Interact CardioVasc Thorac Surg 2008;7:207-211. doi:10.1510/icvts.2007.158899
© 2008 European Association of Cardio-Thoracic Surgery
Institutional report - Cardiopulmonary bypass |
Delayed recovery of human leukocyte antigen-DR expression after cardiac surgery with early non-lethal postoperative complications: only an epiphenomenon?
Axel Frankea,*,
Wolfgang Lantea,
Lothar G. Zoellerb,
Edmund Kurigb,
Christian Weinholda and
Andreas Markewitza
a Department of Cardiovascular Surgery, German Armed Forces Central Hospital, Ruebenacher Str. 170, D-56072 Koblenz, Germany
b Central Institute of the German Armed Forces Medical Service, Koblenz, Germany
Received 5 May 2007;
received in revised form 8 November 2007;
accepted 10 November 2007
 This research was conducted with financial support from the German Armed Forces Medical Service.
*Corresponding author. Tel.: +49-2631-2813736; fax: +49-2631-2813733.
E-mail address: dr.axel.franke{at}t-online.de; axel1franke{at}bundeswehr.org (A. Franke).
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Abstract
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HLA-DR expression on peripheral blood monocytes is reduced after cardiac surgery. Little is known about the reconstitution of HLA-DR expression on peripheral blood monocytes in patients suffering from early non-fatal perioperative complications. We conducted a prospective study to prove whether these complications adversely affect the recovery of HLA-DR expression. Before surgery (d0), on the first (d1), third (d3), fifth (5th) postoperative days, blood samples were collected from 90 patients who underwent elective cardiac surgery with cardiopulmonary bypass (CPB). HLA-DR expression was analysed flow cytometrically. Eleven patients experienced postoperative complications [mechanical ventilation of 24–48 h (n=6); reinstitution of CPB (n=2) intraoperatively; laparotomy (n=1), re-thoracotomy (n=1), re-intubation (n=1) within the first 24 h after surgery]. All patients showed a reduced HLA-DR expression after surgery with nadirs at d1 and d3. Whereas the values increased from d3 to d5 in patients with an uneventful clinical course, HLA-DR expression remained suppressed in patients with complications. HLA-DR expression is reduced after cardiac surgery with CPB. A delayed recovery of HLA-DR expression is seen in patients with early perioperative complications. These non-fatal complications appear to represent a ‘second hit’ resulting in a prolonged deficiency of the innate immune system. This might predispose to further infectious and septic complications.
Key Words: Cardiac surgery; HLA-DR expression; Peripheral blood monocytes; Immune response; Immune dysfunction
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1. Introduction
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In the literature, a decrease in HLA-DR expression on peripheral blood monocytes has been established as an indicator of a reduced activity of these cells. It has been discussed that, as a result, the ability to stimulate the immune response is altered. This is regularly seen in patients who have undergone cardiac surgery with cardiopulmonary bypass (CPB) or extensive surgery as well as in burn and polytrauma patients [1–4]. A persistent decrease in HLA-DR expression on monocytes is indicative of immune paralysis and has been correlated in a number of studies with a poor prognosis and increased mortality in sepsis [1, 2].
In a previous study, we could show that there is a significant correlation for HLA-DR expression on monocytes and IL-12 liberation in stimulated whole blood from patients following cardiac surgery [5]. Since IL-12 is a main stimulus of T-helper cell differentiation and activity [6], monitoring of HLA-DR expression on peripheral blood monocytes might not only give insights into monocyte activity.
To our knowledge, there are no solid data on HLA-DR expression in patients with early postoperative complications without immediate mortality (e.g. mechanical ventilation for more than 24 h, re-thoracotomy, reinstitution of CPB, re-intubation, laparotomy, or cardiopulmonary resuscitation for patients with arrhythmia).
It was the aim of this study to test the hypothesis that in patients with non-fatal complications – such as those described above – reconstitution of HLA-DR expression is delayed in the postoperative course (from the third to the fifth day after surgery). For this reason, we quantitatively analysed HLA-DR expression on peripheral blood monocytes by flow cytometry in patients who had undergone elective cardiac surgery. We compared the results of patients with early non-fatal postoperative complications with the results for patients with an uneventful course.
