Author + information
- Received December 18, 1996
- Revision received March 5, 1997
- Accepted March 12, 1997
- Published online July 1, 1997.
- Nikolaus Marx, MDA,* (, )
- Franz-Josef Neumann, MDA,
- Ilka Ott, MDA,
- Meinrad Gawaz, MDA,
- Werner Koch, PhDA,
- Tobias Pinkau, BScA and
- Albert Schömig, MDA
- ↵*Dr. Nikolaus Marx, Deutsches Herzzentrum und 1. Medizinische Klinik, Technische Universität München, Lazarettstraße 36, 80636 Munich, Germany.
Objectives. This study sought to investigate whether cytokine expression in leukocytes may be induced by plasma from the reperfused heart of patients with an acute myocardial infarction (MI).
Background. Reperfusion in acute MI is associated with deleterious local and systemic inflammatory responses that are regulated by cytokines. Induction of cytokine expression in resident leukocytes could contribute to inflammatory responses of the ischemic and reperfused heart.
Methods. Blood samples of 10 patients with an acute MI were obtained simultaneously from the coronary sinus and the aorta before and 5 min after recanalization of the coronary occlusion. Ten patients with elective percutaneous transluminal coronary angioplasty served as a control group. We incubated leukocytes from healthy donors with plasma samples and analyzed mRNA expression of interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-alpha) by Northern blot analysis.
Results. In patients with an acute MI, plasma obtained from the coronary sinus after recanalization increased the mRNA expression of IL-1betaand IL-8 compared with that of plasma before recanalization (median [quartiles] difference before vs. after recanalization: 34.5 [4, 137], p = 0.017, for IL-1beta; 18.5 [4, 35], p = 0.032, for IL-8) and simultaneously obtained aortic plasma (median [quartiles] coronary sinus–aortic differences after recanalization: 45.5 [−3, 115], p = 0.021, for IL-1beta; 16 [4, 52], p = 0.005, for IL-8). No induction of IL-6 and TNF-alpha expression could be observed. No changes found in the study patients were detectable in the control group.
Conclusions. Plasma from the ischemic and reperfused heart stimulates the expression of IL-1betaand IL-8 in leukocytes. Therefore, leukocyte-derived cytokines may contribute to the regulation of cardiac inflammatory responses in patients with an acute MI.
(J Am Coll Cardiol 1997;30:165–70)
In acute myocardial infarction (MI), early recanalization of the occluded artery can salvage myocardium and improve clinical outcome ([1–5]). Despite these beneficial effects of timely restoration of coronary blood flow, local inflammatory responses of the ischemic and reperfused heart exert deleterious effects on the myocardium ([6–11]). Besides these local reactions, a systemic inflammatory response syndrome develops in patients with acute MI, and a number of clinical studies ([12–14]) have demonstrated an adverse impact of systemic inflammatory response syndromes on clinical outcome.
Several experimental studies ([15–17]) have shown that cytokines are important regulators of inflammatory reactions during reperfusion. Among those, the predominantly leukocyte-derived cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1betaappear to play a key role due to their ability to induce secondary cytokines, such as IL-6 and IL-8 ([18–20]). IL-8 has potent actions in the recruitment and local activation of neutrophils ([21, 22]), whereas IL-6, in concert with IL-1betaand TNF-alpha, induces predominantly systemic inflammatory responses (). In patients with acute MI, we recently found a cardiac release of IL-6 and IL-8 during reperfusion after direct percutaneous transluminal coronary angioplasty (PTCA) of the infarct-related coronary artery, but there was no concomitant release of IL-1betaor TNF-alpha (). However, the lack of detectable transcardiac gradients in TNF-alpha and IL-1betadid not exclude a paracrine release by resident leukocytes and macrophages that may stimulate the endothelial production of IL-6 and IL-8 ([19, 20]).
The present study sought to test the hypothesis that the ischemic and reperfused coronary circulation in acute MI represents a proinflammatory environment, capable of inducing cytokine expression in resident leukocytes. We therefore obtained coronary venous and aortic plasma before and after recanalization of the occluded artery in acute MI and examined its effect on the expression of IL-1beta, IL-6, IL-8 and TNF-alpha in leukocytes.
