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(Circulation. 1995;92:428-432.)
© 1995 American Heart Association, Inc.

Articles

Ischemia-Induced Interleukin-8 Release After Human Heart Transplantation

A Potential Role for Endothelial Cells

Mehmet C. Oz, MD; Hui Liao, MD; Yoshifuma Naka, MD; Alex Seldomridge, MD; David N. Becker; Robert E. Michler, MD; Craig R. Smith, MD; Eric A. Rose, MD; David M. Stern, MD; David J. Pinsky, MD

From the Departments of Surgery (M.C.O., Y.N., A.S., R.E.M., C.R.S., E.A.R.), Physiology (H.L., D.N.B., D.M.S.), and Medicine (D.J.P.), College of Physicians and Surgeons, Columbia University, New York, NY.

Correspondence to Mehmet C. Oz, MD, Columbia University, College of Physicians and Surgeons, Milstein 7-435, 177 Fort Washington Ave, New York, NY 10032. E-mail mco2@columbia.edu.

	Abstract

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Background Interleukin-8 (IL-8) secreted from endothelial cells is a powerful neutrophil chemoattractant and activator. We hypothesized that human endothelial cells deprived of oxygen would secrete IL-8, which might translate into elevated IL-8 production after cardiac ischemia. Furthermore, we hypothesized that coronary sinus (CS) IL-8 levels would be particularly high after cardiac preservation for transplantation, due to extended ischemic times.

Methods and Results Human saphenous vein endothelial cells exposed to a hypoxic environment (PO₂ <20 mm Hg) demonstrated a time-dependent release of IL-8 (measured by ELISA) into the culture supernatant as early as 4 hours after exposure. To determine whether cardiac preservation in humans was associated with IL-8 production, we obtained CS blood samples 5 minutes after reperfusion in a consecutive series of patients after they underwent cardiac transplantation (CTX, n=20) or elective cardiac surgery (non-CTX, n=21). CTX patients demonstrated significantly higher CS IL-8 levels than non-CTX patients (325±123 versus 50±17 ng/mL, respectively, P<.05). Further analysis of the CS samples revealed that a biochemical marker of myocyte injury (myoglobin) was similarly elevated in the CTX patients compared with the non-CTX patients (3340±625 versus 1151±525 ng/mL, respectively, P<.05).

Conclusions These differences may reflect the longer ischemic times of CTX compared with non-CTX hearts (161±10 versus 80±6 minutes, P<.0001) and suggest that the neutrophil chemoattractant/activator IL-8 may contribute to myocyte injury after prolonged hypothermic cardiac ischemia, as occurs during human cardiac transplantation.

Key Words: transplantation • ischemia • endothelium • interleukins

	Introduction

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Endothelial cells are critical homeostatic regulators of the vascular milieu.¹ In the quiescent state, they modulate vascular tone via nitric oxide production,² maintain blood fluidity by displaying an anticoagulant phenotype,³ prevent leakage of intravascular contents,^{3 4} and are relatively nonadhesive for circulating neutrophils.¹ Although human umbilical vein endothelial monolayers can survive a period of oxygen deprivation, under hypoxic conditions, they undergo severe phenotypic modulation, becoming prothrombotic, permeable, and highly adhesive for circulating neutrophils.^{1 3 4 5 6} In addition, they actively synthesize and secrete the proinflammatory cytokine interleukin-1a⁵ and the specific neutrophil chemoattractant interleukin-8 (IL-8).⁶

The recruitment of neutrophils to postischemic myocardium has been implicated as an important cause of myocardial damage after reperfusion.^{7 8 9 10 11 12 13 14 15 16 17 18} Adherent neutrophils can become activated to release numerous cytotoxic compounds, including reactive oxygen intermediates.¹⁹ These compounds can damage cell membranes and result in myocyte death, marked by the leakage of intracellular proteins such as myoglobin.²⁰ If myocardial ischemia is of sufficient severity or duration, myocardial edema²¹ and ventricular dysfunction ensue.²²

This is the clinical scenario often faced immediately after cardiac transplantation, especially after prolonged periods of ischemia. Extreme cases result in early graft failure, a cause of death in 10% of patients who underwent transplantation.²² Longer ischemic time is a highly significant independent variable affecting transplant mortality.^{22 23} Although most efforts to improve the efficiency of organ procurement have concentrated on the changes occurring during the preservation period,²⁴ the deleterious effects of uncontrolled reperfusion are becoming apparent.²⁵ The primacy of the reperfusion period compared with the preservation period in posttransplantation myocardial dysfunction is reflected in the increase in myocardial mass only after the period of reperfusion with relatively little mass change during the period of hypothermic preservation.²¹

