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Circulation. 1995;92:1866-1875

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(Circulation. 1995;92:1866-1875.)
© 1995 American Heart Association, Inc.

Articles

Induction of Interleukin-6 Synthesis in the Myocardium

Potential Role in Postreperfusion Inflammatory Injury

Gilbert L. Kukielka, MD; C. Wayne Smith, MD; Anthony M. Manning, PhD; Keith A. Youker, BA; Lloyd H. Michael, PhD; Mark L. Entman, MD

From the Section of Cardiovascular Sciences, The Methodist Hospital, The DeBakey Heart Center and Department of Medicine, Houston, Tex (G.L.K., K.A.Y., L.H.M., M.L.E.); the Speros P. Martel Section of Leukocyte Biology, Department of Pediatrics and Texas Children's Hospital, Houston, Tex (G.L.K., C.W.S.); and Discovery Research, Upjohn Laboratories, Kalamazoo, Mich (A.M.M.).


*    Abstract
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 *Abstract
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Background Neutrophil-induced injury of myocardial cells requires the expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte surface and is mediated by ICAM-1–CD11b/CD18 adhesion. We have previously shown that interleukin-6 (IL-6) cytokine activity, present in cardiac lymph, induces ICAM-1 on isolated cardiac myocytes. Furthermore, in previous in vivo studies, we have also shown ICAM-1 mRNA induction in the myocardium within the first hour of reperfusion in the previously ischemic viable zone. We hypothesized that induction of IL-6 synthesis in the myocardium was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells.

Methods and Results In this study, cloned canine IL-6 cDNA was used as a molecular probe to study the regulation of IL-6 in an awake canine model of myocardial ischemia and reperfusion. IL-6 mRNA was induced in ischemic and reperfused segments of myocardium preferentially in segments previously exposed to severe ischemia. Peak levels of IL-6 mRNA were reached within 3 hours of reperfusion. At the same time, IL-6 mRNA and ICAM-1 mRNA were found in the same myocardial segments. In contrast to hearts that were ischemic for 1 hour and reperfused for 3 hours, nonreperfused hearts after 4 hours of persistent ischemia demonstrated minimal induction of ICAM-1 or IL-6 despite similar degrees of injury and blood flow reductions during ischemia. After 24 hours of persistent ischemia, levels of IL-6 mRNA were comparable to those observed in hearts that were ischemic for 1 hour and subsequently reperfused for 24 hours.

Conclusions Our results demonstrate induction of IL-6 mRNA in the myocardium and that this synthesis is accelerated by reperfusion. Evidence is also provided to show that peak IL-6 mRNA precedes that of ICAM-1 mRNA. These findings are compatible with our hypothesis that IL-6 is important in the induction of ICAM-1 in the area of ischemia. In addition, these studies suggest that the necessary factors to promote adhesive interactions between transmigrated neutrophils and cardiac myocytes are present in reperfused myocardium.


Key Words: reperfusion • myocardial infarction • interleukin • blood cells


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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Although the association of inflammation with acute myocardial infarction has been described for more than a century,1 its role in injury extension has only recently been proposed.2 The principal focus of this recent interest has related to the potential role of neutrophil infiltration in postreperfusion injury.3 Our laboratory has studied neutrophil-induced injury of isolated adult canine cardiac myocytes as a potential mechanism of inflammation-induced reperfusion injury. Neutrophils adhere to viable cardiac myocytes only when the neutrophil is stimulated with a chemotactic agent and the myocyte has been incubated with a cytokine.4 Neutrophil adhesion to cardiac myocytes is dependent on CD18 integrin activation and also on the induction of ICAM-1 on cardiac myocytes.4 5 Subsequent experiments showed that neutrophil adhesion to cardiac myocytes resulted in lethal myocyte injury mediated by a transfer of reactive oxygen that was dependent on CD11b/CD18–ICAM-1 adhesion.6