The study groupincluded 10 patients with an acute anterior MI who were admitted to the hospital within 3 to 9 h of the onset of pain (median time delay 7 h). The diagnosis of acute MI was based on a history of prolonged ischemic chest pain and characteristic electrocardiographic changes. All patients underwent immediate coronary angiography, which revealed an occluded left anterior descending coronary artery (LAD) that was recanalized successfully by primary PTCA.
The control groupincluded 10 patients with elective PTCA for a LAD stenosis within 19 to 44 days of successful thrombolysis for acute anterior MI. Indication for PTCA was based on a history of stable angina or exercise stress test, or both, in the presence of a significant (>70%) LAD stenosis.
Patients with concomitant noncardiac diseases, such as inflammatory disorders, malignancy or infection, were omitted from the study. The usual medication of the patients was not changed, and no patient was taking antiinflammatory agents, except for aspirin (100 mg twice daily). Table 1shows the baseline characteristics of the study and control groups.
The study was approved by the institutional ethics committee, and informed written consent was obtained from all patients.
1.2 Study Protocol.
Before the study, all patients with an acute MI were given 5,000 IU of unfractionated heparin intravenously, 500 mg of aspirin, one to three injections of 5 mg of metoprolol and one to two injections of 5 mg of morphine, depending on individual response. To obtain a systolic blood pressure between 100 and 120 mm Hg, patients were maintained on intravenous infusions of nitroglycerin (0.24 to 2.4 mg/h). Before coronary angiography, an additional dose of 10,000 IU of heparin was given intraarterially. Coronary angiography was performed by the transfemoral approach. While one operator placed the guiding catheter, a second operator cannulated the coronary sinus with a 7F multipurpose catheter using the brachial approach. If the coronary anatomy was judged suitable for mechanical recanalization, the operators immediately proceeded to PTCA using fixed-wire balloon catheters (ACE, Scimed Life Systems).
Two sets of blood samples were obtained simultaneously from the coronary sinus and the guiding catheter. The first set was taken after the guide wire of the balloon was placed at the proximal end of the occlusion; the second was taken 5 min after the restoration of blood flow in the occluded coronary artery.
In the control patients, PTCA was also performed with the femoral approach, and the coronary sinus was cannulated through an antecubital vein. Identical to the study patients, control patients received an intravenous dose of 500 mg of aspirin and 15,000 IU of heparin intraarterially immediately after femoral arterial access was obtained. Two sets of blood samples were again obtained simultaneously from the guiding catheter and the coronary sinus: the first after the guide wire for the balloon catheter was positioned; the second 5 min after the first balloon inflation.
In both the control and study groups, all blood samples were obtained over a defined period of 1 min and anticoagulated with 1:10 (vol/vol) citrate, phosphate buffer, dextrose and adenine (CPDA) (Greiner, Nürtingen, Germany). The blood samples were put on ice and processed immediately, as indicated below. For immunoassay and incubation, plasma samples were stored at −120°C until final processing.
1.3 Cytokine Enzyme-Linked Immunosorbent Assay.
Concentrations of IL-1beta, IL-6, IL-8 and TNF-alpha were determined by sandwich-type immunoassay (IL-6 and TNF-alpha: IEMA, Immunotech; IL-1beta, IL-8: Quantikine, R&D systems), as described previously. The detection limits were 3.9 ng/liter for IL-1beta, 2.0 ng/liter for IL-6, 3.0 ng/liter for IL-8 and 10 ng/liter for TNF-alpha; the respective intraassay variabilities for the lower assay range were 11%, 7%, 4% and 10%.
1.4 Incubation with Leukocytes.
Unfractionated leukocyte suspensions were prepared from CPDA-anticoagulated blood samples taken from healthy volunteers. Leukocytes were obtained after sedimentation in the presence of 3% dextrane in normal saline and hypotonic lysis of erythrocytes at 4°C. Cells were washed twice in phosphate-buffered saline.
