Anesth Analg 2003;97:1312-1316
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Lidocaine Attenuates Monocyte Chemoattractant Protein-1 Production and Chemotaxis in Human Monocytes: Possible Mechanisms for Its Effect on Inflammation
Chi-Yuan Li, MD MS*,
Chien-Sung Tsai, MD
,
Ping-Ching Hsu, MS,
Sheau-Huei Chueh, PhD
,
Chih-Shung Wong, MD PhD*, and
Shung-Tai Ho, MD MS*
*Departments of Anesthesiology and
Division of Cardiovascular Surgery, Tri-Service General Hospital; and
Departments of Biochemistry, National Defense Medical Center, National Defense University, Taipei, Taiwan, Republic of China
Address correspondence and reprint requests to Chi-Yuan Li, MD, MS, Department of Anesthesiology, #325, Section 2, Cheng-Kung Rd., Taipei, Taiwan, Republic of China. Address e-mail to cyli{at}ndmctsgh.edu.tw
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Abstract
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The recruitment and activation of peripheral blood monocytes are potentially critical regulatory events for the control of inflammation. Increased levels of monocyte chemoattractant protein (MCP)-1 have been reported in several inflammatory disorders. In this study, we examined the effect of lidocaine on lipopolysaccharide-stimulated MCP-1 secretion and MCP-1 induced chemotaxis in a human monocytic cell line, THP-1. Lidocaine inhibited lipopolysaccharide-induced MCP-1 production as well as messenger RNA expression in a dose-dependent manner. Furthermore, we demonstrated that lidocaine suppressed MCP-1-induced chemotaxis and peak cytosolic-free calcium in THP-1 cells. These results suggest that lidocaine may modulate MCP-1 production and MCP-1-induced activation in inflammatory cells.
IMPLICATIONS: Monocyte chemoattractant protein-1 (MCP-1) plays important roles in the inflammatory processes. Lidocaine may modulate MCP-1-induced monocyte response, as reflected by chemotaxis, cytosolic-free calcium, and lipopolysaccharide-induced MCP-1 production by human monocytic THP-1 cells.
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Introduction
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Chemoattracting proteins are involved in the process of leukocyte recruitment during the local immune response to infection or tissue injury. These specialized cytokines, often designated as "chemokines," not only activate chemotaxis of leukocytes, but are also involved in the regulation of adhesion molecules, enzyme release, and oxygen radical formation (1–3). Around 50 chemokines have been identified in humans and are divided into four groups distinguished by the spacing of cysteine residues in the amino-terminal part of the polypeptides; these are CXC, CC, C, and CX3C (4).
Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC subfamily of the chemokine family and exerts strong chemoattractant activities on monocytes, T cells, and natural killer cells (5). In addition to promoting the transmigration of circulating monocytes into tissues, MCP-1 exerts various other effects on monocytes, including superoxide anion induction, chemotaxis, and calcium flux (6). MCP-1 messenger (m)RNA or protein was detected at large levels in the lesions of several diseases including arthritis (7), atherosclerosis (8), and myocarditis (9), strongly suggesting that MCP-1 plays an important role in the inflammatory processes associated with these diseases (5).
Lidocaine is used to provide local or regional anesthesia for intraoperative and postoperative pain relief and to treat or prevent ventricular arrhythmia. In addition to blocking nerve transmission, lidocaine has significant antiinflammatory properties (10). It reduces free radical production by neutrophils, inhibits the phagocytic activity of leukocytes (11,12), and has a profound inhibitory effect on the cytokine responses to endotoxemia (13). Lidocaine also inhibits the migration of leukocytes and interferes with the inflammatory response in wound healing (14). Impairing wound healing in various models by lidocaine may, in part, reflect its ability to affect inflammatory mediator signaling. However, the exact mechanisms underlying these antiinflammatory effects are poorly understood.
Based on the findings of these previous studies, we hypothesized that interference with MCP-1 signaling or production may play a part in the antiinflammatory actions of lidocaine. The aim of this study was to determine the effects of lidocaine on MCP-1-induced monocyte response, as reflected by chemotaxis, cytosolic-free calcium ([Ca2+]i), and lipopolysaccharide (LPS)-induced MCP-1 production by human monocytic THP-1 cells.
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Methods
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Monocytes from the human monocytic cell line THP-1 (American Type Culture Collection, Rockville, MD) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37°C in 5% CO2 in a humidified incubator. Cells were centrifuged and resuspended with fresh medium in 24-well plates at a concentration of 106 cells/mL and incubated for 24 h before experimental use.
