This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gwechenberger, M. Articles by Entman, M. L. Search for Related Content PubMed PubMed Citation Articles by Gwechenberger, M. Articles by Entman, M. L. Pubmed/NCBI databases Substance via MeSH Medline Plus Health Information Heart Attack Related Collections Biochemistry and metabolism Gene expression Growth factors/cytokines

(Circulation. 1999;99:546-551.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Cardiac Myocytes Produce Interleukin-6 in Culture and in Viable Border Zone of Reperfused Infarctions

Marianne Gwechenberger, MD; Leonardo H. Mendoza, MS; Keith A. Youker, PhD; Nikolaos G. Frangogiannis, MD; C. Wayne Smith, MD; Lloyd H. Michael, PhD; Mark L. Entman, MD

From the Section of Cardiovascular Sciences, Department of Medicine, The DeBakey Heart Center, Section of Leukocyte Biology, Department of Pediatrics, and Texas Children's Hospital, Baylor College of Medicine, Houston, Tex.

Correspondence to Mark L. Entman, MD, Department of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, M/S F-602, Houston, TX 77030-3498. E-mail mentman{at}bcm.tmc.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Previous work from our laboratory demonstrated that interleukin (IL)-6 plays a potentially critical role in postreperfusion myocardial injury and is the major cytokine responsible for induction of intracellular adhesion molecule (ICAM)-1 on cardiac myocytes during reperfusion. Myocyte ICAM-1 induction is necessary for neutrophil-associated myocyte injury. We have previously demonstrated the induction of IL-6 in the ischemic myocardium, and the current study addresses the cells of origin of IL-6.

Methods and Results—In the present study, we combined Northern blot analysis and in situ hybridization to demonstrate IL-6 gene expression in cardiac myocytes. Isolated ventricular myocytes were stimulated with tumor necrosis factor-, IL-1ß, lipopolysaccharide, preischemic lymph, and postischemic lymph. Unstimulated myocytes showed no significant IL-6 mRNA expression. Myocytes stimulated with preischemic lymph showed minimal or no IL-6 mRNA expression, whereas myocytes stimulated with tumor necrosis factor-, IL-1ß, lipopolysaccharide, or postischemic lymph showed a strong IL-6 mRNA induction. Northern blot with ICAM-1 probe revealed ICAM-1 expression under every condition that demonstrated IL-6 induction. We then investigated the expression of IL-6 mRNA in our canine model of ischemia and reperfusion. Cardiac myocytes in the viable border zone of a myocardial infarction exhibited reperfusion-dependent expression of IL-6 mRNA within 1 hour after reperfusion. Mononuclear cells infiltrate the border zone and express IL-6 mRNA.

Conclusions—Isolated cardiac myocytes produce IL-6 mRNA in response to several cytokines as well as postischemic cardiac lymph. In addition to its production by inflammatory cells, we demonstrate that IL-6 mRNA is induced in myocytes in the viable border zone of a myocardial infarct. The potential roles of IL-6 in cardiac myocytes in an infarct border are discussed.

Key Words: interleukins • myocardial infarction • reperfusion • myocytes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Reperfusion of infarcted myocardium has been accepted as the treatment of choice for acute myocardial infarction and markedly accelerates the development of an inflammatory reaction in the infarcted tissue. Inflammation plays an important role in the healing of tissue after injury.^{1 2 3 4 5 6 7 8 9} However, there is evidence that the accelerated inflammatory response may also extend tissue injury. Clinical and experimental studies showed that the inflammatory response to myocardial infarction is associated with the induction of cytokines such as tumor necrosis factor (TNF)-, interleukin (IL)-1ß, and IL-6, which are thought to act in a "cascade fashion."^{10 11 12}

Our laboratory has focused on the cellular and molecular basis of the inflammatory reaction resulting from ischemia and reperfusion by using a strategy that integrates the insights from experiments done in cell culture with a disease model of myocardial ischemia and reperfusion in intact animal. Studies in our laboratory demonstrated a potential mechanism of neutrophil-induced myocyte injury. Neutrophils adhere to myocytes in the presence of intracellular adhesion molecule (ICAM)-1 on the myocytes and CD11b/CD18 on the neutrophils^{13 14} and mediate oxidative cytotoxicity for cardiac myocytes.¹⁵ With the use of a canine model in which we cannulated the cardiac lymph duct,^{16 17} we demonstrated that postischemic lymph contained cytokine activity capable of inducing ICAM-1 expression in cardiac myocytes in vitro. This activity could be abolished by neutralizing antibodies to IL-6.¹⁸

In the same model, we demonstrated that reperfusion-dependent induction of ICAM-1 mRNA occurred in the viable myocytes of the border zone of myocardial infarction.^{19 20 21}

Subsequent studies showed an induction of IL-6 in the myocardium of a canine model of ischemia and reperfusion. IL-6 mRNA was found in the ischemic area and appeared to peak earlier on reperfusion. These studies demonstrated that IL-6 synthesis is an integral part of the reaction to injury resulting from ischemia and reperfusion and is associated with induction of ICAM-1 on the myocardial cells.^{21 22} IL-6 is produced by a variety of different cell types, including monocytes/macrophages, fibroblasts, endothelial cells, mast cells, neutrophils, keratinocytes, osteoblasts, and many more.²³ In our previous work, we had reasoned that IL-6 mRNA induction was reperfusion dependent because of its association with the reperfusion-induced influx of leukocytes.^{22 24 25} The current study confirms the induction of IL-6 mRNA in mononuclear cells; however, it also provides the first direct demonstration that cardiac myocytes in vitro and in the viable border zone of a reperfused infarction also produce IL-6 mRNA.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of Cardiac Myocytes
The isolation of cardiac myocytes from healthy mongrel dogs of either sex was performed as described previously.^{13 18} The cells were resuspended with medium A and equilibrated with 95% O₂/5% CO₂. Preparations with a viability of >80% were used in the experiments. Nonmyocyte cells were <10% in all experiments.

