Cardiopulmonary bypass (CPB) induces a systemic inflammatory response, activation of the complement cascade, and profound alteration of the mechanisms of hemostasis (1). There is evidence that avoiding CPB in surgical myocardial revascularization, off-pump coronary artery bypass grafting (OPCAB), results in a lower systemic inflammatory response (2).
Although most patients recover from coronary artery bypass grafting (CABG) without complication, up to 22% (3) develop a systemic inflammatory response syndrome (SIRS), characterized by altered general (fever, tachycardia, edema), inflammatory, hemodynamic, organ, and perfusion variables (4). There is also disruption to hemostasis, and the cardiac, pulmonary, renal, neurological, hematologic, gastrointestinal, and vascular systems can be involved. Although most often self-limiting, multiorgan failure can develop in a proportion of patients with SIRS, and there is an increased rate of mortality in this group (5).
Polymorphonuclear cells (PMNs) have been implicated in the mechanistic link between injury and inflammation. After CPB, both humoral and cellular inflammatory pathways are activated, and this was originally thought to induce PMN priming and hyperactivity (6). Our group has recently shown the effect of CABG with CPB to involve complex changes to normal PMN physiology (7). The most important finding was an attenuated PMN response to in vitro activation that persisted until at least 5 days after CABG. We observed a similar decrease in PMN response in critically ill patients in the intensive care unit (8). Information about the function of PMN in CABG without CPB (OPCAB) is limited to PMN surface marker expression in the first 24 h after surgery.
Polymorphonuclear cell function can be determined using a number of techniques including measuring the expression of cell surface markers and superoxide released from the PMN respiratory burst. Mac-1, consisting of a common β subunit (CD18) noncovalently bonded with CD11b, is a member of the β2 integrin family and essential for normal PMN function, including firm adhesion, migration, and phagocytosis (9). It is constitutively expressed, and the inactive form cannot mediate efficient adhesion, consistent with the quiescent state. When activated, Mac-1 expression is increased, and a high-affinity binding site for intercellular adhesion molecule 1 (ICAM-1), iC3b, and fibrinogen (10) is exposed. This reveals an activation-specific neoepitope within the I domain—CBRM1/5 (11).
CD62L (L-selectin) mediates PMN rolling and tethering on endothelium, before the Mac-1 complex (with other integrins) mediates firm adhesion (12). L-selectin is shed by proteolytic cleavage upon PMN stimulation (13). Integrins are activated by a variety of intracellular signals, including protein kinase C, and the G-protein–coupled receptors (14). Activation of G-protein–coupled receptors by formyl methionyl-leucyl-phenylalanine (fMLF) or other chemokines causes calcium mobilization, protein kinase C activation, and ultimately activation of the NADPH oxidase complex, resulting in respiratory burst, phagocytosis, and bacterial killing (15). Other pathways activated downstream lead to protein phosphorylation influencing adhesive interactions via the integrins.
Platelet-activating factor (PAF) binds to the PAF receptor, and fMLF binds to the formyl-peptide receptor, all seven membrane-spanning G-protein receptors on the surface of PMN (16). These regulate cytoplasmic Ca2+ concentration, phosphatidylinositol turnover, cyclic AMP levels, and phosphorylation of regulatory proteins (16).
To date, PMN function beyond the immediate postoperative period after OPCAB has not been investigated. By assessing PMN response to the stimuli PAF and fMLF in patients after OPCAB, we have investigated whether cardiac surgery itself (without CPB) causes prolonged modulation of PMN physiology.
Patients were eligible for inclusion in the study if they were undergoing elective OPCAB for stable angina at the Cardiothoracic Surgical Unit at Royal Prince Alfred Hospital, Sydney, Australia. None of the patients had recent myocardial infarcts (<14 days) or were converted to CABG with CPB. The study was approved by the Sydney Local Health District Ethics Review Committee. Written, informed consent was obtained from all patients.
