British Journal of Anaesthesia

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British Journal of Anaesthesia, 2001, Vol. 86, No. 6 853-858
© 2001 The Board of Management and Trustees of the British Journal of Anaesthesia

Propofol inhibits FMLP-stimulated phosphorylation of p42 mitogen-activated protein kinase and chemotaxis in human neutrophils

T. Nagata, M. Kansha, K. Irita and S. Takahashi

Department of Anesthesiology and Critical Care Medicine, Kyushu University, Fukuoka, Japan*Corresponding author: 3-1-1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan

Accepted for publication: December 4, 2000

	Abstract

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Propofol is used in the peri-operative setting and may affect some neutrophil functions. The effects of propofol on the function and intracellular signal transduction systems of neutrophils is controversial. Mitogen-activated protein kinase families (MAPKs) are members of the intracellular signal-transducing systems in eukaryotes. MAPKs have been shown to be involved in neutrophil chemotaxis by the use of PD98059, the specific inhibitor of MAPK/ERK kinase (MEK). The effects of propofol in dimethyl sulfoxide on phosphorylation of MAPKs and chemotaxis were investigated in human neutrophils. Isolated neutrophils (2x10⁷ cells per ml) from healthy volunteers were incubated with propofol (2–500 µM) and stimulated by N-formyl-L-methionyl-phenylalanine (FMLP) (100 nM). The effects of propofol on the phosphorylation of p44/42 MAPK were investigated by immunoblotting. The effects of FMLP (1 µM) on chemotaxis were investigated with the under-agarose method. The phosphorylation of p42 MAPK and chemotaxis stimulated by FMLP were both inhibited by propofol at clinically relevant concentrations (10 and 20 µM respectively). PD98059 (50 µM) also inhibited chemotaxis stimulated by FMLP, suggesting the involvement of p42 MAPK in the response. Propofol might therefore inhibit human neutrophil chemotaxis, at least in part, by suppressing the p44/42 MAPK pathway.

Br J Anaesth 2001; 86: 853–8

Keywords: theories of anaesthetic action, signal transduction; blood, neutrophils; toxicity, neutrophil chemotaxis; anaesthetics i.v., propofol

	Introduction

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Neutrophils play an important role in the peri-operative period. In response to inflammatory stimuli, neutrophils exit the microvasculature, move along chemoattractant gradients and ingest and kill invading pathogens. Such microbicidal activity has been established to be an essential component of the host defence system.¹ Furthermore, there is increasing evidence that neutrophils play an important role in the pathogenesis of ischaemia/reperfusion injury as well as in systemic inflammatory response syndrome.² Therefore, controlling neutrophil function during the peri-operative period appears to be an important component of anaesthesia.³

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,^5–7 phagocytosis,^7 8 respiratory burst^9 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 Ras–Raf–MEK–p44/42 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.

View larger version (20K): [in this window] [in a new window]   Fig 1 Possible signal transduction pathway via MAPK in neutrophils. The binding of FMLP to its receptor on the plasma membrane activates the Ras–Raf–MEK–p44/42 MAPK pathway through heterotrimeric GTP binding proteins. MEK is a specific activator of p44/42 MAPK. PD98059 selectively inhibits MEK. This signalling cascade is considered to participate in the activation of various functions of neutrophils. The participation of other MAPK families, such as p38, has been reported recently. In various cells, the activated p44/42 MAPK activates transcription factors by protein phosphorylation, and thus leads to gene expression.

 
In this study, we used human neutrophils to examine the effects of propofol on the phosphorylation of p44/42 MAPK and chemotaxis in response to the chemotactic peptide FMLP.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Materials
FMLP, cytochalasin B, chymostatin, ethylenediamine tetraacetate (EDTA), leupeptin, Nonidet P-40 (Np-40), 2-mercaptoethanol, catalase and agarose (type II) were obtained from Sigma (St Louis, MO, USA). Bromochloroidolyl phosphate (BPB), (p-amidinophenyl) methanesulphonyl fluoride (APMSF) and HEPES were from Wako Pure Chemical Industries (Osaka, Japan). Pepstatin and sodium orthovanadate (Na₃VO₄) were from Nacalai Tesque (Kyoto, Japan). Propofol was from RBI (Natick, MA, USA). Superoxide dismutase (SOD) was from Toyobo (Osaka, Japan). Phospho-p44/42 MAP kinase antibody and PD98059 were from New England Biolabs (Beverly, MA, USA). Anti-ERK1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL^®) Western blotting detection reagents and antibody were obtained from Amersham International (Amersham, UK). Ficoll was from Pharmacia Biotech (Uppsala, Sweden). Conray (sodium iothalamate 60.8% w/v) was from Daiichi Pharmaceutical (Tokyo, Japan). HEPES-buffered saline (HBS), used for phosphorylation of p44/42 MAPK, contained 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.6 mM CaCl₂, 2 mM glucose and 20 mM HEPES (pH 7.4 at 37°C). Radioimmunoprecipitation (RIPA) buffer contained 1 mM EDTA, 150 mM NaCl, 10 mM Tris, 1% Np-40, 0.1% sodium deoxycholate and 0.1% sodium dodecyl sulphate (SDS) (pH 7.4 at 25°C). Just before cell lysis, RIPA buffer was added containing 10 mM sodium fluoride, 25 µM APMSF, 40 µM leupeptin, 50 µM chymostatin, 1.5 mM pepstatin and 1 mM Na₃VO₄ as protease inhibitors. Laemmli buffer contained 62.5 mM Tris, 2% SDS, 10% glycerol, 0.002% BPB, and 5% 2-mercaptoethanol (pH 6.8, 25°C). Medium 199 (with Earle’s salts and L-glutamine without sodium bicarbonate) and fetal bovine serum (FBS) were from Life Technologies (Grand Island, NY, USA). Medium 199 was used in the chemotaxis assay as the Minimum Essential Medium (MEM). MEM was buffered with NaHCO₃ to pH 7.4 at 37°C and was then sterilized by membrane filtration. Just before use in agarose plates, MEM was added containing 10% FBS and 0.5% agarose. FMLP, cytochalasin B, chymostatin, pepstatin, PD98059 and propofol were dissolved in dimethyl sulfoxide (DMSO).