The cell samples were incubated for 60 minutes at 37°C under one of the following conditions: control, TNF- (100 U/mL), IL-1ß (200 U/mL), lipopolysaccharide (LPS) 50 µg/mL, preischemic lymph (1:10 dilution), or postischemic lymph (1:10 dilution). TNF- and IL-1ß were obtained from Genzyme Corp. LPS was obtained from Sigma Chemical Co. After the incubation, the cells were spun again, the supernatant discarded, and the cells were immediately processed for RNA isolation or for immunohistochemical studies.

All dishes and instruments used in the isolation process were pretreated with E-Toxa-Clean (Sigma Chemical Co) before sterilization to eliminate LPS contamination. Supernatants and solutions were periodically tested for LPS contamination with the E-Toxate Limulus LPS detection kit (Sigma Chemical Co), which is sensitive to 0.05 to 0.1 endotoxin units/mL. If the supernatants and solution showed a negative result with this test, they were considered uncontaminated, and only those preparations were used.

Ischemia-Reperfusion Protocols
Healthy mongrel dogs were surgically instrumented as described in detail previously,^{16 17 21} with cannulation of the cardiac lymph duct^{16 17} and placement of a hydraulically occluding device and a Doppler flow probe secured around the circumflex coronary artery just proximal or distal to the first branch. The animals were allowed to recover for 72 hours before occlusion. Coronary artery occlusion was achieved by inflating the coronary cuff occluder until mean flow in the coronary vessel was zero, which was determined by the Doppler flow probe. For subsequent blood flow analysis, radiolabeled microspheres were injected into the left atrium 50 minutes after occlusion. After 1 hour of occlusion, the cuff was deflated and the heart was reperfused for up to 24 hours. Lymph samples used for the myocyte experiment were collected from the cannulas in tubes containing 10 U preservative-free heparin within 3 hours of reperfusion. At the end of the reperfusion period, the heart was stopped by infusion of saturated potassium chloride and removed immediately and processed for RNA isolation or immunohistochemistry and in situ hybridization as previously described.^{20 22} Adjacent sections were used for blood flow determination with radiolabeled microspheres as previously described.^{26 27} Analysis of RNA, blood flow determination, and histopathology were performed independently in different laboratories and in a blinded fashion. Sections used for studies of infarction demonstrated infarction by histopathology and ischemic blood flow of <20%.

All animal protocols were reviewed by the appropriate institutional review committee and conform to institutional guidelines.

Northern Blot Analysis
RNA was isolated from myocytes and myocardial tissue by use of the acid guanidinium phenol chloroform procedure.²⁸ RNA was electrophoresed in 1% agarose gels containing formaldehyde, then transferred to a nylon membrane (Gene screen Plus; New England Nuclear) by standard procedure.²⁹ Loading of RNA was monitored with the use of ethidium bromide staining as well as by probing the nylon membranes with glyceraldehyde 3-phosphate dehydrogenase as previously described.²² Canine IL-6 cDNA and ICAM-1 cDNA were prepared as previously described.^{14 19 21 22}

Immunohistochemical Methods
Samples were fixed in 4% formaldehyde, dehydrated by incubation in increasing concentrations of ethanol with a standard protocol, and then embedded in paraffin; 3- to 5-µm sections were obtained with microtomy. For immunostaining, the slides were rehydrated and incubated with hydrogen peroxide. Immunostaining was performed with the Elite kit (Vector laboratories), which has a peroxidase-based detection system. For myoglobin, the color reaction was performed with AEC tablets from Vector Laboratories as a substrate without counterstaining.

To define viable from nonviable cells, histological criteria such as contraction band necrosis and disrupted cell architecture were supplemented with specific histological stains. To distinguish further between infarcted and noninfarcted areas, serial sections to those used for in situ hybridization were immunostained for myoglobin with an antimyoglobin monoclonal antibody (rabbit anti-human antimyoglobin antibody; DAKO) and were stained for glycogen with PAS (periodic acid–Schiff).^{30 31 32}

In Situ Hybridization
Riboprobes for canine IL-6 and ICAM-1 were prepared as previously described.²⁰ Digoxigenin-labeled probes were prepared by in vitro transcription from a linearized template with the Genius RNA probe labeling kit (Boehringer Mannheim Corp) according to the manufacturer's instructions. A 215-bp [bases 483 to 697 of the published sequence²² ] fragment of canine IL-6 cDNA was obtained with polymerase chain reaction amplification and was subcloned into the polymerase chain reaction plasmid (Invitrogen) so that the use of SP6 polymerase would result in the generation of single-stranded antisense (3'-5') and the use of T7 polymerase would result in the generation of the sense (5'-3') probe. For the ICAM-1 probe, a 150-bp fragment was subcloned in both orientations into PBluescript II Sk+ and PGEM-3 so that use of T7 polymerase would result in the generation of single-stranded antisense (3'-5') and of the sense (5'-3') probe.²⁰ The procedure was the same as described above for the IL-6 riboprobe.

The immunologic detection of the hybridized probe was done with the Genius detection kit from Boehringer Mannheim, following the guidelines of the company as previously described.²⁰ The slides were stained with NBT and x-phosphate and then counterstained with eosin.

