The first step of immune response to infectious agents is mediated primarily by polymorphonuclear leucocytes (PMNs). The combination of anaesthesia and surgery is associated with PMN dysfunction, as indicated by reduced chemotaxis, adherence, phagocytic ability and respiratory burst function [1,2]. A number of previous studies have investigated the effects of intravenous and inhalational anaesthetics on cells involved in the nonspecific and specific immune response [1,3]. The lack of agreement among these studies may be related to several factors: differences among anaesthetic or nonanaesthetic agents; different cell preparation/purification techniques; different stimuli for the respiratory burst; different measurement methods; and other factors such as the effect of the agent's diluent, patient selection criteria, etc. . Immune dysfunction due to anaesthesia and surgery cannot be evaluated properly because of insufficient number of human studies. Most of the studies about anaesthesia-related immune response were either in vitro or in peripheral blood cells. Peripheral blood samples could not reflect immunological activity precisely as the response in the tissue.
Phagocytosis and respiratory burst activity are mainly responsible for bacterial killing of PMNs . Phagocytosis by PMNs constitutes an essential arm of host defence against bacteria and fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent (respiratory burst) and oxygen-independent mechanisms. The objective of this prospective, randomized, double-blinded clinical study was to evaluate the effects of general anaesthesia on the phagocytosis and respiratory burst activity of PMNs in bronchoalveolar lavage (BAL) fluid.
This study was approved by the local ethics committee of the Selcuk University, Meram Medical Faculty. All study patients gave written informed consent. Sixty ASA physical status I adult patients scheduled to have tympanoplasty surgery were studied. Exclusion criteria were cardiovascular or obstructive/restrictive lung disease, malignant or chronic inflammatory disease, anaemia (haemoglobin <10 g l−1), smoking, endocrine and immune system disease, hepatic or renal disease, and chronic therapy with, or abuse of, benzodiazepines or opioids. Patients taking potential immunity-compromising medication, such as steroids or nonsteroidal anti-inflammatory medication, were also excluded. The patients were randomly allocated to receive desflurane (Baxter; Puerto Rico, USA) (group D, n = 20), sevoflurane (Abbott; UK) (group S, n = 20) or propofol (group P, n = 20) anaesthesia by random numbers (Microsoft EXCEL). Patients were premedicated with intramuscular (i.m.) diazepam (10 mg) and atropine (0.5 mg) 1 h before surgery. The heart rate (HR), systolic (SBP) and diastolic blood pressure (DBP), oxygen saturation (SpO2) and end-tidal CO2 (ETCO2) values were continuously monitored. Anaesthesia was induced with 2–3 mg kg−1 propofol (propofol 1%; Fresenius Kabi) and 1 μg kg−1 fentanyl (fentanyl citrate; USP Abbott) administered intravenously (i.v.), and muscle relaxation was achieved with rocuronium bromide (0.6 mg kg−1; Esmeron Organon) then the trachea was intubated. The lungs were mechanically ventilated with Drager Primus (10–12 per min, positive end-expiratory pressure 4 cmH2O, peak airway pressure 15–20 cmH2O). Tidal volume was adjusted to maintain end-tidal pCO2 at 4.5–5.5 kPa. Anaesthesia was maintained with either volatile anaesthetics (1–1.5 MAC desflurane or sevoflurane in 50% O2–air, total flow 4 l min−1) or propofol. For the propofol group, anaesthesia was maintained with 12 mg kg−1 h−1 propofol for the first 20 min, 9 mg kg−1 h−1 for the second 20 min, 6 mg kg−1 h−1 for the third 20 min and titrated doses of 3–6 mg kg−1 h−1 thereafter. In all groups, neuromuscular block was maintained with intermittant doses of rocuronium bromide (0.2 mg kg−1). Nitrous oxide was not used in either group. SBP, DBP, SpO2 and ETCO2 were recorded at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 and 120 min.
BAL was performed from the right middle lobe in all patients immediately after induction of anaesthesia (t0) and after the surgical procedure (t1) by a fibreoptic bronchoscope (Karl Storz 11302 BD1). The procedure was repeated five times, with 100 ml sterile 0.9% saline solution instilled in total. The anaesthetist who performed the BAL procedures was blinded to the anaesthetics being used in this study.
