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Effects of ropivacaine on adhesion molecule CD11b expression and function in human neutrophils
International Immunopharmacology 10 (2010) 662–667
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Effects of ropivacaine on adhesion molecule CD11b expression and function in human neutrophils Xuqin Zhu, Zhiming Tan ⁎, Jiawei Chen, Minmin Zhu, Yajun Xu Department of Anesthesiology, Fudan University Cancer Hospital, No. 270 Dong-an Road, Shanghai, PR China, PC 200032
a r t i c l e
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Article history: Received 25 November 2009 Received in revised form 9 February 2010 Accepted 12 March 2010 Keywords: Local anesthetic Ropivacaine Neutrophil Adhesion molecule
a b s t r a c t Local anesthetics possess a wide range of anti-inflammatory properties through their effects on neutrophils. However, limited information is available on the effects of ropivacaine (a new local anesthetic) on neutrophil function. The aim of this study was to investigate anti-inflammatory properties of ropivacaine with regard to its effects on the expression and function of CD11b in human neutrophils. CD11b expression was examined by flow cytometry and its function was determined by measuring adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs). Ropivacaine inhibited CD11b expression in formyl-methionylleucyl-phenylalanine (fMLP)-activated neutrophils in a concentration-dependent manner, but not in a timedependent manner. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. The up-regulation of CD11b induced by platelet-activating factor (PAF) priming was also inhibited by ropivacaine. fMLP increased adhesion of neutrophils to HUVECs, which was inhibited by ropivacaine. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N´,N´tetraacetic acid (EGTA), a chelator of extracellular Ca2+. However, ropivacaine showed no effect on the fMLPinduced CD11b expression in the presence of butan-1-ol, a blocker of phospholipase D (PLD) pathway, which completely inhibited the fMLP-induced CD11b expression in neutrophils. Our results suggest that ropivacaine exerts anti-inflammatory activity, and this appears to be mediated through inhibiting the expression and function of adhesion molecule CD11b in neutrophils. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Human neutrophils have been implicated in the pathogenesis of a variety of inflammation-related diseases, including adult respiratory distress syndrome (ARDS) and myocardial reperfusion injury [1,2]. In addition, the important role of neutrophils in mechanisms of processing pain and hyperalgesia has also been demonstrated in several inflammatory conditions, including nerve injury, rheumatoid arthritis and allergen-evoked inflammation [3–5]. Besides direct neuronal blocking effects, local anesthetics have significant anti-inflammatory properties [6]. Previous studies have revealed inhibitory effects of local anesthetics on migration [7], chemotaxis [8], oxidative burst and phagocytosis of neutrophils [9]. Furthermore, lidocaine has been introduced in the treatment of burn injuries, ulcerative proctitis and arthritis [6]. The migration of neutrophils into tissues is a crucial event in the inflammatory response, which is dependent on the coordinated regulation of the expression and function of at least three families of
⁎ Corresponding author. Tel.: +86 21 64175590; fax: +81 21 64174774. E-mail address: [email protected] (Z. Tan). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.03.009
cell surface adhesion molecules [10]. CD11b/CD18, a member of the integrin family, appears to be important for the firm adhesion of neutrophils to the endothelium [11]. Moreover, respiratory burst activity is altered following integrin-mediated adhesion, suggesting that the adhesive state of neutrophils is an important determinant of oxygen radical and granule release and thus, potentially, of tissue damage [12]. Ropivacaine is a new long-acting amide local anesthetic with a structure similar to that of bupivacaine. However, ropivacaine is the first local anesthetic as an almost pure S-enantiomer (N99% pure), whereas bupivacaine is a racemic mixture. Although bupivacaine is still widely used for regional anesthetic techniques, the clinical application of ropivacaine is on the increase because it is less likely to induce central nervous system effects or cardiac toxicity compared with bupivacaine [13]. However, previous studies about the effects of ropivacaine on human neutrophils are mainly focused on the respiratory burst activity and some of these findings are inconsistent [8,9,14,15]. No data have been published on whether ropivacaine could modulate the expression and functional activity of adhesion molecule CD11b in formyl-methionyl-leucyl-phenylalanine (fMLP)activated or platelet-activating factor (PAF)-primed neutrophils. Therefore, we performed this study to examine this issue.
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2. Materials and methods 2.1. Materials fMLP, PAF (β-Acetyl-γ-O-alkyl-L-α-phosphatidylcholine), ethylene glycol-bis(2-aminoethylether)-N,N,N´,N´-tetraacetic acid (EGTA), lidocaine hydrochloride monohydrate and bupivacaine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ropivacaine hydrochloride monohydrate and levobupivacaine hydrochloride were gifts from AstraZeneca UK Ltd. Ficoll-Paque was purchased from Biowest (France). Fluorescein isothiocyanate (FITC)-conjugated antibodies against CD11b (Clone ICRF 44) and FITC-conjugated IgG1, κ isotype control were purchased from eBioscience (USA). Redfree FCM lysing solution was purchased from MultiSciences Biotech Co. Ltd (P. R. China). All the other reagents were of the highest quality available. 2.2. Blood samples
Fig. 1. Dot plot of whole blood subjected to flow cytometric analysis for CD11b expression on neutrophils. Neutrophils, monocytes and lymphocytes were defined distinctly. Region A was neutrophils.
After obtaining institutional and ethical committee approval (Fudan University Cancer Hospital, Shanghai, China) and informed consent, human venous blood was obtained from 16 healthy volunteers and immediately anticoagulated with lithium heparinate. All the healthy volunteers had not taken any medication for at least 2 weeks.
micin, Amphotericin-B) and 10% fetal bovine serum (FBS). Cells were grown in a humidified incubator containing 95% air and 5% CO2 at 37 °C with media replenishment every 2 days. On reaching 90% confluence, cells were detached by 0.1% trypsin and subcultured. The fifth passage of HUVECs was used in this study.