Because the presence of neutrophils in the reperfusate is an important determinant of outcome after cardiac preservation¹³ and endothelial cells are important modulators of neutrophil recruitment to the postischemic cardiac vasculature,^{1 26 27} we investigated whether levels of the specific neutrophil chemoattractant and activator IL-8^{28 29 30 31} would be elevated in the human coronary sinus after heart transplantation. Furthermore, we hypothesized that human saphenous vein endothelial cells subjected to period of hypoxia, an important component of the ischemic milieu, would release IL-8.

	Methods

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Patient Selection and Preparation
Informed consent was obtained from all patients before enrollment in the present study, according to a protocol approved by the Institutional Review Board at Columbia-Presbyterian Medical Center. Consecutive patients undergoing heart transplantation between November 1993 and March 1994 were included in the study. During this same period, a group of control patients undergoing routine open heart surgery and matched to the underlying diagnosis of the heart transplant group was studied. Immediately after induction of anesthesia, a 3-mL sample of heparinized blood was drawn, and plasma was obtained and frozen for subsequent analysis. No surgical mortality occurred in either group.

In all patients, a DLP retrograde catheter was inserted into the coronary sinus. The catheter was inserted before placement of the aortic cross-clamp in the routine open heart surgery group and immediately after bringing the donor organ onto the operative field in the heart transplant group. Uniform preservation techniques were used within each group. For the routine open heart cases, an antegrade dose of 4:1 cold blood:high potassium cardioplegia (120 mEq/L KCl, 30 mEq/L NaHCO₃, 12.5 g/L mannitol, and 4.3% dextrose) was administered until a septal temperature of 20°C was achieved. Next, a lower potassium (60 mEq/L) blood cardioplegia mix was administered retrograde, accepting a perfusion pressure of 40 mm Hg until the septal temperature was below 15°C. The cardioplegia was readministered via the coronary sinus catheter every 20 minutes until the procedure was completed. In these patients, ischemic duration was recorded as the time from placement of the aortic cross-clamp to reperfusion with warm blood after removal of the cross-clamp.

The transplanted hearts were harvested using 1 L of 4°C University of Wisconsin solution (Dupont) administered via an antegrade catheter. The hearts were excised and placed into 4°C University of Wisconsin solution for transport to our facility. Ischemic duration was recorded as the time from placement of the donor aortic cross-clamp to reperfusion with warm blood in the recipient. Cardioplegia was administered via the coronary sinus catheter after each anastomosis (left and right atrial and pulmonary arteries) with the last dose administered less than 20 minutes before coronary sinus samples were taken.

Coronary Sinus Blood Sampling
For all patients enrolled in this study, 3 mL of heparinized blood was collected into an EDTA tube from a retrograde coronary sinus catheter 5 minutes after reperfusion. Blood within the coronary sinus catheter was discarded, after which dark coronary sinus blood was collected. Plasma samples were frozen at -80°C until the time of assay.

Saphenous Vein Endothelial Cell Experiments
Unused saphenous vein segments (n=3) were harvested from patients undergoing elective coronary artery bypass graft surgery. The veins were preserved in heparinized blood until the patient was successfully weaned from cardiopulmonary bypass and the operating surgeon released the discarded vein segments for research purposes. One end of each vein was attached to a syringe to allow two rinses with calcium-free HEPES-buffered saline solution. The other end of the vein was then ligated and the vein segment was filled with 0.2% collagenase (GIBCO) for 15 minutes at 37°C. The collagenase solution was then collected with an additional two rinses with HEPES-buffered saline and added to endothelial cell growth medium, with endothelial cells grown as described.^{3 4 5 6} Endothelial cells were grown in T25 cell culture flasks during primary passage. Once cells reached confluence, they were split with trypsin-EDTA and plated into T75 flasks. At confluence, cells were similarly split into 24-well tissue culture plates before normoxic or hypoxic exposure. Immediately before experiments, monolayers were rinsed twice with HEPES-buffered saline, and tissue culture plates were placed into a standard cell culture incubator (humidified, 37°C, 5% CO₂) or in a similar incubator placed in a normobaric hypoxia chamber (environment consisting of a gas mixture containing 5% CO₂, 5% H₂, and 90% N₂). Residual oxygen was eliminated with a palladium catalyst as described.⁵ Under these conditions, the measured PO₂ within the culture medium is 15 to 20 mm Hg.^{3 4 5 6} Aliquots of supernatant (100 µL) were removed at the indicated time intervals and frozen at -80°C until the time of assay, which was performed as described later.