Statistics
Because of the characteristics of the experiments, raw data are presented for each of the pertinent findings along with controls. Each experiment presented was repeated at least 5 times with the same qualitative results.

	Results

Top Abstract Introduction Methods Results Discussion References

 
IL-6 Induction in Isolated Ventricular Myocytes: Northern Blot Analysis
The top panel of Figure 1 shows Northern blot analysis of mRNA isolated from ventricular myocyte preparations under a variety of experimental conditions. Marked IL-6 mRNA induction was noted in myocytes incubated with TNF- (100 U/mL), IL-1ß (200 U/mL), endotoxin (50 µg/mL), or cardiac lymph (diluted 1:10) collected during the first hour after reperfusion of a myocardial infarction. In contrast, cardiac lymph collected before the myocardial infarction (preischemic lymph diluted 1:10) demonstrated minimal or no induction of IL-6 mRNA in cardiac myocytes incubated under identical conditions. All samples were incubated at 37°C for 1 hour. Unstimulated control myocytes kept at room temperature for 1 hour never showed IL-6 mRNA expression. However, after incubation at 37°C for 1 hour, a minimal induction of IL-6 was sometimes observed and was always much lower than in the stimulated myocytes (data not shown).

View larger version (63K): [in this window] [in a new window]   Figure 1. Northern blot analysis of isolated myocytes for IL-6 mRNA (top) and ICAM-1 mRNA (middle). Each lane contains 20 µg of RNA, as demonstrated with ethidium bromide (bottom).

Because of our previous association of IL-6 with induction of myocyte ICAM-1,¹⁹ we also analyzed the same Northern blots for ICAM-1 mRNA induction (Figure 1, bottom). In every case, ICAM-1 mRNA expression matched expression of IL-6 mRNA qualitatively.

In Situ Hybridization Studies of IL-6 mRNA Expression in Isolated Cardiac Myocytes
Serial sections through fixed ventricular myocytes treated identically to those used in the Northern blot analysis were examined for IL-6 induction with a sense and antisense riboprobe as described in "Methods." The unstimulated cardiac myocytes (control) showed no significant staining with the IL-6 antisense probe. Myocytes stimulated with TNF-, IL-1ß, or endotoxin showed a significant amount of staining with the antisense probe; no staining was seen with the use of the IL-6 sense riboprobe as a control (Figure 2). Incubation of cardiac myocytes with the preischemic cardiac lymph showed little or no staining with the IL-6 antisense riboprobe, but postischemic cardiac lymph collected over the first 3 hours of reperfusion in the same dilution was capable of robust induction of IL-6 mRNA in myocytes (Figure 2).

View larger version (49K): [in this window] [in a new window]   Figure 2. In situ hybridization of IL-6 riboprobe in isolated myocytes. Hybridization with antisense IL-6 riboprobe (A through E) and sense IL-6 riboprobe (F) in isolated cardiac myocytes (magnification x100). Myocytes are stimulated with A, postischemic lymph (1:10 dilution); B, TNF-; C, IL-1ß; and D, preischemic lymph (1:10 dilution). E, Unstimulated control myocyte. F, Representative in situ hybridization with sense IL-6 riboprobe.

In Situ Hybridization Assessment of IL-6 mRNA Induction in Intact Myocardium
In Figure 3, serial sections are examined from an experiment of 1 hour of ischemia and 3 hours of reperfusion. In Figure 3D, PAS staining demonstrates an area of predominant glycogen depletion adjoining areas of viable myocardial cells with relatively preserved glycogen on the epicardial aspect of the infarct (arrowheads) and in the innermost layers of the subendocardium (arrows). In situ hybridization studies for IL-6 in panel A show significant IL-6 mRNA induction in viable myocardial cells found in the spared innermost layers of the endocardium (arrows) as well as on the epicardial border zone (arrowheads). The presence of this preserved layer at the innermost portion of the myocardium has been previously reported.³² Higher magnification (Figure 3C) demonstrates that this staining for IL-6 mRNA is seen in both the myocardium as well as (more intensely) in infiltrating mononuclear cells. Panel E (compared with panel A) demonstrates that ICAM-1 mRNA induction is seen in the same regions of viable myocytes on the border zone of the myocardial infarct. The colocalization of IL-6 mRNA and ICAM-1 mRNA induction is almost complete at 3 hours of reperfusion.

View larger version (151K): [in this window] [in a new window]   Figure 3. In situ hybridization of IL-6 riboprobe in serial myocardial sections taken from 1 hour of ischemia and 3 hours of reperfusion. A, In situ hybridization with antisense IL-6 riboprobe: border zone (arrowheads), subendocardial spared cells (solid arrows). No staining in the infarcted area. B, In situ hybridization with sense IL-6 riboprobe. C, Same section as in A at higher magnification (x100). Mononuclear cells (arrows) demonstrate a more intense staining for IL-6 mRNA when compared with cardiac myocytes. D, PAS staining demonstrating relative retention of glycogen in areas of IL-6 mRNA (arrows and arrowheads as in A). Central infarct area is depleted except for occasional regions. E, In situ hybridization with antisense ICAM-1 probe. F, In situ hybridization with sense ICAM-1 probe.

Induction of IL-6 mRNA in myocytes could be demonstrated by 1 hour of reperfusion (Figure 4) and was consistently found in viable areas abutting the area of contraction band necrosis. Note that in addition to the spared subendocardium and epicardial border zone, there is a region (within the infarct) surrounding a small vessel (arrow) that is preserved (retains glycogen) and expresses IL-6 mRNA.