Fifty million leukocytes were incubated in 250 μl of serum-free tissue culture medium (RPMI 1640, Sigma, Deisenhofen, Germany) and 250 μl of patient plasma from CPDA-anticoagulated blood specimen for 2 h at 37°C with 5% CO2. As a positive and negative control, leukocytes were incubated with RPMI medium and pooled plasma of 10 healthy donors with or without lipopolysaccharide (1 mg/liter), respectively.
1.5 RNA Preparation and Northern Blot Analysis.
Total RNA of 5 × 107cells was isolated by the single-step method of Chomczynski and Sacchi ().
Five micrograms of the total RNA of each sample was subjected to electrophoresis on a 1.2% agarose gel that contained 0.1 mol/liter morpholinopropanesulfonis acid (Boehringer, Mannheim, Germany), 40 mmol/liter sodium acetate, 5 mmol/liter ethylenediaminetetraacetic acid (EDTA) and 6% formaldehyde (Merck, Darmstadt, Germany). The RNA was transferred to nylon membrane (Hybond-N, Amersham, Braunschweig, Germany) in 20 × saline sodium citrate (SSC) buffer by capillary blotting overnight. Blots were baked and prehybridized at 42°C in 50% formamide (Merck), 5 × Denhardt’s (Sigma), 5 × SSC, 0.5% sodium dodecyl sulfate (SDS) and 20 mmol/liter salmon sperm DNA (Gibco BRL, Eggenstadt, Germany). Blots were probed with the 1.05-kilobase (kb) PST-I fragment of IL-1betaPBR322 (ATCC, Rockville, Maryland), the 1-kb EcoRI fragment of IL-6 p91023(B) (ATCC), the 0.25-kb EcoRI–HindIII fragment of IL-8 pUC18 (R&D systems, Minneapolis) and the 1.3-kb HindIII–BamHI fragment of TNF-alpha pFC54.t (ATCC) and reprobed with the 0.95-kb PST-I fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pUC18 (gift from Roger Becker, PhD, Toxikologisches Institut, University of Mainz, Germany) to ensure the integrity of total RNA and comparable RNA loading in each lane.
The cDNA probes were radiolabeled by random priming with (alpha-P32) deoxycitosine triphosphate (dCTP) >6,000 Ci/mmol) (Amersham). The blots were washed at 60°C in 1% SDS/2 × SSC and autoradiographed with Kodak X-OMAT film at −70°C with an intensifying screen.
Signal intensity was quantified by densitometry with background reduction using the Eagle-Eye system (Stratagene) and Wincam-software (Windows, Berlin, Germany).
Intensity was expressed in relation to the GAPDH signal to eliminate even minor differences in RNA loading.
1.6 Statistical Analysis.
The Kolkogorow-Smirnov test showed that the data were not normally distributed. Therefore, results are reported as median (interquartile range), and differences were tested by the Wilcoxon matched-pairs signed-rank test, unless otherwise indicated. A value of p < 0.05 was regarded as significant.
2.1 Baseline Characteristics.
There were no differences with respect to gender distribution, age, localization of LAD stenosis and coronary risk factors between the study and control groups (Table 1). In the study group, the delay between onset of symptoms and the start of the intervention ranged from 3 to 9 h, and the median peak plasma creatine kinase concentration was 2,751.5 ± 1,891.1 U/liter.
2.2 Cytokine Contents of Plasma.
The concentration of IL-6 in the coronary sinus blood of patients with an acute MI increased from 49.3 ± 22.89 ng/liter before to 76.29 ± 37.69 ng/liter after recanalization (p = 0.021), and that of IL-8 increased from 56.56 ± 23.09 to 64.38 ± 25.79 ng/liter (p = 0.012). Concentrations of IL-1betaand TNF-alpha were close to the detection limits of the assays at all sampling conditions and did not show significant changes (data not shown). There was no appreciable cytokine release in the control group (data not shown).