Measurement of IL-8 or Myoglobin Levels
IL-8 levels were measured in plasma samples and culture supernatants with a commercially available ELISA (Quantikine ELISA, R&D Systems, Inc). Samples were centrifuged at 10 000g for 10 minutes at 4°C. Supernatants were recovered and diluted 1:1 with the diluent provided with the kit, added to the microtiter plate (wells were precoated with a monoclonal antibody to human IL-8), and allowed to incubate for 2 hours at room temperature. Each well was aspirated and washed three times with washing buffer (diluted 1:25 with distilled water), applied with a manifold dispenser. After we inverted the plate and blotted it with clean paper towels to thoroughly remove excess liquid, 200 µL of a polyclonal anti-human IL-8 antibody conjugated to horseradish peroxidase was added, and the plate was covered with a new adhesive strip and incubated for an additional 2 hours at room temperature. Each well was aspirated, washed three times with washing buffer, and blotted dry. Then, 200 µL of freshly prepared substrate solution (tetramethylbenzidine solution and hydrogen peroxide solution, provided with the kit) was added to each well and allowed to incubate for 20 minutes at room temperature. Next, 50 µL of stop solution (2 N sulfuric acid) was added to each well, and the absorbance of each well at 450 nm was read with a Biotek ELISA plate reader. Sample absorbances were calculated based on those of serial dilutions of a reference standard (human IL-8) that was assayed simultaneously with each sample run. The detection limit of this assay is 30 pg/mL. Assays for myoglobin in the human coronary sinus blood samples were performed similarly as described in detail for IL-8, except that this assay was based on a primary rabbit anti-human myoglobin antibody, with human myoglobin used as the reference standard (reagents provided by Spectral Diagnostics, Inc). The detection limit of this assay is 35 ng/mL. All assays were performed in duplicate, with results expressed as mean±SEM.

Statistical Analysis
Data were evaluated with the Mann-Whitney U test. Values are expressed as mean±SEM, with differences considered statistically significant at P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Because endothelial cells secrete proinflammatory cytokines under conditions of low oxygen tension,^{5 6} we established primary cultures of saphenous vein endothelium obtained at the time of routine coronary artery bypass surgery to determine whether hypoxia stimulates IL-8 release from endothelial cells clinically relevant to cardiac surgery. When these cells were subjected to hypoxia (PO₂ 20 mm Hg), there was a time-dependent release of IL-8 into the culture supernatant that was significantly higher than that of normoxic controls as early as 4 hours after the onset of hypoxia (Fig 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Bar graph of effect of hypoxia on interleukin-8 (IL-8) release by human saphenous vein endothelial cells. Confluent cultures of human saphenous vein endothelial cells were placed in a normobaric hypoxic environment (PO2 in the culture medium <20 mm Hg) or placed in a standard cell culture incubator. IL-8 was measured in the supernatant at the indicated times. Data represent the results of 6 experiments, expressed as mean±SEM, where *P<.05, ***P<.01, for hypoxia vs normoxia.

To identify whether a similar elevation of IL-8 could be detected within the cardiac vasculature after cardiac preservation, we compared coronary sinus levels of IL-8 in a consecutive series of 20 heart transplant patients with levels measured in a control group of 21 patients undergoing elective open heart surgery. Clinical characteristics of these two patient populations were similar in terms of sex and cause of underlying heart disease, although the transplant patients tended to be younger than those undergoing nontransplant heart surgery (Table). When ischemic times were compared between the two groups, that of the transplant patients was notably higher than that of the nontransplant patients (Fig 2).

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

View larger version (12K): [in this window] [in a new window]   Figure 2. Bar graph of relationship between type of surgery (cardiac transplant surgery [n=20], nontransplant elective cardiac surgery [n=21]) and duration of cardiac ischemia. ****P<.0001.

