(Circulation. 1999;99:427-433.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Regulation of CCAAT/Enhancer Binding Protein, Interleukin-6, Interleukin-6 Receptor, and gp130 Expression During Myocardial Ischemia/Reperfusion
From the Division of Cardiology, University of Texas Health Science Center and South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, Tex.
Correspondence to Gregory L. Freeman, MD, Division of Cardiology, University of Texas Health Science Center and South Texas Veterans Health Care System, Audie L. Murphy Division, 7703 Floyd Curl Dr, San Antonio, TX 78284-7872. E-mail freeman{at}uthscsa.edu
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Abstract |
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Background—Interleukin (IL)-6 is elevated in myocardium after ischemia and reperfusion. The IL-6 promoter/enhancer region contains response elements for nuclear factor-
B, AP-1, and CCAAT/enhancer binding
protein (C/EBP). Expression and regulation of C/EBP in rat
myocardium after ischemia and reperfusion has not
been defined, nor has the behavior of the specific IL-6 receptor
(IL-6R) or the signal transducer gp130.
Methods and Results—C/EBP DNA binding activity was not
detected in shams or in previously ischemic tissue at 15
minutes of reperfusion; it was detected at 30 minutes of reperfusion,
increased at 1 hour of reperfusion, and declined by 6 hours of
reperfusion. A supershift was observed with anti–C/EBP-ß but not
with anti-
or anti-
antibodies. mRNA and protein levels of IL-6
and gp130 were detected at low levels in controls, increased at 1 hour
of reperfusion, and remained high until 6 hours of reperfusion. IL-6R
mRNA and protein were not detected in controls, but its mRNA was
induced at 1 hour of reperfusion and its protein at 2 hours of
reperfusion. Although effects of reperfusion were rapid, in
ischemic tissue not reperfused, low levels of C/EBP were
detected at 4 hours, and at 24 hours the levels were slightly elevated.
Significant upregulation in IL-6, IL-6R, and gp130 occurred only at 24
hours of sustained ischemia.
Conclusions—Reperfusion after a brief period of ischemia caused induction of myocardial C/EBP (ß-subunit). The rapid and sustained production of IL-6 with concomitant expression of IL-6 receptor and gp130 suggest that these factors may participate in a local inflammatory cascade after myocardial ischemia and reperfusion.
Key Words: myocardial ischemia • interleukins • receptors • reperfusion
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Introduction |
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Interleukin (IL)-6 is a pleiotropic cytokine with varying effects on cells of the immune system and other tissues. Recently, cardiac myocytes have been shown to produce IL-6. Yamauchi-Takihara et al1 found induction of IL-6 in isolated cardiomyocytes exposed to hypoxia followed by reoxygenation. Kukielka et al2 showed induction of IL-6 in ischemic myocardium of dogs, but not in adjacent nonischemic tissue. We have confirmed these observations on the myocardium, showing increased IL-6 mRNA and protein levels in tissues taken from rat hearts after in vivo ischemia and reperfusion.3 We found that IL-6 levels were upregulated 1 hour after reperfusion, and that unlike tumor necrosis factor (TNF)-
and IL-1ß, which showed a return to low
levels by 3 hours of reperfusion, the levels of IL-6 mRNA were elevated
in a sustained manner for 6 hours after reperfusion. IL-6 exerts its biological activities through binding to an 80-kDa ligand binding subunit, IL-6 receptor (IL-6R). On binding to IL-6R, IL-6 induces interaction and homodimerization of gp130. This leads to the activation of a cytoplasmic tyrosine kinase, phosphorylation of gp130, and subsequent transduction of intracellular signals.4 5 Given the upregulation of IL-6 in postischemic myocardium, the behavior of other aspects of its signaling cascade is of interest. No reports are available demonstrating expression and changes in IL-6R and gp130 during myocardial ischemia and reperfusion.
The promoter/enhancer region of IL-6 contains response elements for
nuclear factor (NF)-B, AP-1, and CCAAT/enhancer binding protein
(C/EBP).6 7 We have reported induction and biphasic
regulation of NF-B and monophasic regulation of AP-1 during
ischemia and reperfusion.8 However, patterns of
expression and role of C/EBP in myocardium, especially
during ischemia and reperfusion, have not been described. Given
the observation that IL-6 mRNA and protein expression follow a pattern
that differs from other proinflammatory cytokines after
myocardial ischemia and reperfusion,3 its
expression may be uniquely controlled. As such, alterations in the
pattern of expression of C/EBP are of interest. In the present
study, we analyzed myocardium for mRNA and protein
levels of IL-6, IL-6R, and gp130 and for C/EBP and C/EBP subunit levels
during reperfusion after a single episode of sublethal
ischemia.