2.3 Induction of Cytokine Expression in Leukocytes.
In 9 of 10 patients with an acute MI, plasma obtained from the coronary sinus after recanalization induced mRNA expression of IL-1betain normal donor leukocytes that was significantly higher than that induced by coronary sinus plasma obtained before recanalization (Fig. 1[top] and 2) ⇓(p = 0.017) and that induced by simultaneously obtained aortic plasma (Table 2) (p = 0.021). No significant differences in induction of IL-1betaexpression could be found between aortic and coronary sinus plasma before recanalization (Fig. 2, Table 2). In none of the sampling conditions did we find significant differences in induction of IL-1betamRNA-expression between aortic plasma of patients with an acute MI and pooled healthy donor plasma (Fig. 2).
We obtained similar results for the induction of IL-8 mRNA expression in normal leukocytes. The induction of IL-8 expression by coronary sinus plasma obtained after recanalization was significantly higher in 8 of 10 patients with an acute MI than that in coronary sinus plasma obtained before recanalization (Fig. 2and Fig. 3[top]) (p = 0.032) and compared with aortic plasma (Table 2) (p = 0.005). No differences in induction of IL-8 expression could be observed between aortic and pooled plasma from healthy donors (Fig. 2).
None of the changes found in patients with an acute MI were found in the control group with elective PTCA (Fig. 1[bottom] and 3 [bottom]). Neither coronary sinus plasma nor aortic plasma from patients with an acute MI induced mRNA expression of IL-6 or TNF-alpha that was different from that induced by pooled healthy donor plasma (Fig. 2). mRNA expression of IL-1betaor IL-8 induced by coronary sinus plasma could not be related to the cytokine contents analyzed.
In the present study we investigated the effect of plasma from the ischemic and reperfused heart on cytokine expression in leukocytes. We found an induction of IL-1betaand IL-8 mRNA expression in leukocytes by coronary sinus plasma obtained after recanalization of the occluded artery. The results of the study indicate that the ischemic and reperfused coronary circulation in acute MI represents a proinflammatory environment that induces cytokine expression in leukocytes.
3.1 Role of Cytokines in Reperfusion.
In our previous study () we revealed neutrophil activation and release of the proinflammatory cytokines IL-8 and IL-6 as evidence of cardiac inflammatory responses in reperfused acute MI. In addition to these results, our present study indicates that leukocytes may serve as a source for IL-8 and IL-1betain the regulation of infarct-related cardiac inflammatory responses. Moreover, even though we do not present direct evidence, our data suggest that IL-1betamay participate in the paracrine regulation of inflammation during ischemia and reperfusion. It was previously shown in animal models that cardiac lymph, collected during ischemia and reperfusion, activates neutrophils ([25, 26]) and induces IL-8 mRNA expression in cultured canine endothelial cells (). To our knowledge the present study is the first to show the induction of cytokine expression in leukocytes by plasma obtained from patients with an acute MI during reperfusion. Thus, the results extend our knowledge about the role of leukocytes in the inflammatory responses in acute MI.
The capability of coronary sinus plasma to induce IL-1betaand IL-8 became apparent only after recanalization of the occluded vessel. No induction of cytokine expression in leukocytes could be observed in the control patients with elective PTCA. Thus, the observed effects cannot be attributed to the procedure itself but appear to be closely related to ischemia and reperfusion of the heart.
The induction of IL-1betaand IL-8 in leukocytes by coronary sinus plasma obtained after recanalization of the infarct-related artery could not be explained by the plasma contents of IL-1beta, IL-6, IL-8 or TNF-alpha. Thus, our study provides evidence that besides the previously investigated cytokines, additional mediators or the metabolic conditions of ischemia and reperfusion, or both, contribute to inflammatory activation of leukocytes. This may affect resident, inflowing and passing leukocytes in the reperfused heart. Hence, the present study suggests a potential role of leukocytes in the regulation of both local and systemic inflammatory responses in patients with an acute MI.