The pertinence of these in vitro studies to reperfusion-associated inflammatory injury in a model of canine myocardial ischemia and reperfusion has been suggested by another line of investigation. In an awake canine model with a permanently cannulated cardiac lymph duct, we demonstrated the presence in postischemic cardiac lymph of chemotactic factors that are neutralized by a C5a antibody7 8 and cytokine activity capable of stimulating ICAM-1 expression on isolated cardiac myocytes.9 10 In these latter experiments, stimulation of myocyte ICAM-1 induction by cardiac lymph was neutralized by a polyclonal antibody to human IL-6.9 These associations led to more direct experiments on ischemic and reperfused myocardial tissue that demonstrated induction of ICAM-1 in vivo.10 More recently, we used in situ hybridization techniques to demonstrate that the induction of ICAM-1 mRNA in jeopardized viable myocardium occurs where maximal inflammatory infiltrate is seen.11 This creates a potential interface between activated neutrophils and myocardial cells, induced to express ICAM-1, that might lead to the inflammatory injury we demonstrated in vitro.4 5 6 The hypothesis of this study was that induction of IL-6 synthesis was an integral part of the reaction to injury resulting from ischemia and reperfusion and was associated with induction of ICAM-1 on myocardial cells. Since the role of myocyte ICAM-1 in neutrophil-induced injury may also be important pathologically, the present study seeks to investigate the mechanism of ICAM-1 induction and examine the correspondence of our in vitro studies with conditions observed after ischemia and reperfusion. This study demonstrates the induction of the IL-6 gene in ischemic and reperfused myocardium. In keeping with our previous observations in cardiac lymph,9 the induction of myocardial IL-6 occurs very rapidly upon reperfusion. Furthermore, this induction is seen only in segments that are severely ischemic. Finally, the relation of IL-6 to ICAM-1 induction is further suggested by the concordance of ICAM-1 mRNA and IL-6 mRNA in the same ischemic and reperfused myocardial segments.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
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Molecular Cloning
A specific canine IL-6 probe was initially prepared by reverse transcription PCR using RNA extracted from LPS-stimulated peripheral blood mononuclear cells. Cells were isolated from citrate-anticoagulated venous blood by dextran sedimentation and Ficoll-Hypaque gradient centrifugation. The banded mononuclear cells were resuspended in RPMI 1640 with 10% FCS. Cells were then stimulated with LPS (Sigma Chemical Co) 100 µg/mL for 4 hours at 37°C. Total RNA was isolated at the end of the 4-hour incubation period with a modification of the acid-guanidinium-thiocyanate-phenol-chloroform extraction method12 and resuspended in diethyl pyrocarbonate–treated water. Reverse transcription protocols were performed with 3 µg of total RNA in each sample. After first-strand synthesis, primed with oligo-dT, aliquots of the reverse transcription reaction were amplified with the following primers: 5' primer, 5'-GATCGGATCCGCTATGAACTCCCTCTCC-3', 3' primer, 5'-GATCAAGCTTAGGTTTCTGACCAGA-3' at a final concentration of 0.025 µmol/L. The nucleotide sequence of the primers was based on a highly conserved sequence of human and porcine IL-6 cDNA13 14 and corresponded to base pairs 30 through 48 and 715 through 729 of the human IL-6 cDNA.13 PCR was performed with 5 U Taq DNA polymerase (Promega Corp) for 25 cycles of 93°C, 1 minute; 60°C, 30 seconds; and 72°C, 30 seconds. The final product was purified, cloned into the BamHI and HindIII restriction endonuclease sites of M13mp19,15 and sequenced on an Applied Biosystems model 373A automated DNA sequencer. Samples were sequenced with Taq DNA polymerase and fluorescently labeled M13 universal and reverse sequencing primers. This PCR product was used to screen two cDNA libraries prepared from LPS or TNF-{alpha}–stimulated CJVECs in the {lambda} ZAP II vector (Stratagene Cloning Systems). The first cDNA library screened was stimulated with LPS and used an oligonucleotide encoding oligo-dT (13 mer) and a Xho I restriction endonuclease recognition site to prime the first-strand cDNA synthesis. The PCR product was labeled with 32P by random-hexanucleotide priming16 and was used to screen 1x106 plaques. Screening was performed by plaque filter hybridization methods on duplicate filters.17 Filters were hybridized in rapid hybridization buffer (Amersham Corp) at 65°C for 2 hours and then washed with 1x SSPE/0.5% SDS at 65°C for 20 minutes followed by 0.5x SSPE/0.5% SDS for 20 minutes. Hybridizing clones were plaque-purified in subsequent rounds of screening and rescued in pBluescript SK(-) (Stratagene Cloning Systems) by the in vivo excision method. The complete nucleotide sequence of both strands of the longest clone (dIL-6-1) was determined on an Applied Biosystems model 373A automated sequencer. Since clone dIL-6-1 did not encode an initiation codon and its 5' end began at the equivalent of residue 14 of the human IL-6 protein, a second cDNA library was screened. This second library used a random-hexamer oligonucleotide strategy to prime the first-strand cDNA synthesis and was made from TNF-{alpha}–stimulated (30 U/mL; 3 hours, 37°C) CJVEC poly (A+) RNA. Approximately 5x105 plaques from this library were screened with a 150-bp probe derived by Rsa I restriction endonuclease digestion of a fragment corresponding to the 5' end of dIL-6-1. Screening was performed under the same conditions as used to screen the oligo-dT–primed cDNA library. Four {lambda} clones were isolated with this probe. These clones were plaque-purified before plasmid rescue in pBluescript SK(-). The 5' and 3' ends of all four clones were sequenced on an Applied Biosystems model 373A automated DNA sequencer by the dideoxynucleotide termination method18 and fluorescently labeled reverse and universal sequencing primers. All four clones contained the initiation codon and extended into the 5' untranslated region. The complete nucleotide sequence of both strands of clone dIL-6-8 was completed by primer walking. DNA sequences were analyzed by the suite of programs within EUGENE and SAM, as supported by the Molecular Biology Information Resource, Baylor College of Medicine, Houston, Tex.