After 16-h treatments with LPS (Serotype 0111:B4; Sigma Chemical Co, St Louis, MO) and various concentrations of lidocaine (3 x 10-7 to 3 x 10-3 M), supernatants were stored at -70°C until assayed. Determination of MCP-1 concentration was performed according to the manufacturer’s recommendations (R&D, Minneapolis, MN) with some modification. Each well of a high binding-efficiency 96-well enzyme-linked immunosorbent assay plate was coated with mouse antihuman MCP-1 monoclonal antibody (100 µL at 1 µg/mL) in phosphate-buffered saline (PBS) at room temperature overnight. The plate was then washed 3 times with PBS containing 0.05% Tween 20 (PBS-T). Residual binding sites were blocked with 1% bovine serum albumin and 5% sucrose (300 µL/well; Sigma) in PBS with 0.05% sodium azide and incubated for 1 h at room temperature. After washing with PBS, standard MCP-1 solution or cell supernatants (100 µL/well) were added in duplicate to the coated wells, incubated for 2 h at room temperature, and washed 3 times with PBS-T. The plates were then incubated with biotinylated goat antihuman MCP-1 detection antibodies (100 µL at 0.1 µg/mL) for 2 h at room temperature. After the wash, 100 µL of streptavidin horseradish-peroxidase (1:2,500 dilution of 1.25 mg/mL solution) was added, and the plates were incubated for 20 min at room temperature. After a triple wash, 100 µL of substrate solution containing 1:1 mixture of H2O2 and tetramethylbenzidine was added, and the plates were incubated for another 20 min at room temperature. The reaction was stopped by adding 50 µL of stop solution (1 M of H2SO4), and the MCP-1 concentrations were measured with a microplate reader (Dynatech, Guernsey, UK).
The THP-1 cells were incubated at 37°C for 4 h with 0.1 µg/mL of LPS in the presence or absence of lidocaine. Total RNA was extracted from cell pellets (1 x 106 cells) using the TRIzol reagent (Gibco BRL, Grand Island, NY) and was reverse transcribed using SuperScript II RNase H Reverse Transcriptase (Gibco BRL). Polymerase chain reaction (PCR) was performed, as previously described (15). Briefly, a minimum of 3 different complimentary DNA concentrations served as the template for amplification through 19–31 cycles of denaturation (60 s at 94°C), primer annealing (60 s at 58°C), and DNA extension (90 s at 72°C) in a Gene Amp PCR System 2400 (Perkin Elmer, Norwalk, CT). Amplification of the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used to produce an internal quality standard. The primer sequences were as follows:
- MCP-1 sense, 5'-GCTCATAGCAGCCACCTTCATTC -3',
- MCP-1 anti-sense, 5'-TGCAGATTCTTGGGTTGTGGAG -3',
- GAPDH sense, 5'-GGGGAGCCAAAAGGGTCATCATCT -3', and,
- GAPDH anti-sense, 5'-GAGGGGCCATCCACAGTCTTCT -3'.
Total RNA from LPS-activated (0.1 µg/mL) THP-1 cells was used as a positive control. The experimental conditions and number of PCR cycles were predetermined to ensure the amount of MCP-1 and housekeeping-gene (GAPDH) fragments were in the linear range of amplification.
Monocyte chemotaxis was measured using a 24-well Micro Chemotaxis Transwell plate (Corning Costar, Cambridge, MA). THP-1 cells were resuspended at 2 x 106 cells/mL and transferred to the upper chamber of the Micro Chemotaxis chamber. The chemoattractant, MCP-1 (10 nM in RPMI medium with 1 mg/mL bovine serum albumin), was added to the lower chamber. The lower and upper chambers were separated by a polycarbonate membrane (5-µm pore size). The THP-1 cells were left to transmigrate for 120 min. After a 2-h incubation at 37°C in a humidified atmosphere with 5% CO2, all cells migrating through the polycarbonate filter in response to MCP-1 were counted using light microscopy.
Calcium flux assays were performed according to the method described previously (16,17). Monocytic THP-1 cells were suspended at a density of 2 x 106 cells/mL in Hank’s balanced salt solution (HBSS) containing 1.3 mM of CaCl2, 10 mM of HEPES (pH value of 7.3), and 1% fetal calf serum (FCS). The cells were loaded with 10 mM of Fura-2 AM (Molecular Probes Inc, Eugene, OR) for 1 h at 30°C with occasional shaking. Loaded cells were washed twice with centrifugation and resuspended at a concentration of 106 cells/mL. The temperature of the cell suspension was kept at 37°C, and immediately before each assay, the cells were collected by centrifugation, resuspended in 2 mL of HBSS/HEPES/FCS, and added to a cuvette in a temperature-controlled holder (37°C) with continuous stirring. For calcium measurements, recombinant human MCP-1 (10 nM) was added with or without lidocaine pretreatment. The changes in [Ca2+]i in treated monocytes were measured by testing the relative ratio of the fluorescence emission intensities (
ex, 505 nm) excited both at em 340 nm and 380 nm, using the AR-CM-MIC Cation Measurement System (Spex Industries Inc, Metuchen, NJ).
