arrowhead). These
cells did not stain with SG8H6, which was used to identify neutrophils
(arrows).
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Postischemic Lymph Induction of IL-6 mRNA Is Inhibited
by an Anti–TNF- Antibody
Canine mononuclear cells were incubated with
preischemic and postischemic cardiac lymph.
After a 2-hour incubation with a 1:10 dilution of the cardiac lymph,
the cells were fixed in ethanol and in situ hybridization for IL-6 was
performed. Cells incubated with recombinant human TNF- were used as
a positive control and demonstrated significant induction of IL-6
(2.29±0.39-fold, P<0.05 compared with mononuclear cells
incubated with medium, n=8), which was inhibited with addition of a
TNF- antibody. The cardiac lymph from 8 consecutive experiments of
myocardial ischemia was used. In 7 of 8 experiments, incubation
with postischemic cardiac lymph significantly increased the
number of IL-6 mRNA–positive cells (1.96±0.29-fold,
P<0.05 compared with control mononuclear cells, n=8). In
contrast, incubation with preischemic cardiac lymph did not
induce IL-6 mRNA in mononuclear cells (0.89±0.19; P>0.5,
n=8). Addition of a neutralizing antibody to TNF- markedly reduced
the percentage of mononuclear cells showing IL-6 mRNA expression
(1.11±0.24, P<0.05 compared with mononuclear cells
stimulated with postischemic cardiac lymph, n=8) (Figure 12). In 2 experiments, inhibition of
IL-6 induction after addition of the antibody to TNF- was partial
(<50% reduction in the number of IL-6–positive cells), and in 1
experiment, no inhibition was noted.
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Figure 12. TNF- is an important upstream cytokine
responsible for IL-6 mRNA induction in canine mononuclear cells
stimulated with cardiac lymph. Mononuclear cells were fixed and stained
for IL-6 mRNA by in situ hybridization after incubation with TNF-,
TNF- plus a neutralizing antibody to TNF-,
preischemic cardiac lymph (1:10 dilution), and
postischemic cardiac lymph (1:10 dilution) with and without
TNF- antibody. Percentage of IL-6–positive cells was counted and
normalized (medium alone=1). Incubation of canine mononuclear cells
with TNF- demonstrated a 2.29±0.39-fold increase in number of
IL-6–positive mononuclear cells (*P<0.05 vs
mononuclear cells incubated with medium only, n=8), which was
neutralized with addition of an antibody to TNF-
(**P<0.05 vs TNF-–stimulated mononuclear cells).
Stimulation with early postischemic cardiac lymph caused a
1.96-fold±0.29 increase in number of IL-6–positive cells
(*P<0.05 vs control mononuclear cells, n=8). Incubation
with a blocking antibody to TNF- significantly decreased number of
IL-6 positive mononuclear cells (PO, 1.96±0.29 vs PO+A, 1.04±0.26;
#P<0.05, n=8), suggesting that TNF- is a significant
factor in postischemic cardiac lymph responsible for IL-6
induction in mononuclear cells. Cardiac lymph from 8 consecutive
experiments of myocardial ischemia was used for these
experiments (n=8). Incubation conditions: medium, TNF- (200 U/mL);
TNF-+anti–TNF- antibody; pre, preischemic lymph
(1:10 dilution); post, postischemic lymph (1:10 dilution);
and post+Ab, postischemic lymph+anti–TNF-
antibody.
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Discussion
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In this report, we investigated the "upstream" components of
the cytokine cascade ultimately responsible for ICAM-1
induction in the reperfused myocardium. The ability of
postischemic cardiac lymph to induce ICAM-1 in myocytes was
neutralized by an antibody to IL-6,9 and IL-6 was
shown to be a potent inducer of ICAM-1 on cardiac myocytes. In
contrast, ICAM-1 expression in canine endothelial cells
stimulated with postischemic cardiac lymph was not
neutralized by anti–IL-6 antibody,9 and IL-6
does not stimulate endothelial ICAM-1 in culture. The
following observations led us to hypothesize the presence of other
cytokines in the cardiac lymph that must be of importance in
acute inflammation after reperfusion of the ischemic
myocardium.