IL-1betastimulates inflammatory cells like leukocytes, fibroblasts and endothelial cells; modulates adhesion molecules; and induces systemic inflammatory reactions like the expression of acute-phase proteins in the liver (). IL-8 is involved in various chemotactic, proinflammatory and reparative functions and is specific for neutrophil activation ([21, 22, 27]). IL-8 is produced by many cell types, even though the predominant source appears to be the endothelium (). Endothelial cells release IL-8 under hypoxic conditions (). Our data suggest that resident and inflowing leukocytes may extend the early release of IL-8 by the vascular endothelium during reperfusion.
IL-6 is a major cytokine in unstable angina and acute myocardial infarction, both locally and systemically ([6, 29]). Nevertheless, in the present study we could not detect a plasma-induced early IL-6 expression in leukocytes. Thus, we conclude that mediators contained in the plasma may not contribute to the elaboration of this cytokine by leukocytes. The IL-6 release in unstable angina () and acute MI () is sufficiently explained by the known hypoxia-induced liberation by endothelial cells and cardiac myocytes ([23, 30]). TNF-alpha has been shown () to be involved in neutrophil–myocyte adhesion after ischemia–reperfusion injury in the canine model. Available clinical data (), including those of the present study, have not been able to suggest an important role for TNF-alpha in the modulation of inflammatory responses after reperfusion.
3.2 Limitations of the Study.
In the present study we investigated the production of cytokines that are known to be regulated transcriptionally ([31, 32]). Therefore, analysis of mRNA expression in leukocytes by Northern blot is an appropriate experimental approach to the study objective. Even though posttranscriptional or posttranslational modulation of cytokine expression in leukocytes has not been shown to play an essential role (), our argument could have been strengthened by assaying the protein levels in the supernatants. However, the critical condition of our patients with an acute MI did not allow us to collect sufficient amounts of blood from the coronary sinus for further incubation experiments.
The limited quantity of coronary sinus blood also prevented us from analyzing subpopulations of leukocytes. We thus present data based on cytokine expression in unfractionated leukocytes. However, it is known that monocytes are one of the most important sources for cytokines ([34, 35]), even though both granulocytes and monocytes are involved in cytokine production ().
Our results demonstrate the induction of cytokine expression by coronary sinus plasma, but we did not investigate the mechanism by which leukocytes are activated. We did not find a correlation of IL-6 and IL-8 content in coronary sinus plasma and the induction of mRNA expression, but these two cytokines represent only a small part of potential mediators. Other reports ([9, 25, 37]) have described the induction of cytokine expression in leukocytes, for example, by complement fractions or platelet-activating factor. Additional mediators, such as leukotrienes or chemokines like MCP-1 or RANTES, may also contribute to the leukocyte-modulated inflammatory reactions ([7, 8, 19, 38]). Likewise, we cannot exclude an effect of traces of IL-1betaand TNF-alpha below the detection limits of our assays. Further experiments with blocking antibodies or heat inactivation of plasma components may help in the identification of the factors responsible for the observed effects.
3.3 Clinical Perspectives.
Early recanalization of the occluded artery in patients with an acute MI reduces mortality and may preserve left ventricular function ([1–5]). However, local and systemic inflammatory reactions of the ischemic and reperfused heart may compromise the clinical outcome ([10–14]). Administration of adjunctive therapies at the time of reperfusion may reduce deleterious inflammatory effects and improve the outcome of reperfusion. Our findings, that leukocyte IL-1betaand IL-8 expression is induced in the ischemic and reperfused heart, may assist in the design of therapeutic strategies aimed at reducing inflammatory responses. Leukocyte-derived cytokines may thus be a promising pharmacologic target for modulating inflammatory responses during reperfusion.
We thank Kathrin Schulz and Tanja Breustedt for excellent technical assistance.
☆ This study was supported by Grant Ne 540/1-1 from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany.
- citrate, phosphate buffer, dextrose and adenine
- glyceraldehyde-3-phosphate dehydrogenase
- left anterior descending coronary artery
- myocardial infarction
- percutaneous transluminal coronary angioplasty
- sodium dodecyl sulfate
- saline sodium citrate
- tumor necrosis factor-alpha
- Received December 18, 1996.
- Revision received March 5, 1997.
- Accepted March 12, 1997.
- The American College of Cardiology
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