Ischemia-Reperfusion Protocols
Healthy mongrel dogs (15 to 25 kg) of either sex were surgically instrumented as previously described.10 Anesthesia was induced with methohexital sodium 10 mg/kg IV (Brevital; Eli Lilly and Co) and maintained with the inhalational anesthetic isoflurane (Anaquest). A midline thoracotomy provided access to the heart and mediastinum, and cannulation of the cardiac lymph duct was then performed as previously described.10 Subsequently, a hydraulically activated occluding device and a Doppler flow probe7 10 were secured around the circumflex coronary artery just proximal or just distal to the first branch. Choice of location depended on the proximity and anatomic arrangement of lymphatic vessels so that subsequent dissection would not damage the lymphatic system. In animals selected for experimental assessment of cardiac lymph, intact lymphatic vessels draining the regions of ischemic myocardium were identified by Evans blue dye (0.05 mL) injected into the free wall of the left ventricle after the occluder and flow probe were in place. The appearance of Evans blue dye in the cardiac lymph cannula confirmed the patency of the lymph vessels. Indwelling cannulas placed in the right atrium, left atrium, and femoral artery allowed blood sampling and pressure monitoring as needed. 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 inflating the coronary cuff occluder until mean flow in the coronary vessel was zero, as determined by the Doppler flow probe. After 50 minutes of occlusion, radiolabeled microspheres (for subsequent blood flow determinations) were injected into the left atrium. For most experiments (total of 22, see below), at the end of 1 hour the cuff was deflated and the myocardium was reperfused. Reperfusion intervals ranged from 1 to 24 hours. In 8 experiments, reperfusion was not instituted. In 4 of these 8, the period of ischemia was extended to 4 hours, and in the remaining 4, it was extended to 24 hours. Circumflex blood flow, arterial blood pressure, heart rate, and ECG (standard limb II) were recorded continuously. Analgesia was achieved with pentazocine (Talwin; Winthrop Pharmaceuticals) 0.1 to 0.2 mg/kg IV. Lymph samples were collected from the cannulas in tubes containing 10 U preservative-free heparin. The samples were spun in a tabletop centrifuge at 13 000g for 5 minutes. Aliquots of supernatants were immediately frozen in liquid nitrogen and stored at -80°C until ready to use. For all incubation protocols, lymph samples, once thawed, were used immediately. After the reperfusion periods, hearts were stopped by the infusion of saturated potassium chloride, removed from the chest, and sectioned from apex to base into four transverse rings {approx}l cm thick. The posterior papillary muscle and the posterior free wall were identified. Transmural myocardial samples (1.0 g) were isolated from myocardial rings and labeled as control (obtained from the anterior wall) or infarcted (obtained from the posterior papillary muscle and posterior free wall) on the basis of anatomic location within the distribution of the circumflex artery and visual inspection. Myocardial samples were then minced into smaller pieces: first a transmural section was taken from the middle of the sample and fixed in 10% buffered formalin for histological studies, then the top and bottom halves were minced into smaller pieces and divided into two halves. The first half was used for blood flow determinations with radiolabeled microspheres as previously described.10 19 20 The remaining portions of each sample were immediately frozen in liquid nitrogen. Frozen tissue samples were homogenized and processed for RNA studies. Analyses of RNA, blood flow determinations, and histopathological examinations of samples obtained from each experiment were conducted independently (in separate laboratories) and in a blinded fashion. Once the independent analyses of the data were completed, the information was gathered and a final analysis performed. The first step in this final analysis was to determine the presence or absence of a myocardial infarct in each separate experiment. The presence of a myocardial infarct was based on light-microscopic examination of hematoxylin-eosin–stained tissue sections. Basic fuchsin staining (Titus Lie stain) was also used to aid in the identification of an infarct.10 11 The presence of a myocardial infarct in experiments with short reperfusion intervals (in which myocardial necrosis is not easily discernible) was defined in hematoxylin-eosin–stained tissue sections by findings of contraction bands, increased eosinophilia, "wavy fibers," interstitial edema, and infiltration of neutrophils, all in segments displaying markedly reduced blood flow during the ischemic period. For experiments lasting 24 hours after the start of the ischemic insult, the presence of histological elements characteristic of myocyte necrosis was added to the required criteria. A subsequent step in the final analysis of each sample was to correlate the level of IL-6 mRNA with the blood flow quantified by microspheres. This final analysis allowed for the classification of myocardial segments into two groups: control (no evidence of necrosis, normal blood flow) and ischemically injured myocardium (by methods mentioned above and reductions in blood flow). The data in this article describe the findings in these two groups of segments. Segments that were initially identified as infarcted in our initial blind assessment but had normal blood flow during occlusion did not have detectable levels of IL-6 or ICAM-1 mRNA and therefore were analogous to control segments (data not shown).

Data for ischemia-reperfusion studies were derived from 22 experiments. For all 22 experiments, the circumflex artery was occluded for 1 hour, and subsequently the myocardium was reperfused for 1 (7 experiments), 3 (8 experiments), or 24 (7 experiments) hours. In 8 additional experiments, the myocardium was rendered ischemic and reperfusion was not instituted. In 4 of these experiments, the myocardium remained ischemic for 4 hours, and in the other 4, the period of persistent ischemia was extended to 24 hours. To examine the relation of IL-6 induction to reperfusion, levels of IL-6 mRNA in ischemic and reperfused myocardial segments were compared directly with the levels found in persistently ischemic segments. All animal protocols were approved by the appropriate institutional review committee and conformed to institutional guidelines.

Isolation and Culture of CJVECs
Cells were obtained by modification of the method of Ford.21 Jugular veins were everted on glass rods and incubated in collagenase solution (Boehringer Mannheim type A) for 20 minutes. Cells were collected by centrifugation and suspended in DMEM containing 5% FCS, 5% bovine calf serum, 50 µg/mL endothelial cell growth factor (Collaborative Research), 50 U/mL heparin, 1 mmol/L sodium pyruvate, and antibiotics. Cells were seeded in Primaria flasks (Becton Dickinson). After 2 to 4 days of incubation at 37°C in a CO2 incubator, areas of cells with "cobblestone" morphology were collected by scraping, transferred to gelatin-coated flasks (0.1%, Difco), and grown to confluence. For incubation experiments with cardiac lymph, aliquots of lymph obtained before coronary occlusion and at designated times during reperfusion were diluted 1:7 and incubated with CJVEC monolayers for 2 hours at 37°C. Cells were then lysed, and RNA was isolated.

RNA Isolation and Northern Blot Analysis
RNA was isolated from myocardial tissue segments or cultured CJVECs by the acid-guanidinium-thiocyanate-phenol-chloroform procedure.12 RNA (5 to 20 µg) was electrophoresed in 1% agarose gels containing formaldehyde, then transferred to a nylon membrane (Gene Screen Plus; New England Nuclear) by standard procedures.17 Loading of RNA was monitored with ethidium bromide staining as well as by probing of the nylon membranes with human GAPDH. Membranes were hybridized in QuikHyb hybridization buffer (Stratagene Cloning Systems) at 68°C for 2 hours with 1x106 cpm/mL random-hexamer 32P-labeled canine ICAM-1 or IL-6 cDNA probe.10 16 Canine ICAM-1 cDNA probe was prepared as previously described,10 and the canine IL-6 cDNA probe was generated by BstXI and Ssp I restriction endonuclease digestion of clone dIL-6-1. This cDNA fragment was 415 bp long and did not include any nucleotide sequences from pBluescript SK(-) or the poly (A) tail. For certain experiments, the same membrane was probed with more than one cDNA. In these experiments, each probe was stripped from the membrane before subsequent hybridizations. Filters were washed with 2xSSPE at 68°C for 20 minutes, with 1xSSPE/1% SDS at 68°C for 15 minutes, and with 1xSSPE at 21°C for 15 minutes and were then exposed to Hyperfilm-MP (Amersham) for 24 to 96 hours. Analyses of radioactivity were performed on a Betascope 603 blot analyzer (Betagen).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
down arrowReferences
 