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 Conray–Ficoll 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 2–500 µM. After pretreatment with propofol, cells were stimulated with cytochalasin B (5 µg ml^–1) 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).

View larger version (33K): [in this window] [in a new window]   Fig 2 (A) Schematic representation of the under-agarose method. Four series of three wells, measuring 3 mm in diameter and spaced 6 mm apart, were cut into 60x15 mm agarose plates. The centre well received 10 µl of the prepared cell suspension. The outer wells received 10 µl FMLP dissolved in MEM and the inner wells received 10 µl MEM. (B) Chemotaxis pattern of neutrophils. The number of neutrophils was counted in 1x1 mm square areas, which were 2 mm from the edges of the neutrophil wells.

 
Statistical analysis
All data were analysed by the Mann–Whitney test and are presented as mean (SEM). Differences were considered to be significant when P<0.01.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Effects of propofol on FMLP-induced phosphorylation of p44/42 MAPK
Initially, we examined the effect of FMLP with cytochalasin B, which primes neutrophils, thus enhancing the response to chemoattractants, on the phosphorylation of p44/42 MAPK. As shown in Fig. 3, only p42 MAPK was phosphorylated. In an analysis of the molecular weight, upper thin bands were observed as part of the phosphorylated p42 MAPK. As there were more phosphorylated residues than in the lower bands of phosphorylated p42 MAPK, the molecular weight of MAPK in these thin bands was thought to be a little higher. As shown in the lower blot of Fig.4A, the bands of p42 MAPK were thicker than the bands of p44 MAPK, indicating that the amount of p42 MAPK was greater than that of p44 MAPK. p42 MAPK has also been reported to be predominant in human neutrophils.²²

View larger version (25K): [in this window] [in a new window]   Fig 3 Time course of phosphorylation of p44/42 MAPK of human neutrophils. The cells were stimulated with 5 µg ml–1 cytochalasin B and 100 nM FMLP, as described in Materials and methods. The arrow indicates the bands of phosphorylated p42 MAPK. The band of phosphorylated p44 MAPK is not shown in this blot. Phosphorylation of p42 MAPK reached a maximum 1 min after FMLP stimulation.

 
Phosphorylation of p42 MAPK in human neutrophils activated by 100 nM FMLP reached a maximum at 1 min and disappeared within 5 min. Accordingly, the FMLP stimulation time for the phosphorylation of MAPKs was set at 1 min in the subsequent experiments (Fig. 3).

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).

View larger version (53K): [in this window] [in a new window]   Fig 4 (A) Effect of propofol on p42 MAPK phosphorylation. A typical blot is shown. Cells were stimulated with 5 µg ml–1 cytochalasin B and 100 nM FMLP, as described in Materials and methods. The protein blots were probed with phospho-p44/42 MAP kinase antibody or anti-ERK1 antibody on the same samples. The arrows indicate the bands of phosphorylated p42 MAPK and p44/42 MAPK. nc=negative control sample that was stimulated only with cytochalasin B. (B) Densitometric analysis of p42 MAPK phosphorylation. Phosphorylation is expressed relative to the phosphorylation produced by 5 µg ml–1 cytochalasin B and 100 nM FMLP without pretreatment (positive control). Means (SEM) *P<0.001 vs positive control; P<0.01 was considered to indicate significance. Negative control, FMLP 0 µM.

 
Phosphorylation of p42 MAPK induced by FMLP was also observed in the presence of 100 µg ml^–1 SOD or 40 µg ml^–1 catalase, indicating that phosphorylation is not secondary to the production of superoxide and hydrogen peroxide (results not shown).

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.

View larger version (16K): [in this window] [in a new window]   Fig 5 Effect of propofol and PD98059 on neutrophil chemotaxis. The under-agarose method was used to study chemotaxis as described in Materials and methods. Chemotaxis is expressed as the percentage of chemotaxis caused by 1 µM FMLP without pretreatment (positive control). Mean (SEM); *P<0.0001 vs positive control; P<0.01 was considered to indicate significance.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
There have been various reports of the inhibitory effects of propofol on neutrophil activity. Various concentrations of propofol, ranging from 11 to 280 µM, were reported to be effective in these studies. In most of these reports, propofol inhibited neutrophil activity at a clinically achievable concentration, which has been reported to be approximately 30 µM (5 µg ml^–1) for surgical anaesthesia and 15 µM (3 µg ml^–1) for sedation. It is important to rule out the effect of the solvent of propofol (10% Intralipid), as Heine and colleagues²³ reported that the inhibitory effect on respiratory burst was caused by the solvent. Intralipid affects neutrophil activity in a concentration-dependent manner.²⁴ In our study, we used propofol dissolved in DMSO to examine the effect of pure propofol on neutrophil activity. Although the IC₅₀ value required for propofol to inhibit the chemotaxis [88.0 (2.5) µM] exceeded those seen clinically, we did demonstrate that propofol significantly inhibited chemotaxis at clinically achievable concentrations (20 µM). Direct inhibitory effects of propofol on respiratory burst,⁹ polarization,⁴ phagocytosis⁸ and chemotaxis⁶ have also been reported.

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 O’Flaherty¹⁷ 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 O’Flaherty¹⁷ 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

	References

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