2.3. Expression of adhesion molecule CD11b
2.5. Isolation of human neutrophils
Whole blood (100 µl) was preincubated at 37 °C in the absence or presence of local anesthetics for the indicated duration. Thereafter, samples were activated with fMLP (1 µM) for 15 min or primed with PAF (1 µM) for 5 min at 37 °C, because in our preliminary experiments activation effect by fMLP (1 µM) has been shown to be maximal after 15 min and priming effect by PAF (1 µM) has been shown to be maximal after 5 min. To test the effects of ropivacaine on overactive state of neutrophils, samples were primed with PAF (1 µM) for 5 min before activation with fMLP (1 µM) for 15 min. In order to determine the effects of ropivacaine on CD11b expression in neutrophils which have been activated in advance, whole blood was initially activated with fMLP (1 µM) for 15 min prior to the incubation of ropivacaine. In addition, to further investigate the mechanisms of the effects of ropivacaine on CD11b expression, samples were incubated with 0.3% butan-1-ol or EGTA (5 mM) for 3 min prior to treatment with ±fMLP following ropivacaine treatment. The reactions were stopped by the addition of cold phosphate buffered saline (PBS) and samples were kept on ice for 5 min. Then each sample was incubated for 30 min on ice with FITC-conjugated monoclonal antibodies against CD11b, and the erythrocytes were lysed using Redfree FCM lysing solution. Samples were then centrifuged, washed, and resuspended in PBS containing 1% paraformaldehyde and stored at 4 °C in the dark until flow cytometric analysis. Flow cytometric analysis was performed using flow cytometer (Coulter, Epics ALTRA) and FITC-conjugated IgG1, κ isotype control was used to define the cutoff for positive fluorescence. Neutrophils, monocytes and lymphocytes were discriminated as shown in Fig. 1. Data were acquired and processed using EXPO32 ADC Analysis. For each sample, 20,000 neutrophils were analyzed. The mean fluorescence intensity (MFI) of antibody-stained neutrophils was related to the quantity of CD11b, thus MFI was determined as a measure of CD11b expression.
Human neutrophils were prepared as described previously with minor modifications [16]. Briefly, venous blood was mixed with 4.5% dextran saline and left to sit vertically for 40 min. The supernatant was taken and placed gently on the same volume of Ficoll-Paque. By centrifugation (400 g) for 20 min, neutrophils were pelletted at the bottom of the tube. Contaminating erythrocytes were lysed with hypotonic ammonium chloride solution (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA.Na2). Following centrifugation, neutrophils were washed and resuspended to a concentration of 106 cells/ml in the culture medium. The purity of the neutrophils was assessed microscopically (N90%). The viability of cells before and after incubation with local anesthetics was N95% as determined by the trypan blue exclusion test. 2.6. Adhesion of neutrophils to HUVECs Isolated neutrophils were preincubated at 37 °C with or without ropivacaine (1 mM) for the indicated duration and then stimulated with fMLP (1 µM) for 15 min. Thereafter, neutrophils were added to the HUVECs and incubated at 37 °C for 1 h. The cells were then washed 3 times with Hanks' balanced salt solution (HBSS), and observed under a phase-contrast microscope. Adherent cells were counted in 10 different fields in 5 separate culture dishes. In addition, to exclude the effects of ropivacaine on HUVECs in the process of adhesion of neutrophils to HUVECs, HUVECs were incubated with or without ropivacaine (1 mM) for 1 h at 37 °C and neutrophils were stimulated with fMLP (1 µM) for 15 min. Then ropivacaine was washed off the HUVECs before the addition of neutrophils. After incubation at 37 °C for 1 h, the cells were washed and observed as described above. Adherent cells were then counted in 10 different fields in 3 separate culture dishes. 2.7. Statistical analysis
2.4. Human umbilical vein endothelial cells (HUVECs) culture The initial batch of HUVECs was purchased from Clonetics (Cat# C2517A), and cultured in endothelial basal medium containing vascular endothelial growth factor, fibroblast growth factor, insulinlike growth factor-1, hydrocortisone, ascorbic acid, GA-1000 (Genta-
Data are presented as mean ± SEM. Statistical significance was determined by using Student's paired t-test for comparison between two groups or by using ANOVA (analysis of variance) followed by Student–Newman–Keuls test for comparisons among three or more groups. A value of P b 0.05 was considered statistically significant.
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3. Results 3.1. Ropivacaine inhibited CD11b expression in fMLP-activated neutrophils To verify the effects of ropivacaine on CD11b expression, we incubated whole blood with ropivacaine (1 mM) for 0, 5, 15, 30 and 60 min before activation with fMLP. As shown in Fig. 2A, ropivacaine significantly inhibited CD11b expression after 5 min as compared with fMLP-activated control cells (P b 0.05), however, this effect was not time dependent. Maximal inhibition was achieved at 15 min, therefore, this incubation time was used in the following experiments. Then, we incubated whole blood with different concentrations of ropivacaine (1 µM to 1000 µM) for 15 min before activation with fMLP. Ropivacaine ≥ 100 µM significantly reduced CD11b expression as compared with fMLP-activated control cells (P b 0.05, Fig. 2B). 3.2. Ropivacaine inhibited CD11b expression in neutrophils primed with PAF To test the effects of ropivacaine on CD11b expression in different states of neutrophils, we stimulated neutrophils with different stimuli following ropivacaine treatment. As shown in Fig. 3, CD11b expression was inhibited significantly to 78 ± 1% of control (P b 0.05) compared with fMLP-activated control cells and to 70 ± 1% of control (P b 0.05) compared with PAF-primed control cells. Whereas CD11b expression was only inhibited to 87 ± 3% of control (P N 0.05) compared with PAF-primed-fMLP-activated control cells. 3.3. Ropivacaine showed a similar inhibition of CD11b expression compared with bupivacaine and levobupivacaine, but was more potent than lidocaine To determine whether the inhibitory potency of local anesthetics on CD11b expression was dependent on chemical properties, we used
Fig. 3. Inhibitory effects of ropivacaine on CD11b expression in different states of neutrophils. Whole blood was preincubated with or without ropivacaine (1 mM) for 15 min before adding different stimuli. MFI of untreated cells was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs fMLP-activated control; # P b 0.05 vs PAF-primed control; P N 0.05 vs PAF-fMLP control.