Molecular Cloning of Canine IL-6
An LPS-stimulated, oligo-dT–primed, CJVEC cDNA library was screened with a specific canine IL-6 PCR probe encoding the equivalent of nucleotides 30 to 729 of the human IL-6 cDNA.13 This probe was generated by use of oligonucleotides encompassing highly conserved sequences of human and porcine IL-6 as primers for the amplification reaction.13 14 Thirty-four {lambda} cDNA clones hybridized and contained inserts of 400 bp to 1 kb that upon Southern blotting demonstrated strong hybridization signals. Clone dIL-6-1, the largest of these clones, contained an insert 1034 bp in length. However, clone dIL-6-1 did not encode an initiation codon and extended from the equivalent of residue 14 of the human IL-6 primary protein to the poly (A) tail. In an attempt to isolate a clone encoding the remaining 5' sequences of the IL-6 cDNA, we screened a random-primed cDNA library with a probe representing the 5' end of clone dIL-6-1. This library was prepared from TNF-{alpha}–stimulated endothelial cell poly (A+) RNA and was screened under the same conditions as the oligo-dT–primed library. Four {lambda} clones were isolated and subjected to further analyses. All four clones encoded the remainder of the IL-6 signal peptide, contained the initiation codon, and extended into the 5' untranslated region. One of these clones, dIL-6-8, was sequenced in its entirety. Nucleotide sequences derived from clones dIL-6-1 and dIL-6-8 were used to compile the complete canine IL-6 cDNA (Fig 1Down). The nucleotide sequences of the 5' untranslated region, along with the first four codons and the first nucleotide of the fifth codon derived from dIL-6-8, were added to those of clone pdIL-6-1 to construct the complete canine IL-6 cDNA. DNA sequence and restriction endonuclease analysis established the relation of each of these cDNA clones (Fig 2Down). Sequence analysis revealed that the canine IL-6 cDNA is 1104 bp in length, consisting of a 57-nucleotide-long 5' untranslated region followed by a single open-reading frame of 621 bp and a 3' untranslated region of 426 bp excluding the poly (A) tail. The open-reading frame begins with the AUG start codon at position 58 and ends with the TAG stop codon at position 679. The length of the 3' untranslated region of canine IL-6 is similar to that reported for human, porcine, and mouse IL-6.13 14 22 Furthermore, an 85% nucleotide sequence identity is present between human and canine 3' untranslated regions (Fig 3Down). Within the 3' untranslated region of canine IL-6 are five copies of the pentamer AUUUA found in many cytokines and chemokines. These sequences are capable of reducing mRNA stability23 24 and accelerating nuclear transport.25 Four of these pentamers are completely conserved in human IL-6 (Fig 3Down). Finally, a eukaryotic consensus polyadenylation hexamer, AATAAA, is followed 9 bp downstream by a poly (A) tail.26



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  		Figure 1. Complete cDNA and deduced amino acid sequence of canine IL-6. Both strands of clone pdIL6-1 were completely sequenced by the dideoxynucleotide termination method and automated DNA sequencing (see "Methods"). The complete sequence of a single cDNA clone encoding the 5' end of the IL-6 cDNA (pdIL-6-8) was also determined as above. The nucleotide sequences of the 5' untranslated region along with the first four codons and the first nucleotide of the fifth codon derived from clone pdIL-6-8 were added to those of clone pdIL-6-1 and used to construct the complete canine IL-6 cDNA sequence. The N-terminal methionine is underlined, and the consensus polyadenylation hexamer is double underlined. This nucleotide sequence has been submitted to Genbank under the accession number U12234.



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  		Figure 2. Structure of the canine IL-6 cDNA and clones characterized in this study. The IL-6 cDNA is depicted with the 5' untranslated region as a solid bar, the coding region as a dark hatched bar, and the 3' untranslated region as an open bar. The relative positions of selected restriction endonucleases are shown at top; nucleotides are numbered in the 5' to 3' direction, starting with the first nucleotide of the complete IL-6 cDNA. The two cDNA clones that originated from the endothelial cell cDNA libraries are shown as hatched bars (dIL-6-1 and dIL-6-8). The relative positions of restriction endonuclease cleavage sites are shown. The PCR-derived cDNA fragment used to isolate these clones is depicted at the bottom as a solid bar.



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  		Figure 3. Comparison of the canine and human cDNA 3' untranslated regions. Nucleotides are numbered from 5' to 3', beginning with the TAG stop codon. Nucleotide identity is indicated with a dash (-). Dots indicate a gap in the sequence. The stop codon is boldface, and the polyadenylation consensus signal is underlined.