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Methods |
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Experimental Animals and Induction of Ischemia With or Without Reperfusion
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 to 23, revised 1985). Wistar-Kyoto rats weighing
200 g were used for these studies (a total of 104 rats).
Preparation of animals, induction of ischemia and reperfusion,
and tissue collection were carried out as described
earlier.3 8 Reperfusion was initiated after 15 minutes of
ischemia for 15 minutes, 30 minutes, and 1, 2, 3, and 6 hours
(n=8/group). Sham-operated animals euthanized at 15 minutes and 3 and 6
hours after surgery (n=8/group) were used as controls. Four animals
selected at random were used for mRNA, protein analyses, and
immunocytochemistry, and the other 4 were used for electrophoretic
mobility shift assay (EMSA) and supershift assay. In a separate group of animals (n=32), ischemia alone (without reperfusion) was induced by ligating the left anterior descending coronary artery for 15 minutes and 1, 4, or 24 hours (n=8/group). After the ischemic period, the heart was excised and processed as described above. Four animals selected at random were used for mRNA and protein analyses, and the other 4 were used for EMSA.
Electrophoretic Mobility Shift Assay
All steps were carried out at 4°C unless otherwise noted.
Nuclei from frozen myocardium were prepared as described by
Muller et al.9 The ischemic and
nonischemic portions of myocardium adjacent to the
ischemic zone (4 hearts [900 mg], pooled at each time
period) were pulverized in the presence of liquid nitrogen. They were
homogenized in lysis buffer (10 mmol/L HEPES, pH 7.4,
5 mmol/L KCl, 10 mmol/L MgCl2, 5
mmol/L 2-mercaptoethanol) containing 0.32 mol/L sucrose and 0.1%
Triton X-100. The homogenate was vacuum-filtered through a
nylon filter (200 µm mesh) and centrifuged at
2000g for 10 minutes. Pellets were resuspended in lysis
buffer containing 2.2 mol/L sucrose without detergent and
centrifuged for 90 minutes at 100 000g. To verify
and estimate the yield of nuclei, DNA content of the pellet was
determined spectrophotometrically at 260 nm.
Proteins were extracted for 45 minutes from the nuclei as described by Dignam et al.10 Protein concentration was determined by BCA protein assay reagent (Pierce). The EMSA was performed as described by Dent and Latchman,11 with some modifications.3 Consensus double-stranded oligonucleotides (5'-TGC AGA TTG CGC AAT CTG CA-3'; Santa Cruz Biotechnology, Inc) containing the binding site for C/EBP (TTGCGCAA) were used as a probe. In competition experiments, a 100-fold molar excess of unlabeled consensus or mutant C/EBP oligonucleotides (mutant, 5'-TGC AGA GAC TAG TCT CTG CA-3') was added to the reaction mixture for 20 minutes, followed by the addition of labeled consensus probe. Binding reactions, separation of free DNA and DNA-protein complexes, autoradiography, and densitometry were carried out as described earlier.3
In the gel supershift assay, the nuclear protein extract (20 µg) was
preincubated for 40 minutes on ice with either anti-, anti-ß, or
anti- polyclonal antibodies (0.2 µg) before the addition of
32P-labeled C/EBP consensus oligo.
Subunit-specific rabbit anti-rat antibodies were from Santa Cruz
Biotechnology, Inc.
Total RNA Isolation and Northern Blot Analysis
Total RNA isolation, Northern blotting,
autoradiography, and densitometry were carried out as
described earlier.12 13 14 15 The following probes were used:
IL-6 (American Type Culture Collection, Rockville, Md; IL-6R, and gp130
(a kind gift from Dr G.M. Fuller, Professor, Department of Cell Biology
and Anatomy, The University of Alabama at Birmingham). 28S rRNA
probe (Oncogene Science, Inc, Uniondale, NY) was used as internal
control.