bupivacaine, lidocaine and levobupivacaine (the S-(−)-enantiomer of bupivacaine) to compare with ropivacaine. We found that CD11b expression was attenuated significantly (to 74 ± 2% of control for ropivacaine, 72 ± 2% of control for bupivacaine and 72 ± 2% of control for levobupivacaine, P b 0.05) as compared with fMLP-activated control cells. Ropivacaine, bupivacaine and levobupivacaine were similar in their inhibitory effects (P N 0.05). However, lidocaine (84 ± 3% of control, P b 0.05) was found to inhibit CD11b expression less effectively (P b 0.05) than ropivacaine (Fig. 4). 3.4. Ropivacaine showed no effect when added after the increase of CD11b expression When neutrophils were activated with fMLP before incubation with ropivacaine (1 mM) for 15 min, CD11b expression was not affected by ropivacaine (P N 0.05, Fig. 5). 3.5. Ropivacaine inhibited adhesion of neutrophils to HUVECs induced by fMLP To further characterize the modulation of CD11b function by ropivacaine, we investigated the effects of ropivacaine on adhesion of neutrophils to HUVECs. As shown in Fig. 6A, fMLP increased the adhesion of neutrophils to HUVECs (P b 0.05), which was significantly reduced when neutrophils were pretreated with ropivacaine (15 min) (P b 0.05). However, when HUVECs were pretreated with ropivacaine, ropivacaine showed no effect on HUVECs in the process of adhesion of neutrophils to HUVECs induced by fMLP (P N 0.05, Fig. 6B).
Fig. 2. Inhibitory effects of ropivacaine on CD11b expression in fMLP-activated neutrophils. (A) Time course for the effects of ropivacaine on CD11b expression. (B) Ropivacaine inhibited CD11b expression in a concentration-dependent manner. MFI of fMLP-activated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs control.
Fig. 4. Inhibitory effects of local anesthetics on CD11b expression in fMLP-activated neutrophils. Whole blood was preincubated with or without 1 mM one of the four local anesthetics for 15 min before activation with fMLP. MFI of fMLP-activated neutrophils in the absence of local anesthetics (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs control; # P b 0.05 for lidocaine vs ropivacaine. Ropi: Ropivacaine, Bupi: Bupivacaine, Levobupi: Levobupivacaine, Lido: Lidocaine.
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Fig. 5. Effects of ropivacaine on CD11b expression when added after fMLP. MFI of fMLPactivated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate.
3.6. Ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of EGTA To investigate the role of Ca2+ in the effects of ropivacaine, we used EGTA to chelate extracellular Ca2+. As shown in Fig. 7A, EGTA significantly inhibited CD11b expression to 75 ± 1% of control as compared with fMLP-activated control cells (P b 0.05). When neutrophils were pretreated with ropivacaine (1 mM) for 15 min, CD11b expression was further inhibited to 64 ± 3% of control in the presence of EGTA as compared with fMLP-activated control cells (P b 0.05). Moreover, there existed significant difference between CD11b expression with and without ropivacaine in the presence of EGTA (P b 0.05).
Fig. 7. Effects of ropivacaine on CD11b expression in fMLP-activated neutrophils in the presence of EGTA (A) or butan-1-ol (B). MFI of fMLP-activated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs fMLP-activated control; # P b 0.05 vs EGTA + fMLP; P N 0.05 vs butan-1-ol+ fMLP.
3.7. Ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol We also investigated the role of phospholipase D (PLD) in the effects of ropivacaine by using butan-1-ol as a pseudosubstrate to block PLD signaling pathway. As shown in Fig. 7B, butan-1-ol completely inhibited the fMLP-induced CD11b expression (P b 0.05), which was not affected when neutrophils were pretreated with ropivacaine (1 mM) for 15 min (P N 0.05). 4. Discussion
Fig. 6. Effects of ropivacaine on adhesion of neutrophils to HUVECs. Results were expressed as numbers of adherent neutrophils per 100 HUVECs. (A) Ropivacaine inhibited adhesion of neutrophils to HUVECs. Neutrophils were pretreated with ropivacaine before activation with fMLP, and then added to HUVECs. Data represent the means ± SEM of five different experiments. * P b 0.05 vs untreated cells (white bar); # P b 0.05 vs fMLP-activated control (grey bar). (B) Ropivacaine showed no effect on HUVECs in the process of adhesion. HUVECs were pretreated with ropivacaine, and then fMLP-activated neutrophils were added to HUVECs following that ropivacaine was washed off HUVECs. Data represent the means ± SEM of three different experiments. * P b 0.05 vs untreated cells (white bar); P N 0.05 vs fMLP-activated control (grey bar).
The present study demonstrated that ropivacaine not only inhibited CD11b expression in fMLP-activated or PAF-primed human neutrophils, but also inhibited adhesion of neutrophils to HUVECs induced by fMLP. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of EGTA. However, ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol. Neutrophil adhesion is tightly regulated during inflammatory process and it is likely that perturbation of adhesion mechanisms will influence the progression of inflammatory response [12]. fMLP is a physiological chemotactic peptide that arises during bacterial protein processing and activates neutrophils [17]. Activation of neutrophils with fMLP is accompanied by the translocation of CD11b from the intracellular storage pool, increasing cell surface expression [18]. PAF, an endogenous inflammatory mediator, mediates priming neutrophils. Previous studies have reported that exposure of neutrophils to priming agents [such as PAF, tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF)] induced rapid up-regulation of CD11b expression, but had no direct stimulatory effects upon O2− generation, suggesting that priming agents may have specific regulatory effects on adhesion process in neutrophils
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[12,15,19]. As shown in Fig. 3, CD11b expression was markedly increased by PAF-fMLP when compared with fMLP activation alone or PAF priming alone. This state of neutrophils seems to play a pivotal role in the “overstimulation” of inflammatory pathways, which then results in tissue damage rather than protect the host [20]. In the present study, ropivacaine inhibited CD11b expression induced by fMLP or PAF, indicating that ropivacaine interferes with both priming and activation process of neutrophils. To our knowledge, the effects of ropivacaine on CD11b expression in PAF-primed-fMLP-activated neutrophils have not been reported previously. In this study ropivacaine showed only a nonsignificant trend to reduce CD11b expression in PAF-fMLP neutrophils. Ropivacaine, which is structurally similar to levobupivacaine and bupivacaine but similar in lipophilicity to lidocaine, showed no significant difference in inhibitory potency as compared with bupivacaine and levobupivacaine. Interestingly, it was more potent than lidocaine in inhibiting CD11b expression. These findings suggest that the inhibitory