View larger version (136K): [in this window] [in a new window]   Figure 4. Serial sections taken from 1 hour of occlusion and 1 hour of reperfusion. A, In situ hybridization with antisense IL-6 riboprobe. B, PAS staining. Glycogen stains the same areas as IL-6 mRNA.

Figure 5 represents serial sections taken from an animal after 1 hour of ischemia and 24 hours of reperfusion. The architectural features are almost identical to those seen at 3 hours of reperfusion, although there is greater infiltration of mononuclear cells into the infarct, and these cells produce IL-6 mRNA as described above (data not shown). The figure again demonstrates the preserved subendocardial cells expressing IL-6 mRNA.

View larger version (107K): [in this window] [in a new window]   Figure 5. Relation between in situ hybridization with IL-6 riboprobe, PAS staining, and immunohistochemistry for myoglobin in serial sections taken from ischemic (A through D) and control (E through H) areas after 1 hour of occlusion and 24 hours of reperfusion (x10 magnification). A and E, Immunohistochemistry for myoglobin. Infarcted area shows no staining for myoglobin. Viable myocytes demonstrate staining for myoglobin in spared subendocardium (arrows) and epicardial border zone (arrowheads). B and F, PAS staining. Cells of the border zone show relatively preserved glycogen; control myocardium shows preserved glycogen. C, In situ hybridization with antisense IL-6 riboprobe stains only viable cells of border zone and subendocardial cells. G, Section taken from the control area shows no significant IL-6 mRNA staining. D and H, In situ hybridization with sense IL-6 riboprobe.

In the absence of reperfusion, no significant staining of IL-6 mRNA was detected either at 1 hour, 2 hours (data not shown), or 4 hours (Figure 6) of ischemia. This was compatible with our previous observation of reperfusion dependence of IL-6 mRNA induction.^{19 22}

View larger version (119K): [in this window] [in a new window]   Figure 6. Serial sections taken from ischemic (A through C) and control (D through F) tissue samples from 4 hours of occlusion without reperfusion. A and D, In situ hybridization with antisense IL-6 riboprobe. No significant IL-6 mRNA expression is observed. B and E, In situ hybridization with sense IL-6 riboprobe. C and F, PAS staining demonstrating glycogen depletion in part of the section taken from the ischemic area and preserved glycogen in the section from the control area.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our study combines in vitro data with isolated cardiac myocytes and in vivo data with our canine ischemia/reperfusion model and clearly demonstrates that myocytes can express message for IL-6 in our experimental setting. Various cytokines as well as postischemic lymph are capable of inducing IL-6 mRNA in the isolated myocytes. To the best of our knowledge, this is the first direct histological demonstration of IL-6 mRNA production in the adult cardiac myocyte, both in vitro and in vivo. These data are compatible with a recent observation with Northern blots,³³ suggesting that cultured neonatal cardiac myocytes respond to hypoxic stress by IL-6 production.

Guillen et al¹⁰ demonstrated the sequential increase in IL-1ß and IL-6 after myocardial infarction, suggesting an inducer function of IL-1ß for IL-6. TNF- is also upregulated in myocardial infarction and may also induce IL-6 production.^{34 35} In previous work, we have demonstrated that there is a rapid increase in TNF- in postischemic lymph and that TNF- is released from preformed stores in cardiac mast cells.³⁶ In our model, we found no evidence for the early presence of IL-1.³⁶ TNF- is a known inducer of IL-6 in endothelial cells, fibroblasts, and mononuclear cells,^{36 37 38 39} and, in the present study, we could demonstrate that it also induces IL-6 gene expression in cardiac myocytes. We suggested that TNF- may act as an upstream cytokine inducer of IL-6 in ischemia/reperfusion.

Investigation of IL-6 induction demonstrated reperfusion dependence. Northern blot analysis suggested that IL-6 induction preceded ICAM-1 induction in the reperfused infarct.^{19 22} Because both IL-6 and ICAM-1 induction were reperfusion dependent within the first 6 hours, we postulated that IL-6 production was most likely occurring in infiltrating leukocytes that would not be present in the absence of reperfusion. Indeed, we have demonstrated that mononuclear cells found within postischemic cardiac lymph demonstrate IL-6 mRNA induction within 15 minutes of reperfusion.^{36 40} The current studies demonstrate that mononuclear cells in tissue stained intensely in an in situ hybridization reaction with an IL-6 riboprobe (Figure 3C). However, it became obvious that IL-6 mRNA was being induced (at lower intensity) in cardiac myocytes in the viable border zone.

Relation of Cellular Source of IL-6 to ICAM-1 Induction
The exceptional colocalized induction of IL-6 and ICAM-1 at 3 hours invites consideration of an autocrine mechanism for some of the observed ICAM-1 mRNA induction. It is unlikely, however, that all of the ICAM-1 induction is autocrine. In Figure 5, examination of reperfusion after 24 hours shows that IL-6 mRNA remains confined to cardiac myocytes on the border zone of the myocardial infarction. In contrast, in our previous studies, examination of the reperfused myocardial infarction after 24 hours of reperfusion demonstrated ICAM-1 mRNA in all of the cardiac myocytes.²⁰ This correlated with our observation that postischemic cardiac lymph retained its ability to induce ICAM-1 for up to 72 hours after reperfusion and was quite high at 24 hours. The induction of ICAM-1 mRNA in the "normal" myocardium might appear to result from IL-6 in the extracellular fluid and blood as observed by us and other laboratories.^{10 11 12 18} An autocrine contribution to early ICAM-1 induction in the border zone remains a possibility.

