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DeBakey Heart Center, Section of Cardiovascular Sciences, Department of Medicine, The Methodist Hospital, Houston, TX 77030; and
Speros P. Martel Laboratory, Section of Leukocyte Biology, Department of Pediatrics, Texas Children’s Hospital, Baylor College of Medicine, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release, IL-6
induction, and subsequent neutrophil-mediated cytotoxic injury. Because
inflammation is also an important factor in cardiac repair, we
hypothesized the presence of components of the inflammatory reaction
with a possible role in suppressing acute injury. Thus, we investigated
the role of IL-10, an anti-inflammatory cytokine capable of
modulating extracellular matrix biosynthesis, following an experimental
canine myocardial infarction. Using our canine model of myocardial
ischemia and reperfusion, we demonstrated significant up-regulation of
IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10
expression was first detected at 5 h and peaked following 96–120
h of reperfusion. In contrast, IL-4 and IL-13, also associated with
suppression of acute inflammation and macrophage deactivation, were not
expressed. In the ischemic canine heart, CD5-positive lymphocytes were
the predominant source of IL-10 in the myocardial infarct. In the
absence of reperfusion, no significant induction of IL-10 mRNA was
noted. In addition, IL-12, a Th1-related cytokine associated with
macrophage activation, was not detected in the ischemic myocardium. In
vitro experiments demonstrated late postischemic cardiac-lymph-induced
tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in
isolated canine mononuclear cells. This effect was inhibited when the
incubation contained a neutralizing Ab to IL-10. Our findings suggest
that lymphocytes infiltrating the ischemic and reperfused myocardium
express IL-10 and may have a significant role in healing by modulating
mononuclear cell phenotype and inducing TIMP-1
expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies from our laboratory have demonstrated that monocyte chemoattractant protein (MCP)3-1 is markedly induced in the ischemic and reperfused myocardium (4), suggesting a significant role for MCP-1 in mononuclear cell (monocyte and lymphocyte) trafficking in the reperfused myocardium (5). In the absence of reperfusion, no significant MCP-1 mRNA induction is seen (4). Monocytes and lymphocytes chemotactically attracted in the reperfused myocardium may have an important role in ventricular repair by modulating the inflammatory response.
It is generally accepted that T lymphocytes mediate immunologic
responses by release of cytokines. Mossman et al. (6)
identified two very distinct cytokine secretion patterns among a panel
of mouse T cell clones, introducing the concept of Th1 and Th2 cells.
Th1 cells produce macrophage-activating cytokines, whereas Th2 cells
augment humoral responses and inhibit Th1 responses (7).
Th1 and Th2 responses have been implicated as regulators of
cell-mediated immunity, as important mediators in clearance of many
infectious organisms (8), and in the immunopathology of
organ-specific autoimmune diseases (9, 10). IL-10
(6, 11), a cytokine initially described as cytokine
synthesis inhibitory factor (12), is primarily a product
of activated Th2 cells and endotoxin-stimulated monocytes
(11). Among the different cell types affected by IL-10,
monocyte macrophages appear to be particularly modified in regard to
their function, morphology, and phenotype (11). IL-10
inhibits the production of IL-1, IL-1ß, TNF-, IL-6, and IL-8 by
LPS-activated monocytes (13, 14), suppressing the
inflammatory response. IL-10 also suppresses expression of IL-12
(11), a cytokine primarily produced by activated monocytes
and a dominant factor in directing Th1-type responses (15, 16). Furthermore, IL-10 may have a significant role in
extracellular matrix formation by modulating expression of
metalloproteinases and tissue inhibitor of metalloproteinases (TIMP)
(17, 18).
In this study, we investigated the regulation of T cell-derived lymphokines using a canine model of experimental myocardial ischemia and reperfusion. We found significant numbers of infiltrating CD5-positive T cells in the reperfused myocardium. We provide the first evidence that IL-10 is induced in the ischemic and reperfused myocardium; over the same time period, IL-6 mRNA expression is down-regulated. IL-10 induction was highly dependent upon reperfusion of the previously ischemic myocardium. In contrast, the Th1-related cytokine, IL-12, was not detected. We identified infiltrating T lymphocytes and a subset of macrophages as the predominant source of IL-10 in the healing myocardium. Furthermore, we demonstrated that IL-10 is responsible for cardiac-lymph-mediated TIMP-1 up-regulation in isolated canine mononuclear cells. Our findings suggest a potential role for lymphocyte-derived IL-10 in suppressing proinflammatory cytokine synthesis by macrophages infiltrating the ischemic myocardium and in modulating scar formation and maturation following experimental myocardial infarction. This may represent an important mechanism explaining the benefit associated with late reperfusion of the ischemic myocardium when significant myocardial salvage cannot be expected (19, 20, 21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy mongrel dogs (15–25 kg) of either sex were surgically
instrumented as previously described (22, 23, 24). Anesthesia
was induced i.v. with 10 mg/kg methohexital sodium (Brevital, Eli
Lilly, Indianapolis, IN) and maintained with the inhalational
anesthetic isoflurane (Anaquest, Madison, WI). A midline thoracotomy
provided access to the heart and mediastinum, and cannulation of the
cardiac lymph duct was then performed. Subsequently, a hydraulically
activated occluding device and a Doppler flow probe were secured around
the circumflex coronary artery just proximal or just distal to the
first branch. Choice of location depended on the proximity and
anatomical arrangement. Indwelling catheters placed in the right
atrium, left atrium, and femoral artery allowed blood sampling and
pressure monitoring as needed. After surgery, the animals were allowed
to recover for 72 h before occlusion. Coronary artery occlusion
was achieved by inflating 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 h, the cuff was deflated and the myocardium was
reperfused. Reperfusion intervals ranged from 1 h to 7 days.