Protein Extraction and Western Blot Analysis
Extraction of protein homogenates, detection of
IL-6, IL-6R, and gp130, and densitometry were carried out as
described.12 13 14 15 These antibodies were used: anti-rat
(IL-6 [R-19]) or anti-mouse (IL-6R [M-20], gp130 [M-20];
cross-reactive with rat). Antibodies were from Santa Cruz
Biotechnology, Inc (affinity-purified polyclonal antibodies).
Immunohistochemistry
At 6 hours of reperfusion, the left ventricle was divided into
ischemic and nonischemic zones, embedded in OCT,
snap-frozen in liquid nitrogen, and stored for not more than 3 days at
-82°C. Cryosections of 6-µm thickness were prepared for
immunostaining with the use of an immunoenzymatic
staining kit (For IL-6, Universal DAKO PAP kit, K0 549; for IL-6R and
gp130, DAKO LSAB2 kit, peroxidase for use on rat specimen, K0 609;
DAKO).13 16 Omission of primary antibody, rabbit/goat
preimmune serum in place of primary antibody, and primary antibody
after neutralization with its peptide antigen served as controls.
Immunohistochemistry staining was evaluated by light microscopy.
Specific staining was graded on a semiquantitative scale from 0 to 3
(0=none, 1=weak, 2=intermediate, 3=strong).
Statistical Analysis
Comparisons between control (15 minutes sham-operated) and each
of the 7 reperfusion time periods and between 15 minutes of
ischemia and 1-, 4-, and 24-hour ischemic periods were
performed for measures of mRNA and protein for IL-6, IL-6R, and gp130
by ANOVA with post hoc Dunnett's t tests. F
tests and Dunnett's t tests with values of
P<0.05 were considered statistically significant. Since
IL-6R mRNA and protein values were undetectable for control and
durations shorter than 1 hour of reperfusion for mRNA and 2 hours of
reperfusion for protein, 1-sample Student's t tests were
performed. Similarly for immunohistochemistry, because no positive
immunoreactivity was detected for IL-6 and IL-6R, significance was
obtained by 1-sample Student's t tests, and for gp130,
ANOVA was used with post hoc Dunnett's t tests.
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Results |
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C/EBP and C/EBP Subunit Levels
Figure 1
shows C/EBP levels in the
ischemic (no reperfusion), postischemic, and
control (sham-operated) myocardium. At baseline, no
specific C/EBP DNA binding activity was detected in the nuclear protein
extracts from sham-operated hearts (at 15 minutes and 3 and 6 hours
after surgery; Figure 1, lanes 22 to 24) or nonischemic
regions adjacent to the ischemic zone (data not shown). In
postischemic tissue, C/EBP was not detected at 15 minutes
of reperfusion. It was, however, readily detected at 30 minutes of
reperfusion. Its levels increased, being maximum at 2 hours of
reperfusion, then declined by 6 hours of reperfusion (Figure 1, lanes 11 to 16). The specificity of the signal was verified by
preincubating the nuclear protein extract with 100-fold molar excess
cold consensus oligo (lane 5), which abolished the signal. There was
also no interference in the signal obtained from labeled consensus
oligo when the extract was preincubated with a 100-fold molar excess of
cold mutant oligo (lane 4). Furthermore, no signal was
obtainedwhen the reaction mixture did not contain protein
extract (lane 2) or when labeled mutant oligo was used in place of
labeled consensus oligo (lane 3).
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To define which subunit or subunits participated in the elevated C/EBP
levels, we analyzed the nuclear protein extract at the time it
peaked (2 hours of reperfusion) for C/EBP subunit levels by gel
supershift assay with the use of subunit-specific polyclonal
antibodies. As seen in Figure 1
(lanes 25 to 27), no further
shift was observed when nuclear protein extracts were preincubated with
anti- and anti- antibodies before the addition of labeled
consensus C/EBP oligo. However, supershift was observed when anti-ß
antibodies were used, indicating that the ß-subunit was the moiety
elevated at this time.