The complete IL-6 cDNA encodes a 207–amino acid precursor protein. The nucleotide sequences in the vicinity of the AUG provide the context for translation initiation and are in agreement with many of the known consensus sequences present in eukaryotes.27 28 Furthermore, the canine IL-6 initiation motif CAGCTAUGA is identical to the human,13 porcine,14 and bovine29 IL-6 homologues. After the N-terminal methionine, there is a 23–amino acid region typical of a signal peptide.30 This N-terminal signal peptide is highly homologous to those of human, porcine, and bovine IL-6.13 14 29 However, all of the clones isolated from both cDNA libraries contained a 15-nucleotide deletion corresponding to amino acids 8 through 12 of the human IL-6 primary protein. Interestingly, this region corresponds to the junction of the first and second exons in the genomic structure of human31 and mouse IL-6.32 Nucleotide deletions in the IL-6 signal peptide have also been reported for murine and rat IL-6 cDNA.22 33 The canine IL-6 protein deduced from the cDNA sequence displayed 60% overall identity to human IL-6. It also displayed high levels of amino acid homology to porcine IL-6, being 70% identical (Fig 4Down). In total, 109 amino acids are identical between the three species, and all four cysteine residues present in human and porcine IL-6 are conserved. There is also remarkable similarity with human and porcine IL-6 in terms of the signal peptide sequence and total length of the proteins.13 14 Canine IL-6 does not contain potential N-linked glycosylation sites.



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  		Figure 4. Comparison of the deduced amino acid sequences of human, porcine, and canine IL-6. Canine, human,18 and porcine19 IL-6 cDNA sequences were translated and compared. Identical amino acids are indicated with a dash (-). Conserved cysteine residues are indicated by asterisks (*). Percent amino acid identities to canine IL-6 are 60% and 70% for human and porcine IL-6, respectively.

Regulation of IL-6 Expression in CJVECs
Regulation of IL-6 gene expression was first studied in vitro in cultured CJVECs. Northern analysis using the canine IL-6 cDNA as a probe demonstrated that cultured CJVECs were capable of expressing IL-6 mRNA (Fig 5Down). The main transcript was identified as a 1.1-kb hybridizing band. Under the specific culture conditions, we occasionally detected minimal levels of IL-6 mRNA as a single species in untreated control samples. Incubation of CJVECs with TNF-{alpha} rapidly elicited a marked increase in IL-6 mRNA. Kinetic studies of the induction of IL-6 mRNA demonstrated peak levels after only 30 minutes of stimulation. Steady-state levels of IL-6 mRNA remained elevated for several hours, returning to baseline after 5 hours of stimulation. These results are consistent with the known induction of IL-6 on human endothelial cells by cytokines in vitro.34



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  		Figure 5. Regulation of IL-6 mRNA expression in CJVECs by TNF-{alpha}. Total cellular RNA was isolated from T-75 flasks of CJVECs after incubation in the presence or absence of TNF-{alpha} (30 U/mL) for the indicated times (0 to 5 hours). Levels of IL-6 mRNA were assayed by Northern analysis and hybridization with a 32P-labeled canine IL-6 cDNA probe. The time course of steady-state CJVEC IL-6 mRNA is demonstrated. Each lane contained 5 µg of RNA, as shown by the ethidium bromide staining. The migration position of the 18S rRNA band is marked.

Induction of IL-6 mRNA in CJVECs by Postischemic Cardiac Lymph
Cardiac lymph, representing myocardial extracellular fluid, was collected via cannulation of the cardiac lymphatic system and assayed for its ability to induce IL-6 mRNA in CJVECs. As shown in Fig 6Down, postreperfusion cardiac lymph elicited IL-6 mRNA when incubated with CJVECs in all samples tested. In contrast, preischemic lymph did not elicit significant levels of IL-6 mRNA. Peak stimulatory activity for IL-6 was present in the first hour after reperfusion of the previously ischemic myocardium.



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  		Figure 6. Induction of IL-6 mRNA in canine endothelial cells by postischemic cardiac lymph. Aliquots of cardiac lymph, representing myocardial extracellular fluid, were collected before coronary artery occlusion or at designated times during reperfusion. The ability of cardiac lymph (1:7 dilution) to induce IL-6 mRNA was assessed by incubation with canine endothelial cell monolayers for 2 hours at 37°C and compared with that of TNF-{alpha} (30 U/mL; 2 hours at 37°C). Media (lane 2) represents the tissue culture media used for the endothelial cell cultures (see "Methods"). This tissue culture medium was used to dilute both TNF-{alpha} and cardiac lymph. After incubation, total cellular RNA was isolated and subjected to Northern analysis and hybridization with an IL-6 cDNA probe. Each lane contained 10 µg of total RNA, as shown by the ethidium bromide staining. The migration position of the 18S rRNA band is marked.

Regulation of IL-6 mRNA in Ischemic and Reperfused Myocardium
The expression of IL-6 mRNA after experimental circumflex coronary artery occlusion and reperfusion was assessed in ischemic and reperfused myocardial segments. These segments were characterized for their blood flow during occlusion by use of radiolabeled microspheres. Representative experiments in animals subjected to 1 hour of coronary occlusion and 1 hour of reperfusion are shown in Fig 7Down. In the experiment illustrated on the right (lanes C through G), the animal sustained a large myocardial infarction. Significant induction of IL-6 mRNA was observed in all ischemic and reperfused myocardial samples analyzed (lanes D through G). IL-6 mRNA was not detectable in normally perfused anterior ventricular wall segments (lane C). Contrasting findings were evident in the experiment illustrated on the left (lanes A and B), which demonstrated no evidence of significant blood flow reduction after identical coronary occlusion and reperfusion protocols (lane B). In this experiment, IL-6 mRNA was not elicited in any of the myocardial tissue segments analyzed. Similar findings were observed in an additional series of experiments in which the reperfusion interval was extended to 3 hours. Highest levels of IL-6 mRNA were observed in the most ischemic myocardial segments (Fig 8Down, lanes C and D). IL-6 mRNA was also detected in segments with more modest blood flow reductions but was much less intense than observed with more severe degrees of ischemia. No detectable levels of IL-6 mRNA were present in normally perfused myocardial segments (lanes A and B). Coronary occlusions not associated with significant levels of ischemia, presumably because of collateral blood flow, did not elicit measurable levels of IL-6 mRNA despite identical occlusion and reperfusion intervals (Fig 8Down, lanes F and G).