Because myocardial PO₂ declines after cardiac ischemia^{32 33} to a similar degree as that observed to cause the release of IL-8 from saphenous vein endothelial cells, we hypothesized that blood obtained from the cardiac vasculature after cardiac preservation would show elevated levels of IL-8. As a baseline value, coronary sinus IL-8 levels obtained immediately after initiation of cardiopulmonary bypass in patients undergoing elective open heart procedures was 17±8 ng/mL (this value was not obtained in donor hearts removed for transplantation because a coronary sinus catheter could not be justifiably placed in these patients before cardiac harvest). After ischemia and reperfusion, coronary sinus IL-8 levels were found to be significantly higher in the transplant group than in the nontransplant control subjects (325±123 versus 50±17 ng/mL, respectively; P<.05) (Fig 3). Within the transplant recipients, no direct correlation between ischemic time and IL-8 levels could be found.

View larger version (11K): [in this window] [in a new window]   Figure 3. Bar graph of coronary sinus interleukin-8 levels after cardiac preservation. *P<.05.

To determine whether the increased ischemic times of the transplanted hearts would be associated with release of an intracellular marker of myocyte injury (myoglobin), coronary sinus myoglobin levels were measured with ELISA in these same two groups of patients. Patients undergoing heart transplantation had significantly elevated coronary sinus levels of myoglobin compared with the nontransplant cardiac surgical control subjects (3340±625 versus 1151±525 ng/mL, respectively; P<.05) (Fig 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. Bar graph of coronary sinus myoglobin levels after cardiac preservation. *P<.05. There was no surgical mortality in either group.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cardiac transplantation has become an accepted therapy for end-stage heart failure; however, the field has been limited by the need to minimize organ ischemic time.^{22 23} Most current techniques for prolonging organ ischemic time have emphasized improved preservation.²⁴ However, the important role of reperfusion in limiting posttransplant cardiac function has become increasingly apparent.^{34 35} Left ventricular mass assessment during organ procurement reveals that most of the edema noted in transplanted human hearts occurs after reperfusion, not during the period of cold ischemia.²¹

Endothelial cells lining the endovascular lumen play an important role in orchestrating the complex vascular changes that occur during the critical period early after cardiac reperfusion.¹ Among the many changes in endothelial phenotype that occur during the ischemic and postischemic periods, the propensity for endothelial cells to secrete neutrophil chemoattractants⁶ and express neutrophil adhesion molecules^{5 36} is likely to be significantly deleterious during the vulnerable early period after cardiac preservation. Activated neutrophils release numerous cytotoxic/cytolytic compounds and generate a spectrum of reactive oxygen intermediates,^{10 19} which are likely to damage both the vasculature and surrounding myocytes. Experimental strategies designed to deplete neutrophils from the reperfusion milieu, either by leukocyte filtration^{13 14} or by stimulating the cAMP or nitric oxide/cGMP pathways^{26 27} (both prevent neutrophil adhesion), have been shown to be beneficial after hypothermic myocardial preservation.

To mirror an important component of the ischemic vascular environment, many investigators have exposed endothelial cells to hypoxia to study ischemia-driven mechanisms of vascular dysfunction.^{3 4 5 6 36} Endothelial cells exposed to hypoxia synthesize and secrete the proinflammatory cytokine interleukin-1a, which can augment the expression of neutrophil adhesion molecules such as E-selectin and ICAM-1 at the endothelial cell surface in an autocrinal fashion.⁵ Platelet-activating factor, a lipid that also participates in neutrophil adhesion, is also formed by endothelial cells after a period of hypoxia³⁶ and may participate in neutrophil recruitment after ischemia.³⁷ Human umbilical veins exposed to a hypoxic environment demonstrate increased transcription of IL-8 mRNA and release of chemotactically active IL-8 protein into culture supernatants.⁶ This information is similar to that demonstrated in the present study, in which increased IL-8 levels could be detected in supernatants from hypoxic human saphenous vein endothelial cells, although in the present study, increased IL-8 levels were detected somewhat earlier (at 4 hours).