IL-6, IL-6R, and gp130 Levels
Figure 2 shows total RNA from
control and postischemic myocardium by Northern
blotting. Confirming our earlier observations, low level expression of
a single transcript (1.4 kb) of IL-6 was detected in control
myocardium at 15 minutes. It remained at this level in
reperfused myocardium until 30 minutes of reperfusion; its
level rose at 1 hour of reperfusion and remained high until 6 hours of
reperfusion. Densitometric analysis of the
autoradiographic bands (Figure 2) revealed that
compared with sham-operated controls, the levels were significantly
higher at 1 hour of reperfusion (1.66-fold; P<0.0001;
Figure 3). The levels rose further by 2
hours of reperfusion (2.78-fold, P<0.005), increased
further by 3 hours (3.19-fold; P<0.0001), and declined at 6
hours of reperfusion (3.01-fold; P<0.0001). IL-6R was not
detected under steady-state conditions in control
myocardium, nor was it present in
postischemic myocardium until 30 minutes of
reperfusion (Figures 2 and 3). However, it was readily
detected at 1 hour of reperfusion (0.31±0.012; P<0.01),
and its levels increased by 2 hours of reperfusion (0.46±0.026) and
remained high until 6 hours of reperfusion (3 hours of reperfusion,
0.50±0.015; 6 hours, 0.53±0.23; all P<0.0001). Similar to
IL-6, gp130 mRNA (a single 5.1 kb transcript) was detected at low
levels in control and postischemic myocardium.
It remained at a low level until 30 minutes of reperfusion (Figures 2 and 3), rose by 1 hour of reperfusion (2.23-fold,
P<0.0001), and remained elevated until 6 hours of
reperfusion (2 hours of reperfusion, 1.92-fold; 3 hours, 1.99-fold; 6
hours, 2.17-fold; all P<0.0001).
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P<0.0001 (vs
control).
As a separate control, we tested both sham-operated and
postischemic myocardium for the presence of
IL-1 mRNA. Results from these experiments confirmed our earlier
observations that no signal was obtained in postischemic
tissue, even after 8 days of autoradiographic exposure
(data not shown).
Immunoblots for all 3 proteins are shown in Figure 4, with associated semiquantitation in
Figure 5. Following the pattern of mRNA,
both IL-6 and gp130 proteins were detected in low levels in controls.
The levels remained low until 30 minutes of reperfusion, then increased
gradually. IL-6 levels were significantly elevated at 1 hour of
reperfusion (2.31-fold; P<0.0001) and remained high until 6
hours of reperfusion (2.4-fold at 2, 3, and 6 hours of reperfusion;
P<0.0001). Levels of gp130 were increased by 2.64-fold at 1
hour of reperfusion (P<0.0001), increased further at 2
hours of reperfusion (3.37-fold, P<0.0001), and remained
elevated throughout the 6-hour study period (3 hours of reperfusion,
2.67-fold, P<0.005; 6 hours, 3.23-fold;
P<0.0001). In contrast to IL-6 and gp130, IL-6R protein was
not detected in controls at steady state nor in
postischemic myocardium up to 1 hour of
reperfusion. However, its levels were readily detected at 2 hours of
reperfusion (169±11.2; P<0.0001) and progressively
increased until 6 hours of reperfusion (3 hours of reperfusion,
192±7.5; 6 hours, 196±5.71; all P<0.0001; Figures 4 and 5).
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Immunohistochemistry
Immunohistochemical staining for IL-6, IL-6R, and gp130 was
carried out in myocardium from sham-operated animals, in
normal tissue adjacent to ischemic zone, and in
postischemic myocardium at 6 hours of
reperfusion (Figure 6). We chose this
time period because high levels of IL-6, IL-6R, and gp130 protein were
detected by Western blotting (Figure 4 and 5). No
immunoreactivity for IL-6 was observed in shams (Figure 6A) or
in normal tissue adjacent to the ischemic zone (Figure 6B). However, in postischemic
myocardium, IL-6 immunoreactivity was readily detected,
localized to the cytoplasm, and in a diffuse manner (control versus 6
hours of reperfusion; P<0.0001; Figure 6C). Similar
to IL-6, IL-6R immunoreactivity was detected neither in shams nor in
normal tissue adjacent to ischemic zone (data not shown).
