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Ann Thorac Surg 2002;73:123-129
© 2002 The Society of Thoracic Surgeons
a Department of Clinical and Experimental Medicine, Unit of Cardiac Surgery, Medical School of Catanzaro, Catanzaro, Italy
b Department of Clinical and Experimental Medicine, Unit of Internal Medicine, Medical School of Catanzaro, Catanzaro, Italy
Accepted for publication June 26, 2001.
* Address reprint requests to Dr Chello, Department of Cardiac Surgery, Via S. Giacomo dei Capri 29, 80128 Naples, Italy
e-mail: chello{at}unicz.it
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Abstract |
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Methods. Twenty patients undergoing coronary operation with CPB were studied. Patients undergoing off-pump (OP) coronary bypass and healthy subjects served respectively as stressed and normal groups. Interleukin-6 (IL-6), IL-8, and tumor necrosis factor-
were assessed on plasma collected preoperatively, at the end of CPB, and after intervals of 4, 8, 12, and 24 hours. Neutrophil apoptosis was detected by light microscopy as well as by the annexin-V assay on postoperative samples. The polymorphonuclear leukocyte (PMN) apoptotic receptors, Fas and FasL, were studied together with the activity of caspase 3 in postoperative neutrophils.
Results. Spontaneous apoptosis was significantly delayed in PMNs from CPB patients when compared with either the stressed or control patients. Neutrophils were activated, as indicated by increased surface expression of CD11b. Western blot analysis showed a normal expression of the apoptotic receptors Fas and FasL. Caspase 3 activity was found to be significantly reduced in neutrophils from CPB patients after 18 and 24 hours of culture. When control neutrophils were cultured in the presence of postoperative plasma from OP and CPB patients, apoptosis was significantly delayed. Depleting surgical plasma of IL-6 and IL-8 completely abolished this antiapoptotic effect.
Conclusions. Inflammatory mediators during CPB prolong the functional lifespan of neutrophils through modulation of apoptosis, and potentiate the inflammatory response observed after coronary bypass operation.
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Introduction |
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Neutrophils have the shortest half-life among leukocytes and normally survive for less than a day in the circulation [10] before undergoing morphologic and functional changes characteristic of apoptosis. During culture, 50% to 70% of neutrophils undergo constitutive apoptosis by 20 hours [11]. As neutrophils undergo apoptosis, they lose surface adhesion molecules and the ability to secrete granular contents [12, 13], and are subsequently ingested rapidly by macrophages before losing membrane integrity. Thus, apoptosis provides a way of removing polymorphonuclear leukocytes (PMNs) from an area of inflammation with minimal damage to the surrounding tissue. Although little is known about the mechanisms involved in regulating neutrophil survival and death, there are indications that the expression of neutrophil-programmed cell death can be modulated by environmental signals. In vitro studies have now shown that a variety of proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor, tumor necrosis factor- (TNF-), interleukin-8 (IL-8), and IL-6 inhibit the process of apoptosis in granulocytes [14–17] in hyperinflammatory disease states, including adult respiratory distress syndrome [18] and systemic inflammatory response syndrome [19]. Cytokines that activate neutrophils after CPB would also be expected teleologically to increase their lifespan through inhibition of apoptosis. Therefore, the desired therapy for truncating the inflammatory response would be to promote apoptosis.
The aim of the present study was to evaluate the effects induced by CPB on neutrophil apoptosis in vitro.
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Material and methods |
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Cardiopulmonary bypass procedure
The same standard anesthesia protocol was used for all patients. Anesthesia was induced with sodium thiopental, and muscle relaxation was achieved with pancuronium bromide; analgesia was obtained with fentanyl. The pump (Sarns roller pumps and Dideco hollow-fiber oxygenators) was primed with 2,000 mL of Ringer’s lactate. The heart was exposed through a median sternotomy, and heparin sodium (300 U/kg) was administered intravenously before CPB to produce an activated clotting time of longer than 400 seconds. The hematocrit level was maintained between 20% and 25%, and pump flows were kept between 2.0 and 2.5 L · min-1 · m-2 during moderate hypothermia to maintain a mean arterial pressure between 50 and 70 mm Hg. Cardioplegia was achieved with ice-cold St. Thomas’ Hospital solution infused into the ascending aorta. After completion of the distal anastomosis, the aortic cross-clamp was removed, and the aortovenous anastomoses were performed while the patient was being rewarmed to 37°C. After decannulation, protamine sulfate (10 mg/mL) (Eli Lilly, Indianapolis, IN) was administered intravenously to neutralize the heparin.