IL-6 Effect on Cardiac Function
Recent studies suggest that "inflammatory cytokines" may exert primary effects on myocardial function. Finkel and his coworkers^{41 42} have demonstrated that IL-6 may act as a nitric oxide–dependent cardiac depressant and may be associated with stunned myocardium.⁴² IL-6 has produced a nitric oxide–mediated reduction in calcium flux and contractility in chick ventricular myocytes.⁴³

IL-6 is a member of a class of cytokines whose receptor mechanisms share the presence of a common protein, gp130.⁴⁴ Two members of this family, cardiotrophin and leukemia inhibitory factor, have been shown to induce cardiac hypertrophy.^{45 46} These agents work through the JAK/STAT pathway induced by gp130. Transgenic mice double overexpressing IL-6 and IL-6 receptor also demonstrate myocardial hypertrophy.⁴⁷ In addition to their effect on hypertrophy, these cytokines have been shown to be cytoprotective against apoptosis. Cardiotrophin prevents apoptotic cell death induced by serine depletion in neonatal rat cardiac myocytes.^{48 49} Overexpression of IL-6 and IL-6 receptor has been demonstrated to be cytoprotective in cell systems through induction of the antiapoptotic gene bcl-xL, and LIF has been demonstrated to exert an antiapoptotic effect in cardiac myocytes through induction of the same gene.^{50 51} It is interesting to speculate that these 3 responses, reduced contractility, positive protein balance (hypertrophy), and antiapoptosis, might favorably influence myocardial cells and allow them to survive in an area of jeopardy.

	Acknowledgments

 
This work was supported by NIH grant HL-42550. The authors wish to thank Concepcion Mata and Sharon Malinowski for their editorial assistance with the manuscript and Peggy Jackson, Alida Evans, and Etai Funk for their outstanding technical expertise.

	Footnotes

 
Guest Editor for this article was Robert Engler, MD, Collateral Therapeutics, San Diego, Calif.

Received March 4, 1998; revision received August 25, 1998; accepted September 16, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Go LO, Murry CE, Richard VJ, Weischedel GR, Jennings RB, Reimer KA. Myocardial neutrophil accumulation during reperfusion after reversible or irreversible ischemic injury. Am J Physiol. 1988;255:H1177–H1198.
   
2. Mullane KM, Smith CW. In: Piper, HM, ed. The Role of Leukocytes in Ischemic Damage, Reperfusion Injury and Repair of the Myocardium: Pathophysiology of Severe Ischemic Myocardial Injury. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1990:239–267.
   
3. Dreyer WJ, Michael LH, West MS, Smith CW, Rothlein R, Rossen RD, Anderson DC, Entman ML. Neutrophil accumulation in ischemic canine myocardium: insights into the time course, distribution and mechanism of localization during early reperfusion. Circulation. 1991;84:400–411.[Abstract/Free Full Text]
   
4. Entman ML, Smith CW. Postreperfusion inflammation: a model for reaction to injury in cardiovascular disease. Cardiovasc Res. 1994;28:1301–1311.[Free Full Text]
   
5. Hawkins HK, Entman ML, Zhu JY, Youker KA, Berens K, Dore M, Smith CW. Acute inflammatory reaction after myocardial ischemic injury and reperfusion: development and use of a neutrophil-specific antibody. Am J Pathol. 1996;148:1957–1969.[Abstract]
   
6. Baumgarten W. Infarction of the heart. Am J Physiol.. 1899;2:243–265.
   
7. Gruppo Italiano per lo studio della Streptochinasi nell'Infarto Miocardico. Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet. 1986;1:397–401.[Medline] [Order article via Infotrieve]
   
8. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both or neither among 17,187 cases of suspected myocardial infarction: ISIS-2. Lancet. 1988;2:349–360.[Medline] [Order article via Infotrieve]
   
9. Neuhaus KL, Feuerer W, Jeep Tebbe S, Niederer W, Vogt A, Tebbe U. Improved thrombolysis with a modified dose regimen of recombinant tissue type plasminogen activator. J Am Coll Cardiol. 1989;14:1566–1569.[Abstract]
   
10. Guillen I, Blanes M, Gomez-Lechon MJ, Castell JV. Cytokine signaling during myocardial infarction: sequential appearance of IL-1 beta and IL-6. Am J Physiol. 1995;269:R229–R235.[Abstract/Free Full Text]
    
11. Ikeda U, Ohkawa F, Seino Y, Yamamoto K, Hidaka Y, Kasahara T, Kawai T, Shimada K. Serum Interleukin-6 becomes elevated in acute myocardial infarction. J Mol Cell Cardiol. 1992;24:579–584.[Medline] [Order article via Infotrieve]
    
12. Neumann FJ, Ott I, Gawaz M, Richardt G, Holzapfel H, Jochum M, Schoemig A. Cardiac release of cytokines and inflammatory responses in acute myocardial infarction. Circulation. 1995;92:748–755.[Abstract/Free Full Text]
    
13. Entman ML, Youker KA, Shappell SB, Siegel C, Rothlein R, Dreyer WJ, Schmalstieg FC, Smith CW. Neutrophil adherence to isolated adult canine myocytes: evidence for a CD18 dependent mechanism. J Clin Invest. 1990;85:1497–1506.
    