However, IL-6R immunoreactivity was detected in a granular fashion in
postischemic myocardium, localized to both
membranous and cytoplasmic compartments (control versus 6 hours of
reperfusion; P<0.0001). Though Western blotting revealed
low levels of gp130 in sham-operated animals (Figure 4),
immunohistochemistry revealed a very weak staining for gp130 (data not
shown) in these tissues. However, gp130 immunoreactivity was detected
in postischemic myocardium, localized to the
cytoplasm as well as to the perinuclear region (4.6-fold;
P<0.0001), confirming previous results from a study in
which we found a similar pattern of gp130 immunoreactivity in
myocardium from animals infected with Trypanosoma
cruzi.17 Omitting primary antibody and replacing
it with respective preimmune sera abolished the signals (data not
shown). Also, no immunoreactivity was detected when primary antibodies
were neutralized with their respective peptide antigens. As an example,
in Figure 4D, no IL-6 immunoreactivity was detected when
anti–IL-6 antibodies were used after neutralization with their peptide
antigen.
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C/EBP, IL-6, IL-6R, and gp130 Levels After Sustained
Ischemia Without Reperfusion
To determine the impact of ischemia alone on induction of
C/EBP, IL-6, IL-6R, and gp130, we studied tissue after coronary
ligation was sustained for 15 minutes 1, 4, or 24 hours, and tissue was
harvested without reperfusion. Figure 1 (lanes 6 to 9) shows
C/EBP levels by EMSA in nuclear protein extracts taken from
myocardium in the ischemic zone. C/EBP DNA binding
activity was not detected at 15 minutes or 1 hour of ischemia.
However, at 4 hours a weak signal was detected, and by 24 hours the
levels increased modestly. These levels were considerably lower than
the levels detected in previously ischemic tissue after
reperfusion (Figure 1, lanes 12 to 16). Figures 7 and 8
show mRNA and protein levels of IL-6, IL-6R, and gp130 in
ischemic myocardium. Northern blots revealed low
levels of IL-6 mRNA at 15 minutes and 1 and 4 hours of
ischemia, but significantly higher levels were detected at 24
hours (Figures 7 and 8; densitometry; 15 minutes versus
24 hours; IL-6, 3.79 fold, P<0.0001). Its protein levels
followed a similar trend (15 minutes versus 24 hours; 2.01-fold,
P<0.0001). Similar to IL-6, gp130 mRNA was detected at low
levels at 15 minutes (0.19±0.011) and 1 hour (0.19±0.01) of
ischemia, rose moderately at 4 hours (1.36-fold;
P<0.05), and increased further at 24 hours (2.95-fold;
P<0.005). However, its protein levels (Figures 7B and 8B) were found elevated only at 24 hours of ischemia
(2.33-fold; P<0.0001). In contrast to IL-6 and gp130, IL-6R
mRNA and protein levels were undetectable at 15 minutes and 1 and 4
hours. They were detected only after 24 hours of ischemia (mRNA
and protein, 15 minutes versus 24 hours, P<0.0001).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our results show for the first time that in myocardial tissue, reperfusion after brief sublethal ischemia leads to increased nuclear C/EBP levels, with a predominant impact on the ß-subunit. Although IL-6R was not present under baseline conditions, and only very low levels of gp130 were found, both were readily detected in the postischemic tissue. Furthermore, as we and others have previously shown, both mRNA and protein levels of IL-6 were upregulated in postischemic tissue. Thus, there appears to be coordinated expression of IL-6 and other factors that participate in its activity after ischemia and reperfusion in the myocardium.
A key observation in the present study is the detection of
increased levels of C/EBP in the nuclear extracts. Specific C/EBP-DNA
protein complexes were not detected under control conditions and were
not found in reperfused myocardium until 30 minutes of
reperfusion. Furthermore, a supershift was observed when the protein
extracts were preincubated with anti–C/EBP-ß but not with - or
-antibodies, suggesting that C/EBP-ß may play an important role in
postischemic myocardium. C/EBP is an inducible
transcription factor, which is also known as IL-6 DNA binding protein
(IL6DBP), liver-enriched transcriptional activator protein
(LAP), and nuclear factor-IL-6. Whereas some C/EBP proteins are
expressed constitutively and their levels are altered on cell
differentiation, others, such as C/EBP-ß and C/EBP-, are inducible
in response to inflammatory mediators. The best-characterized inducible
C/EBP protein is C/EBP-ß. In cultured endothelial
cells, during hypoxic conditions, induction of IL-6 was observed
through activation of C/EBP-ß.18 In addition to
induction of IL-6, C/EBP has been shown to participate in the induction
of other proinflammatory cytokines, including IL-1, as well as
participating in TNF-–induced IL-1ß induction.19
This factor binds to the transcriptional regulatory regions in TNF-,
IL-8, and G-CSF, suggesting a role for C/EBP in inflammation and the
acute phase response.