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  		Figure 7. Regulation of myocardial IL-6 mRNA after 1 hour of coronary occlusion and 1 hour of reperfusion. Representative results from experimental animals that were exposed to 1 hour of coronary occlusion and 1 hour of reperfusion are shown. Two representative experiments are shown in this figure. One developed a myocardial infarction (lanes C through G), whereas the other did not (lanes A and B). Induction of IL-6 mRNA was assessed in RNA isolated from control (C) or ischemic and reperfused (I) myocardial segments. RNA was processed for Northern blots and hybridized with a 32P-labeled IL-6 cDNA as described in "Methods." Blood flow to each individual myocardial segment is indicated. Each lane contained 20 µg of RNA, as shown by the ethidium bromide staining at bottom. Top, Graphical representation of the signal obtained by analysis of the radioactivity measured by a Betascope blot analyzer in each lane.



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  		Figure 8. Regulation of myocardial IL-6 mRNA after 1 hour of coronary occlusion and 3 hours of reperfusion. Representative results from experimental animals that were exposed to 1 hour of coronary occlusion and 3 hours of reperfusion are shown. Two representative experiments are shown in this figure. The first developed a myocardial infarction (lanes A through E), but the second did not (lanes F and G). Induction of IL-6 mRNA was assessed in RNA isolated from control (C) or ischemic and reperfused (I) myocardial segments in Northern blots hybridized with an IL-6 cDNA probe. Blood flow to each myocardial segment is indicated. Each lane contained 20 µg of RNA, as shown by the ethidium bromide staining (bottom). Top, Graphical representation of the signals obtained by analysis of radioactivity measured by a Betascope blot analyzer in each lane.

To directly assess the relation of time on induction of myocardial IL-6 mRNA, a series of experiments with various intervals of reperfusion were compared. In all 5 experiments shown in Fig 9Down, the circumflex coronary artery was occluded for 1 hour, and subsequently the myocardium was reperfused for 1, 3, or 24 hours. A time-dependent induction of the IL-6 gene was present in ischemic and reperfused segments of these 5 animals. Steady-state levels of IL-6 mRNA peaked 3 hours after reperfusion and remained elevated for 24 hours after reperfusion. IL-6 mRNA levels were inversely related to regional blood flow during ischemia, with the highest levels being detected in the myocardial segments displaying the greatest degree of ischemia. Concurrent assessments of ischemic blood flow in 22 experiments with intervals of reperfusion ranging from 1 to 24 hours demonstrated that only segments with reductions in blood flow of >50% demonstrated measurable levels of IL-6 mRNA. Segments with blood flow reductions >80% generally displayed the highest levels of IL-6 gene induction compared with less ischemic myocardial segments.



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  		Figure 9. Regulation of myocardial IL-6 mRNA: relation to time of reperfusion. Representative results from experimental animals that were exposed to 1 hour of coronary occlusion and 1, 3, or 24 hours of reperfusion are shown. Five representative experiments are shown in this figure. Each separate experiment is identified by brackets at bottom. Myocardial infarcts were present in all five experiments. Myocardial segments are labeled C for control and I for ischemic and reperfused. Blood flow to each myocardial segment is indicated. Each lane contained 20 µg of RNA, as shown by the ethidium bromide staining (bottom).

To examine whether ICAM-1 mRNA was detectable during early reperfusion, in the same segments in which IL-6 induction was found, levels of mRNA for both genes were assessed in the same ischemic and reperfused myocardial segments. Fig 10Down shows experiments in which the myocardium was ischemic for 1 hour and subsequently reperfused for 3 hours. Four separate experiments are shown, each one identified by brackets at the bottom of Fig 10Down. Consistent with our previous observations after 3 hours of reperfusion, ICAM-1 is induced in ischemic and reperfused segments of myocardium.10 In all 4 experiments shown, ICAM-1 mRNA was detected in the same ischemic and reperfused segments in which IL-6 mRNA was found. This finding was invariably present during the first 3 hours of reperfusion after 1 hour of ischemia.



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  		Figure 10. IL-6 and ICAM-1 are induced in the same ischemic and reperfused myocardial segments. Four representative experiments are shown. Experimental animals were exposed to 1 hour of coronary occlusion and 3 hours of reperfusion. Each separate experiment is identified by brackets at bottom. Myocardial infarcts were present in all four experiments. Control segments are labeled C, and ischemic and reperfused segments are labeled I. The same nylon membrane was sequentially probed with 32P-labeled canine ICAM-1 (top) and 32P-labeled canine IL-6 (middle) to assess the expression of both genes in the same myocardial segments. Subsequently, the membrane was probed with human 32P-labeled GAPDH (bottom) as loading control.

Reperfusion Accelerates the Induction of Myocardial IL-6 mRNA
The role of reperfusion in the myocardial induction of IL-6 mRNA was directly assessed in a subsequent series of experiments comparing experiments with and without reperfusion (Figs 11Down and 12Down). In contrast to the high levels of IL-6 mRNA observed in the ischemic and reperfused myocardium, in nonreperfused segments after 4 hours of persistent ischemia without reperfusion, we detected only minimal levels of IL-6 mRNA (Fig 11Down). These findings were evident in the presence of comparable degrees of myocardial blood flow reductions. In the absence of reperfusion, minimal levels of IL-6 mRNA were observed during the first 4 hours of ischemia in four separate experiments. In these four experiments, a total of 14 separate segments were examined. Six of the 14 segments studied had relatively low but detectable levels of IL-6 mRNA. No detectable levels of IL-6 mRNA were found in the remaining 8 segments in this series of studies. Furthermore, when the radioactivity in each of the lanes representing the levels of IL-6 mRNA in each myocardial segment was quantified, it did not exceed 15% of equally ischemic but reperfused segments that originated from six separate experiments that were ischemic for 1 hour and reperfused for 3 hours (data not shown). As previously shown in Fig 10Up, a consistent finding in this type of experiment was the induction of ICAM-1 mRNA in the same myocardial segments in which IL-6 mRNA was found. The role of ischemia and reperfusion on ICAM-1 induction has been previously described.10 11