1. With regard to the induction of endothelial ICAM-1,
most data suggested IL-1 or TNF- to be the potential
cytokine; with respect to the cytokine activity of the
cardiac lymph, we demonstrated that the presence of excess IL-1
inhibitory activity precludes its effect in inducing ICAM-1
in cultured endothelial cells.9
Furthermore, immunohistochemical experiments indicated that IL-1ß was
not constitutively expressed in the control heart (Figure 4) and was
not found in ischemic segments after 1 hour of reperfusion.
This led us to postulate that TNF- was a likely source of
cytokine activity in the cardiac lymph that might be
responsible for induction of ICAM-1 in the
endothelium.
2. Because of the relationship between IL-6 and cardiac myocyte ICAM-1
induction, we evaluated the induction of IL-6 and found that it
occurred very early on reperfusion. IL-6 mRNA was found after
reperfusion of severely ischemic segments only and, during the
first 3 hours of reperfusion, was completely dependent on reperfusion
of the previously ischemic myocardium. This led us
to hypothesize that IL-6 induction is related to the influx of
leukocytes into the ischemic myocardium on
reperfusion.12 The data in this study confirm
this hypothesis and demonstrate the early induction of IL-6 mRNA in
mononuclear cells found in the reperfused myocardium,
compatible with the early appearance of IL-6 activity in the cardiac
lymph capable of myocyte ICAM-1 induction.9 The
experiments shown in Figure 12 support the hypothesis that preformed
TNF- may be an important upstream cytokine responsible for
the rapid induction of IL-6 mRNA in the ischemic and reperfused
myocardium.
Potential Role for Mast Cells
The presence of mast cells in the heart has been previously
described in several species; in a recent report, Patella et
al26 isolated and characterized human heart mast
cells by demonstrating the presence of chymase and tryptase. They also
showed that incubation of human heart mast cells with C5a caused a
rapid and dose-dependent release of histamine.26
Furthermore, Ito et al15 demonstrated in pig
heart that cardiac mast cells, when exposed to C5a, rapidly
degranulated and released histamine and thromboxane
B2. In previous studies,27
we have shown the presence of both C5a and thromboxane
B2 in postischemic cardiac lymph.
This suggested to us that mast cell degranulation might be an important
part of the ischemia/reperfusion process. The discovery that
mast cells are an important source of cytokines has suggested
new ways in which mast cell activation may influence inflammatory
responses.14 28 Significant evidence in the
literature indicates that preformed cytokines, specifically
TNF-, exist in various mast cell populations and that
cytokine release can be induced by Fc
RI
ligation.13 14 28 Recently, evidence has arisen
that mast cells might also release cytokines in response to
anaphylatoxins during a reverse passive Arthus
reaction.29
Evidence for Degranulation of Cardiac Mast Cells in the
Ischemic and Reperfused Myocardium
We used 2 independent methods to assess and characterize
mast cell degranulation during ischemia and reperfusion. Each
of these methods has limitations that are partially addressed by the
other; together, they provide strong evidence for a role of mast cells
in the release of TNF- during the acute inflammatory injury
accompanying reperfusion of the ischemic
myocardium.