Our surgical technique for OPCAB has been described previously (17). In brief, general anesthesia was induced by propofol, fentanyl and a nondepolarizing muscle relaxant. An endotracheal tube was placed, and positive pressure ventilation established. A pulmonary artery catheter and radial artery catheter were used to monitor systemic and pulmonary hemodynamics. After sterile preparation of the operative field, median sternotomy was performed. The right pericardial fat pad was partially harvested, and the pericardium incised laterally at the diaphragm and superior vena cava/right atrial junction to allow rotation of the beating heart about the caval axis, improving exposure of the inferior and lateral walls for complete myocardial revascularization. Revascularization was achieved using all-arterial grafts. Patients were routinely commenced on a milrinone infusion intraoperatively for 24 h to optimize arterial graft vasodilation and cardiac output. Normothermia was maintained throughout. At completion of the procedure, patients were transferred to the intensive care unit—cessation of sedation and extubation followed once hemodynamic and respiratory functions were satisfactory. Patients were commenced on subcutaneous heparin (5,000 units twice daily) on the evening of the first postoperative day and continued this until discharge. β-Blockers were introduced as tolerated from day 1.
Blood was collected in 0.109 M sodium citrate vacutainer tubes (Becton-Dickinson, Franklin Lakes, NJ) on the morning before and on days 1, 3, and 5 after surgery. On postoperative days 3 and 5, blood was collected before the morning dose of heparin was administered. Polymorphonuclear cells were isolated within 4 h of blood collection, by the method described previously (7). Briefly, using sterile, endotoxin-free equipment and reagents, PMNs were isolated using dextran for red cell sedimentation and Percoll at concentrations of 1.077 g/mL and 1.114 g/mL (GE Healthcare, Uppsala, Sweden) for double-density gradient separation. Isolated PMNs were then washed and resuspended in Hanks buffered salt solution (HBSS; Invitrogen, Carlsbad, Calif) before being quantified (Cell-Dyn Sapphire; Abbott Diagnostics, Abbott Park, Ill). Isolated neutrophil solutions were of greater than 95% purity. Once quantified, PMNs were resuspended in HBSS solution at a concentration of 10 × 106 PMNs/mL and assayed immediately.
Polymorphonuclear cells stimulated after an initial priming stimulus have a greater response than those activated alone, according to the “two-hit” hypothesis (6, 18). Four stimulation combinations were examined using a combination of buffer (HBSS with magnesium and calcium), PAF (priming agent at final concentration 1 μmol/L), and fMLF (activator at final concentration 1 μmol/L): (a) buffer only, (b) PAF given at 10 min after incubation in buffer, (c) fMLF given at 20 min after buffer, and (d) PAF given at 10 min after buffer, then fMLF given at 20 min to simulate the “two-hit.”
Flow cytometric analysis of surface marker expression
Analysis of the PMN surface markers CD11b (Dako, Glostrup, Denmark), CD18, CD62L (San Jose, Calif), and CBRM1/5 (eBioscience, San Diego, Calif) was performed on PMNs for the four stimulation conditions at each time point. Antibodies were aliquoted to a final volume of 67.5 μL (including 62.5 μL of resting/stimulated PMNs at a concentration of 1 × 106 /mL). These were incubated for 10 min on ice and fixed with 0.16 % paraformaldehyde in HBSS. The leukocyte population was identified by expression of CD45, and acquisition of the PMN population was based on gated light-scatter characteristics on a BD FACSCalibur (Becton-Dickinson).
Respiratory burst assay
The maximal rate of superoxide (O2 −) (nmol/2 × 105 PMN/min) was measured as previously described (7). Polymorphonuclear cells (total 4 × 105) were stimulated as described above, and the change in optical density at 550 nm reflected reduction of cytochrome C. The maximal rate of O2 − production was calculated using the steepest slope of at least five data points and the molar extinction coefficient 21.1 × 103 mol/L for cytochrome C (8).