Off-pump coronary artery bypass procedure
A median sternotomy was performed and the left internal mammary artery (LIMA) was dissected as a pedicle by the usual technique. The myocardial surface was stabilized at the site of anastomosis to the coronary vessel with a special retractor (Octopus II, Medtronic, Minneapolis, MN). After harvesting the LIMA, heparin (1 mg/kg) was administered, and the pericardium was opened. Before the anastomosis was performed, the coronary vessels were occluded proximally and distally using two 4-0 Prolene (Ethicon, Somerville, NJ) sutures. The vessel was incised between these sutures, and the LIMA or saphenous vein graft anastomosis was constructed in an end-to-side fashion. Heparin was neutralized with protamine sulfate after all anastomoses were completed.
Sampling points
Blood samples for measurement of IL-6, IL-8, TNF-, blood gases, and full blood counts were taken at the following time points: at 5 minutes before induction of anesthesia, at the end of the operation, and then 4, 8, 12, and 24 hours postoperatively. Neutrophil apoptosis was evaluated on neutrophil from the 18-hour postoperative samples.
Cytokine assay
The blood specimen were collected in different phlebotomy tubes and immediately centrifuged for 10 minutes (3,000 rpm); all plasma samples were frozen in aliquots at -70°C until analysis. Interleukin-6, IL-8, and TNF- were measured by solid-phase sandwich enzyme-linked immunosorbent assay kits using commercially available antibodies (Genzyme, Cambridge, UK), according to the manufacturer’s directions. In the CPB group, the plasma values have been corrected for hemodilution.
Neutrophil isolation and culture
Polymorphonuclear leukocytes were isolated by dextran sedimentation and centrifugation through a discontinuous Ficoll gradient. Preparations of isolated neutrophils were maintained in RPMI medium 1640 supplemented with 10% autologous plasma and 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/L amphotericin B, at a concentration of 5 x 10 6/mL in tissue culture dishes at 37°C in a humidified CO2 incubator (5% CO2, 95% air).
CD11b assessment
Neutrophil CD11b expression was detected by indirect immunofluorescence and flow cytometry as described previously [20]. In the flow cytometry studies, the logarithmic mean fluorescence values were calculated from the histograms.
Morphologic criteria for apoptosis
Neutrophils cell morphology was analyzed in duplicate independently by 2 blinded investigators under oil immersion light microscopy. The PMNs were considered apoptotic if they showed dense condensation of chromatin in the form of either a single nucleus or nuclear fragments not connected by strands. At least 500 cells were counted per slide, and the results were expressed as the percentage of PMNs on each slide that met the criteria for apoptosis.
Annexin V-fluorescein isothiocyanate binding
The assay of PMN apoptosis was performed using fluorescein isothiocyanate-(FITC)-labeled recombinant human annexin V that binds to phosphatidylserine exposed on the surface of apoptotic cells. Stock annexin V was diluted 1:200 with binding buffer and then 25 µL was added to 75 µL of the recovered cell samples. After a 5-minute incubation at room temperature, these samples were fixed by the addition of 100 µL of 3% formaldehyde in phosphate-buffered saline before analysis using a dual filter fluorescence microscope.
Western blot analysis
For sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole extracts, 2 x 106 PMNs were sedimented, resuspended in 150 µL of lysing solution, and boiled for 10 minutes. Western blot analyses using commercially available primary antibodies (Cell Signaling, New England Biolabs) were performed with cellular lysate after 18 hours of incubation to examine the following proteins: Fas, Fas-L, CPP32, and ICE. Bands were detected by chemiluminescence with the Enhanced Chemi-Luminescence Kit (Amersham) and exposed with Hyperfilm MP (Amersham), and optical density readings were obtained by Quantity One software for IBM.
Assessment of caspase activity
Caspase activity was measured in vitro by the cleavage of the fluorogenic substrate Asp-Glu-Val-Asp (DEVD)-7-amino-4-methyl-coumarin (DEVD-AMC) in a continuous fluorometric assays as described by Fadeel and coworkers [21]. For preparation of cytoplasmic extracts, neutrophils (1 x 10 7) were lysed in 20 µL lysis buffer (50 mmol/L KCl, 50 mmol/L PIPES, pH 7.0, 10 mmol/L EGTA, 2 mmol/L MgCl2, 1 mmol/L dithiothreitol, 20 µmol/L cytochalasin B, 100 µmol/L phenylmethyl sulfonyl fluoride, and 1 mg/mL each of chymostatin, leupeptin, antipain, and pepstatin). For real-time recording of caspase activity, lysates were mixed with 100 µL of reaction buffer and the reaction was measured by the release of AMC from 100 µmol/L DEVD-AMC using a fluorometric microplate reader with excitation and emission wavelengths of 355 and 460 nm, respectively.