effects are independent of lipophilicity of the compounds. Recently, less cardio- and neurotoxicity of levobupivacaine has also been reported [21,22]. In a previous study, levobupivacaine exerted significantly less inhibitory actions on neutrophil function compared to R-(+) and racemic bupivacaine, but the differences between R-(+) and levobupivacaine were small compared to the overall changes [23]. However, our data demonstrated that there was no significant difference between bupivacaine and levobupivacaine. Taken together, these findings indicate that enantiomer-specific effects may play a minor role in inhibition of neutrophil function. Ropivacaine concentrations of 6 mM and higher were required for analgesia and anesthesia in clinical practice, however, plasma concentration of about 8 µM was observed after epidural administration [24]. Therefore, we used ropivacaine over a wide range of concentrations according to clinical practice. Martinsson et al. [25] reported that ropivacaine ≥ 100 µM inhibited up-regulation of CD11b expression induced by TNF-α. In the present study, we obtained the similar inhibitory effects of ropivacaine at the same concentrations. Although the concentrations at which these effects take place are 1001000 times higher than those clinically observed in plasma [24], our data at least suggest the possibility of clinically relevant effects on neutrophil function at sites with high ropivacaine concentrations. Ropivacaine exerted an inhibitory action on the fMLP-stimulated up-regulation of CD11b expression when added to neutrophils before fMLP. However, it showed no effect when added after the increase of CD11b expression, suggesting that ropivacaine does not promote reinternalization of CD11b. Firm adhesion of neutrophil to endothelial cell is mainly mediated through CD11b/CD18 and its ligand intercellular cell adhesion molecule-1 (ICAM-1) on the endothelial cell surface [26]. Because adhesion molecule CD11b/CD18 can be expressed at the cell surface in an inactive form, expression does not correlate with functional activity [12]. We therefore assessed CD11b function by measuring adhesion of neutrophils to HUVECs. In this study, dramatic upregulation of adhesion of neutrophils to HUVECS was induced by fMLP, which was also observed by stimulation with TNF in a previous study [27]. The up-regulation was likely to be due to the effects both on neutrophils and HUVECs. As shown in Fig. 6A, ropivacaine significantly inhibited adhesion of neutrophils to HUVECs, which might be mainly mediated through reduction of CD11b expression in neutrophils. Since Lan et al. [28] reported that ICAM-1 expression in activated HUVECs was attenuated by lidocaine, it could be speculated that ropivacaine inhibits adhesion of neutrophils to HUVECs by interfering with ICAM-1 expression in the endothelial cells. However, our finding that ropivacaine showed no effect on HUVECs in the process of adhesion does not support this hypothesis. In some cases, ropivacaine showed weak or lack of inhibitory effects on neutrophil function [9,14,29], which is not in agreement with the previous findings [8,15,25] and ours. The inconsistency may
have arisen from a different methodology including different drug concentrations tested and different stimuli used. The precise mechanism of the inhibitory actions of ropivacaine on neutrophil function still remains to be elucidated. Sodium channel blockade can be ruled out, because sodium channels are not present in human neutrophils [30]. It has been reported that increases in intracellular Ca2+ concentration play a major role in neutrophil activation [31] and up-regulation of CD11b is also Ca2+-dependent [32]. This was confirmed in our finding that fMLP-induced CD11b expression in neutrophils was markedly inhibited when the extracellular Ca2+ was chelated by EGTA. We also found that ropivacaine pretreatment further inhibited CD11b expression to 64% of control in the presence of EGTA. Here EGTA still exerted an approximately 14% inhibition of fMLP-activated neutrophils in addition to the 22% inhibition by ropivacaine (Fig. 7A). This effect of EGTA is less than that in the absence of ropivacaine, suggesting that ropivacaine interferes with the same signaling pathway as EGTA. Based on this point, we hypothesized that the effects of ropivacaine on neutrophil function were, at least in part, mediated through suppression of the calcium pathway. Moreover, we observed significant difference between CD11b expression with and without ropivacaine in the presence of EGTA, and it indicates that there exist other possible mechanisms for ropivacaine on neutrophils. Another site of ropivacaine actions is likely to be located in PLD, which plays an important role in the regulation of neutrophil functions of phagocytosis, degranulation, oxidative burst, adhesion and chemotaxis [33]. PLD is involved in cellular signaling pathways primarily through the production of the messenger lipid, phosphatidic acid (PA). We found that inhibition of PA production with butan-1-ol completely inhibited CD11b expression in fMLP-activated neutrophils, and it indicates that CD11b expression is regulated by PLD. Furthermore, no significant difference between CD11b expression with and without ropivacaine in the presence of butan-1-ol was observed, which suggests that most effects of ropivacaine are hidden behind the effects of butan-1-ol or that ropivacaine interferes with the same signaling pathway as butan-1-ol. Thus, it could be speculated that the main target site for ropivacaine is PLD. A previous study has demonstrated that local anesthetics (tetracaine, bupivacaine, lidocaine and procaine) suppressed PLD activation in differentiated HL60 cells either by preventing the membrane translocation of PLDactivating factors and/or by direct inhibition of the enzyme [34]. Therefore, further studies are required to confirm our hypothesis. In conclusion, ropivacaine pretreatment inhibited CD11b expression and functional activity in fMLP-activated human neutrophils, and it also inhibited CD11b expression induced by PAF priming. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. Therefore, our findings suggest that ropivacaine exerts anti-inflammatory activity through its effects on neutrophils. Furthermore, it could also be speculated that the effects of ropivacaine on neutrophils are mainly mediated through inhibiting PLD pathway. Acknowledgements This work was supported by the National Nature Foundation of China (Grant# 30772078), by Institutes of Biomedical Sciences, Fudan University, and by Department of Anatomy Histology and Embryology, Shanghai Medical College, Fudan University. References [1] Henson PM, Johnston Jr RB. Tissue injury in inflammation: oxidants, proteinases, and cationic proteins. J Clin Invest 1987;79:669–74. [2] Simpson PJ, Lucchesi BR. Free radicals and myocardial ischemia and reperfusion injury. J Lab Clin Med 1987;110:13–30. [3] Perkins NM, Tracey DJ. Hyperalgesia due to nerve injury: role of neutrophils. Neuroscience 2000;101:745–57.