Circumflex blood flow, arterial blood pressure, heart rate, and
electrocardiogram (standard limb II) were recorded continuously.
Analgesia was accomplished with 0.1–0.2 mg/kg i.v. administered
pentazocine (Talwin, Winthrop Pharmaceuticals, New York, NY). After the
reperfusion periods, hearts were stopped by the rapid i.v. infusion of
30 milliequivalent KCl and removed from the chest for sectioning from
apex to base into four transverse rings 
1 cm in thickness. The
posterior papillary muscle and the posterior free wall were identified.
Tissue samples were isolated from infarcted or normally perfused
myocardium based on visual inspection. Myocardial segments were fixed
in 10% buffered formalin, Carnoy’s fixative, or B*5 fixative
(25) for histological analysis or immediately frozen,
homogenized, and processed for RNA extraction. Duplicate adjacent
samples were also processed for blood flow determinations using
radiolabeled microspheres as previously described (22).
Six ischemia/reperfusion experiments were performed for each
reperfusion interval.
The presence of a myocardial infarct was based on light-microscopic examination of hematoxylin-eosin-stained tissue sections by findings of contraction bands, "wavy fibers," interstitial edema, and neutrophil infiltration, all in segments displaying markedly reduced blood flow (<25% control) during the ischemic period. For experiments lasting 24 h or more after the start of the ischemic insult, the presence of histological elements characteristic of myocyte necrosis and fibrosis was added to the required criteria. Samples described as ischemic were all from areas where ischemic blood flow was <25%. Samples of control tissues were taken from the anterior septum and had normal blood flow during coronary occlusion.
Tissue samples from endotoxemic animals were used as positive controls for Northern hybridization and immunohistochemical experiments. Endotoxemia was achieved by i.v. injection of 500 µg/kg Escherichia coli endotoxin (Sigma, St. Louis, MO). Animals were sacrificed 3 h later, and the spleen, heart, lung, liver, and kidney were used for mRNA extraction and histological studies.
Immunohistochemistry and histology
For histological study of cardiac tissue, sections taken from endocardium to epicardium and sections taken parallel to the wall of the heart were fixed in 10% phosphate-buffered formalin or B*5 fixatives and embedded in paraffin. Immunohistochemistry for IL-10 was successful only with the use of the B*5 fixative. Sequential 3- to 5-µm sections were cut by microtomy. Immunostaining was performed using the Elite rabbit or mouse kit (Vector Laboratories, Burlingame, CA). Briefly, sections were pretreated with a solution of 3% hydrogen peroxide to inhibit endogenous peroxidase activity and incubated with 2% goat serum to block nonspecific protein binding. Subsequently, they were incubated with the primary Ab for 2 h at room temperature. After rinsing with PBS, the slides were incubated for 30 min with the secondary Ab. The slides were rinsed with PBS and incubated for 30 min in ABC reagent. Peroxidase activity was detected using diaminobenzidine with nickel. Slides were counterstained with eosin.
The following primary Abs were used for immunohistochemistry: rabbit anti-human polyclonal Ab to IL-10 (Genzyme, Cambridge, MA), rabbit anti-human Ab to TIMP-1 (Chemicon, Temecula, CA), monoclonal anti-bovine Ab to TIMP-1 (Chemicon), monoclonal anti-CD5 Ab (VMRD, Pullman, WA), rabbit anti-human CD3 Ab (Dako, Carpinteria, CA), mouse anti-human CD79a clone HM57 Ab (Dako), and monoclonal anti-human macrophage Ab AM-3K (26, 27, 28) (a generous gift from Dr. Takahashi, Kumamoto University, Japan). For T and B lymphocyte immunolabeling, staining with Abs to CD5 and CD3 with known cross-reactivity to canine species (29) was used. Sections from the spleen of an endotoxin-stimulated animal were used as positive controls. Fluorescent immunohistochemistry was achieved by using appropriate rhodamine- or Cy3-labeled secondary Abs (Sigma). Appropriate positive and negative controls were used for each Ab. Dual immunohistochemistry was performed by combining peroxidase-based immunostaining for CD5 with fluorescent immunohistochemistry for IL-10 rhodamine-labeled secondary Abs.