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  		Figure 11. Induction of IL-6 and ICAM-1 is accelerated by reperfusion. Experimental animals were exposed to 1 hour of coronary occlusion followed by 3 hours of reperfusion (left) or 4 hours of ischemia without reperfusion (right). Myocardial infarcts were present in both experiments. Myocardial segments are labeled C for control and I for ischemic and reperfused. Blood flow to each myocardial segment is indicated. The Northern blot was first hybridized with a 32P-labeled IL-6 cDNA probe (bottom), the probe was then stripped, and the membrane was subsequently hybridized with a 32P-labeled canine ICAM-1 cDNA probe (top) to assess the expression of both genes in the same myocardial segments.



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  		Figure 12. IL-6 is induced in the myocardium after 24 hours of ischemia. Experimental animals were exposed to 1 hour of coronary occlusion followed by 24 hours of reperfusion (right, labeled I/R 1h/24h) or 24 hours of ischemia without reperfusion (left, labeled 24 h Ischemia). Each separate experiment is identified by brackets at bottom. Myocardial infarcts were present in both experiments. Myocardial segments are labeled C for control and I for ischemic or ischemic and reperfused. Blood flow to each myocardial segment is indicated. The Northern blot was first hybridized with a 32P-labeled IL-6 cDNA probe (top), the probe was then stripped, and the membrane was subsequently hybridized with a 32P-labeled human GAPDH probe (bottom) as loading control.

Contrasting findings were evident in the experiments illustrated in Fig 12Up. After persistent ischemia for 24 hours, levels of IL-6 mRNA were present in ischemic segments and were comparable to those observed in segments that were ischemic for 1 hour and reperfused for 24 hours (Fig 12Up). These findings are representative of four separate experiments. In agreement with the experiments in which reperfusion was instituted, concurrent assessment of myocardial blood flow indicated that the degree of ischemia was a major determinant regulating the levels of induction of IL-6 mRNA. Highest levels of induction were present in segments demonstrating the lowest levels of blood flow, whereas IL-6 mRNA was not detectable in normally perfused segments. IL-6 mRNA was detected in ischemic segments with moderately diminished levels of blood flow but was less than observed with more severe blood flow reductions.


*    Discussion
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up arrowResults
*Discussion
down arrowReferences
 
In this study, we describe for the first time the isolation and characterization of cDNA clones encoding the canine IL-6 gene. We used this cDNA as a molecular probe to investigate the regulation of IL-6 in the context of myocardial ischemia and reperfusion. We have provided direct evidence that IL-6 is rapidly induced in the myocardium after reperfusion of the previously ischemic heart. This report extends our previous observations that postreperfusion cardiac lymph contains cytokine activity.9 This activity is capable of inducing ICAM-1 mRNA expression and ICAM-1–dependent adhesion of neutrophils to cardiac myocytes, both of which are reduced with a polyclonal antibody directed against human IL-6.9 10 It also strengthens our insights into the induction of the ICAM-1 gene in the myocardium10 11 by providing molecular evidence for the induction of a cytokine likely to be a potential stimulus of myocyte ICAM-1 synthesis in vivo.9

On the basis of nucleotide sequence analysis and comparisons of the deduced protein sequence, we established that the cDNA clones isolated contain the complete open-reading frame of the canine homologue of IL-6. The canine IL-6 protein is 70% identical with its corresponding porcine sequence and 60% identical with human IL-6. While the degree of similarity between human and canine IL-6 proteins is surprisingly low, in view of the evidence that recombinant human IL-6 acts on canine cardiac myocytes,9 a similar phenomenon has been observed between human and rat IL-6. Human IL-6 is only 58% identical to rat IL-6, yet human IL-6 acts on rat liver cells, in which it must be recognized by the IL-6 receptor.33 Quite possibly, a receptor binding domain of IL-6 is conserved across these species.33 Such a proposal is further supported by the finding that all cysteine residues present in human IL-6 are conserved in both canine and rat IL-6, predicting the same disulfide bond pattern and perhaps similar tertiary structure.

A striking feature of the IL-6 gene compared across species is the high degree of nucleotide sequence identity in the 5' and 3' flanking regions, indicating that the transcriptional controls have been evolutionarily conserved.31 32 The high degree of nucleotide sequence homology in the 3' untranslated regions of human and canine IL-6 cDNA suggests that mechanisms controlling mRNA degradation23 24 and nuclear transport25 are conserved even when the coding regions have significantly diverged. Such a high level of regulation and control is likely to be important in IL-6 in light of the pleiotropic nature of its actions, the ubiquitous distribution of IL-6 receptors, and its involvement in orchestrating the response of the organism to incipient tissue injury or necrosis, otherwise known as the acute-phase response.35 36 37

The present studies document the upregulation of IL-6 mRNA in the previously ischemic myocardium, demonstrating that part of the inflammatory response that ensues as a result of ischemia and reperfusion is an increase in IL-6 mRNA expression. This induction occurs preferentially in ischemic and reperfused myocardial segments under conditions of significant blood flow reduction, while sparing the normally perfused control segments. The highest levels of induction observed were present in the most ischemic myocardial segments regardless of the reperfusion interval. Furthermore, an additional level of specificity was afforded by the observation that IL-6 was not induced in animals when coronary occlusion was not associated with blood flow reductions. These findings are consistent with previously reported patterns of induction of ICAM-1 mRNA under similar experimental conditions10 and with the time course of IL-6 cytokine activity present in cardiac lymph.9 Our findings are also consistent with the observed elevated levels of plasma IL-6 bioactivity in models of ischemia and reperfusion in other tissues.38