Immunodepletion experiments
To determine which factors in the plasma modulate apoptosis of PMN, we immunodepleted the CAB plasma of each of the following cytokines: TNF-, IL-6, and IL-8. Neutralizing polyclonal antibodies were added to postoperative plasma (4 hour postoperatively) for 4 hours at 4°C. The concentration of anti-IL-6, anti-IL-8, and anti-TNF- antibodies was chosen to achieve more than 90% neutralization, based on the measured circulating cytokine plasma concentrations and neutralizing bioassays performed by the manufacturer using factor-dependent cell lines. Normal neutrophils (1 x 106/mL) were then incubated in complete media supplemented with autologous plasma, postoperative plasma (18 hour postoperatively) from patients of both CPB and OP groups, and neutralized postoperative plasma. Apoptosis was quantified by dual staining with annexin V-FITC and propidium iodide after 24 hours’ culture.
Statistical analysis
Data are expressed as the mean ± the standard error of the mean. A repeated-measures analysis of variance followed by Scheffé multiple-comparison analysis was used to test for significant changes over the time course of the study, both within and between groups. The unpaired Student’s t test and Mann–Whitney U test were used when appropriate. A p value of less than 0.05 was considered significant.
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Tumor necrosis factor-
Although TNF- levels tended to increase (Fig 1C), there was no statistically significant change in TNF- levels either during or after CAB procedure in either the CPB or the OP group.
CD11b expression
CD11b expression (log mean fluorescence) was significantly higher on neutrophils from both OP (52.5 ± 4.4) and CPB (7 1.3 ± 4.7) patients compared with control patients (38.7 ± 3.4). Additionally, CD11b expression was significantly higher on neutrophils from CPB patients than from OP patients (p = 0.01).
Neutrophil apoptosis
Morphologic examination revealed that most normal neutrophils cultured in vitro maintained the cell membrane integrity until 12 hours of culture, whereas after 24 hours, two thirds of the cultured neutrophils obtained from normal donor showed typical apoptotic morphology with the expected decrease in DNA contents. These results were confirmed by annexin-V analysis (Fig 2).
At 12 hours of culture, almost 50% of control neutrophils exhibited apoptotic changes, compared with 20% and 30% of neutrophils from the CPB and OP groups, respectively. At 24 hours of culture almost 70% of neutrophils from normal donors were apoptotic, compared with 28% and 37% of neutrophils from the OP and CPB groups, respectively. The percentage of apoptotic cells was significantly higher in the OP compared with the CPB group at both 12 (p < 0.05) and 24 hours (p < 0.05) of culture.
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had no significant effect on neutrophil apoptosis.
Fas and FasL expression
Expression of Fas and Fas ligand on neutrophil membrane was evaluated in a subset of patients from CPB (n = 10) and OP (n = 10) groups. Western blot analysis showed no significant difference in the expression of these proteins compared with controls (n = 10).
ICE and CPP32 expression
Expression of the proapoptotic caspases, ICE and CPP32, was evaluated in a subset of patients from CPB (n = 8) and OP (n = 8) groups. Western blot analysis showed no significant difference in the expression of these proteins compared with controls (n = 8).