Quantitative analysis of immunohistochemical experiments
Stained sections were photographed with a Leaf MicroLumina digital camera (Leaf Systems, Southboro, MA) mounted on a Zeiss microscope (Oberkochen, Germany). Multiple digital images were taken and stored for each sample. Staining was analyzed by Zeiss image-analysis software.
Molecular cloning
Specific canine cDNA clones for IL-4, IL-10, IL-13, IL-12 p40, and TIMP-1 were prepared by reverse transcription using RNA extracted from Con A plus PMA-stimulated mononuclear cells (for IL-4 and IL-13), or the spleen of an endotoxin-stimulated animal (for IL-10, IL-12 p40, and TIMP-1). RT-PCR protocols were performed using the following primers: IL-10 sense primer, 5'-CTTGCTCGAGGACTTTAAGG-3'; IL-10 antisense primer, 5'-CTTGCTCTTGTTCTCACAGGGC-3'; IL-4 sense primer, 5'-ATCACCTTACAAGAGATCATCAA-3'; IL-4 antisense primer, 5'-CTTGGCTTCATTCACAGAACAG-3'; IL-13 sense primer, 5'-GCCCTCAGGGAGCTCATTGAGGAGCTG-3'; IL-13 antisense primer, 5'-ACATTTTTGACATCAGAAAGAAAAATGA-3'; IL-12 p40 sense primer, 5'-CTCACCTGTGACACCCCTGAAG; IL-12 p40 antisense primer, 5'-GCAGAGAGTGTAGCAGCTCCAC; TIMP-1 sense primer, 5'-TCGTCATCAGGGCCAAGTTCGTGGGGA-3'; and TIMP-1 antisense primer, 5'-AGCTGGTCCGTCCACAAGCAATGA-3'. The nucleotide sequence of the primers was based on areas of the published sequence for their human homologues that showed a high degree of interspecies conservation. Reverse transcription protocols were performed with 5 µg of total RNA. After first-strand synthesis was primed with the antisense primer, aliquots of the reverse-transcription reaction were amplified using 5 U Taq DNA polymerase (Promega, Madison, WI) for 30 cycles at 93°C for 1 min, 55°C for 2 min, and 72°C for 3 min. The resulting fragments were purified, cloned in the PCR vector (Invitrogen, San Diego, CA), and sequenced.
RNA isolation
RNA isolation from myocardial tissue segments was performed using the acid-guanidinium-phenol-chloroform procedure. RNA (20 µg) was electrophoresed in 1% agarose gels containing formaldehyde and then transferred to a nylon membrane (Gene Screen Plus; New England Nuclear, Boston, MA) by standard procedures.
Northern hybridization
Membranes were hybridized in QuikHyb (Stratagene, La Jolla, CA) at 68°C for 2 h with 1 x 106 dpm random hexamer 32P-labeled canine cDNA probes for IL-10, IL-4, IL-13, IL-12 p40, IL-6, and TIMP-1. Filters were washed with 2x standard saline citrate phosphate/EDTA (SSPE) at 68°C for 20 min, with 1x SSPE plus 1% SDS at 68°C for 15 min twice, and with 1x SSPE at 21°C for 15 min with constant shaking, and were exposed to Hyperfilm (Amersham, Arlington Heights, IL). Quantitation of the Northern hybridization results was performed using densitometry. Relative density was normalized to the intensity of the 28S ribosomal RNA as previously described (30, 31) and expressed as the following ratio: (IL-10 relative density: 28S relative density) x 100.
Mononuclear cell isolation and stimulation
Canine mononuclear cells were isolated by use of a Ficoll-Hypaque gradient and resuspended in PBS as previously described (32). For incubation experiments with cardiac lymph, aliquots of lymph were obtained before coronary occlusion and during reperfusion. Mononuclear cells were incubated for 18 h at 37°C in the presence or absence of 50–100 ng/ml recombinant human IL-10 (R&D Systems, Minneapolis, MN). The postischemic lymph used for these experiments was collected following 24–48 h of reperfusion. Blocking studies were performed with the addition of 5 µl/ml polyclonal neutralizing Ab to human IL-10 (R&D Systems). After incubation, RNA was extracted from the mononuclear cells as previously described, and Northern hybridization for TIMP-1 was performed. Four experiments, using the cardiac lymph obtained from four different animals that demonstrated significant ischemia, were performed.
Statistical analysis
The statistical significance of the findings was assessed by ANOVA. This was followed by a Student’s t test and corrected for multiple comparisons (Bonferroni). In histological studies, each experiment and time point was analyzed as a function of time of reperfusion after 1 h of occlusion. The findings described all that occurred in at least three consecutive experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using RT-PCR techniques, we obtained partial clones for canine IL-4, IL-10, IL-12 p40, IL-13, and TIMP-1. The partial clones for IL-4 (245 bp), IL-10 (206 bp), IL-12 p40 (403 bp), and TIMP-1 (405 bp) showed excellent homology with the previously reported canine sequences (33, 34, 35, 36). The percent identity with their respective human homologues was 75% for IL-4, 87% for IL-10, 87% for IL-12 p40, and 80% for TIMP-1. The partial clone for IL-13 (387 bp) showed a 79% identity with its human homologue (37).