Cardiac Lymph
We have demonstrated a rapid increase in the level of histamine
and TNF- in postischemic cardiac lymph (Figures 5 and 9). These studies used a model developed in our laboratory that allows
collection of cardiac lymph from chronically instrumented animals in
which all the inflammatory sequelae of the instrumentation surgery have
dissipated.10 30 Although this model has been
used extensively in our research, 2 important articles require review
here to understand the significance of the present findings. First,
in previous experiments,27 we have demonstrated a
prompt rise of thromboxane B2 during
early reperfusion, which may provide additional evidence for mast cell
degranulation during the ischemia and reperfusion events. The
second important point stems from one of our earliest studies, in which
we measured the appearance of creatine kinase and
phosphorylase b in the cardiac
lymph.31 Because these enzymes egress the
infarcted myocardium regardless of the presence of
reperfusion, it was of interest that we could measure rises in their
activity only on reperfusion of the ischemic
myocardium. It became obvious that the absence of perfusion
of the ischemic bed markedly diminished its contribution to
cardiac lymph so that the time course of creatine kinase or
phosphorylase appearance in the cardiac lymph probably
represented, at least in part, the washout of previously
existing enzymes in the extracellular fluid.31
Washout of a dye injected into the infarct during occlusion followed a
similar time course. This is pertinent to the present experiments
because the time course for egress of histamine and TNF- (as well as
thromboxane B227) is
very rapid, with significant activity seen within the first 15 minutes
of reperfusion. We would interpret these data as suggesting that at
least some of the mast cell degranulation antecedes reperfusion, which
suggests that degranulation may be initiated by preexisting autacoids
found in the ischemic myocardium.
Immunohistochemistry
Our initial studies established the presence of preformed TNF-
in cardiac mast cells in normal myocardium in both control
hearts (Figures 2 and 3) and control areas of ischemic and
reperfused dog heart. Although no method can absolutely rule out the
presence of some TNF- in other cells, the intense staining of
TNF- observed in mast cells (identified as tryptase and
FITC-avidin–positive cells) in control myocardial sections in the
absence of any other staining certainly suggests that the mast cell
must be a highly significant source of preformed TNF-.
To investigate degranulation, we used the FITC-avidin staining
technique as a simple method for identifying mast cells and
immunohistochemical techniques to evaluate TNF- egress from mast
cells. Both of these experiments provided the same information.
Degranulating mast cells were seen only in the area of necrosis and the
viable area bordering the infarct. In an effort to demonstrate this,
Figure 7 shows a transmural segment of myocardium of
300 µm that spans a subendocardial myocardial infarct with
mast cell degranulation and a normal area toward the epicardium with
fully granulated mast cells. Infiltrating leukocytes and
endothelium in this area are also stained, although
much less intensely. In the more external area of the section, where
degranulation is not seen, there are no infiltrating leukocytes and the
endothelium is not stained. The less intense TNF-
staining of the infiltrating leukocytes and endothelium
is most likely a result of binding of the secreted TNF (Figure 8); new
synthesis in these cell types cannot be ruled out. It should be
emphasized, however, that histochemical determination of mast cell
degranulation is not sensitive enough to detect mast cells undergoing a
slower degranulation process in cardiac mast cells, for which the term
"piecemeal degranulation" has been used.32
For that, we have also relied on the cardiac lymph studies described
above.
Initiation of Mast Cell Degranulation
The data in this article do not speak to the stimulus for mast
cell degranulation, but several candidates are obvious. C5a is known to
induce degranulation in cardiac mast cells26 and
is present exclusively in the area surrounding the myocardial
injury before initiation of reperfusion.33 In
addition, adenosine has been shown to induce mast cell
degranulation through an A3
receptor34 and would be expected to be increased
in ischemic areas. Reactive oxygen has been shown to induce
mast cell degranulation35 and might be an
important factor in early reperfusion when production of
reactive oxygen is highest. Finally, the C-C chemokine monocyte
chemoattractant protein-1 has been shown to be a stimulator of mast
cell degranulation36; we have shown this
chemokine to be induced in the previously ischemic
myocardium during reperfusion,37 but
its presence is not important until hours 2 and 3 of reperfusion. In
view of the piecemeal degranulation seen in cardiac mast cells in other
systems,32 it is possible that several of these
autacoids are important.