14. Smith CW, Entman ML, Lane CL, Beaudet AL, Ty TI, Youker KA, Hawkins HK, Anderson DC. Adherence of neutrophils to canine cardiac myocytes in vitro is dependent on intercellular adhesion molecule-1. J Clin Invest. 1991;88:1216–1223.
    
15. Entman ML, Youker KA, Shoji T, Kukielka GL, Shappell SB, Taylor AA, Smith CW. Neutrophil induced oxidative injury of cardiac myocytes: a compartmented system requiring CD11b/CD18-ICAM-1 adherence. J Clin Invest. 1992;90:1335–1345.
    
16. Michael L, Lewis R, Brandon T, Entman M. Cardiac lymph flow in conscious dogs. Am J Physiol. 1979;237(Heart Circ Physiol 6):H311–H317.
    
17. Michael LH, Hunt JR, Weilbacher D, Perryman MB, Roberts R, Lewis RM, Entman ML. Creatine kinase and phosphorylase in cardiac lymph: coronary occlusion and reperfusion. Am J Physiol. 1985;248:H350–H359.
    
18. Youker KA, Smith CW, Anderson DC, Miller D, Michael LH, Rossen RD, Entman ML. Neutrophil adherence to isolated adult cardiac myocytes: induction by cardiac lymph collected during ischemia and reperfusion. J Clin Invest. 1992;89:602–609.
    
19. Kukielka GL, Youker KA, Hawkins HK, Perrard JL, Micheal LH, Ballantyne CM, Smith CW, Entman ML. Regulation of ICAM-1 and IL-6 in the myocardial ischemia: effects of reperfusion. Ann N Y Acad Sci. 1994;723:258–270.[Medline] [Order article via Infotrieve]
    
20. Youker KA, Hawkins HK, Kukielka GL, Perrard JL, Michael LH, Ballantyne CM, Smith CW, Entman ML. Molecular evidence for induction of intercellular adhesion molecule 1 in the viable border zone associated with ischemia reperfusion injury of the dog heart. Circulation. 1994;89:2736–2746.[Abstract/Free Full Text]
    
21. Kukielka GL, Hawkins HK, Michael LH, Manning AM, Lane CL, Entman ML, Smith CW, Anderson DC. Regulation of intercellular adhesion molecule-1 (ICAM-1) in ischemic and reperfused canine myocardium. J Clin Invest. 1993;92:1504–1516.
    
22. Kukielka GL, Smith CW, Manning AM, Youker K, Michael LH, Entman ML. Induction of interleukin-6 synthesis in the myocardium: potential role in postreperfusion inflammatory injury. Circulation. 1995;92:1866–1875.[Abstract/Free Full Text]
    
23. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1–78.[Medline] [Order article via Infotrieve]
    
24. Kukielka GL, Youker KA, Michael LH, Kumar AG, Ballantyne CM, Smith CW, Entman ML. Role of early reperfusion in the induction of adhesion molecules and cytokines in previously ischemic myocardium. Mol Cell Biochem. 1995;147:5–12.[Medline] [Order article via Infotrieve]
    
25. Birdsall HH, Green DM, Trial J, Youker KA, Burns AR, MacKay CR, LaRosa GL, Hawkins HK, Smith CW, Michael LH, Entman ML, Rossen RD. Complement C5a, TGF-ß1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion. Circulation. 1997;95:684–692.[Abstract/Free Full Text]
    
26. Heymann MA, Payne BD, Hoffmann JIE, Rudolph AM. Blood flow measurements with radionuclide labeled particles. Prog Cardiovasc Dis. 1977;20:55–78.[Medline] [Order article via Infotrieve]
    
27. Goddard-Finegold J, Michael LH. Cerebral blood flow and experimental intraventricular hemorrhage. Pediatr Res. 1984;18:7–11.[Medline] [Order article via Infotrieve]
    
28. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
    
29. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989:545.
    
30. Meerbaum S, Haendchen RV, Corday E, Povzhitkov M, Fishbein MC, Y-Rit J, Land T, Uchiyama T, Aosaki N, Broffman J. Hypothermic coronary venous phased retroperfusion: a closed-chest treatment of acute regional myocardial ischemia. Circulation. 1982;65:1435–1445.[Abstract/Free Full Text]
    
31. Fishbein MC, Hare CA, Gissen SA, Spadaro J, Maclean D, Maroko PR. Identification and quantification of histochemical border zones during the evolution of myocardial infarction in the rat. Cardiovasc Res. 1980;14:41–49.[Medline] [Order article via Infotrieve]
    
32. Block ML, Said JW, Siegel RJ, Fishbein MC. Myocardial myoglobin following coronary artery occlusion: an immunohistochemical Study. Am J Pathol. 1983;111:374–379.[Abstract]
    
33. Yamauchi-Takihara K, Ihara Y, Ogata A, Yoshizaki K, Azuma J, Kishimoto T. Hypoxic stress induces cardiac myocyte-derived interleukin-6. Circulation. 1995;91:1520–1524.[Abstract/Free Full Text]
    
34. Hirschl MM, Gwechenberger M, Binder T, Graf S, Stefenelli T, Rauscha F, Laggner AN, Sochor H. Assessment of myocardial injury by tumor necrosis factor alpha measurements in acute myocardial infarction. Eur Heart J. 1996;17:1852–1859.[Abstract/Free Full Text]
    
35. Lissoni P, Pelizzoni F, Mauri O, Perego M, Pittalis S, Barni S. Enhanced secretion of tumor necrosis factor in patients with myocardial infarction. Eur J Med. 1992;1:277–280.[Medline] [Order article via Infotrieve]
    