IL-10 and not IL-4 or IL-13 is induced following myocardial ischemia/reperfusion
The expression of IL-10 mRNA after experimental circumflex
coronary artery occlusion and reperfusion was assessed using Northern
hybridization with a 32P-labeled canine IL-10
probe. IL-10 mRNA was not detected in normally perfused myocardial
segments. In contrast, high levels of IL-10 mRNA expression were noted
in experiments of 1 h of ischemia and 4 days of reperfusion, with
the highest levels observed in the most ischemic myocardial segments
(Fig. 1
A). A trend to
increased induction of IL-10 mRNA was first noted after 3 h of
reperfusion (Figs. 2A and
3). IL-10 mRNA expression in ischemic
segments peaked at 24–96 h (Figs. 2B and 3), with levels
decreasing toward baseline after 7 days (Figs. 2C and 3).
Northern hybridization was performed using the IL-4 and IL-13 cDNA
clones. No induction of IL-4 and IL-13 was observed in the canine
myocardium (data not shown).
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We assessed the role of reperfusion in IL-10 mRNA induction
following myocardial ischemia by comparing experiments with and without
reperfusion. In contrast to the relatively high levels of IL-10 mRNA
expression noted in the ischemic and reperfused myocardium, in
nonreperfused experiments undergoing 24 h and 96 h (Fig. 4) of coronary occlusion, we detected
minimal levels of IL-10 mRNA, in the presence of comparable degrees of
myocardial blood flow reductions.
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Previous studies from our laboratory (24) have
demonstrated early induction of IL-6 mRNA (peaking at 3 h of
reperfusion, Ref. 24), which was markedly enhanced during
the first 6 h by reperfusion of the previously ischemic
myocardium; however, by 24 h, IL-6 induction was also
observed in nonreperfused infarcts. Our current experiments suggested
that IL-6 mRNA expression, which is maximal at 3 h of reperfusion,
is down-regulated after 24 h of reperfusion in the same ischemic
segments in which IL-10 mRNA induction is found (Fig. 4
). In contrast,
animals with permanent occlusion showed minimal IL-10 mRNA
up-regulation and high IL-6 mRNA expression, which persisted for
96 h.
IL-10 immunoreactivity in the ischemic and reperfused myocardium
To examine the localization of IL-10 immunoreactivity in the
ischemic myocardium, we performed immunohistochemical studies using a
rabbit anti-human polyclonal Ab to IL-10. Sections from the spleen
of an endotoxin-stimulated animal were used as a positive control (Fig. 5A). In the ischemic
myocardium, some cells with IL-10 immunoreactivity were identified
first after 5 h of reperfusion (8.23 ± 1.89
cells/mm2, Fig. 5B) and became more
numerous with longer reperfusion intervals (72–120 h), peaking after 5
days of reperfusion (22.5 ± 2.7 cells/mm2,
Fig. 5, C and D).
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Using immunohistochemical techniques, we demonstrated a striking
infiltration of the ischemic and reperfused myocardium with
AM-3K-positive macrophages (Fig. 6A) and T lymphocytes labeled
with Abs to CD3 and CD5 (Fig. 6B). A resident macrophage
population was identified in the canine heart as previously
demonstrated (28). A much smaller resident T lymphocyte
population was also found. In contrast, CD79-positive B lymphocytes
were very rare in control and ischemic canine myocardium. Macrophage
and lymphocyte accumulation started as early as 5 h of reperfusion
and was markedly increased after 72–120 h of reperfusion (Fig. 6, C and D). Following 5 days of reperfusion, there
was an 8-fold increase of macrophage numbers and a similar increase of
lymphocyte numbers in ischemic segments vs the respective controls
(Fig. 6, C and D).
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IL-10-positive cells demonstrated characteristics of mononuclear
cells. Staining of serial sections and dual immunohistochemistry
localized IL-10 immunoreactivity in lymphocytes identified by their
positive staining for the T cell markers CD3 (Fig. 7) and CD5 (Fig. 8) at as early as 5 h of reperfusion
(Fig. 7). A subset of macrophages also produced IL-10. However, the
majority of the macrophages did not demonstrate IL-10 immunoreactivity
(Fig. 7).
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Northern hybridization studies demonstrated negligible amounts of
IL-12 p40 mRNA (the inducible chain of IL-12) in control canine heart
and in ischemic segments from experiments of canine ischemia and
reperfusion (Fig. 9). RNA extracted from
the spleen of an endotoxin-stimulated animal was used as a positive
control and showed robust expression of IL-12.
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Isolated canine mononuclear cells were stimulated with human
recombinant IL-10 and pre- and postischemic cardiac lymph. IL-10 and
late postischemic cardiac lymph collected from 24 to 48 h of
reperfusion were capable of up-regulating TIMP-1 mRNA expression (Fig. 10). Furthermore, incubation with a
neutralizing Ab to human IL-10 significantly inhibited the ability of
postischemic cardiac lymph to induce TIMP-1 in isolated mononuclear
cells (postischemic cardiac lymph plus Ab 1.36 ± 0.15 vs
postischemic cardiac lymph 3.58 ± 0.88; p <
0.05, n = 4) (Fig. 10B).
