Ann Thorac Surg 2004;77:951-955
© 2004 The Society of Thoracic Surgeons
Original article: cardiovascular
Monocyte Fc gamma receptor expression in patients undergoing coronary artery bypass grafting
Demetrios C. Stefanou, FRCSa,
George Asimakopoulos, FRCSa,
Darshna R. Yagnik, MSa,
Dorian O. Haskard, FRCPa,
Jon R. Anderson, FRCSa,
Pandelis Philippidis, FRCSa,
R. Clive Landis, PhDa,
Kenneth M. Taylor, MD*a
a British Heart Foundation, Cardiothoracic Surgery Unit and Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, London, England, UK
Accepted for publication September 5, 2003.
* Address reprint requests to Dr Taylor, Cardiothoracic Surgery Unit, NHLI, B Block (2nd floor), Hammersmith Hospital Campus, Du Cane Rd, London W12 0NN, UK
e-mail: k.m.taylor{at}ic.ac.uk
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Abstract
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BACKGROUND: Cardiopulmonary bypass is associated with an inflammatory response with potential deleterious effects. The white cell subpopulation mostly investigated so far is the neutrophil. To date very little has been investigated regarding the role of the monocyte/macrophage. This study focuses on the expression of Fc gamma receptors I, II, and III by monocytes in patients undergoing cardiopulmonary bypass.
METHODS: We studied the surface expression of Fc gamma receptors I, II, and III by flow cytometry on gated monocyte subpopulations in the whole blood of adult patients undergoing elective coronary artery bypass grafting. Blood samples were drawn preoperatively and at 15 minutes, 1, 2, 4, 24, 48, and 72 hours, and 6 days postoperatively. A second group of patients undergoing lung resection surgery were studied in a similar fashion.
RESULTS: Neither Fc receptor I nor receptor II expression were significantly changed throughout the time points studied. Fc receptor III expression was reduced at 2 and 4 hours (p = 0.016 and 0.002) and increased at 24, 48, and 72 hours after commencement of CPB on a selected subpopulation (15%–35%) of monocytes (p = 0.004, < 0.001, and < 0.001, respectively). This expression returned to preoperative levels by the sixth postoperative day. There were no statistically significant changes in the lung resection group.
CONCLUSIONS: Our study demonstrated that cardiopulmonary bypass is associated with a biphasic Fc gamma receptor III expression on a subpopulation of peripheral blood monocytes up to 3 days postoperatively.
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Introduction
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Cardiopulmonary bypass (CPB) is associated with an inflammatory response which involves interactions between leukocytes, platelets, and the endothelium. The first cell to become activated is the neutrophil. As early as 15 minutes after commencement of CPB, neutrophil expression of the integrin CD11b, which mediates adhesion to the endothelium, increases [1]. Less known is the role of the monocyte and the regulatory mechanisms involved in its functions during CPB. Monocyte expression of CD11b increases to peak levels at 24 hours and returns to base line levels at 72 hours [2]
Fc gamma receptors (FcR) are transmembrane/surface glycoprotein complexes that bind IgG and are expressed on leukocytes, including monocytes. They mediate receptor-specific phagocytosis of IgG-opsonized particles [3] or endocytosis of immune complexes [4]. There are three families of Fc receptors: FcRI or CD64, FcRII or CD32, and FcRIII or CD16.
Monocyte Fc gamma receptor cross-linking over immobilized human IgG induces interleukin (IL)-8 production [5]. This induction is the result of two separate pathways involving Fc gamma receptor I (CD64) and III (CD16) [6].
Fc gamma receptor III+/ve monocytes recognize immune complexes and induce tumor necrosis factor (TNF) alpha production in rheumatoid arthritis [7]. The role of monocyte subpopulations expressing Fc gamma receptor III+/ve phenotypes has been studied in fields such as rheumatology [8] and AIDS [9]. Our project was aimed at characterizing the changes on Fc receptor expression on monocytes isolated from patients undergoing routine coronary artery surgery. We also included a group of lung resection surgery patients in order to establish the impact of nature of procedure on our results
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Material and methods
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Patients
Ten patients were recruited in order to study the patterns of CPB-related FcR expression during hospital stay. Our project had written approval from Hammersmith Hospitals NHS Trust Ethical Committee and informed consent was obtained from all patients. Patients were excluded if they met any of the following criteria: unstable angina and myocardial infarction within the 6 preceding weeks, cerebrovascular accident within 3 months preceding the operation, combined valve procedure, infective endocarditis, coagulopathy, use of steroids or nonsteroidal agents, use of anticoagulants or aspirin within the week before the operation, serum creatinine in excess of 177 µmol/L, presence of malignancy, more than 75% carotid obstruction as shown by carotid Doppler scan, or previous use of aprotinin. Table 1 describes the clinical data of the patients
Additionally, 10 patients undergoing lobectomy for stage T1N0 mol/L0 lung cancer were recruited. None of these patients had any significant past medical history and fulfilled the inclusion criteria of our cardiac group.