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Effects of ropivacaine on adhesion molecule CD11b expression and function in human neutrophils Xuqin Zhu, Zhiming Tan ⁎, Jiawei Chen, Minmin Zhu, Yajun Xu Department of Anesthesiology, Fudan University Cancer Hospital, No. 270 Dong-an Road, Shanghai, PR China, PC 200032
a r t i c l e
i n f o
Article history: Received 25 November 2009 Received in revised form 9 February 2010 Accepted 12 March 2010 Keywords: Local anesthetic Ropivacaine Neutrophil Adhesion molecule
a b s t r a c t Local anesthetics possess a wide range of anti-inflammatory properties through their effects on neutrophils. However, limited information is available on the effects of ropivacaine (a new local anesthetic) on neutrophil function. The aim of this study was to investigate anti-inflammatory properties of ropivacaine with regard to its effects on the expression and function of CD11b in human neutrophils. CD11b expression was examined by flow cytometry and its function was determined by measuring adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs). Ropivacaine inhibited CD11b expression in formyl-methionylleucyl-phenylalanine (fMLP)-activated neutrophils in a concentration-dependent manner, but not in a timedependent manner. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. The up-regulation of CD11b induced by platelet-activating factor (PAF) priming was also inhibited by ropivacaine. fMLP increased adhesion of neutrophils to HUVECs, which was inhibited by ropivacaine. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N´,N´tetraacetic acid (EGTA), a chelator of extracellular Ca2+. However, ropivacaine showed no effect on the fMLPinduced CD11b expression in the presence of butan-1-ol, a blocker of phospholipase D (PLD) pathway, which completely inhibited the fMLP-induced CD11b expression in neutrophils. Our results suggest that ropivacaine exerts anti-inflammatory activity, and this appears to be mediated through inhibiting the expression and function of adhesion molecule CD11b in neutrophils. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Human neutrophils have been implicated in the pathogenesis of a variety of inflammation-related diseases, including adult respiratory distress syndrome (ARDS) and myocardial reperfusion injury [1,2]. In addition, the important role of neutrophils in mechanisms of processing pain and hyperalgesia has also been demonstrated in several inflammatory conditions, including nerve injury, rheumatoid arthritis and allergen-evoked inflammation [3–5]. Besides direct neuronal blocking effects, local anesthetics have significant anti-inflammatory properties [6]. Previous studies have revealed inhibitory effects of local anesthetics on migration [7], chemotaxis [8], oxidative burst and phagocytosis of neutrophils [9]. Furthermore, lidocaine has been introduced in the treatment of burn injuries, ulcerative proctitis and arthritis [6]. The migration of neutrophils into tissues is a crucial event in the inflammatory response, which is dependent on the coordinated regulation of the expression and function of at least three families of
⁎ Corresponding author. Tel.: +86 21 64175590; fax: +81 21 64174774. E-mail address: [email protected] (Z. Tan). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.03.009
cell surface adhesion molecules [10]. CD11b/CD18, a member of the integrin family, appears to be important for the firm adhesion of neutrophils to the endothelium [11]. Moreover, respiratory burst activity is altered following integrin-mediated adhesion, suggesting that the adhesive state of neutrophils is an important determinant of oxygen radical and granule release and thus, potentially, of tissue damage [12]. Ropivacaine is a new long-acting amide local anesthetic with a structure similar to that of bupivacaine. However, ropivacaine is the first local anesthetic as an almost pure S-enantiomer (N99% pure), whereas bupivacaine is a racemic mixture. Although bupivacaine is still widely used for regional anesthetic techniques, the clinical application of ropivacaine is on the increase because it is less likely to induce central nervous system effects or cardiac toxicity compared with bupivacaine [13]. However, previous studies about the effects of ropivacaine on human neutrophils are mainly focused on the respiratory burst activity and some of these findings are inconsistent [8,9,14,15]. No data have been published on whether ropivacaine could modulate the expression and functional activity of adhesion molecule CD11b in formyl-methionyl-leucyl-phenylalanine (fMLP)activated or platelet-activating factor (PAF)-primed neutrophils. Therefore, we performed this study to examine this issue.
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2. Materials and methods 2.1. Materials fMLP, PAF (β-Acetyl-γ-O-alkyl-L-α-phosphatidylcholine), ethylene glycol-bis(2-aminoethylether)-N,N,N´,N´-tetraacetic acid (EGTA), lidocaine hydrochloride monohydrate and bupivacaine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ropivacaine hydrochloride monohydrate and levobupivacaine hydrochloride were gifts from AstraZeneca UK Ltd. Ficoll-Paque was purchased from Biowest (France). Fluorescein isothiocyanate (FITC)-conjugated antibodies against CD11b (Clone ICRF 44) and FITC-conjugated IgG1, κ isotype control were purchased from eBioscience (USA). Redfree FCM lysing solution was purchased from MultiSciences Biotech Co. Ltd (P. R. China). All the other reagents were of the highest quality available. 2.2. Blood samples
Fig. 1. Dot plot of whole blood subjected to flow cytometric analysis for CD11b expression on neutrophils. Neutrophils, monocytes and lymphocytes were defined distinctly. Region A was neutrophils.
After obtaining institutional and ethical committee approval (Fudan University Cancer Hospital, Shanghai, China) and informed consent, human venous blood was obtained from 16 healthy volunteers and immediately anticoagulated with lithium heparinate. All the healthy volunteers had not taken any medication for at least 2 weeks.
micin, Amphotericin-B) and 10% fetal bovine serum (FBS). Cells were grown in a humidified incubator containing 95% air and 5% CO2 at 37 °C with media replenishment every 2 days. On reaching 90% confluence, cells were detached by 0.1% trypsin and subcultured. The fifth passage of HUVECs was used in this study.