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Immunohistochemical experiments using two different Abs to TIMP-1
demonstrated significant TIMP-1 protein expression in healing
myocardial infarcts after 72–120 h of reperfusion (Fig. 11). TIMP-1-expressing cells were rare
after 5 h of reperfusion. Most TIMP-1-positive cells were
identified as macrophages using serial section staining with the
macrophage-specific Ab, AM-3K (Fig. 11, D and E).
In addition, some spindle-shaped myofibroblast-like cells also
expressed TIMP-1 (Fig. 11). TIMP-1 immunoreactive cells were
predominantly located in the border zone of reperfused myocardial
infarcts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release
(32) and IL-6 mRNA up-regulation (24). IL-6,
predominantly induced in infiltrating mononuclear cells
(32), may be a crucial factor in mediating myocyte ICAM-1
expression in the ischemic border zone of reperfused myocardial
infarcts (39). IL-10 inhibits synthesis of various
cytokines (including IL-1, TNF-, IL-6, GM-CSF, and IL-8) by
stimulated monocyte/macrophages, regulates matrix metalloproteinase
expression by mononuclear cells (17) and fibroblasts
(18), enhances monocyte growth and differentiation
(40), modulates neutrophil function and apoptosis
(41, 42, 43, 44), and induces mast cell growth (45, 46). In the present work, we show the first evidence that IL-10
is induced following myocardial ischemia and reperfusion. We
demonstrated that infiltrating lymphocytes are the predominant cell
type that is positive for IL-10 in the healing myocardium. Our findings
suggest that IL-10 may be an important factor in up-regulating TIMP-1
induction in infiltrating mononuclear cells, adding a new aspect to its
potential beneficial effects in promoting healing of the injured
myocardium.
IL-4, IL-10, and IL-13 have potent macrophage deactivating effects,
markedly inhibiting cytokine expression (47, 48, 49).
Endogenous IL-10 production has been described in models of endotoxemia
(10, 11, 50), atherosclerotic plaques (51),
myocarditis (52), and allograft rejection (53, 54). Furthermore, increased levels of plasma IL-10 were noted in
patients with complicated acute myocardial infarction (Ref.
55 , Killip class II–IV). Our experiments suggest
endogenous induction of IL-10 in the ischemic heart following
experimental canine myocardial ischemia and reperfusion. IL-10 mRNA
(
Figs. 1–3) and protein (Figs. 5 and 7) were expressed in the ischemic
areas only as early as the first 5 h of reperfusion and could be
detected for 7 days. Maximal IL-10 expression was seen from 24 to
120 h of reperfusion (Figs. 2 and 3). In contrast, no IL-4 and
IL-13 expression was noted in the ischemic myocardium. IL-10 can be
produced by monocytes/macrophages, lymphocytes, and keratinocytes
(11). We demonstrated that IL-10 immunoreactivity is
predominantly localized in lymphocytes infiltrating the reperfused
myocardium (Figs. 7 and 8). In addition, a small subset of macrophages
was stained for IL-10. IL-10 strongly inhibits its own synthesis by
macrophages (14); in part, this may explain its limited
expression in macrophages of the healing heart. Furthermore, IL-10
production by T cells and monocytes is differentially regulated; IL-6
can induce the expression of IL-10 by stimulated T cells, whereas
TNF- stimulates IL-10 synthesis by monocytes (56). Data
from our laboratory have described the sequential release of TNF-
from mast cells followed by induction of IL-6 in early reperfusion
(32).
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). We postulate that this results from increased
lymphocyte access to the ischemic region and from the
reperfusion-dependent up-regulation of cytokines potentially
responsible for IL-10 expression (such as IL-6, Ref. 56)
and mononuclear cell chemotaxis (such as MCP-1, Ref.
4).
Macrophages infiltrating the ischemic myocardium can support a number
of different responses depending on the local microenvironment.
Monocyte/macrophages are the predominant source of IL-6 mRNA in the
reperfused myocardium and may have a significant role in mediating the
inflammatory response associated with myocardial ischemia/reperfusion.
IL-10 is capable of modulating the phenotype and functional activity of
monocyte/macrophages. The production of IL-1, TNF-, IL-6, GM-CSF,
IL-12 (11), IFN-
-inducible protein-10, and
macrophage-inflammatory protein-1 (57) by
endotoxin-stimulated macrophages can be inhibited by IL-10. Kukielka et
al. (24) have previously demonstrated marked IL-6
up-regulation in the ischemic and reperfused myocardium, peaking after
1–3 h of reperfusion. Furthermore, Chandrasekar et al.
