Department of Anesthesiology and Critical Care Medicine, Kyushu University, Fukuoka, Japan*Corresponding author: 3-1-1 Maidashi, Higashi-ku, Fukuoka 8128582, Japan
Accepted for publication: December 4, 2000
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Abstract |
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Br J Anaesth 2001; 86: 8538
Keywords: theories of anaesthetic action, signal transduction; blood, neutrophils; toxicity, neutrophil chemotaxis; anaesthetics i.v., propofol
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Introduction |
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Propofol has been widely used in the induction and maintenance of anaesthesia and the sedation of patients in intensive care units. Accordingly, it is important to determine the effects of this agent on the immune system. It has been reported in an in vitro study that propofol inhibits such neutrophil functions as polarization,4 chemotaxis,57 phagocytosis,7 8 respiratory burst9 10 and bactericidal activity6 at clinically achievable concentrations. However, the degree of these inhibitory effects varies considerably. Furthermore, some studies have failed to observe an inhibitory effect of propofol on neutrophil function.6 7 11 Therefore, the effects of propofol on neutrophils remain unclear. Moreover, there have been few mechanistic studies.7
Mitogen-activated protein kinases (MAPKs) are members of the serine/threonine protein kinase family, and mediate signal transduction from the cell surface to the nucleus. They are highly conserved in many types of eukaryotes from yeast to mammalian cells, including human neutrophils, suggesting that the signalling cascade involving MAPKs may be very important in signal transduction in eukaryotes.12 13 Recently, three mammalian MAPKs have been identified,14 including extracellular signal-regulated kinase (ERKs or p44/42 MAPK), c-Jun N-terminal kinase (JNK) and p38 MAPK. MEK (MAPK/ERK kinase) is the specific kinase of p44/42 MAPK and the phosphorylated form of p44/42 MAPK is active.15 MEK is directly activated by Raf-1 kinase and Raf-1 kinase is modulated by Ras. In human neutrophils, p44/42 MAPK has been reported to be activated rapidly in response to various stimuli, including chemotactic factors,16 while p44/42 MAPK has been reported to participate in various neutrophil functions, such as chemotaxis,17 18 adhesion, phagocytosis, granule secretion and respiratory burst. N-Formyl-L-methionyl-phenylalanine (FMLP), a chemoattractant, activates the RasRafMEKp44/42 MAPK pathway19 through the FMLP receptor activating the trimeric GTP-binding protein (Fig. 1). Downstream activity of p44/42 MAPK in the intracellular signal transduction pathway has not been elucidated.
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Materials and methods |
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Isolation of neutrophils
Neutrophils were isolated from healthy human volunteers. The majority of erythrocytes in heparinized whole blood were removed by dextran sedimentation at room temperature. The sample was then cooled to 4°C to prevent neutrophil activation. The remaining erythrocytes were eliminated by hypotonic lysis. Monocytes and lymphocytes were removed by ConrayFicoll centrifugation.20 The isolated neutrophils were washed twice with 0.9% sodium chloride and suspended in HBS or MEM. Cell viability after incubation with drugs was estimated using a trypan blue dye exclusion test.
Phosphorylation of MAPKs
Neutrophils, suspended in HBS at 2x107 cells per ml, were incubated for 30 s with propofol or DMSO. The final concentration of DMSO in all sample was 3%. Propofol was prepared at final concentrations of 2500 µM. After pretreatment with propofol, cells were stimulated with cytochalasin B (5 µg ml1) for 5 min followed by FMLP (100 nM) for 1 min at 37°C. The reaction was terminated by adding cold 0.9% NaCl. The cells were immediately lysed in RIPA buffer at 4°C and then centrifuged (10 min, 4°C, 13 000 g), and the cell lysates were analysed by immunoblotting. All samples were diluted with Laemmli buffer and boiled for 5 min before separation by SDS/10% polyacrylamide gel electrophoresis. Proteins within the gel were transferred to nitrocellulose membranes and immunoblotted with phospho-p44/42 MAP kinase antibody or anti-ERK1 antibody. Phospho-p44/42 MAP kinase antibody produced by immunizing rabbits with synthetic peptide corresponding to human p44 MAPK detects phosphorylation of both p44 and p42 MAPK, and anti-ERK1 antibody detects both p44 and p42 MAPK. Antibody binding was detected by ECL. Visualized bands on films were captured and stored as images on a computer using a scanner (Opal Ultra; Druckmaschina, Heidelberg, Germany), and analysed with NIH Image (version 1.61), a public domain image analysis program from the National Institutes of Health (Bethesda, MD, USA), as the densitometric analyser.