2.3. Expression of adhesion molecule CD11b
2.5. Isolation of human neutrophils
Whole blood (100 µl) was preincubated at 37 °C in the absence or presence of local anesthetics for the indicated duration. Thereafter, samples were activated with fMLP (1 µM) for 15 min or primed with PAF (1 µM) for 5 min at 37 °C, because in our preliminary experiments activation effect by fMLP (1 µM) has been shown to be maximal after 15 min and priming effect by PAF (1 µM) has been shown to be maximal after 5 min. To test the effects of ropivacaine on overactive state of neutrophils, samples were primed with PAF (1 µM) for 5 min before activation with fMLP (1 µM) for 15 min. In order to determine the effects of ropivacaine on CD11b expression in neutrophils which have been activated in advance, whole blood was initially activated with fMLP (1 µM) for 15 min prior to the incubation of ropivacaine. In addition, to further investigate the mechanisms of the effects of ropivacaine on CD11b expression, samples were incubated with 0.3% butan-1-ol or EGTA (5 mM) for 3 min prior to treatment with ±fMLP following ropivacaine treatment. The reactions were stopped by the addition of cold phosphate buffered saline (PBS) and samples were kept on ice for 5 min. Then each sample was incubated for 30 min on ice with FITC-conjugated monoclonal antibodies against CD11b, and the erythrocytes were lysed using Redfree FCM lysing solution. Samples were then centrifuged, washed, and resuspended in PBS containing 1% paraformaldehyde and stored at 4 °C in the dark until flow cytometric analysis. Flow cytometric analysis was performed using flow cytometer (Coulter, Epics ALTRA) and FITC-conjugated IgG1, κ isotype control was used to define the cutoff for positive fluorescence. Neutrophils, monocytes and lymphocytes were discriminated as shown in Fig. 1. Data were acquired and processed using EXPO32 ADC Analysis. For each sample, 20,000 neutrophils were analyzed. The mean fluorescence intensity (MFI) of antibody-stained neutrophils was related to the quantity of CD11b, thus MFI was determined as a measure of CD11b expression.
Human neutrophils were prepared as described previously with minor modifications [16]. Briefly, venous blood was mixed with 4.5% dextran saline and left to sit vertically for 40 min. The supernatant was taken and placed gently on the same volume of Ficoll-Paque. By centrifugation (400 g) for 20 min, neutrophils were pelletted at the bottom of the tube. Contaminating erythrocytes were lysed with hypotonic ammonium chloride solution (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA.Na2). Following centrifugation, neutrophils were washed and resuspended to a concentration of 106 cells/ml in the culture medium. The purity of the neutrophils was assessed microscopically (N90%). The viability of cells before and after incubation with local anesthetics was N95% as determined by the trypan blue exclusion test. 2.6. Adhesion of neutrophils to HUVECs Isolated neutrophils were preincubated at 37 °C with or without ropivacaine (1 mM) for the indicated duration and then stimulated with fMLP (1 µM) for 15 min. Thereafter, neutrophils were added to the HUVECs and incubated at 37 °C for 1 h. The cells were then washed 3 times with Hanks' balanced salt solution (HBSS), and observed under a phase-contrast microscope. Adherent cells were counted in 10 different fields in 5 separate culture dishes. In addition, to exclude the effects of ropivacaine on HUVECs in the process of adhesion of neutrophils to HUVECs, HUVECs were incubated with or without ropivacaine (1 mM) for 1 h at 37 °C and neutrophils were stimulated with fMLP (1 µM) for 15 min. Then ropivacaine was washed off the HUVECs before the addition of neutrophils. After incubation at 37 °C for 1 h, the cells were washed and observed as described above. Adherent cells were then counted in 10 different fields in 3 separate culture dishes. 2.7. Statistical analysis
2.4. Human umbilical vein endothelial cells (HUVECs) culture The initial batch of HUVECs was purchased from Clonetics (Cat# C2517A), and cultured in endothelial basal medium containing vascular endothelial growth factor, fibroblast growth factor, insulinlike growth factor-1, hydrocortisone, ascorbic acid, GA-1000 (Genta-
Data are presented as mean ± SEM. Statistical significance was determined by using Student's paired t-test for comparison between two groups or by using ANOVA (analysis of variance) followed by Student–Newman–Keuls test for comparisons among three or more groups. A value of P b 0.05 was considered statistically significant.
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3. Results 3.1. Ropivacaine inhibited CD11b expression in fMLP-activated neutrophils To verify the effects of ropivacaine on CD11b expression, we incubated whole blood with ropivacaine (1 mM) for 0, 5, 15, 30 and 60 min before activation with fMLP. As shown in Fig. 2A, ropivacaine significantly inhibited CD11b expression after 5 min as compared with fMLP-activated control cells (P b 0.05), however, this effect was not time dependent. Maximal inhibition was achieved at 15 min, therefore, this incubation time was used in the following experiments. Then, we incubated whole blood with different concentrations of ropivacaine (1 µM to 1000 µM) for 15 min before activation with fMLP. Ropivacaine ≥ 100 µM significantly reduced CD11b expression as compared with fMLP-activated control cells (P b 0.05, Fig. 2B). 3.2. Ropivacaine inhibited CD11b expression in neutrophils primed with PAF To test the effects of ropivacaine on CD11b expression in different states of neutrophils, we stimulated neutrophils with different stimuli following ropivacaine treatment. As shown in Fig. 3, CD11b expression was inhibited significantly to 78 ± 1% of control (P b 0.05) compared with fMLP-activated control cells and to 70 ± 1% of control (P b 0.05) compared with PAF-primed control cells. Whereas CD11b expression was only inhibited to 87 ± 3% of control (P N 0.05) compared with PAF-primed-fMLP-activated control cells. 3.3. Ropivacaine showed a similar inhibition of CD11b expression compared with bupivacaine and levobupivacaine, but was more potent than lidocaine To determine whether the inhibitory potency of local anesthetics on CD11b expression was dependent on chemical properties, we used
Fig. 3. Inhibitory effects of ropivacaine on CD11b expression in different states of neutrophils. Whole blood was preincubated with or without ropivacaine (1 mM) for 15 min before adding different stimuli. MFI of untreated cells was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs fMLP-activated control; # P b 0.05 vs PAF-primed control; P N 0.05 vs PAF-fMLP control.