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2. Patients, materials and methods
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Our study was approved by the local Ethics Committee. Only patients without known local or systemic infection or inflammation (fever, leucocytosis, tachycardia or tachypnoea), immune or central nervous system dysfunctions, congestive heart failure, exogenous hormone therapy or malnutrition were enrolled in this study.
HLA-DR expression was assayed in whole blood samples that were obtained from study patients preoperatively (d0) and on the 1st (d1), 3rd (d3) and 5th (d5) postoperative days at 8:00 am and anticoagulated with ethylene diamine tetraacetic acid (EDTA). We used a FACSCalibur®-flow cytometer to analyse peripheral blood monocytes obtained from elective cardiac surgery patients. Data were collected in the years 2003 and 2004. We compared the results for 11 patients who experienced non-fatal complications after surgery and the results for 79 patients with an uncomplicated clinical course.
2.1. Patient management
We used the same cardiopulmonary bypass and perioperative management procedures as described previously [7]. Anaesthesia was induced with etomidate and sufentanyl. After endotracheal intubation and muscle relaxation with pancuronium, anaesthesia was maintained using sufentanyl, midazolam and isoflurane.
The cardiopulmonary bypass equipment included non-pulsatile roller pumps and membrane oxygenators (Quadrox®, Jostra, Hirrlingen, Germany). The pump was primed with a standard electrolyte solution containing 5000 IU heparin, 1000 ml Ringer's lactate, 500 ml NaCl 0.9%, 500 ml Biseko® (Biotest, Dreieich, Germany) and 250 ml of a 15% mannitol solution (Osmofundin 15%®, Braun Melsungen, Melsungen, Germany). Heparin (300 IU/kg) was administered just before vascular cannulation. After the institution of cardiopulmonary bypass at a flow rate of 2–2.4 l/m2/min, the aorta was cross-clamped and a cold crystalloid cardioplegic solution was injected. After CPB, protamine was infused. All patients received 2,000,000 IU of aprotinin before the onset and 1,000,000 IU at the end of CPB to preserve platelet function. Cefazolin (3x1.5 g) was used for perioperative antibiotic prophylaxis.
2.2. HLA-DR expression on peripheral blood monocytes
HLA-DR expression was analysed using the QuantiBRITE Anti-HLA-DR PE/Anti-monocyte PerCP-Cy 5.5 system and a FACSCalibur® flow cytometer (Becton Dickinson, Heidelberg, Germany). We used whole blood samples that were cooled and anticoagulated with EDTA in order to minimise in vitro activation.
A forward scatter – PerCP-Cy 5.5 plot was used to identify peripheral blood monocytes. Five thousand monocyte events were acquired. The system was set up and calibrated before using QuantiBRITE PE beads in accordance with the manufacturer's recommendations. The QuantiBRITE system uses calibrated standardized phycoerythrin (PE) – conjugated beads to measure and calculate of fluorophore bound per cell. These calibration beads with known numbers of PE molecules per bead provide an easy and sensitive means of quantifying PE-stained cells with a flow cytometer.
Using four groups of beads with different numbers of PE molecules per bead and the QuantiQUEST software (Becton Dickinson, Heidelberg, Germany) a linear standard curve is generated relating fluorescence to PE molecules. The number of PE molecules bound per cell is determined from the linear PE fluorescence value of the cell, using QuantCALC software and a linear regression equation according to the supplier's manual. Results are calculated in the number of PE molecules (x1000) bound per cell and represent the amount of antibody molecules bound, because of a given PE to HLA-DR antibody ratio of 1:1. The number of anti-HLA-DR antibodies bound per monocyte (mAb/cell) was expressed as mean±standard error of the mean.
2.3. Statistical analysis
Data were collected and analysed using standard computer software (Statview 5.0, Abacus concepts, Berkeley, CA). An unpaired Student's t-test was used to identify differences within groups (time points) and between groups (patients with vs. without complications). A P-value of <0.05 was considered significant. The results are expressed as mean±standard error of the mean, unless otherwise indicated.