(58) have recently demonstrated a delayed induction of
IL-6 following occlusion without reperfusion of rat myocardium. Our
current experiments showed decreased expression of IL-6 mRNA following
24–96 h of reperfusion when IL-10 is maximally induced (Fig. 4). In
addition, experiments of coronary occlusion without reperfusion
demonstrated minimal induction of IL-10 mRNA. In contrast, IL-6
expression, which was delayed to 24 h in permanently occluded
studies (Ref. 24 , Fig. 4), persisted for 96 h. We
speculate that reperfusion-dependent IL-10 expression modulates the
IL-6 response and may be an important factor regulating proinflammatory
cytokine synthesis in the ischemic myocardium.
Recently, Lacraz et al. (17) demonstrated that IL-10
inhibits metalloproteinase and stimulates TIMP-1 production in human
macrophages. This alteration of the proteinase/antiproteinase balance
in favor of matrix preservation may be important in suppressing the
inflammatory response following myocardial ischemia. The importance of
IL-10 in modulating collagen metabolism was suggested by a recent study
demonstrating a very low percentage of collagen in atherosclerotic
lesions of IL-10-deficient mice (59). Our experiments
demonstrate that IL-10 is an important factor in enhancing TIMP-1
expression in mononuclear cells stimulated with late postischemic
cardiac lymph (Fig. 10). Significant numbers of TIMP-1-positive
macrophages are noted in the healing scar after 72–120 h (Fig. 11) of
reperfusion when IL-10 expression is maximal. These findings suggest an
important role for IL-10 in scar formation and maturation following an
experimental myocardial infarction. Other studies (40)
suggest that IL-10 may act as a cytokine, enhancing monocyte survival,
growth, and differentiation into macrophages by cooperating with M-CSF.
Recent experiments from our laboratory demonstrated induction of M-CSF
following myocardial ischemia and reperfusion (60). These
effects may be of significance in the maturation of monocytes
chemotactically attracted in the ischemic myocardium by MCP-1.
It is generally accepted that activated T cells mediate immunologic
responses by differentiating into one of two subsets, Th1 and Th2,
characterized by the release of distinct types of cytokines; Th1 cells
activate proinflammatory effector mechanisms and are associated with
macrophage activation, whereas Th2 cells inhibit Th1 responses and
down-regulate local inflammation. IL-12 and IL-10 are two important
cytokines regulating the Th1 and Th2 responses. IL-12 is a 70-kDa
protein heterodimer (termed p70), of which the p40 component is the
major inducible chain. It is primarily produced by activated monocytes
and selectively induces the cytolytic activity of a number of effector
cells, stimulating the Th1-cytokine pattern, which is associated with
macrophage activation. In contrast, IL-10 facilitates Th2 responses and
down-regulates IL-12 production. Our studies demonstrated marked IL-10
mRNA and protein induction but no significant IL-12 p40-mRNA (Fig. 9)
expression in the reperfused myocardium. Following myocardial ischemia
and reperfusion, IL-10 may be important in down-regulating IL-12
expression and in shifting the T cell response toward a Th2
direction.
In summary, our studies demonstrated a marked reperfusion-dependent induction of IL-10 following myocardial ischemia. IL-10 was predominantly localized in lymphocytes infiltrating the reperfused myocardium. We suggest a potential role for infiltrating lymphocytes in suppressing the acute inflammatory response by modulating macrophage-cytokine synthetic activity and function, and in promoting metabolic steps associated with tissue repair. This may represent an important aspect of the beneficial effect of reperfusion in healing of the injured myocardium.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nikolaos G. Frangogiannis, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza M/S F-602, Houston, TX 77030.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; TIMP, tissue inhibitor of metalloproteinase; SSPE, standard saline citrate phosphate/EDTA.
Received for publication January 24, 2000. Accepted for publication June 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation 98:699. and IL-6 by human monocytes. Blood 76:1392.