Anesthetic and operative techniques
Standardized techniques were applied for anesthesia and CPB. Anesthetic premedication included morphine (10 mg) and hyoscine (0.3 mg) administered intramuscularly on the morning of the operation. Anesthesia was commenced with midazolam (100–200 µg/kg), fentanyl (150–200 µg), and pancuronium (50–100 µg/kg) and sustained with propofol (5–10 mg · kg-1 · h-1). The CPB circuit consisted of a roller pump (Stockert Instruments, Munich, Germany), a Bard William Harvey HF-750 membrane oxygenator (C.R. Bard, Crawley, England), and polyvinyl chloride tubing. Pulsatile extracorporeal circulation was used at 2.4–2.8 L/m2/min. Moderate hypothermia of 32–34°C was applied in all patients. Myocardial protection was administered with a Bard cardioplegia delivery system using cold blood antegrade cardioplegia.
Flow cytometric analysis of monocyte Fc gamma receptor expression
Central venous or peripheral venous samples (5 ml) were drawn from patients and immediately placed into heparin-containing tubes at the following time points: (1) before skin incision (pre-CPB), (2) 15 minutes after initiation of CPB, (3) 60 minutes after initiation of CPB, (4) 2 hours, (5) 4 hours, (6) 24 hours, (7) 48 hours, (8) 72 hours, and (9) 6 days postoperatively. Tubes containing blood were placed on ice and flow-cytometric analysis was carried out within 2 hours from blood sampling. For analysis, blood samples were placed in 12 x 75 mm polystyrene tubes (Falcon, Becton Dickinson, Cowley, UK) using 90 µL whole blood and 10 µL primary antibody (100 µg/mL) directed against the unique Fc gamma receptors of monocytes. Primary incubations were carried out on ice for 15 minutes. Each antibody was matched at each time point to class-matched mouse IgG1 control MOPC-21 (BD PharMingen, Oxford, UK). After two washes with phosphate-buffered saline, fluorescein isothiocyanate-conjugated secondary antibodies were added at the manufacturer's recommended concentration (Sigma Chemical Co., Dorset, UK) and a further incubation of 15 minutes was carried out. This was followed by red cell lysis for 30 seconds (1 ml of lysing reagent) and the application of 250 µL of fixative solution (Coulter Electronics Ltd., Luton, UK). The samples were then analyzed immediately on a flow cytometer (EPICS XL, Coulter Electronics Ltd., Luton, UK). Monocytes were identified by their characteristic cytometry profile marked by the use of anti-CD14 antibody.
Statistical analysis
Data are expressed as mean ± standard deviation. Values at all time points were compared with pre-CPB levels by analyzing our results with one way analysis of variance (ANOVA) and with the Dunnett post test. Statistical significance was declared at p values < 0.05.
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Results
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Intraoperative and postoperative characteristics of patients
All cardiac patients received 3–4 coronary artery conduits, one of which was always the internal thoracic artery grafted to the left anterior descending coronary artery. The rest of the conduits were venous. The thoracic patients underwent lobectomy and tumor excision was complete. There were no major intraoperative or postoperative complications in either group.
Expression of fc gamma receptors on circulating monocytes
Fc gamma receptors I and II exhibited normal distribution on monocytes and expression was therefore measured as a relative fluorescence intensity (RFI). This is defined as the ratio of the mean fluorescence of test antibody versus the fluorescence of class-matched control antibody. Figures 1 and 2 demonstrate that Fc gamma receptor I and II expression remained unchanged throughout the procedure and early recovery (p > 0.05).
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Fig 1. Fc gamma receptor I expression on peripheral blood monocytes during and after cardiopulmonary bypass. Upper and lower limits, means and standard deviations, box, and whisker plots. Onset of bypass at 0 minutes. (Pre = pre-bypass; RFI = relative fluorescence intensity.)
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Fig 2. Fc gamma receptor II expression on peripheral blood monocytes during and after cardiopulmonary bypass. Upper and lower limits, means and standard deviations, box, and whisker plots. Onset of bypass at 0 minutes. (Pre = pre-bypass; RFI = relative fluorescence intensity.)
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Fc gamma receptor III exhibited a biphasic distribution thus indicating that expression was confined to a subset of monocytes (Fig 3). The Fc gamma receptor III histograms show that Fc gamma receptor III was downregulated at 2 and 4 hours after CPB (p = 0.016 and 0.002, respectively), upregulated at 24, 48, and 72 hours (p = 0.04, < 0.0001, and < 0.0001), and returned to baseline values by the sixth postoperative day. Expression of Fc gamma receptor III is shown in Figure 4 as the percentage of cells positive for Fc gamma receptor III expression (15 to 35%).