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3. Results
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3.1. Clinical course
Initially, we included samples from a total of 91 patients. On the fifth postoperative day, however, one patient developed sepsis and was therefore excluded from the study. Eleven of the remaining 90 patients experienced different types of complications.
As a result of global respiratory insufficiency, six patients required mechanical ventilation for 24–48 h. One of these six patients had to be resuscitated from ventricular fibrillation immediately after surgery. Two patients required reinstitution of cardiopulmonary bypass after having been removed from the heart-lung machine. Both patients were extubated within the first 36 h after surgery. One patient underwent re-thoracotomy for bleeding within the first 24 h after surgery. One patient required laparotomy for an acute abdomen 36 h after surgery (X-rays had shown free air). The insertion of a mediastinal chest tube had caused peritoneal injury but not organ injury. After successful extubation, one patient had to be re-intubated for global respiratory insufficiency within 24 h after surgery. All patients were successfully treated without further postoperative complications. Table 1 and 2 provide an overview of preoperative and intraoperative data for both patient groups.
3.2. HLA-DR expression
In both groups, the absolute numbers of HLA-DR on peripheral blood monocytes significantly decreased during the entire postoperative course. We observed the lowest values on the first and third postoperative day.
Whereas these values remained low on the fifth postoperative day in patients with a complicated course, they began to increase again at this time point in patients with an uneventful course. The values observed for patients without complications were significantly higher at d5 than those obtained at d3 and d5 for patients with complications (Fig. 1).
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Fig. 1. HLA-DR expression (number of anti-HLA-DR antibodies bound per monocyte) before surgery and during the first five postoperative days after CABG (d0 – before surgery; d1 – first postoperative day; d3 – third postoperative day; d5 – fifth postoperative day). Displayed is mean±standard error of the mean (S.E.M.). *P<0.05 vs. d0; **P<0.001 vs. d0; #P<0.05 in between-group comparisons (same time point).
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Preoperative HLA-DR expression differed a little between the study groups, therefore postoperative changes were also expressed in percentage terms. In both groups, HLA-DR expression decreased to below 30% when compared with preoperative baseline values and increased again at d3 in patients with an uncomplicated course. The decrease, when expressed in percentage of baseline, was significantly lower at d3 and d5 in patients with an uncomplicated course than in patients with perioperative complications (Fig. 2).
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Fig. 2. HLA-DR expression (number of anti-HLA-DR antibodies bound per monocyte) in percentage of baseline before surgery and during the first five postoperative days after CABG (d0 – before surgery; d1 – first postoperative day; d3 – third postoperative day; d5 – fifth postoperative day). Displayed is mean±standard error of the mean (S.E.M.). *P<0.05 vs. d0; **P<0.001 vs. d0; #P<0.05 in between-group comparisons (same time point).
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4. Discussion
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In our prospective study on HLA-DR expression in patients who had either a complicated or an uncomplicated course after elective cardiac surgery with CPB, we observed a significant decrease in postoperative HLA-DR expression on peripheral blood monocytes in all patients. A quantitative analysis of HLA-DR molecules expressed on the cells revealed no differences between the two study groups until the third postoperative day. At d5, however, HLA-DR expression recovered in patients with an uncomplicated course whereas it remained depressed in patients with perioperative complications. Since preoperative HLA-DR expression was slightly higher in the group of patients with a complicated course, postoperative changes were also expressed in percentage terms. A comparison of percentages showed a significant difference between the two patient groups as early as on the third postoperative day when we obtained lower values for patients with a complicated course.