The levels of hypoxia used in the present in vitro studies appear to be physiologically relevant for cardiac preservation. Intramyocardial PO₂ declines rapidly after the onset of normothermic cardiac ischemia.³² Although cardiac metabolism is presumably low during the period of hypothermic cardiac preservation, the decline in intramyocardial pH during this period suggests that some degree of metabolism continues, albeit at a lower rate.³⁸ Although during the course of cardiac surgery in humans it is difficult to obtain pure cardiac venous blood without admixture with other blood or ambient oxygen, the low PO₂ values (<20 mm Hg) observed after cardiac preservation in rats²⁶ further suggest that cardiac metabolism continues during hypothermic preservation. This level of metabolism may be sufficient for the synthesis of IL-8, which can thereby be released into the coronary sinus. Although coronary sinus IL-8 levels may be even higher at later times after cardiac preservation, ethical considerations do not permit prolonged monitoring of coronary sinus blood. Therefore, in these in vivo experiments in humans, we were unable to identify the peak of coronary sinus IL-8 production.

Our findings extend the observations of others^{39 40 41} that plasma IL-8 levels are elevated after elective cardiac surgery, although in previous studies, coronary sinus blood was not obtained and transplant patients were not evaluated. Although our data do not identify a coronary origin for secreted IL-8, measurements of coronary sinus IL-8 levels are most likely to reflect levels within the cardiac vasculature during the preservation period. Although no direct correlation between ischemic time and IL-8 was identified within the transplant group, several explanations for this observation may exist. In addition to the small number of patients studied, which makes statistical comparison within groups difficult, many factors other than ischemic time may influence the efficacy of preservation. Issues such as age and hemodynamic status of the donor may play a large role in the response to cold preservation. There are also potential variations in warm ischemic times, which are difficult to control for in a clinical study.

Because our endothelial cell studies showed a time-dependent release of IL-8 after exposure to hypoxia, we measured IL-8 levels in two groups of patients whose underlying causes of cardiac disease were similar but whose ischemic durations differed markedly. Not unexpectedly, due to the logistics of organ harvest and transport, ischemic times were significantly longer in patients undergoing heart transplantation than in the control group of patients who underwent elective cardiac surgery. Although the heart transplant patients presumably represent a sicker group of patients, preoperatively there was no difference between these two groups in terms of baseline IL-8 levels (38±12 ng/mL for transplant recipients versus 39±16 ng/mL for nontransplant recipients, P=NS). Compared with these baseline levels, coronary sinus IL-8 levels were elevated in both groups of patients after cardiac ischemia (Fig 3). When compared with each other, there was a significant difference in the IL-8 levels between the two groups, with higher values found in those patients receiving cardiac transplants. Although this difference is likely to reflect the longer cardiac ischemic duration in this same group of patients, it is possible that other factors, such as the myocardial preservation solution or the type of surgery, may have influenced IL-8 levels.

For several reasons, it is likely that IL-8 participates in cardiac reperfusion injury. Neutrophils are deleterious in the setting of cardiac reperfusion after either normothermic or hypothermic ischemia,^{7 8 9 10 11 12 13 14 15 16 17 18} contributing directly to both tissue damage and the no-reflow phenomenon⁴² by plugging the postischemic microvasculature.⁴³ IL-8 is a potent neutrophil chemoattractant²⁸ as well as activator,²⁹ stimulating activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils³¹ and promoting neutrophil emigration from the vasculature.³⁰ In addition, antibody to IL-8 has been shown to be protective in a rabbit model of pulmonary ischemia.⁴⁴ Although a causal relationship between IL-8 release and myocardial injury cannot be proved in the absence of a strategy to block IL-8 levels or activity, the concordance among prolonged ischemic duration, elevated IL-8 levels, and increased levels of a sensitive marker of myocyte injury (myoglobin) suggests a causal relationship. To place our data in the proper perspective, however, it must be recognized that neutrophil accumulation after cardiac preservation is likely to be multifactorial, as neutrophil adhesion molecules such as P-selectin may also contribute to cardiac reperfusion injury.⁴⁵ Overall, our findings are consistent with the clinical observation that prolonged organ storage time is a risk factor for significant ventricular dysfunction²² as well as mortality²³ after cardiac transplantation. Taken together, our data contribute to the growing body of evidence suggesting a detrimental role of neutrophils after cardiac preservation. Pharmacological strategies targeted at interfering with neutrophil-endothelial interactions may significantly enhance cardiac preservation in the future.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid from the American Heart Association and by grants from the US Public Health Service (HD-13063, HL-42507, and HL-50629). Dr Oz is an Irving Scholar of the College of Physicians and Surgeons, Columbia University (NIH MOI RR00645), and Dr Pinsky is a Clinician-Scientist of the American Heart Association.

	Footnotes

 
This work was presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 1994.

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