Caspase activity
Asp-Glu-Val-Asp (DEVD)-7-amino-4-methyl-coumarin is a specific substrate for caspase-3-like proteases and represents the common cleavage site for this class of enzymes. Freshly isolated neutrophils were lysed at various times (0 to 24 hours) after the initiation of the in vitro culture and the maximum linear rate of AMC release was measured. Figure 4
shows that a clear induction of caspase activity, with peak values at 24 hours was seen during constitutive apoptosis in the three groups. In neutrophils from CPB patients, DEVD cleavage was significantly reduced compared with the control group after 18 (17.1 ± 2 versus 26.4 ± 2.4, p < 0.05) and 24 hours of incubation (24.7 ± 2.8 versus 36.6 ± 4.7, p < 0.05), whereas no significant difference was observed between OP and the control group throughout the time course of the experiment.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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The major finding of our study was the significant reduction of apoptosis of PMN from CPB compared with PMN from either OP or control patients, accompanied by a concomitant state of activation of PMNs as demonstrated by the increased expression of CD11b. Of neutrophils from healthy controls, 67.5% displayed signs of apoptotic changes after 24 hours in culture, whereas fewer than 30% of the neutrophils from CPB patients showed these changes. However, delayed apoptosis was not specific for CPB, as similar changes were observed at a lesser level in neutrophils from patients after OP-coronary bypass. These present findings confirm previous studies in which neutrophil apoptosis was delayed because of evident surgical stress. Fanning and coworkers [23] demonstrated that spontaneous apoptosis of neutrophils was significantly inhibited after elective operation with an effect evident within an hour of surgical incision. Neutrophil apoptosis was significantly inhibited 24 hours after operation, and at that point, patients’ plasma significantly inhibited spontaneous apoptosis of normal neutrophils. Jiminez and colleagues [19] reported that PMNs from patients with systemic inflammatory response syndrome and from patients undergoing major operation had delayed apoptosis. In addition, they were able to delay apoptosis of normal PMNs with plasma from both patient groups. In the above-cited studies, the delay in neutrophil apoptosis has been strictly linked to the increased plasma concentrations of IL-6 and IL-8. In the present report, we observed a significant increase of plasma cytokine levels in both OP and CPB groups over the whole time course of the study, with peak values in the postoperative period significantly higher in CPB patients. Interestingly, plasma from both CPB and OP patients was effective in delaying apoptosis of control neutrophils, and the maximum apoptosis inhibition was achieved with plasma from CPB patients. Moreover, immunodepletion of IL-6 significantly reduced the antiapoptotic effect of the OP plasma, but only moderately affected the effect of CPB plasma on neutrophil apoptosis. Conversely, the immunodepletion of IL-8 was associated with the most significant reduction in the antiapoptotic effect of CPB-plasma. Finally, the addition of plasma depleted for both cytokines had no effect on the apoptosis of control neutrophils. Depleting surgical plasma of TNF- had no significant effect on neutrophil apoptosis.
Taken together, these results lead to two conclusions: (1) the surgical stress, independently of CPB, is able to induce a decrease of neutrophil apoptosis, and this effect is probably mediated by elevated plasma concentrations of IL-6, and (2) the significant decrease of neutrophil apoptosis after CPB is mainly a consequence of the increased plasma concentrations of IL-8 more than of IL-6.
The autocrine interactions between Fas and Fas ligand on the neutrophil membrane have been demonstrated to play an important role in the induction of apoptosis in human PMN, which constitutively express not only Fas, but also FasL [10, 24]. In the attempt to elucidate the mechanisms of apoptosis control in the context of coronary operations, we evaluated the expression of PMN Fas and Fas ligand in a subset of 10 patients. However, we could not find any significant difference in the expression of these receptors on PMN from either CPB or OP compared with PMN in the control group, suggesting that the Fas/FasL system is not involved in the delay of apoptosis triggered by either surgical stress or CPB. In this context, our findings are in keeping with the report by Jimenez and coworkers [19], which showed no changes in the Fas expression of PMN in systemic inflammatory syndrome stress and with the observation by Liles and coworkers [24] that the incubation of PMN with IL-8, IL-6, and TNF- in vitro was able to suppress Fas-mediated apoptosis but had no influence on the Fas expression.
Fas and TNFR1 signal for apoptotic cell death by downstream activation of proteins of the ICE family of proteases, including ICE and CPP32 [8, 9]. Caspases are activated in constitutive and Fas/APO-1-mediated apoptosis. In a separate set of experiments we examined whether reduced PMNs apoptosis induced by coronary operation was the result of decreased caspase activity. We found a considerable less caspase activity, as determined by DEVD-AMC cleavage, in freshly isolated neutrophils from CPB patients compared with the control patients. A decreased caspase activity was also found in neutrophils from the OP group compared with controls, but the difference failed to reach statistical significance. Finally, it is noteworthy that lower levels of caspases activity were also found at 18 and 24 hours in neutrophils from CPB patients compared with OP patients, although the difference failed to reach statistical significance. Considering these results, we can hypothesize that a major role is played by IL-8, compared with IL-6, in suppressing caspase activity in neutrophils triggered to undergo apoptosis.
In this study we evaluated the role of CPB on neutrophil apoptosis and observed that the rate of programmed cell death was markedly reduced after coronary bypass operation with CPB compared with that of both healthy individuals and of OP patients. We found that IL-8 and IL-6 plasma levels play an important role in reducing cell death, by mechanisms involving a reduction in caspase3 activity as determined by DEVD-AMC cleavage, in the constitutive and Fas/APO-1 triggered models. Thus, prolonged neutrophil survival in the postoperative period can cause an unbalanced tissue load of PMN and uncontrolled release of toxic metabolites, which results in tissue damage, activation, and unwanted migration of neutrophils to the tissues.
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= off pump; • = cardiopulmonary bypass. *p < 0.05 versus off pump.)