The group of patients who had a complicated course is inhomogeneous with respect to the causes of their perioperative complications. In a number of patients, there was a second hit caused by a further surgical trauma after initial surgery (n=2) or by the reinstitution of CPB during surgery (n=2). In the majority of patients, perioperative complications were secondary to the failure of an organ system (global respiratory insufficiency). Although a within-group comparison is limited due to the small number of patients, perioperative complications resulted in all cases in a uniform and persistent decrease in HLA-DR expression, irrespective of the underlying causes. This confirms the results reported by Oczenski et al. who showed that there were no significant differences in HLA-DR expression between patients with and without complications during the first 24 h after cardiac surgery [1]. A significant decrease in HLA-DR expression after cardiac surgery has been reported in a number of previous studies [5, 8–11]. Other studies investigating patients who had undergone non-cardiac surgery were able to correlate the expression of HLA-DR on peripheral blood monocytes with morbidity and mortality levels [1–3]. These studies found a higher number of septic complications in patients with a persistent decrease in HLA-DR expression and hypothesised that a reduced activity of peripheral blood monocytes might indicate a paralysis of the innate immune system and thus may predispose to these complications.
Moreover, there is some evidence suggesting that a constantly low HLA-DR expression of monocytes is associated with an increased risk of subsequent sepsis [2]. Aside from the relationship between HLA-DR expression and the immunocompetence of patients with sepsis, which can develop as a later complication after cardiac surgery, the question arises as to the extent to which HLA-DR expression is influenced by immediate postoperative complications that are not primarily associated with an increased mortality.
The complications that were seen in our patients were found to go hand in hand with delayed recovery of the innate immune system, as revealed by the development of HLA-DR expression. On the basis of the aforementioned results reported in the literature, this finding might explain the clinical experience that early perioperative complications often cause further septic and in particular infectious conditions.
There are a wide variety of mechanisms that may contribute to the delay in the recovery of HLA-DR expression in the group of patients with perioperative complications. A longer period of sedation, the anaesthetic procedure and a longer operating time can modulate both the release of pro- and anti-inflammatory cytokines and the expression of HLA-DR [12]. Different studies have shown that especially interleukin (IL)-10 and prostaglandin E2 (PGE2), which are released in response to surgical trauma, cause a decrease in HLA-DR expression on monocytes [13]. The reduction in HLA-DR expression could thus be a late effect of the intraoperative release of these mediators. Cardiac surgery with CPB regularly leads to an activation of the innate immune system and thus not only to an increase in the peripheral blood levels of monocyte derived cytokines, like IL-6, IL-8 and tumour-necrosis factor (TNF)-alpha [7, 14]. Therefore, it may also be hypothesised that an intraoperative overstimulation of peripheral blood mononuclear cells postoperatively leads to a depression of monocytes, or in other words to exhaustion of these cells. Our data, however, can neither prove nor disprove this hypothesis.
Furthermore, the small number of patients makes it impossible for us to confirm the widely discussed clinical experience that patients with early non-fatal complications develop sepsis more frequently after cardiac surgery than other patients. Moreover, the patients in our study did not experience infectious complications. For this reason, it remains to be established whether the persistent decrease in HLA-DR expression after cardiac surgery represents a risk factor for developing infectious complications – or is simply an epiphenomenon without causal relationship.
Against this background, it should be investigated whether the postoperative decrease in HLA-DR expression after cardiac surgery and at the onset of sepsis reflect the same pathophysiology.
In summary, our results demonstrate that the recovery of HLA-DR expression after cardiac surgery in patients with early non-fatal complications is delayed. This might indicate a depression of the innate immune response. Both the reason behind and the consequences of this observation, however, remain unclear. Further studies are needed to prove if these findings result in a higher susceptibility to postoperative infectious complications.
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5. Conclusions
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- Cardiac surgery induces a significant decrease of HLA-DR expression on peripheral blood monocytes.
- The recovery of HLA-DR expression is delayed in patients with early intraoperative and postoperative complications. As a result, the activity of innate immune system and thus the ability to combat bacterial infection might be reduced postoperatively.
- Therefore, from an immunological point of view, monitoring of HLA-DR expression is important and further research should be undertaken to investigate the underlying cause of the delayed recovery of HLA-DR expression and its clinical implications after the fifth postoperative day.
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Acknowledgements
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The authors gratefully acknowledge the excellent assistance provided by B. Isenberg, L. Herter and Y. Wyss.
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References
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Abstract
1. Introduction