B (NF-B) activation in human monocytes. IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J. Biol. Chem. 270:9558.. J. Immunol. 157:12.[Abstract]
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D. G. Souza, C. T. Fagundes, F. A. Amaral, D. Cisalpino, L. P. Sousa, A. T. Vieira, V. Pinho, J. R. Nicoli, L. Q. Vieira, I. M. Fierro, et al. The Required Role of Endogenously Produced Lipoxin A4 and Annexin-1 for the Production of IL-10 and Inflammatory Hyporesponsiveness in Mice J. Immunol., December 15, 2007; 179(12): 8533 - 8543. [Abstract] [Full Text] [PDF]
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 P. Zymek, D.-Y. Nah, M. Bujak, G. Ren, A. Koerting, T. Leucker, P. Huebener, G. Taffet, M. Entman, and N. G. Frangogiannis Interleukin-10 is not a critical regulator of infarct healing and left ventricular remodeling Cardiovasc Res, May 1, 2007; 74(2): 313 - 322. [Abstract] [Full Text] [PDF]
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K. Tsujita, K. Kaikita, T. Hayasaki, T. Honda, H. Kobayashi, N. Sakashita, H. Suzuki, T. Kodama, H. Ogawa, and M. Takeya Targeted Deletion of Class A Macrophage Scavenger Receptor Increases the Risk of Cardiac Rupture After Experimental Myocardial Infarction Circulation, April 10, 2007; 115(14): 1904 - 1911. [Abstract] [Full Text] [PDF]
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 P. Zymek, M. Bujak, K. Chatila, A. Cieslak, G. Thakker, M. L. Entman, and N. G. Frangogiannis The Role of Platelet-Derived Growth Factor Signaling in Healing Myocardial Infarcts J. Am. Coll. Cardiol., December 5, 2006; 48(11): 2315 - 2323. [Abstract] [Full Text] [PDF]
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 T.B. Corcoran, A. Engel, H. Sakamoto, A. O'Shea, S. O'Callaghan-Enright, and G. D. Shorten The effects of propofol on neutrophil function, lipid peroxidation and inflammatory response during elective coronary artery bypass grafting in patients with impaired ventricular function Br. J. Anaesth., December 1, 2006; 97(6): 825 - 831. [Abstract] [Full Text] [PDF]
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 D.M. Green, P.C. Noble, J.R. Bocell Jr., J.S. Ahuero, B.A. Poteet, and H.H. Birdsall Effect of Early Full Weight-Bearing After Joint Injury on Inflammation and Cartilage Degradation J. Bone Joint Surg. Am., October 1, 2006; 88(10): 2201 - 2209. [Abstract] [Full Text] [PDF]
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 K. Nishihira, T. Imamura, A. Yamashita, K. Hatakeyama, Y. Shibata, Y. Nagatomo, H. Date, T. Kita, T. Eto, and Y. Asada Increased expression of interleukin-10 in unstable plaque obtained by directional coronary atherectomy Eur. Heart J., July 2, 2006; 27(14): 1685 - 1689. [Abstract] [Full Text] [PDF]
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N. G. Frangogiannis, G. Ren, O. Dewald, P. Zymek, S. Haudek, A. Koerting, K. Winkelmann, L. H. Michael, J. Lawler, and M. L. Entman Critical Role of Endogenous Thrombospondin-1 in Preventing Expansion of Healing Myocardial Infarcts Circulation, June 7, 2005; 111(22): 2935 - 2942. [Abstract] [Full Text] [PDF]
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O. Dewald, P. Zymek, K. Winkelmann, A. Koerting, G. Ren, T. Abou-Khamis, L. H. Michael, B. J. Rollins, M. L. Entman, and N. G. Frangogiannis CCL2/Monocyte Chemoattractant Protein-1 Regulates Inflammatory Responses Critical to Healing Myocardial Infarcts Circ. Res., April 29, 2005; 96(8): 881 - 889. [Abstract] [Full Text] [PDF]
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 S. L. Karackattu, M. H. Picard, and M. Krieger Lymphocytes Are Not Required for the Rapid Onset of Coronary Heart Disease in Scavenger Receptor Class B Type I/Apolipoprotein E Double Knockout Mice Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 803 - 808. [Abstract] [Full Text] [PDF]
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 M. Dobaczewski, S. Akrivakis, K. Nasser, L. H. Michael, M. L. Entman, and N. G. Frangogiannis Vascular Mural Cells in Healing Canine Myocardial Infarcts J. Histochem. Cytochem., August 1, 2004; 52(8): 1019 - 1029. [Abstract] [Full Text] [PDF]
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E. Falk Widespread Targets for Friendly Fire in Acute Coronary Syndromes Circulation, July 6, 2004; 110(1): 4 - 6. [Full Text] [PDF]
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 I. Y. P. Wan, A. A. Arifi, S. Wan, J. H. Y. Yip, A. D. L. Sihoe, K.H. Thung, E. M. C. Wong, and A. P. C. Yim Beating heart revascularization with or without cardiopulmonary bypass: Evaluation of inflammatory response in a prospective randomized study J. Thorac. Cardiovasc. Surg., June 1, 2004; 127(6): 1624 - 1631. [Abstract] [Full Text] [PDF]
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 J. M. Pearl, S. M. Schwartz, D. P. Nelson, C. J. Wagner, J. M. Lyons, S. M. Bauer, and J. Y. Duffy Preoperative glucocorticoids decrease pulmonary hypertension in piglets after cardiopulmonary bypass and circulatory arrest Ann. Thorac. Surg., March 1, 2004; 77(3): 994 - 1000. [Abstract] [Full Text] [PDF]