The association of myocyte ICAM-1 induction with IL-6 was originally made by our observations that the synthesis of ICAM-1 in cardiac myocytes stimulated by postreperfusion cardiac lymph was neutralized by a polyclonal antibody against human IL-6.9 Several lines of evidence presented in the study at hand strengthen this association. IL-6 mRNA is induced in the myocardium in the same ischemic segments in which ICAM-1 mRNA is found. IL-6 mRNA peaks in the first 3 hours of reperfusion, whereas ICAM-1 mRNA continues to increase for up to 24 hours.10 Thus, peak IL-6 mRNA levels in the myocardium precede those for ICAM-1 mRNA.10 The accelerating effect of reperfusion appears to be equally important for the early induction of both ICAM-1 and IL-6 genes in experimentally defined myocardial segments. In this regard, it is important to emphasize that this conclusion is supported by comparison with persistently ischemic myocardial segments. The role of IL-6 in the induction of ICAM-1 appears to be cell specific. Cardiac myocytes appear to be particularly sensitive to IL-6 stimulation9 : the concentrations of IL-6 required to induce ICAM-1 on myocytes are up to two orders of magnitude lower than those required for induction on liver cells and melanocytes.9 39 40 Furthermore, we have found that most of the ICAM-1 produced by liver cells was released into the supernatant; consequently, only minimal amounts remained on the cell surface.39 These findings, coupled with the inability of IL-6 to induce ICAM-1 on endothelial or epithelial cells,9 40 suggest that IL-6 may be a selective inducer of ICAM-1, with effects limited to specific parenchymal cells.

Expression of ICAM-1 on cardiac myocytes is possibly an important mechanism facilitating the cytotoxic potential of neutrophils for myocytes, since activated neutrophils are capable of directly damaging cardiac myocytes through CD11b/CD18–ICAM-1–mediated adhesion.6 This adhesive mechanism can be directly stimulated by the ability of recombinant IL-6 to induce ICAM-1 on cardiac myocytes.9 In addition, IL-6 effects may extend beyond the induction of ligand-specific adhesion of neutrophils to cardiac myocytes. IL-6 is devoid of a direct effect on neutrophils, but it has been shown to prime and enhance the neutrophil oxidative burst in response to chemotactic stimulation.41 This priming effect represents a potential complementary mechanism for IL-6 to facilitate neutrophil cytotoxic behavior in reperfused myocardium, in which the presence of complement-derived chemotactic factors and IL-8 has been demonstrated.7 8 41 42 43 44 Finally, recent reports have demonstrated elevated levels of IL-6 in patients with acute myocardial infarction45 46 and that clinically achievable levels of IL-6 could potentially cause reversible myocardial depression in the human heart.47

Our data imply that events initiated by reperfusion of the previously ischemic myocardium are responsible for the rapid and localized cytokine generation that ultimately results in highly localized ICAM-1 mRNA expression.10 11 It is well known that one of the factors induced by reperfusion is a marked influx of leukocytes into the ischemic area, resulting in dense margination in cardiac venules followed by neutrophil migration.3 10 48 49 50 We have proposed that cytokine induction in the myocardium is dependent on the postreperfusion influx of blood leukocytes.51 Both neutrophils and monocytes are known to produce IL-6 in response to stimulation by cytokines such as TNF-{alpha} or IL-1,52 53 and recent data also suggest that C5a stimulation induces interleukin-6 synthesis in mononuclear cells.54 This proposal is supported by the finding of IL-6 induction after 24 hours of persistent ischemia. By this time, in contrast to permanent occlusions of only 4 hours, leukocytes have densely infiltrated the infarcted area, even in the absence of reperfusion.55 56 Although the augmented influx of inflammatory cells may be responsible for IL-6 production, the data suggest that the cytokine control of this process may involve a more complex construct. Interestingly, the ability of cardiac lymph to induce ICAM-1 in canine endothelial cells was not inhibited by anti–IL-6 antibodies,9 leading us to postulate the appearance of more than one cytokine in cardiac lymph during reperfusion. The present results are best explained by the presence of an "upstream" inducer capable of stimulating IL-6 synthesis; this would account for the ability of postischemic cardiac lymph to induce IL-6 mRNA in endothelial cells (Fig 6Up). The identity and source of this putative "upstream" inducer remains to be determined. IL-6 induction may result from a complex cell physiological event that ultimately allows neutrophil-myocyte interactions via CD11b/CD18–ICAM-1 adhesion.


*    Selected Abbreviations and Acronyms
 
CJVECs = canine jugular vein endothelial cells
ICAM = intercellular adhesion molecule
IL = interleukin
LPS = lipopolysaccharide
PCR = polymerase chain reaction
TNF = tumor necrosis factor


*    Acknowledgments
 
These studies were supported by NIH grant HL-42550, by grants from The Methodist Hospital Foundation and Baylor College of Medicine (Dr Kukielka), and by the Lillie Frank Abercrombie Foundation (Dr Kukielka). The authors wish to acknowledge the assistance of J.L. Slightom and R.F. Drong with DNA sequencing and the assistance of Leonardo Mendoza, Lisa T. Thurmon, Peggy Jackson, Gary Liedtke, and Wei Chen with animal experiments and tissue culture.


*    Footnotes
 
Reprint requests to Gilbert L. Kukielka, MD, Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498.

Guest editor for this article was Robert L. Engler, MD, San Diego (Calif) VA Medical Center.

Received July 18, 1994; revision received January 17, 1995; accepted April 5, 1995.


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