JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawasaki, T.
Right arrow Articles by Shigematsu, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawasaki, T.
Right arrow Articles by Shigematsu, A.
Related Collections
Right arrow Pharmacology

Anesth Analg 2004;98:1024-9
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000104480.04856.93

ANESTHETIC PHARMACOLOGY

The Effect of Local Anesthetics on Monocyte mCD14 and Human Leukocyte Antigen-DR Expression

Takashi Kawasaki, MD, Chika Kawasaki, MD, Masanori Ogata, MD, and Akio Shigematsu, MD

Department of Anesthesiology, University of Occupational and Environmental Health, Kitakyushu, Japan

Address correspondence and reprint requests to Masanori Ogata, MD, Department of Anesthesiology, University of Occupational and Environmental Health, 1-1-1 Iseigaoka Yahatanishiku Kitakyushu, 807-8555, Japan. Address e-mail to mogata{at}med.uoeh-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
It has been demonstrated that local anesthetics have several effects on the immune system. Monocytes and macrophages are essential components of the host response to microbial infection; however, the effect of local anesthetics on monocyte surface receptor expression remains unclear. We designed this study to investigate the effects of local anesthetics on monocyte mCD14 and human leukocyte antigen (HLA)-DR expression and lipopolysaccharide (LPS)-induced or staphylococcal enterotoxin B (SEB)-induced tumor necrosis factor (TNF)-{alpha} production. Blood samples were obtained from 10 healthy volunteers. The effects of local anesthetics on LPS- or SEB-induced TNF-{alpha} production were determined by using an enzyme-linked immunosorbent assay. After different doses of local anesthetics were added, the blood was stimulated with LPS (10 ng/mL) or SEB (10 µg/mL) for 4 h. The effects of local anesthetics on monocyte mCD14 and HLA-DR expression were measured by dual monoclonal antibody staining and flow cytometry. Local anesthetics showed no effect on LPS- or SEB-induced TNF-{alpha} production in human whole blood. Local anesthetics suppressed monocyte HLA-DR expression in a dose-dependent manner (P < 0.05) but had no effect on monocyte mCD14 expression. This study demonstrated that local anesthetics suppress HLA-DR expression on the surface of human monocytes.

IMPLICATIONS: Monocyte surface receptors have a crucial role in the host response to microbial infection. We investigated the effects of local anesthetics on monocyte mCD14 and human leukocyte antigen (HLA)-DR expression. Our results show that local anesthetics suppress HLA-DR expression on the surface of human monocytes.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Regional anesthetics are used widely in clinical medicine—not only for acute pain, such as during surgery, but also for the management of chronic and refractory pain. In patients with extradural catheter placement, regional anesthetics provide a sufficient analgesic effect. Although an epidural abscess is an extremely rare complication, infection is very common, and care must be taken to prevent this complication in patients with long-term epidural catheter placement. In addition, it has been reported that, after surgery and in patients who are experiencing pain, there is an increased risk of the development of infection and sepsis as a result of immune suppression.

Regional anesthetics have antibacterial actions (1,2) and beneficial effects on inflammatory responses such as inflammatory lung injury, increased microvascular permeability, and myocardial ischemia/reperfusion injury (3). However, they have also been shown to impair immune function (4–6). Thus, it is possible that local anesthetics participate in bacterial infection. Previous studies demonstrated that local anesthetics, such as mepivacaine, bupivacaine, and lidocaine, induce innate immune system dysfunction, as indicated by reduced chemotactic ability, phagocytic ability, and superoxide anion production by neutrophils (5,6). Furthermore, local anesthetics also impair the activity of natural killer cells (7).