Immune function tests were performed by a single investigator who was blinded to the anaesthesia management. Leucocyte respiratory burst and phagocytic activity in BAL were determined using commercially available kits (Bursttest, Phagotest Orpegen Pharma, Heidelberg, Germany) and flow cytometric analysis of gated leucocyte populations within 2 h. Briefly, the PMNs in the BAL fluid were incubated with 5 μl of opsonized and fluorescein-marked (FITC) Escherichia coli (1 × 109 bacteria ml−1) to assess phagocytic activity at 37°C for 10 min. Negative controls were kept on ice. The samples were centrifuged after addition of quenching solution (100 μl). After additional washing, lysing solution (50 μl) was added and incubated for another 10 min. After repeated washing and addition of DNA staining solution (200 μl), the samples were analysed in a flow cytometer. To assess the respiratory burst activity, the PMNs in the BAL fluid were incubated with 5 μl of opsonized and fluorescein-marked (FITC) E. coli (1 × 109 bacteria ml−1) and phorbol 12-myristate 13-acetate (PMA) at 37°C for 10 min. Negative controls were kept on ice. The samples were centrifuged after addition of substrate solution (20 μl). After additional washing, lysing solution (50 μl) was added and incubated for another 10 min. After repeated washing and addition of DNA staining solution (200 μl), the samples were analysed in a flow cytometer. The PMN gate was set using the routine position for PMNs in the sideways scatter and forward scatter (Fig. 1). A total of 10 000 PMNs per sample were analysed with flow cytometry (FACS Calibur II; Becton Dickinson, California, USA) using green excitation light (488 nm argon laser). All leucocyte function studies were performed after induction of anaesthesia and after surgery. The results of leucocyte functions were expressed as mean fluorescence intensity (MFI).
Data are expressed as the means ± SD. Kruskal–Wallis analysis was used to analyse differences in age, weight, duration of anaesthesia and surgery. Sex was analysed by chi-squared test. The distribution of MFI data was analysed by the Shapiro–Wilk test. The MFI data for respiratory burst activity were compared using the paired t-test and MFI data for phagocytic activity were compared using the Wilcoxon Signed Rank Test. When we compared the differences between the three groups, one-way ANOVA and Tukey HSD were used for respiratory burst activity and Kruskal–Wallis one-way ANOVA was used for phagocytic activity. The SPSS for Windows statistical package was used. A P value less than 0.05 was considered as statistically significant.
The characteristics of the patients and duration of anaesthesia/surgery were similar in all groups. The characteristics of the patients and duration of anaesthesia are summarized in Table 1. There were no significant differences among the groups in the HR, SBP, DBP, SpO2 and ETCO2 values.
Perioperative changes in respiratory burst function of PMNs are shown in Table 2. The MFI showed an increase after surgery in group P (P < 0.05), whereas the changes in MFI in groups D and S were not statistically significant. When we compared the differences between the three groups, we found the difference in MFI to be statistically significant between group P and group S (Fig. 2).
Changes in phagocytic activity of PMNs in BAL fluid after induction of anaesthesia and after surgery, expressed as MFI, are shown in Table 3. There were no significant differences in phagocytic activity of PMNs within and between the groups.
The main results of this study are that the increase in MFI of PMNs showing respiratory burst activity was found to be statistically significant in the propofol group, the difference in MFI was found to be statistically significant between groups P and S, and desflurane, sevoflurane and propofol had no significant effects on the phagocytic activity of PMNs in BAL fluid. These results showed that propofol had increased the respiratory burst activity of PMNs in BAL fluid.
In previous in-vitro and in-vivo studies, the immunomodulatory effects of anaesthetics, especially on PMN function, have been extensively described [1,5–8]. Several investigators have found an in-vitro decrease in PMN free radical production [9,10] and a decrease in phagocytosis in vitro[11,12] and in vivo[13–15] in peripheral blood. Other studies have not confirmed these changes in PMN functional variables [16–19]. As a result of such contradictory data, useful conclusions have been difficult to obtain . As the changes in peripheral blood may be affected by multiple factors, how these changes in the peripheral blood are reflected in the tissue is unpredictable. In the present study, we aimed to evaluate the effects of desflurane, sevoflurane and propofol in BAL fluid, which is a tissue-derived sample. Experiments conducted on tissue fluids show the direct effects of the agents and may be considered more valuable than peripheral blood samples. To our knowledge, this is the first study comparing the effects of propofol, sevoflurane and desflurane on the PMN functions in BAL.