bupivacaine, lidocaine and levobupivacaine (the S-(−)-enantiomer of bupivacaine) to compare with ropivacaine. We found that CD11b expression was attenuated significantly (to 74 ± 2% of control for ropivacaine, 72 ± 2% of control for bupivacaine and 72 ± 2% of control for levobupivacaine, P b 0.05) as compared with fMLP-activated control cells. Ropivacaine, bupivacaine and levobupivacaine were similar in their inhibitory effects (P N 0.05). However, lidocaine (84 ± 3% of control, P b 0.05) was found to inhibit CD11b expression less effectively (P b 0.05) than ropivacaine (Fig. 4). 3.4. Ropivacaine showed no effect when added after the increase of CD11b expression When neutrophils were activated with fMLP before incubation with ropivacaine (1 mM) for 15 min, CD11b expression was not affected by ropivacaine (P N 0.05, Fig. 5). 3.5. Ropivacaine inhibited adhesion of neutrophils to HUVECs induced by fMLP To further characterize the modulation of CD11b function by ropivacaine, we investigated the effects of ropivacaine on adhesion of neutrophils to HUVECs. As shown in Fig. 6A, fMLP increased the adhesion of neutrophils to HUVECs (P b 0.05), which was significantly reduced when neutrophils were pretreated with ropivacaine (15 min) (P b 0.05). However, when HUVECs were pretreated with ropivacaine, ropivacaine showed no effect on HUVECs in the process of adhesion of neutrophils to HUVECs induced by fMLP (P N 0.05, Fig. 6B).
Fig. 2. Inhibitory effects of ropivacaine on CD11b expression in fMLP-activated neutrophils. (A) Time course for the effects of ropivacaine on CD11b expression. (B) Ropivacaine inhibited CD11b expression in a concentration-dependent manner. MFI of fMLP-activated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs control.
Fig. 4. Inhibitory effects of local anesthetics on CD11b expression in fMLP-activated neutrophils. Whole blood was preincubated with or without 1 mM one of the four local anesthetics for 15 min before activation with fMLP. MFI of fMLP-activated neutrophils in the absence of local anesthetics (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs control; # P b 0.05 for lidocaine vs ropivacaine. Ropi: Ropivacaine, Bupi: Bupivacaine, Levobupi: Levobupivacaine, Lido: Lidocaine.
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Fig. 5. Effects of ropivacaine on CD11b expression when added after fMLP. MFI of fMLPactivated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means ± SEM of three different experiments, each carried out in duplicate.
3.6. Ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of EGTA To investigate the role of Ca2+ in the effects of ropivacaine, we used EGTA to chelate extracellular Ca2+. As shown in Fig. 7A, EGTA significantly inhibited CD11b expression to 75 ± 1% of control as compared with fMLP-activated control cells (P b 0.05). When neutrophils were pretreated with ropivacaine (1 mM) for 15 min, CD11b expression was further inhibited to 64 ± 3% of control in the presence of EGTA as compared with fMLP-activated control cells (P b 0.05). Moreover, there existed significant difference between CD11b expression with and without ropivacaine in the presence of EGTA (P b 0.05).
Fig. 7. Effects of ropivacaine on CD11b expression in fMLP-activated neutrophils in the presence of EGTA (A) or butan-1-ol (B). MFI of fMLP-activated neutrophils in the absence of ropivacaine (control) was expressed as 100%. Data represent the means± SEM of three different experiments, each carried out in duplicate. * P b 0.05 vs fMLP-activated control; # P b 0.05 vs EGTA + fMLP; P N 0.05 vs butan-1-ol+ fMLP.
3.7. Ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol We also investigated the role of phospholipase D (PLD) in the effects of ropivacaine by using butan-1-ol as a pseudosubstrate to block PLD signaling pathway. As shown in Fig. 7B, butan-1-ol completely inhibited the fMLP-induced CD11b expression (P b 0.05), which was not affected when neutrophils were pretreated with ropivacaine (1 mM) for 15 min (P N 0.05). 4. Discussion
Fig. 6. Effects of ropivacaine on adhesion of neutrophils to HUVECs. Results were expressed as numbers of adherent neutrophils per 100 HUVECs. (A) Ropivacaine inhibited adhesion of neutrophils to HUVECs. Neutrophils were pretreated with ropivacaine before activation with fMLP, and then added to HUVECs. Data represent the means ± SEM of five different experiments. * P b 0.05 vs untreated cells (white bar); # P b 0.05 vs fMLP-activated control (grey bar). (B) Ropivacaine showed no effect on HUVECs in the process of adhesion. HUVECs were pretreated with ropivacaine, and then fMLP-activated neutrophils were added to HUVECs following that ropivacaine was washed off HUVECs. Data represent the means ± SEM of three different experiments. * P b 0.05 vs untreated cells (white bar); P N 0.05 vs fMLP-activated control (grey bar).
The present study demonstrated that ropivacaine not only inhibited CD11b expression in fMLP-activated or PAF-primed human neutrophils, but also inhibited adhesion of neutrophils to HUVECs induced by fMLP. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of EGTA. However, ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol. Neutrophil adhesion is tightly regulated during inflammatory process and it is likely that perturbation of adhesion mechanisms will influence the progression of inflammatory response [12]. fMLP is a physiological chemotactic peptide that arises during bacterial protein processing and activates neutrophils [17]. Activation of neutrophils with fMLP is accompanied by the translocation of CD11b from the intracellular storage pool, increasing cell surface expression [18]. PAF, an endogenous inflammatory mediator, mediates priming neutrophils. Previous studies have reported that exposure of neutrophils to priming agents [such as PAF, tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF)] induced rapid up-regulation of CD11b expression, but had no direct stimulatory effects upon O2− generation, suggesting that priming agents may have specific regulatory effects on adhesion process in neutrophils
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[12,15,19]. As shown in Fig. 3, CD11b expression was markedly increased by PAF-fMLP when compared with fMLP activation alone or PAF priming alone. This state of neutrophils seems to play a pivotal role in the “overstimulation” of inflammatory pathways, which then results in tissue damage rather than protect the host [20]. In the present study, ropivacaine inhibited CD11b expression induced by fMLP or PAF, indicating that ropivacaine interferes with both priming and activation process of neutrophils. To our knowledge, the effects of ropivacaine on CD11b expression in PAF-primed-fMLP-activated neutrophils have not been reported previously. In this study ropivacaine showed only a nonsignificant trend to reduce CD11b expression in PAF-fMLP neutrophils. Ropivacaine, which is structurally similar to levobupivacaine and bupivacaine but similar in lipophilicity to lidocaine, showed no significant difference in inhibitory potency as compared with bupivacaine and levobupivacaine. Interestingly, it was more potent than lidocaine in