36. Frangogiannis NG, Lindsey ML, Michael LH, Youker KA, Bressler RB, Mendoza LH, Spengler RN, Smith CW, Entman ML. Resident cardiac mast cells degranulate and release preformed TNF- initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation.. 1998;98:699–710.[Abstract/Free Full Text]
    
37. Sanceau J, Kaisho T, Hirano T, Wietzerbin J. Triggering of the human interleukin-6 gene by interferon gamma and tumor necrosis factor alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NfkB and Sp1 transcription factors. J Biol Chem. 1995;270:27920–27931.[Abstract/Free Full Text]
    
38. Podor TJ, Jirik FR, Loskutoff DJ, Carson DA, Lotz M. Human endothelial cells produce IL-6: lack of responses to exogenous IL-6. Ann N Y Acad Sci. 1989;557:374–385.[Abstract]
    
39. Elias JA, Lentz V. IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA. J Immunol. 1990;145:161–166.[Abstract]
    
40. Youker KA, Frangogiannis N, Lindsey M, Smith CW, Entman ML. Adhesion molecule induction and expression in neutrophil-induced myocardial injury. In: Schultheiss HP, Schwimmbeck P, eds. The Role of Immune Mechanisms in Cardiovascular Disease. Berlin: Springer-Verlag; 1997:125–137.
    
41. Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effect of cytokines on the heart mediated by nitric oxide. Science. 1992;257:387–389.[Abstract/Free Full Text]
    
42. Finkel MS, Hoffman RA, Shen L, Oddis CV, Simmons RL, Hattler BG. Interleukin-6 (IL-6) as a mediator of stunned myocardium. Am J Cardiol. 1993;71:1231–1323.[Medline] [Order article via Infotrieve]
    
43. Kinugawa K, Takahashi T, Kohmoto O, Yao A, Aoyagi T, Momomura S, Hirata Y, Serizawa T. Nitric oxide–mediated effects of interleukin-6 on [Ca²⁺]_i, and cell contraction in cultured chick ventricular myocytes. Circ Res. 1994;75:285–295.[Abstract/Free Full Text]
    
44. Kishimoto T, Taga T, Akira S. Cytokine signal transduction. Cell. 1994;76:253–262.[Medline] [Order article via Infotrieve]
    
45. Pennica D, King KL, Shaw KJ, Luis E, Rullamas J, Luoh SM, Darbonne WG, Knutzow DS, Yen R, Chien KR. Expression cloning of cardiotrophin-1, a cytokine that induces cardiac myocyte hypertrophy. Proc Natl Acad Sci U S A. 1995;92:1142–1146.[Abstract/Free Full Text]
    
46. Kodama H, Fukuda K, Pan J, Makino S, Baba A, Hori S, Ogawa S. Leukemia inhibitory factor, a potent hypertrophic cytokine, activates the JAK/STAT pathway in rat cardiomyocytes. Circ Res. 1997;81:656–663.[Abstract/Free Full Text]
    
47. Hirato H, Yoshida K, Kishimoto T, Taga T. Continuous activation of gp 130, a signal transducing receptor component for interleukin-6 related cytokines, causes myocardial hypertrophy in mice. Proc Natl Acad Sci U S A.. 1995;92:4862–4866.[Abstract/Free Full Text]
    
48. Sheng Z, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development. 1996;122:419–428.[Abstract]
    
49. Sheng Z, Knowlton K, Chen J, Hoshijiama M, Brown JH, Chien KR. Cardiotrophin-1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein-kinase dependent pathway: divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem. 1997;272:5783–5791.[Abstract/Free Full Text]
    
50. Schwarze MMK, Hawley RG. Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin-6 mediated upregulation of bcl-xL. Cancer Res.. 1995;55:2262–2265.[Abstract/Free Full Text]
    
51. Fujio Y, Kunisada K, Hirota H, Yamauchi-Takihara K, Kishimoto T. Signals through gp130 upregulate bcl-x gene expression via STAT-1 binding cis-element in cardiac myocyte. J Clin Invest. 1997;99:2898–2905.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				M. Andrassy, H. C. Volz, J. C. Igwe, B. Funke, S. N. Eichberger, Z. Kaya, S. Buss, F. Autschbach, S. T. Pleger, I. K. Lukic, et al. High-Mobility Group Box-1 in Ischemia-Reperfusion Injury of the Heart Circulation, June 24, 2008; 117(25): 3216 - 3226. [Abstract] [Full Text] [PDF]

				W. D. Gilson, F. H. Epstein, Z. Yang, Y. Xu, K.-M. R. Prasad, M.-C. Toufektsian, V. E. Laubach, and B. A. French Borderzone Contractile Dysfunction Is Transiently Attenuated and Left Ventricular Structural Remodeling Is Markedly Reduced Following Reperfused Myocardial Infarction in Inducible Nitric Oxide Synthase Knockout Mice J. Am. Coll. Cardiol., October 30, 2007; 50(18): 1799 - 1807. [Abstract] [Full Text] [PDF]

				E. L. Olivares, M. P. Marassi, R. S. Fortunato, A. C. M. da Silva, R. H. Costa-e-Sousa, I. G. Araujo, E. C. Mattos, M. O. Masuda, M. A. Mulcahey, S. A. Huang, et al. Thyroid Function Disturbance and Type 3 Iodothyronine Deiodinase Induction after Myocardial Infarction in Rats A Time Course Study Endocrinology, October 1, 2007; 148(10): 4786 - 4792. [Abstract] [Full Text] [PDF]