Volatile anaesthetics had been shown to produce variable immunomodulatory effects. The studies revealed the agent-dependent and time-dependent effects on neutrophil function [2,20]. Fröhlich et al.  reported that halothane, enflurane and sevoflurane decreased and desflurane increased the neutrophil response significantly in the peripheral blood. Cullen  could not detect a significant depression in phagocytic activity following in-vitro equilibration of leucocytes with halothane and nitrous oxide. Isoflurane anaesthesia had only a minimal effect on the bacterial phagocytosis of E. coli. An ex-vivo study revealed that anaesthesia with isoflurane time dependently decreased phagocytosis . In the present study, the respiratory burst activity and phagocytic activity were found to be unchanged by desflurane and sevoflurane anaesthesia in BAL. These different effects suggested that volatile anaesthetic agents may act differently on PMN functions in BAL fluid and peripheral blood.
The accumulation of activated neutrophils in the lung is an early signal in the pulmonary inflammatory process. It was reported that propofol anaesthesia resulted in an increase in the alveolar granulocyte fraction . Propofol may influence neutrophil activation by inhibition of the respiratory burst in the peripheral blood [5,7,22–24]. Propofol significantly decreased PMN chemotaxis, whereas phagocytosis (E. coli) was found to be unaffected [5,25]. In contrast, an ex-vivo study revealed that anaesthesia with propofol time-dependently decreased phagocytosis . Propofol has been shown to inhibit a variety of functions of neutrophils in vitro, but there is a lack of in-vivo data. For instance, propofol concentrations that yielded a marked suppression in vitro did not alter the neutrophil oxidative response during cataract surgery in vivo. The discrepancies between in-vivo and in-vitro effects in general may be due to the great pool of extravascular PMNs in the organism . In this study, we found an increase in MFI of PMNs with respiratory burst activity in the propofol group. This result suggested that propofol might have different effects in different body tissues such as peripheral blood and BAL. We considered that these activated PMNs might have circulated from peripheral blood to the alveoli by propofol anaesthesia.
In conclusion, we have demonstrated that propofol increased the respiratory burst function of PMNs in BAL fluid; however, it still remains unclear how far these results are applicable to the clinical setting. Further studies with larger numbers of patients may offer further clarification of the influence of the anaesthetic agents on PMN functions.
The author would like to thank Professor Said Bodur for his excellent help and support in statistics. This study was supported by the Selcuk University Research Foundation.
1 Davidson JA, Boom SJ, Pearsall FJ, et al
. Comparison of the effects of four i.v. anaesthetic agents on polymorphonuclear leucocyte function. Br J Anaesth 1995; 74:315–318.
2 Schilling T, Kozian A, Kretzschmar M, et al
. Effects of propofol
anaesthesia on the alveolar inflammatory response to one-lung ventilation. Br J Anaesth 2007; 99:368–375.
3 Schneemilch CE, Hachenberg T, Ansorge S, et al
. Effects of different anaesthetic agents on immune cell function in vitro. Eur J Anaesthesiol 2005; 22:616–623.
4 Zhao J, Juettner B, Scheinichen D, et al
. Respiratory burst activity
of polymorphonuclear cells is dependent on the cell preparation technique. Acta Anaesthesiol Scand 2003; 47:702–706.
5 Heine J, Jaeger K, Osthaus A, et al
. Anaesthesia with propofol
decreases FMLP-induced neutrophil respiratory burst but not phagocytosis
compared with isoflurane. Br J Anaesth 2000; 85:424–430.
6 Fröhlich D, Rothe G, Schwall B, et al
. Thiopentone and propofol
, but not methohexitone nor midazolam, inhibit neutrophil oxidative responses to the bacterial peptide FMLP. Eur J Anaesthesiol 1996; 13:582–588.