All data are presented as mean ± SEM. One-way analysis of variance was used for all statistical comparisons, and the Student-Newman-Keuls test was used for multiple comparisons. A value of P < 0.05 was considered indicative of a significant difference between groups. SigmaStat software (Jandel Scientific; Erkrath, Germany) was used for all statistical analysis.
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Results
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LPS-induced MCP-1 production by THP-1 cells (1774.2 ± 648.9 pg/mL) was significantly inhibited by lidocaine in a dose-dependent manner; the 50% effective concentration was 6.1 x 10-4 M (Fig. 1). At the largest test concentration of lidocaine (3 x 10-3 M), MCP-1 production was reduced by 83% relative to paired controls. Treatment of THP-1 cells with LPS (0.1 µg/mL) induced the appearance of mRNA for MCP-1, as measured 4 h after stimulation (Fig. 2). Lidocaine attenuated MCP-1 mRNA accumulation in a dose-dependent manner (Fig. 2). However, there was no change in GAPDH mRNA levels after treatment.
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Figure 1. Effect of lidocaine on monocyte chemoattractant protein-1 (MCP-1) production by THP-1 cells at 16 h after lipopolysaccharide (LPS) treatment (0.1 µg/mL). Inhibition of MCP-1 production by various concentrations of lidocaine has been shown as a percentage compared with the LPS-stimulated group. Data are expressed as mean ± SEM for separate experiments performed in duplicate (n = 5 or 6). **P < 0.01 indicates significant suppression of MCP-1 in comparison to the LPS-stimulated group.
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Figure 2. Lidocaine suppressed monocyte chemoattractant protein-1 (MCP-1) messenger RNA expression. Human monocytic THP-1 cells were stimulated with lipopolysaccharide (LPS, 0.1 µg/mL) with medium alone (control) or LPS with lidocaine (30–3000 µM). Reverse transcriptase-polymerase chain reaction (RT-PCR) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and MCP-1 primers were performed on total RNA extracts 4 h after initiation of culture; b.p. = base pairs; Control = culture medium; LPS = LPS alone; L+x30–x3000 = lidocaine treatment (30 µM–3000 µM) with LPS; x3000 = lidocaine alone (3000 µM).
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Cell migration was 11.8 ± 3.3 x 104 cells/mL in the absence of MCP-1 (random migration) and increased to 26.6 ± 4.0 x 104 cells/mL after MCP-1 stimulation. Lidocaine (3 x 10-5 to 3 x 10-3M) significantly reduced THP-1 cell migration induced by MCP-1 in a concentration-dependent manner; the 50% effective concentration was 1.7 x 10-3 M (Fig. 3). After stimulation of cells with MCP-1 (10 nM), [Ca2+]i increased to a peak value by 15 s and then gradually diminished. Treatment with lidocaine dramatically reduced the peak of [Ca2+]i induced by MCP-1 in THP-1 cells (Fig. 4). Lidocaine reduced the increase of [Ca2+]i induced by MCP-1 starting at the 1 x 10-4 M concentration.
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Figure 3. Effect of lidocaine on MCP-1–induced monocyte chemotaxis. THP-1 monocytic cells were incubated with medium alone or increasing concentrations of lidocaine at 37°C for 120 min in the upper compartment of a Micro Chemotaxis chamber. MCP-1 (10 nM) was added in the lower compartment. The number of monocytes that migrated to the lower compartment was counted using light microscopy. Inhibition percentage is expressed as mean ± SEM for separate experiments performed in duplicate (n = 5 or 6). *P < 0.05; **P < 0.01 indicates significant suppression of MCP-1–induced chemotaxis.
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Figure 4. Effect of lidocaine on MCP-1-induced cytosolic-free calcium ([Ca2+]i) change in human THP-1 monocytic cells. The cells were loaded with fura-2, and MCP-1 (10 nM) was added with or without lidocaine pretreatment. Stimulus-dependent [Ca2+]i change was calculated from real-time fluorescence recording. A rapid, transient, and concentration-dependent increase in [Ca2+]i was observed with MCP-1 treatment (10 nM). Treatment with lidocaine (100 µM–3 mM) reduced MCP-1-induced peak [Ca2+]i in THP-1 cells.