Preoperative and postoperative data were compared by repeated-measures analysis of variance with Dunnett post hoc test (SPSS, IBM, Armonk, NY). Neutrophil counts were compared by paired t test using GraphPad Prism 5 (GraphPad Software, La Jolla, Calif). Significance was determined for P < 0.05. Data are expressed as mean (±SEM) of n patients, where n = 15 unless otherwise indicated.
A total of 15 patients with coronary artery disease underwent OPCAB for stable angina. Preoperative characteristics and postoperative outcomes are described in Table 1. No patients had clinical signs consistent with the SIRS in the preoperative or postoperative periods. Results did not differ significantly in those who received a blood transfusion (data not shown).
Expression of Mac-1–related cell surface markers after stimulation of days 3 and 5 neutrophils
Polymorphonuclear cells were sampled at four time points: preoperative and days 1, 3, and 5 postoperative. Expression of Mac-1 heterodimers CD11b and CD18 on PMNs was measured after exposure to four stimulation conditions. Polymorphonuclear cells treated with PAF and/or fMLF had increased expression of CD11b (Fig. 1) and CD18 (Fig. 2). Those first primed with PAF and subsequently activated with fMLF demonstrated the greatest upregulation (preoperative CD11b relative to unstimulated baseline: PAF +64%, fMLF +160%, and PAF/fMLF +272%) (Fig. 1).
The expression of CD11b in buffer-treated PMNs (basal, unstimulated conditions) did not change with surgery, suggesting minimal activation in vivo. Polymorphonuclear cells stimulated with fMLF on postoperative days 3 and 5 demonstrated blunted upregulation of CD11b compared with PMNs isolated preoperatively (preoperative, +160%; day 3, +119%; day 5, 75%; Fig. 1). Similarly, PMNs primed with PAF then stimulated with fMLF also demonstrated blunted upregulation of CD11b on days 3 and 5 compared with preoperative baseline (upregulation compared with unstimulated baseline: preoperative +272%, day 3 +228% vs. day 5 +156%).
The expression of CD18, the β subunit of Mac-1, differed from its non–covalently bonded α subunit CD11b (Fig. 2). With fMLF stimulation, it was not significantly different from preoperative baseline in the postoperative period.
We also measured expression of the isotype of Mac-1 activation CBRM1/5 (Fig. 3). Results for CBRM1/5 reflected those of CD11b. That is, expression after stimulation was significantly decreased on days 3 and 5 (upregulation with fMLF compared with unstimulated baseline: preoperative +468%, day 3 +252%, day 5 +300%).
Expression of CD62L after stimulation of days 3 and 5 neutrophils
The expression of CD62L on PMNs decreases when the cells are stimulated (7, 8). Under resting conditions, there was no difference in CD62L expression between PMNs sampled before and after surgery (Fig. 4). As expected, shedding of CD62L occurred after PMNs were stimulated. However, PMNs stimulated on days 3 and 5 with PAF, then fMLF demonstrated less shedding than those activated preoperatively (shedding with PAF/fMLF compared with baseline: preoperative −88% vs. day 3 −79% vs. day 5 −81%). When CD62L expression at each time point was expressed as a ratio of its unstimulated baseline, there was a trend for decreased shedding for fMLF and PAF/fMLF–treated PMNs on days 3 and 5 that did not reach statistical significance.
Respiratory burst impaired after stimulation on days 3 and 5
Measuring the production of superoxide after stimulation gives further insight into PMN function after OPCAB. Compared with treatment with buffer, PMNs had greater maximum production of O2 − when treated with fMLF alone and greatest maximum production of O2 − when treated with PAF then fMLF (fMLF: +142%, PAF/fMLF: +625%; Fig. 5). Platelet-activating factor alone induced a small and insignificant increase in the maximum rate of O2 − production, whereas PAF followed by fMLF induced maximum production of O2 −, confirming that PAF stimulation was priming, rather than activating PMNs.
There was no difference in the maximum rate of O2 − production over time in PMNs treated with buffer. However, the increase in the maximum rate of O2 − production achieved by the combination of PAF and fMLF was significantly blunted on day 3 (preoperative +641% vs. day 3 +363%) and nonsignificantly blunted on day 5 (+430%) compared with preoperative baseline (Fig. 5).