inhibiting CD11b expression. These findings suggest that the inhibitory effects are independent of lipophilicity of the compounds. Recently, less cardio- and neurotoxicity of levobupivacaine has also been reported [21,22]. In a previous study, levobupivacaine exerted significantly less inhibitory actions on neutrophil function compared to R-(+) and racemic bupivacaine, but the differences between R-(+) and levobupivacaine were small compared to the overall changes [23]. However, our data demonstrated that there was no significant difference between bupivacaine and levobupivacaine. Taken together, these findings indicate that enantiomer-specific effects may play a minor role in inhibition of neutrophil function. Ropivacaine concentrations of 6 mM and higher were required for analgesia and anesthesia in clinical practice, however, plasma concentration of about 8 µM was observed after epidural administration [24]. Therefore, we used ropivacaine over a wide range of concentrations according to clinical practice. Martinsson et al. [25] reported that ropivacaine ≥ 100 µM inhibited up-regulation of CD11b expression induced by TNF-α. In the present study, we obtained the similar inhibitory effects of ropivacaine at the same concentrations. Although the concentrations at which these effects take place are 1001000 times higher than those clinically observed in plasma [24], our data at least suggest the possibility of clinically relevant effects on neutrophil function at sites with high ropivacaine concentrations. Ropivacaine exerted an inhibitory action on the fMLP-stimulated up-regulation of CD11b expression when added to neutrophils before fMLP. However, it showed no effect when added after the increase of CD11b expression, suggesting that ropivacaine does not promote reinternalization of CD11b. Firm adhesion of neutrophil to endothelial cell is mainly mediated through CD11b/CD18 and its ligand intercellular cell adhesion molecule-1 (ICAM-1) on the endothelial cell surface [26]. Because adhesion molecule CD11b/CD18 can be expressed at the cell surface in an inactive form, expression does not correlate with functional activity [12]. We therefore assessed CD11b function by measuring adhesion of neutrophils to HUVECs. In this study, dramatic upregulation of adhesion of neutrophils to HUVECS was induced by fMLP, which was also observed by stimulation with TNF in a previous study [27]. The up-regulation was likely to be due to the effects both on neutrophils and HUVECs. As shown in Fig. 6A, ropivacaine significantly inhibited adhesion of neutrophils to HUVECs, which might be mainly mediated through reduction of CD11b expression in neutrophils. Since Lan et al. [28] reported that ICAM-1 expression in activated HUVECs was attenuated by lidocaine, it could be speculated that ropivacaine inhibits adhesion of neutrophils to HUVECs by interfering with ICAM-1 expression in the endothelial cells. However, our finding that ropivacaine showed no effect on HUVECs in the process of adhesion does not support this hypothesis. In some cases, ropivacaine showed weak or lack of inhibitory effects on neutrophil function [9,14,29], which is not in agreement with the previous findings [8,15,25] and ours. The inconsistency may
have arisen from a different methodology including different drug concentrations tested and different stimuli used. The precise mechanism of the inhibitory actions of ropivacaine on neutrophil function still remains to be elucidated. Sodium channel blockade can be ruled out, because sodium channels are not present in human neutrophils [30]. It has been reported that increases in intracellular Ca2+ concentration play a major role in neutrophil activation [31] and up-regulation of CD11b is also Ca2+-dependent [32]. This was confirmed in our finding that fMLP-induced CD11b expression in neutrophils was markedly inhibited when the extracellular Ca2+ was chelated by EGTA. We also found that ropivacaine pretreatment further inhibited CD11b expression to 64% of control in the presence of EGTA. Here EGTA still exerted an approximately 14% inhibition of fMLP-activated neutrophils in addition to the 22% inhibition by ropivacaine (Fig. 7A). This effect of EGTA is less than that in the absence of ropivacaine, suggesting that ropivacaine interferes with the same signaling pathway as EGTA. Based on this point, we hypothesized that the effects of ropivacaine on neutrophil function were, at least in part, mediated through suppression of the calcium pathway. Moreover, we observed significant difference between CD11b expression with and without ropivacaine in the presence of EGTA, and it indicates that there exist other possible mechanisms for ropivacaine on neutrophils. Another site of ropivacaine actions is likely to be located in PLD, which plays an important role in the regulation of neutrophil functions of phagocytosis, degranulation, oxidative burst, adhesion and chemotaxis [33]. PLD is involved in cellular signaling pathways primarily through the production of the messenger lipid, phosphatidic acid (PA). We found that inhibition of PA production with butan-1-ol completely inhibited CD11b expression in fMLP-activated neutrophils, and it indicates that CD11b expression is regulated by PLD. Furthermore, no significant difference between CD11b expression with and without ropivacaine in the presence of butan-1-ol was observed, which suggests that most effects of ropivacaine are hidden behind the effects of butan-1-ol or that ropivacaine interferes with the same signaling pathway as butan-1-ol. Thus, it could be speculated that the main target site for ropivacaine is PLD. A previous study has demonstrated that local anesthetics (tetracaine, bupivacaine, lidocaine and procaine) suppressed PLD activation in differentiated HL60 cells either by preventing the membrane translocation of PLDactivating factors and/or by direct inhibition of the enzyme [34]. Therefore, further studies are required to confirm our hypothesis. In conclusion, ropivacaine pretreatment inhibited CD11b expression and functional activity in fMLP-activated human neutrophils, and it also inhibited CD11b expression induced by PAF priming. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. Therefore, our findings suggest that ropivacaine exerts anti-inflammatory activity through its effects on neutrophils. Furthermore, it could also be speculated that the effects of ropivacaine on neutrophils are mainly mediated through inhibiting PLD pathway. Acknowledgements This work was supported by the National Nature Foundation of China (Grant# 30772078), by Institutes of Biomedical Sciences, Fudan University, and by Department of Anatomy Histology and Embryology, Shanghai Medical College, Fudan University. References [1] Henson PM, Johnston Jr RB. Tissue injury in inflammation: oxidants, proteinases, and cationic proteins. J Clin Invest 1987;79:669–74. [2] Simpson PJ, Lucchesi BR. Free radicals and myocardial ischemia and reperfusion injury. J Lab Clin Med 1987;110:13–30. [3] Perkins NM, Tracey DJ. Hyperalgesia due to nerve injury: role of neutrophils. Neuroscience 2000;101:745–57.
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