Chemotaxis
Neutrophils suspended in MEM at 1x107 cells per ml were incubated at 37°C for 30 min with propofol, 50 µM PD98059 (specific inhibitor of MEK) or 0.1% DMSO. Propofol concentrations ranged from 10 to 200 µM. To prepare the agarose plates for the under-agarose method,21 5 ml of agarose medium was added to each 60x15 mm dish. Four series of three wells, 3 mm in diameter and spaced 6 mm apart, were cut in each plate (Fig. 2A). Each well received 10 µl of solution. Neutrophils (1x105 cells) incubated with the reagents mentioned above were added to the centre well of each three-well series. The outer and inner wells received MEM with or without 1 µM FMLP (the final concentration of DMSO was 0.1%). Plates were then incubated at 37°C in 5% carbon dioxide for 3 h. After incubation, the neutrophils were fixed by addition of 5 ml absolute methanol overnight and the gels were then hardened by addition of 3 ml 47% formalin for 30 min. After fixation, the gels were gently removed and the neutrophils that were fixed on the dishes were stained with Giemsa stain. Micrographs (x40) of the cell migration patterns were taken, scanned and stored on a computer. The number of neutrophils in a square area (1x1 mm) 2 mm from the edge of the centre well, towards the outer well, was counted using the particle-counter function of NIH Image (Fig. 2B).
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Results |
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FMLP-stimulated phosphorylation of p42 MAPK was inhibited by propofol at concentrations above 10 µM (n=6), whereas the quantity of p42 MAPK was not affected. The 50% inhibitory concentration (IC50) for the inhibition of p42 MAPK phosphorylation by propofol was 20 (12) µM. The inhibitory effect of propofol on p42 MAPK phosphorylation seemed to be concentration-dependent, and was observed at clinically achievable concentrations (Fig. 4A and B).
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Effects of propofol on FMLP-induced chemotaxis
As shown in Fig. 5, propofol inhibited FMLP-stimulated chemotaxis of neutrophils at concentrations above 20 µM (n=12) in a concentration-dependent manner. The IC50 for the inhibition of neutrophil chemotaxis by propofol was 88.0 (2.5) µM. PD98059 (50 µM) also inhibited the chemotaxis of neutrophils, indicating that the p44/42 MAPK pathway is involved in regulating the chemotaxis of neutrophils. These concentrations of propofol were clinically achievable, and were identical with the concentrations required to inhibit the phosphorylation of p42 MAPK induced by FMLP.
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Discussion |
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We have also shown that propofol at clinically achievable concentrations (10 µM) inhibited the phosphorylation of p42 MAPK. Although some investigators have reported the relationship of neutrophil activity with the p44/42 MAPK pathway, this subject remains controversial. Kuroki and OFlaherty17 reported that PD98059, a specific inhibitor of MEK, inhibited FMLP-induced chemotaxis of neutrophils. Hii et al.18 observed partial inhibition of chemotaxis and marked inhibition of chemokinesis by PD98059. These inhibitory effects of PD98059 on neutrophil chemotaxis suggest the involvement of the p44/42 MAPK pathway. However, in their studies, after almost completely inhibiting phosphorylation and the activity of p44/42 MAPK, PD98059 (50 µM) only partially inhibited chemotaxis. In our studies, the inhibitory effect of PD98059 (50 µM) on chemotaxis was also partial. These results suggest multiple pathways in FMLP-induced chemotaxis. On the other hand, Zu et al.25 reported that PD98059 did not inhibit FMLP-induced chemotaxis. These authors suggested the involvement of p38 MAPK in FMLP-induced chemotaxis of neutrophils, because the specific inhibitors of p38 MAPK (SB20358) inhibited chemotaxis.25 Furthermore, Coffer et al.26 reported the possibility that p38 MAPK participates in neutrophil chemotaxis. They also reported that PD98059 did not inhibit FMLP-induced chemotaxis. This discrepancy may be explained partly by methodological differences. Kuroki and OFlaherty17 and Hii et al.18 used the same agarose method as in the present study, whereas Zu et al.25 and Coffer et al.26 used the filter method. The agarose method may be more sensitive than a filter method in evaluating cell adhesion, as neutrophils should adhere horizontally to the plate for migration.27 MAPK is also known as microtubule-associated protein kinase. An association of p44/42 MAPK with cytoskeletal alteration has also been observed, for example, in NIH 3T3 mouse fibroblasts.28 In addition, propofol was found to inhibit the polarization of neutrophils,4 which is an expression of cytoskeletal alteration and is essential for chemotaxis.
Although p44/42 MAPK has been reported to activate transcription factors in neutrophils,29 it is not known how these transcription factors are involved in neutrophil activity. Because some neutrophil responses to stimuli such as chemotaxis are induced immediately, the signal transmitted by activated p44/42 MAPK does not necessarily induce gene expression.
In conclusion, our observations indicate the possibility that some of the inhibitory effects of propofol on neutrophil activity may be mediated by inhibition of the p44/42 MAPK pathway. The clinical significance of propofol-induced inhibition of neutrophil activity, however, is not clear. Whether such an inhibition is beneficial or detrimental should also be evaluated in a clinical setting. For example, tourniquet-induced ischaemia/reperfusion injury has been shown to be lessened by the administration of propofol in orthopaedic patients.30
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