We report persistent hyporesponsive PMN function after cardiac surgery performed without CPB. Polymorphonuclear cell response to stimulus is impaired for a period of at least 5 days after OPCAB, as suggested by lower increase in both expression of CD11b and the neoepitope of its high-affinity binding site CBRM1/5. This is supported by lower maximum superoxide production on days 3 and 5 after PMN priming and activation. The pattern of impairment over time is similar to that observed previously after CABG (7). A validation study on a small cohort of CABG patients (n = 5) confirmed that the techniques and results generated from this study were comparable to those of the initial CABG study (data not shown) (7). Together, these results suggest that the cause of PMN hyporesponsiveness may be attributed to the surgical insult or perioperative management rather than CPB itself.
Impaired PMN response to in vitro stimulus has been reported in patients after CABG (7), with sepsis (19, 20), and with SIRS (8). An animal model of sepsis (cecal ligation and puncture) demonstrated impaired PMN migration and impaired PMN/endothelial adhesion (21). We speculate that the significantly lower postoperative CD11b and CBRM1/5 expression after stimulation may represent either incompetence of the innate immune response or a protective mechanism tempering a potential excessive PMN response because of the associated neutrophilia.
In stimulated PMNs from patients after OPCAB, we observed independent increased expression of the CD18 and CD11b components of the Mac-1 heterodimer, as has been previously described (7).
In vitro stimulation of PMNs typically involves L-selectin shedding (12, 13). We observed no changes to CD62L expression on buffer-treated PMNs before and after OPCAB but a resistance to shedding of CD62L in stimulated cells on postoperative days 3 and 5. We have previously described no changes to CD62L after CABG with similar stimulation conditions to the present study (7, 12). However, in a population of critically ill patients, CD62L was resistant to shedding with stimulation compared with healthy controls (8). Decreased shedding of CD62L after OPCAB observed in our study supports the hypothesis of partial hyporesponsiveness and attenuated PMN function.
The mechanism of immunocompromise after injury and sepsis remains unclear. Chemoattractant activators bind with G-protein–coupled receptors on the cell surface to cause calcium release and effect PMN activation, but the individual downstream pathways of integrin, selectin, and respiratory burst activation differ (12, 22). The pathways regulating Mac-1 and L-selectin expression are overlapping but not identical; evidence of this includes the different responses to various tyrosine kinase inhibitors (22) and the presence of a subpopulation of conformationally active Mac-1 complex on PMNs without shedding of L-selectin after cardiac surgery (12). Binding of ligands to L-selectin increases adhesive function of β2 integrins and release of secretory granules via an “outside-in” p38 MAPK pathway (23). The pathway of respiratory burst activation differs from that of the integrins and selectins. NADPH oxidase is the enzyme responsible for production of the superoxide anion, and the p47phox subunit is thought to be responsible for moving cytosolic granules of NADPH oxidase to the cell surface. Once assembled with flavocytochrome b558 at the cell membrane, the active enzyme is formed, and the respiratory burst occurs (15). Platelet-activating factor stimulation causes partial phosphorylation of the regulatory cytosolic phox proteins via MAPK results in “priming” of the PMN (24); subsequent activation by fMLF causes complete phosphorylation, rapid movement of cytosolic components to the membrane, and exocytosis of the reactive oxygen species (15).
In patients after OPCAB surgery, we describe impairment of PMN selectin, integrin, and respiratory burst activation, which may suggest changes to a part of the common pathway of activation—for example, the G-protein–coupled receptor. Some studies have suggested downregulation of G-protein–coupled receptors on the cell surface as a mechanism of PMN impairment (25). More recently in septic patients, Arraes and colleagues (26) demonstrated no change in chemokine receptor expression but upregulation of G-protein receptor kinases, which phosphorylate the G-protein–coupled receptor and attenuate its responsiveness. This suggests that endogenous mediators may continually activate PMNs, inducing desensitization to chemoattractants via G-protein–coupled receptor kinase activation, rather than via receptor downregulation (26). Studies in both OPCAB and CABG cohorts document elevated cytokines and acute phase proteins for at least 8 days after surgery (27). Therefore, the post-OPCAB hyporesponsive PMN phenotype observed may be associated with G-protein–coupled receptor desensitization in the persistent inflammatory environment following surgery. However, this is yet to be investigated in cardiac surgical patients.