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 O. Dewald, G. Ren, G. D. Duerr, M. Zoerlein, C. Klemm, C. Gersch, S. Tincey, L. H. Michael, M. L. Entman, and N. G. Frangogiannis Of Mice and Dogs: Species-Specific Differences in the Inflammatory Response Following Myocardial Infarction Am. J. Pathol., February 1, 2004; 164(2): 665 - 677. [Abstract] [Full Text] [PDF]
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 C. A. Rivera, M. H. Tcharmtchi, L. Mendoza, and C. W. Smith Endotoxemia and hepatic injury in a rodent model of hindlimb unloading J Appl Physiol, October 1, 2003; 95(4): 1656 - 1663. [Abstract] [Full Text] [PDF]
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P. Gessler, J. Pfenninger, J.-P. Pfammatter, T. Carrel, O. Baenziger, and C. Dahinden Plasma levels of interleukin-8 and expression of interleukin-8 receptors on circulating neutrophils and monocytes after cardiopulmonary bypass in children J. Thorac. Cardiovasc. Surg., September 1, 2003; 126(3): 718 - 725. [Abstract] [Full Text] [PDF]
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 N. G. Frangogiannis, L. H. Mendoza, G. Ren, S. Akrivakis, P. L. Jackson, L. H. Michael, C. W. Smith, and M. L. Entman MCSF expression is induced in healing myocardial infarcts and may regulate monocyte and endothelial cell phenotype Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H483 - H492. [Abstract] [Full Text] [PDF]
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 D. R. Jeyarajah, M. L. Kielar, N. Frantz, G. Lindberg, and C. Y. Lu Infection by Gram-Negative Organisms via the Biliary Route Results in Greater Mortality than Portal Venous Infection Clin. Vaccine Immunol., July 1, 2003; 10(4): 664 - 669. [Abstract] [Full Text] [PDF]
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M. Qing, K. Schumacher, R. Heise, M. Woltje, J. F. Vazquez-Jimenez, T. Richter, M. Arranda-Carrero, J. Hess, G.o. von Bernuth, and M.-C. Seghaye Intramyocardial synthesis of pro- and anti-inflammatory cytokines in infants with congenital cardiac defects J. Am. Coll. Cardiol., June 18, 2003; 41(12): 2266 - 2274. [Abstract] [Full Text] [PDF]
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D. G. Souza, R. Guabiraba, V. Pinho, A. Bristow, S. Poole, and M. M. Teixeira IL-1-Driven Endogenous IL-10 Production Protects Against the Systemic and Local Acute Inflammatory Response Following Intestinal Reperfusion Injury J. Immunol., May 1, 2003; 170(9): 4759 - 4766. [Abstract] [Full Text] [PDF]
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N. G. Frangogiannis, S. Shimoni, S. Chang, G. Ren, O. Dewald, C. Gersch, K. Shan, C. Aggeli, M. Reardon, G. V. Letsou, et al. Active interstitial remodeling: an important process in the hibernating human myocardium J. Am. Coll. Cardiol., May 1, 2002; 39(9): 1468 - 1474. [Abstract] [Full Text] [PDF]
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N. G. Frangogiannis, S. Shimoni, S. M. Chang, G. Ren, K. Shan, C. Aggeli, M. J. Reardon, G. V. Letsou, R. Espada, M. Ramchandani, et al. Evidence for an Active Inflammatory Process in the Hibernating Human Myocardium Am. J. Pathol., April 1, 2002; 160(4): 1425 - 1433. [Abstract] [Full Text] [PDF]
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G. Ren, L. H. Michael, M. L. Entman, and N. G. Frangogiannis Morphological Characteristics of the Microvasculature in Healing Myocardial Infarcts J. Histochem. Cytochem., January 1, 2002; 50(1): 71 - 80. [Abstract] [Full Text]
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 N. KOTAJIMA, T. KIMURA, T. KANDA, A. KUWABARA, Y. FUKUMURA, M. MURAKAMI, and I. KOBAYASHI Reciprocal increase of circulating interleukin-10 and interleukin-6 in patients with acute myocardial infarction Heart, December 1, 2001; 86(6): 704 - 705. [Full Text]
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T. O. Nossuli, N. G. Frangogiannis, P. Knuefermann, V. Lakshminarayanan, O. Dewald, A. J. Evans, J. Peschon, D. L. Mann, L. H. Michael, and M. L. Entman Brief murine myocardial I/R induces chemokines in a TNF-alpha -independent manner: role of oxygen radicals Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2549 - H2558. [Abstract] [Full Text] [PDF]
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J. F. Vazquez-Jimenez, M. Qing, B. Hermanns, B. Klosterhalfen, M. Woltje, R. Chakupurakal, K. Schumacher, B. J. Messmer, G.o. von Bernuth, and M.-C. Seghaye Moderate hypothermia during cardiopulmonary bypass reduces myocardial cell damage and myocardial cell death related to cardiac surgery J. Am. Coll. Cardiol., October 1, 2001; 38(4): 1216 - 1223. [Abstract] [Full Text] [PDF]
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A. Tedgui and Z. Mallat Anti-Inflammatory Mechanisms in the Vascular Wall Circ. Res., May 11, 2001; 88(9): 877 - 887. [Abstract] [Full Text] [PDF]
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J. H. Choi, H. M. Ko, J.-W. Kim, H.-K. Lee, S. S. Han, S.-B. Chun, and S. Y. Im Platelet-Activating Factor-Induced Early Activation of NF-{{kappa}}B Plays a Crucial Role for Organ Clearance of Candida albicans J. Immunol., April 15, 2001; 166(8): 5139 - 5144. [Abstract] [Full Text] [PDF]
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