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Discussion
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Chemokines play an important role in controlling leukocyte activation and regulating leukocyte trafficking in several immune-mediated and inflammatory disorders (3). Inappropriate expression of chemokines can lead to leukocyte infiltrates and tissue damage. Growing evidence suggests that MCP-1 is an important participant in the inflammatory process of wound healing (18). In this study, we demonstrated that lidocaine causes a dose-dependent inhibition of MCP-1 secretion in the human monocytic cell line, THP-1. Moreover, we found that lidocaine suppressed LPS-induced MCP-1 mRNA expression, suggesting that it inhibits MCP-1 production at a transcriptional level.
Previous in vitro and animal studies have indicated that lidocaine could modulate inflammatory mediators. Lidocaine has been reported to inhibit the release of leukotriene B4 and interleukin (IL)-1 from human leukocytes (19) and to attenuate the release of tumor necrosis factor and IL-1 in bronchoalveolar lavage fluid in endotoxin-induced lung injury (20). Taniguchi et al. (13) found that lidocaine had a profound inhibitory effect on the production of IL-6 and IL-8 in a rabbit endotoxin model. There is growing evidence that suggests that lidocaine has direct effects on G protein-coupled receptors, such as the lysophosphatidic acid receptor (21), which is involved in platelet activation, inflammation, and wound healing, as well as the thromboxane receptor (22), which is involved in platelet aggregation and release of granule contents. The mechanisms related to the antiinflammatory effects of lidocaine, however, have not been completely established.
MCP-1 plays a critical role in the regulation of human monocyte function and has largely been associated with modulating monocyte migration in response to inflammation (5). In this study, we found that MCP-1–induced monocytes migration was attenuated by lidocaine. Similarly, Sasagawa (23) reported that lidocaine has an inhibitory effect on neutrophil chemotaxis. A significant decrease in MCP-1–induced chemotaxis may therefore decrease the amount of inflammatory cells attracted to the site of inflammation and prevent further tissue damage. In addition to directed migration, MCP-1 also induced characteristic monocyte responses, such as [Ca2+]i changes and respiratory burst (6). The increase of [Ca2+]i is an important response after cytokine stimulation. Lidocaine was reported to inhibit [Ca2+]i accumulation and decrease calcium influx in mast cells (24). In this study, lidocaine inhibited MCP-1 stimulated calcium influx in THP-1 cells. Taken together, these observations suggest lidocaine may suppress monocyte activation triggered by MCP-1 in inflammatory processes. The effects demonstrated at the cellular level may explain the protective effects of lidocaine obtained in vivo. Protection against chemical-, hyperoxia-, or endotoxin-induced lung injury (20,25,26) and attenuation of endotoxin-induced alterations in leukocyte-endothelial cell adhesion and macromolecular leakage (27) are examples of beneficial effects of lidocaine.
Knowledge of the effects of lidocaine on MCP-1 production and MCP-1–induced monocyte activation may be important for understanding the interactions between local anesthetics and wound healing. Lidocaine is often used in the treatment of patients with surgical wounds. The administration of lidocaine in the surgical wound reduced leukocyte migration and metabolic activation in the wound area (14). After local application or tissue infiltration, tissue concentrations of lidocaine are typically in the millimolar range, and similar concentrations are present around the spinal nerves after epidural or spinal administration of lidocaine. Our study is the first to demonstrate that lidocaine attenuates MCP-1–induced monocyte activation, such as chemotaxis and [Ca2+]i as well as LPS-induced production of MCP-1 in THP-1 cells. These results suggest that the effects of lidocaine on wound healing may act, at least in part, by modulating MCP-1 production and MCP-1–induced activation in inflammatory cells.
In conclusion, this study demonstrated that lidocaine inhibits MCP-1 production in human monocytic THP-1 cells in vitro and that this regulation occurs, at least in part, at the transcriptional level. Moreover, lidocaine suppresses MCP-1–induced chemotaxis and peak [Ca2+]i in monocytes. Nonetheless, the exact mechanisms responsible for these inhibitory effects require further investigation.
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Acknowledgments
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Supported, in part, by grants from the Tri-Service General Hospital (TSGH-C91–78) and the National Science Council (NSC 91–2314-B-016–088), Taiwan, Republic of China.
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Accepted for publication May 28, 2003.
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Abstract
Introduction