7 Heine J, Leuwer M, Scheinichen D, et al
. Flow cytometry
evaluation of the in vitro influence of four i.v. anaesthetics on respiratory burst of neutrophils. Br J Anaesth 1996; 77:387–392.
8 Murphy PG, Ogilvy AJ, Whiteley SM. The effect of propofol
on the neutrophil respiratory burst. Eur J Anaesthesiol 1996; 13:471–473.
9 Nakagawara M, Takeshige K, Takamatsu J, et al
. Inhibition of superoxide production and Ca2+ mobilization in human neutrophils by halothane, enflurane and isoflurane. Anesthesiology 1986; 64:4–12.
10 White IWC, Gelb AW, Wexler HR, et al
. The effects of intra-venous anaesthetic agents on human neutrophil chemiluminescence. Can Anaesth Soc J 1983; 30:506–511.
11 Gelb AW, Lok P. Etomidate reversibly depresses human neutrophil chemiluminescence. Anesthesiology 1987; 66:60–63.
12 Moudgil GC. Effects of premedicants, intravenous anaesthetic agents and local anaesthetics on phagocytosis
in vitro. Can Anaesth Soc J 1981; 28:597–602.
13 Bardosi L, Tekeres M. Impaired metabolic activity of phagocytic cells after anaesthesia and surgery. Br J Anaesth 1985; 57:520–523.
14 Busoni P, Sarti A, De Martino M, et al
. The effect of general and regional anesthesia on oxygen dependent microbicidal mechanisms of polymorphonuclear leukocytes in children. Anesth Analg 1988; 67:453–456.
15 Barth J, Petermann W, Entzian P, et al
. Modulation of oxygen free radicals from human leukocytes during halothane and enflurane induced general anaesthesia. Acta Anaesthesiol Scand 1987; 31:740–743.
16 Cullen BF. The effect of halothane and nitrous oxide on phagocytosis
and human leukocyte metabolism. Anesth Analg 1974; 53:531–536.
17 Nunn JF, Sturrock JE, Jones AJ, et al
. Halothane does not inhibit human neutrophil function in vitro
. Br J Anaesth 1979; 51:1101–1108.
18 Heberer M, Zbinden AM, Ernst M, et al
. The effect of surgery and anaesthetic agents on granulocyte chemiluminescence in whole blood. Experienta 1985; 41:342–346.
19 Perttila J, Salo M, Rajamaki A. Granulocyte microbicidal function in patients undergoing major abdominal surgery under balanced anaesthesia. Acta Anaesthesiol Scand 1987; 31:100–103.
20 Fröhlich D, Rothe G, Schwall B, et al
. Effects of volatile anaesthetics on human neutrophil oxidative response to the bacterial peptide FMLP. Br J Anaesth 1997; 78:718–723.
21 Kotani N, Hashimoto H, Sessler DI, et al
. Intraoperative modulation of alveolar macrophage function during isoflurane and propofol
anesthesia. Anesthesiology 1998; 89:1125–1132.
22 O'Donnell NG, McSharry CP, Wilkinson PC, Asbury AJ. Comparison of the inhibitory effect of propofol
, thiopentone and midazolam on neutrophil polarization in vitro in the presence or absence of human serum albumin. Br J Anaesth 1992; 69:70–74.
23 Mikawa K, Akamatsu H, Nishina K, et al
inhibits human neutrophil functions. Anesth Analg 1998; 87:695–700.
24 Weiss M, Birkhahn A, Krone M, Schneider EM. Do etomidate and propofol
influence oxygen radical production of neutrophils? Immunopharmacol Immunotoxicol 1996; 18:291–307.
25 Skoutelis A, Lianou P, Papageorgiou E, et al
. Effects of propofol
and thiopentone on polymorphonuclear leukocyte functions in vitro. Acta Anaesthesiol Scand 1994; 38:858–862.
26 Fröhlich D, Trabold B, Rothe G, et al
. Inhibition of the neutrophil oxidative response by propofol
: preserved in vivo function despite in vitro inhibition. Eur J Anaesthesiol 2006; 23:948–953.
27 Heine J, Jaeger K, Weingaertner N, et al
. Effects of different preparations of propofol
, diazepam, and etomidate on human neutrophils in vitro. Acta Anaesthesiol Scand 2001; 45:213–220.