				Z. Guo, Z. Xia, J. Jiang, and J. H. McNeill Downregulation of NADPH oxidase, antioxidant enzymes, and inflammatory markers in the heart of streptozotocin-induced diabetic rats by N-acetyl-L-cysteine Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1728 - H1736. [Abstract] [Full Text] [PDF]

				S. Zhang, E. Shpall, J. T. Willerson, and E. T.H. Yeh Fusion of Human Hematopoietic Progenitor Cells and Murine Cardiomyocytes Is Mediated by {alpha}4{beta}1 Integrin/Vascular Cell Adhesion Molecule-1 Interaction Circ. Res., March 16, 2007; 100(5): 693 - 702. [Abstract] [Full Text] [PDF]

				H. Tada, Y. Kagaya, M. Takeda, J. Ohta, Y. Asaumi, K. Satoh, K. Ito, A. Karibe, K. Shirato, N. Minegishi, et al. Endogenous erythropoietin system in non-hematopoietic lineage cells plays a protective role in myocardial ischemia/reperfusion Cardiovasc Res, August 1, 2006; 71(3): 466 - 477. [Abstract] [Full Text] [PDF]

				P. J. Lafontant, A. R. Burns, E. Donnachie, S. B. Haudek, C. W. Smith, and M. L. Entman Oncostatin M differentially regulates CXC chemokines in mouse cardiac fibroblasts Am J Physiol Cell Physiol, July 1, 2006; 291(1): C18 - C26. [Abstract] [Full Text] [PDF]

				Q.-Y. Zhang, J.-B. Ge, J.-Z. Chen, J.-H. Zhu, L.-H. Zhang, C.-P. Lau, and H.-F. Tse Mast Cell Contributes to Cardiomyocyte Apoptosis after Coronary Microembolization J. Histochem. Cytochem., May 1, 2006; 54(5): 515 - 523. [Abstract] [Full Text] [PDF]

				N. Smart, M. H. Mojet, D. S. Latchman, M. S. Marber, M. R. Duchen, and R. J. Heads IL-6 induces PI 3-kinase and nitric oxide-dependent protection and preserves mitochondrial function in cardiomyocytes Cardiovasc Res, January 1, 2006; 69(1): 164 - 177. [Abstract] [Full Text] [PDF]

				M. Wang, B. M. Tsai, M. W. Turrentine, Y. Mahomed, J. W. Brown, and D. R. Meldrum p38 Mitogen Activated Protein Kinase Mediates Both Death Signaling and Functional Depression in the Heart Ann. Thorac. Surg., December 1, 2005; 80(6): 2235 - 2241. [Abstract] [Full Text] [PDF]

				S. Sezaki, S. Hirohata, A. Iwabu, K. Nakamura, K. Toeda, T. Miyoshi, H. Yamawaki, K. Demircan, S. Kusachi, Y. Shiratori, et al. Thrombospondin-1 Is Induced in Rat Myocardial Infarction and Its Induction Is Accelerated by Ischemia/Reperfusion Experimental Biology and Medicine, October 1, 2005; 230(9): 621 - 630. [Abstract] [Full Text] [PDF]

				S.-Y. Park, Y.-R. Cho, B. N. Finck, H.-J. Kim, T. Higashimori, E.-G. Hong, M.-K. Lee, C. Danton, S. Deshmukh, G. W. Cline, et al. Cardiac-Specific Overexpression of Peroxisome Proliferator-Activated Receptor-{alpha} Causes Insulin Resistance in Heart and Liver Diabetes, September 1, 2005; 54(9): 2514 - 2524. [Abstract] [Full Text] [PDF]

				A. Deten, G. Marx, W. Briest, H. Christian Volz, and H.-G. Zimmer Heart function and molecular biological parameters are comparable in young adult and aged rats after chronic myocardial infarction Cardiovasc Res, May 1, 2005; 66(2): 364 - 373. [Abstract] [Full Text] [PDF]

				D. H. Freed, R. H. Cunnington, A. L. Dangerfield, J. S. Sutton, and I. M.C. Dixon Emerging evidence for the role of cardiotrophin-1 in cardiac repair in the infarcted heart Cardiovasc Res, March 1, 2005; 65(4): 782 - 792. [Abstract] [Full Text] [PDF]

				M. Wang, L. Baker, B. M. Tsai, K. K. Meldrum, and D. R. Meldrum Sex differences in the myocardial inflammatory response to ischemia-reperfusion injury Am J Physiol Endocrinol Metab, February 1, 2005; 288(2): E321 - E326. [Abstract] [Full Text] [PDF]

				M. Wang, B. M. Tsai, A. Kher, L. B. Baker, G. M. Wairiuko, and D. R. Meldrum Role of endogenous testosterone in myocardial proinflammatory and proapoptotic signaling after acute ischemia-reperfusion Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H221 - H226. [Abstract] [Full Text] [PDF]

				A. Deten, H. C. Volz, S. Clamors, S. Leiblein, W. Briest, G. Marx, and H.-G. Zimmer Hematopoietic stem cells do not repair the infarcted mouse heart Cardiovasc Res, January 1, 2005; 65(1): 52 - 63. [Abstract] [Full Text] [PDF]

B. Dawn, Y.-T. Xuan, Y. Guo, A. Rezazadeh, A. B. Stein, G. Hunt, W.-J. Wu, W. Tan, and R. Bolli IL-6 plays an obligatory role in late preconditioning via JAK-STAT signaling and upregulation of iNOS and COX-2 Cardiovasc Res, October 1, 2004; 64(1): 61 - 71. [Abstract] [Full Text] [PDF]