In addition to C/EBP, the IL-6 promoter region also contains binding
sites for AP-1 and NF-B, transcription factors regulated by the
redox status of the cell. All of these sites have been shown to be
essential for the induction of IL-6. We have previously shown increased
NF-B and AP-1 levels in postischemic reperfused
myocardium.3 8 We observed low and
consistent levels of NF-B p50 and increased levels of p65 in
reperfused myocardium. We speculate that the mechanism
underlying the fact that the appearance of IL-6 follows a different
pattern of expression than either TNF- or IL-1ß may be the more
prominent role of C/EBP in transcriptional regulation of IL-6.
Another important observation in the present study is the
induction of IL-6R and upregulation of gp130 in reperfused
myocardium. Whereas IL-6R was not detected under basal
conditions, it was induced at 1 hour of reperfusion and remained at
significantly higher levels throughout the 6-hour study period. On the
other hand, both mRNA and protein levels of gp130 were detected at low
levels under basal conditions, were significantly elevated at 1 hour of
reperfusion, and remained elevated up to 6 hours of reperfusion.
Whereas IL-6 exerts its biological effects through binding to IL-6R,
IL-6R by itself cannot transduce signals intracellularly. This requires
dimerization of gp130, which activates a cytoplasmic tyrosine
kinase5 20 and subsequent transduction of intracellular
signals. Proinflammatory cytokines such as IL-1ß and TNF-
have also been shown to upregulate gp130 in amnion (UAC) and hepatoma
(Hep3B) cell lines.21 Our data show a concurrent
expression of IL-6 and its receptor system, which probably are primary
events not induced by cytokines but rather a direct result of
the ischemia and reperfusion process.
To evaluate the importance of reperfusion on the patterns of gene expression, we studied groups of animals with persistent ischemia and no reperfusion. Prior studies on how sustained ischemia affects expression of IL-6 have been inconsistent. Herskowitz et al22 showed that IL-6 mRNA levels were increased in hearts of rats after 1 and 3 hours of ischemia but not after 24 hours. On the other hand, Kukielka et al2 showed that there was no expression of IL-6 mRNA after a 4-hour period of ischemia in hearts of dogs but that it was present if ischemia persisted for 24 hours. Our results support the observation of Kukielka et al2 that IL-6 is not expressed by tissue rendered ischemic in the absence of reperfusion at early time points, appearing only after 24 hours. In addition to IL-6, we also studied changes in C/EBP levels and expression of IL-6R and gp130 in ischemic tissue. C/EBP DNA binding activity was not detected either at 15 minutes or 1 hour of ischemia; it was detected at very low levels in ischemic tissue at 4 and 24 hours. Furthermore, both mRNA and protein levels of IL-6R and gp130 were only increased after 24 hours of ischemia. The levels were low compared with their expression in reperfused postischemic tissue, and overall our results suggest that changes in IL-6 and its receptors can occur very rapidly and that they are more pronounced after reperfusion than after ischemia alone. Although we did not study reperfusion after periods of ischemia longer than 15 minutes, both Herskowitz et al22 and Kukielka et al2 showed that after ischemic durations sufficient to cause cell death (35 minutes or 1 hour, respectively), IL-6 was expressed in reperfused myocardium. Under those conditions, the picture is more complex because cell death by necrosis or apoptosis is taking place. Whether cytokine expression is mediated by myocardium or by inflammatory cells, either in the early stages or at 24 hours, is not known and will require further study.
In conclusion, the present study demonstrates for the first time that reperfusion after a brief period of myocardial ischemia leads to the upregulation of C/EBP as well as IL-6, IL-6R, and gp130. This is a unique and temporally synchronized tissue response, consistent with the expression of other proinflammatory cytokines under these conditions. The fact that all of the components of the signaling cascade are induced in a coordinated fashion strongly suggests that there is a defined role for this protein in the postischemic heart. Full delineation of this role will require further studies.
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Acknowledgments |
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This research was supported by the Research Service of the Department of Veterans Affairs. The authors thank Danny Escobedo for excellent technical assistance.
Received May 29, 1998; revision received August 31, 1998; accepted September 16, 1998.
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