Catecholamines, heparin, and phosphodiesterase-3 inhibitors are all used routinely in the perioperative period. Each has been implicated in PMN immunocompetence and may be present in the plasma in the postoperative period (12, 28, 29). Plasma concentrations of catecholamines (endogenous and exogenous), heparin, and milrinone are likely to be highest intraoperatively and within the very early perioperative period. Our results suggest that PMNs are not yet hyporesponsive on day 1 but only on days 3 and 5. Although it is likely that catecholamines, heparin, and milrinone influence neutrophil function to some extent, it is unlikely that they are primarily responsible for the hyporesponsive PMN phenotype that we have observed on days 3 and 5.
Neutrophilia, an important part of the acute inflammatory response, is common after surgery. It is caused by a number of humoral factors including cytokines, chemokines, complement fragments, endotoxin (30). There is mobilization of the PMN “marginal pool,” delayed apoptosis (31), and release of immature cells from the bone marrow (30). Other groups have demonstrated impairment of immature neutrophils. Taneja and colleagues (32) showed that immature PMNs (identified by nuclear morphology) isolated from patients with sepsis have impaired phagocytosis and calcium signaling; similarly, immature PMNs from patients with cancer have impaired superoxide production and decreased expression of chemokine receptors (CXC-1/2) (33). Orr and colleagues (30) reported that immature cells (absent CD10, low CD16 expression) represented 50% of circulation PMNs after CABG. As the maturity of PMNs was not determined in our study, it is not possible to speculate whether PMN maturity might contribute to functional impairment observed at later time points in our study.
A decreased systemic inflammatory response after OPCAB compared with CABG has been reviewed extensively (3, 34). Coronary artery bypass grafting involves exposure of the blood to nonendothelialized surfaces, thereby producing an inflammatory response. The milder inflammatory response after OPCAB has been held largely responsible for the lower rates of renal failure, blood transfusion, atrial fibrillation, and for improved outcomes after surgery in high-risk and elderly patients (35). The results from this study demonstrate that PMN responsiveness to stimulation follows a pattern similar to that previously described after CPB (7). This implies that impaired PMN responsiveness is not responsible for the differences in systemic inflammation between CABG and OPCAB.
The absence of a direct comparison between the CABG group with CPB and OPCAB group limits our ability to assess the specific role of the CPB circuit on neutrophil function. This, however, was not our aim, and we have reported the results of this cohort previously (7).
In this study, only circulating PMNs were investigated. These may not reflect the phenotype of marginated and noncirculating PMNs in various organs, also important in the pathogenesis of SIRS. In addition, the relatively small group of patients may have prevented detection of a statistical difference in some PMN surface markers.
Decreased PMN response to stimuli persists for at least 5 days after cardiac surgery without CPB. Altered expression of adhesion molecules and decreased superoxide production with stimulation imply dampening of PMN function. The results of this study demonstrate that CPB is not required to alter the PMN function in CABG and suggest that surgical insult may be primarily responsible. The consistent modulation of host PMN function in both CABG and OPCAB may represent an autoprotective mechanism to avoid inappropriate systemic inflammatory response. Whether this downregulation jeopardizes the patient’s ability to mount an adequate physiological innate immune system response to infectious pathogens is not clear. Future studies should investigate the mechanisms linking tissue injury with inflammation and neutrophil function.
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Neutrophils; off-pump coronary artery bypass grafting; systemic inflammatory response syndrome; cardiopulmonary bypass; immunocompromise