Cellular Origin of IL-6
We have previously demonstrated that induction of IL-6 occurs in
the ischemic injured myocardium and requires
reperfusion of the previously ischemic
myocardium.12 Our present studies
showed that IL-6 mRNA could be detected only in SG8H6-negative
mononuclear cells (Figure 10). In addition, similar studies in cardiac
lymph cells demonstrated the presence of IL-6–positive mononuclear
cells in the cardiac extracellular fluid within 15 minutes of
reperfusion (Figure 11). These findings suggest a very potent inducing
stimulus present from the initiation of reperfusion (because IL-6
induction did not occur in the absence of reperfusion). Our data
suggest a major role for TNF- as an upstream cytokine
inducer after myocardial ischemia.
Hypothesized Cytokine Cascade
In summary, the experiments allow us to refine our hypothesis
regarding the role of mast cells in the cellular responses to injury
governed by cytokine induction. We have demonstrated mast cell
degranulation by 2 independent techniques and shown that it is confined
to the injured area of the ischemic and reperfused
myocardium. This degranulation results in rapid release of
preformed TNF- into the ischemic area. TNF- is known to
induce IL-6 in endothelial
cells,38 fibroblasts,39
neutrophils,40 and mononuclear
cells41–43; our experiments indicate that the
primary cell responsible for IL-6 mRNA expression in early reperfusion
of the previously ischemic myocardium is the
infiltrating mononuclear cell and suggest that TNF- released from
preformed stores in cardiac mast cells has a crucial role in inducing
IL-6 in infiltrating mononuclear cells. As shown in Figure 13, several stimuli pertinent to
ischemia may induce mast cell degranulation; it is possible
that >1 of these stimuli are important. Histamine may also be an
important autacoid in the reaction to injury ensuing on reperfusion of
the ischemic myocardium. Histamine has been shown
to enhance cytokine-induced IL-6 synthesis via activation
of H2 receptors.44 In
addition, histamine can induce leukocyte rolling in vivo by stimulating
surface expression of P-selectin from Weibel-Palade bodies in venular
endothelium.45 The role of
P-selectin in leukocyte margination during early ischemia has
been proposed by several investigators.45 46 47
Mast Cells in Ischemia and Reperfusion
The role of mast cells in the injury associated with
myocardial ischemia and reperfusion was initially suggested by
Jolly and coworkers.48 Furthermore, a growing
body of evidence supports a role for mast cells in leukocyte
recruitment associated with intestinal
ischemia/reperfusion.49 Recent
experiments suggest mast cell degranulation after intestinal
ischemia and reperfusion,50 closely
associated with leukocyte rolling and adhesion.51
Oxidants and anaphylatoxins49 were suggested as
potential factors responsible for mast cell activation. This report
seeks to further identify the mechanisms by which mast cells
participate in ischemia-reperfusion injury.
Mast Cells in the Healing Phase of a Reperfused Myocardial
Infarct
In the companion article,52 a
potential role for mast cells in the later cellular events after
myocardial infarction and reperfusion is described. Taken together,
these reports suggest that the mast cell may play a critical role in
the reaction to myocardial injury.
|
Selected Abbreviations and Acronyms
|
|---|
| DAB |
= |
diaminobenzidine |
| ICAM-1 |
= |
intercellular adhesion molecule-1 |
| IL |
= |
interleukin |
| TNF- |
= |
tumor necrosis factor- |
|
|
Acknowledgments
|
|---|
This work was supported by NIH grant HL-42550. Merry L. Lindsey
is supported by the Baylor College of Medicine Graduate Program in
Cardiovascular Sciences training grant HL-07816. The
authors wish to thank Concepcion Mata and Sharon Malinowski for their
editorial assistance with the manuscript and Evelyn Brown, Peggy
Jackson, Gary Liedtke, Richard Chou, Alida Evans, and Margaret Jones
for their outstanding technical assistance.
|
Footnotes
|
|---|
Guest editor for this article was Benedict R. Lucchesi, MD, PhD, University of Michigan, Ann Arbor.
Received December 17, 1997;
revision received February 18, 1998;
accepted March 1, 1998.
|
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Abstract
Introduction