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Fig 3. Fc gamma receptor III expression on a subset of monocytes. Flow cytometry analysis fluorescent histograms of Fc gamma receptor III expression. The open areas show Fc gamma receptor III expression and the filled ones show expression of class matched control antibody. (Pre = pre-bypass.)
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Fig 4. Percentage of positivity (15%–35%) for Fc gamma receptor III expression on peripheral blood monocytes during and after cardiopulmonary bypass. Onset of bypass at 0 minutes. (Pre = pre-bypass.)
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Fig 5. Fc gamma receptor III expression on peripheral blood monocytes during and after lung resection surgery. Upper and lower limits, means and standard deviations, box, and whisker plots. Onset of operation at 0 minutes. (Pre = pre-bypass.)
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Lung resection surgery patients had a similar initial reduction at 2 and 4 hours (p = 0.3461 and 0.2524, respectively), followed by increases at 24 and 48 hours (p = 0.3914 and 0.2346), and reduction at 6 days (p = 0.3174). These changes were not statistically significant (Fig 5).
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Comment
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Fc gamma receptor III expression was downregulated at 2 and 4 hours after CPB. This coincides with observations of monocyte activation by other studies [10] and is most likely due to margination of activated monocytes in target organs [11].
Our results also demonstrate an increase in the expression of monocyte Fc gamma receptor III at 1, 2, and 3 days after CPB before returning to preoperative values on the sixth postoperative day. The inflammatory cascades activated during CPB are a result of exposure to the artificial surfaces of the CPB circuit and ischemia reperfusion. We know that a number of events take place activating cells such as neutrophils, platelets, and endothelial cells.
Previous research has shown that, during this period, there is an increased production of IL-6, IL-8, and TNF-alpha [12]. TNF-alpha is locally produced in the myocardium during cardiopulmonary bypass [13]. Fc gamma receptor I cross-linking produces IL-8 in patients with severe trauma [14]. The increased expression of monocyte chemoattractant factor around the same time [15] suggests that the monocyte may well be a contributor to the events observed.
Fc gamma receptor III expression on monocytes is associated with a more differentiated stage of development of this cell type. Fc gamma receptor III+/ve monocytes are considered to bear characteristics similar to mature tissue macrophages including the production of TNF-alpha [16]. Alveolar macrophages are Fc gamma receptor III +/ve [17] and, during CPB, alveolar macrophages produce higher levels of TNF-alpha and IL-8 and express more CD11b than peripheral blood monocytes [18]. It has also been suggested that IL-10 induces monocyte maturation toward the Fc gamma receptor III +/ve subpopulation and this induction is prevented by the application of anti-IL-10 antibodies [19]. Additionally IL-10 is known to possess anti-inflammatory properties in rheumatoid arthritis synovium mononuclear cells and increases expression of Fc gamma receptor III and I [8]. Fc gamma receptor III +/ve cells are observed in patients with AIDS dementia [9] and sepsis [20]. This suggests that mobilization of the Fc gamma receptor III+/ve monocyte subpopulation is part of the stress response. AIDS patients have an IL-10-enriched environment that shifts monocyte Fc receptor expression toward the Fc gamma receptor III+/ve phenotype [21]. In other immune complex-mediated diseases, it has been postulated that Fc gamma receptor cross-linking may be responsible for IL-8-induced recruitment of locally destructive neutrophils [6].
A study of 12 patients recovering from valve surgery produced the observation that CD14+/Fc gamma receptor III+ monocytes were expanded in the patients with high APACHE II scores [22]. Another study showed that monocytes isolated 20 hours after CPB express more tissue factor and are more excitable in vitro [23]. Glucocorticoids selectively deplete CD14+/Fc gamma receptor III+ monocytes [24] and increase the production of the anti-inflammatory IL-10 [25].
Our findings suggest that CPB induces changes on peripheral blood monocytes as late as 3 days postoperatively. There is a Fc gamma receptor III+/ve subpopulation during this period of time susceptible to the anti-inflammatory effects of IL-10 and steroids and with a profile very similar to mature committed alveolar macrophages.
Further research into the role of the monocyte during CPB is needed to establish the importance of Fc gamma receptor cross-linking in relation to pro-inflammatory (IL-6, IL-8, and TNF-alpha) and anti-inflammatory cytokines (such as IL-10). Ex vivo isolated peripheral blood monocyte studies may elucidate the role of specific subtypes of Fc receptor such as Fc gamma receptor III during the periods of most significant upregulation.
A percentage of patients who undergo routine coronary surgery—especially those with comorbidity—go on to develop complications such as acute respiratory distress syndrome (ARDS), multiorgan failure, and sepsis. Further knowledge of the prevailing profile of monocytes within such groups will provide us with valuable information about inflammation and CPB in general. Additionally new inflammatory end points may be identified that can be used as targets to measure the efficacy of existing and new anti-inflammatory strategies.
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Abstract
Introduction