Monocytes are important in immune reactions, such as cytokine production and antigen presentation. However, the effects of local anesthetics on cytokine production and monocyte surface receptor expression are still unclear. Therefore, we investigated the influence of local anesthetics on lipopolysaccharide-(LPS) or staphylococcal enterotoxin B (SEB)-induced tumor necrosis factor (TNF)-{alpha} production and on expression of monocyte membrane surface receptor CD14 and human leukocyte antigen (HLA)-DR.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After we received approval from our human investigations committee, informed consent was obtained from 10 healthy male volunteers who were taking no medication. First, we investigated the effects of local anesthetics on TNF-{alpha} production in human whole blood. Because LPS contained in the outer membrane of Gram-negative bacteria and SEB produced by Gram-positive bacilli are potent mitogens for cytokine production by monocytes, we stimulated whole blood with these inflammatory stimuli. The effects of local anesthetics on LPS- or SEB-induced TNF-{alpha} production in whole blood were determined. Briefly, blood samples were drawn into a heparinized syringe and diluted with five volumes of RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan). The diluted blood (990 µL) was then placed into a 24-well plate (Becton Dickinson, Lincoln Park, NJ). Different doses of local anesthetics were added to each well, and whole blood was stimulated with SEB (10 µL) or LPS (10 µL) at a final concentration of 10 µg/mL or 10 ng/mL, respectively. After incubation at 37°C in 95% air/5% CO2 for 6 h, the blood was centrifuged at 700g for 10 min to remove blood cells. The supernatants were collected and stored at -80°C until they were assayed. The plasma TNF-{alpha} concentration was measured in duplicate by using a commercially available enzyme-linked immunosorbent assay (Medgenix; BioSource Europe S.A., Fleurus, Belgium). According to the manufacturer’s data sheet, cross-reactivity with other cytokines is negligible in this assay.

Because monocyte mCD14 plays an essential role in transmitting LPS signals intracellularly and ultimately activates TNF-{alpha} production (8,9), we investigated whether local anesthetics affect monocyte mCD14 expression. Furthermore, staphylococcal enterotoxins function as superantigens that activate T cells, monocytes, and macrophages by cross-linking the external domains of HLA-DR on antigen-presenting cells (10). The superantigen activity of staphylococcal enterotoxins results in the synthesis of a variety of cytokines, including interleukin (IL)-1, IL-2, IL-6, IL-8, interferon-{gamma}, and TNF-{alpha} (11–13). Therefore, we also investigated whether local anesthetics affect monocyte HLA-DR expression.

The effects of local anesthetics on monocyte HLA-DR expression and mCD14 expression were measured by dual monoclonal antibody staining and flow cytometry. After incubation with different doses of each local anesthetic for 30 min, 100 µL of anticoagulated whole blood was mixed with 20 µL of fluorescein isothiocyanate-coupled RMO52 monoclonal antibody (IMO645; Coulter Immunology, Hialeah, FL) and with 20 µL of phycoerythrin-coupled B8.12.2 monoclonal antibody (IMO464; Coulter Immunology). After incubation in the dark at room temperature for 45 min, erythrocytes were lysed by the addition of Immunolyse medium (Coulter Immunology), and the samples were incubated for a further 10 min. The samples were then washed twice and fixed with 0.2% paraformaldehyde. All samples were analyzed immediately by using a flow cytometer and XL software (EPICS-XL; Coulter Electronics, Luton, UK). The results are presented as the mean fluorescence intensity. The monocyte gate was set by using the routine position for monocytes in side scatter and forward scatter for mononuclear cells.

To assess the effects of local anesthetics on leukocyte viability, different doses of each local anesthetic were added to diluted human whole blood, followed by incubation for 6 h at 37°C under an atmosphere of 95% air/5% CO2. After incubation, the blood was centrifuged at 700g for 10 min. Buffy coats were isolated, and NH4Cl lysis of red blood cells was performed. Leukocytes were resuspended in RPMI 1640 medium containing 5% fetal calf serum, and the cells were stained with 0.2% trypan blue. The cell survival rate was assessed by microscopy.

All data are presented as mean ± SEM. The paired Student’s t-test was used for statistical analysis to compare the results with control values. P < 0.05 was taken to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
After different doses of local anesthetics were added (lidocaine 1–100 µg/mL, mepivacaine 1–100 µg/mL, and bupivacaine 0.25–25 µg/mL), whole blood was stimulated with LPS (10 ng/mL) or SEB (10 µg/mL) for 6 h. Figure 1 shows the effects of local anesthetics on LPS-induced TNF-{alpha} production. Plasma TNF-{alpha} concentration of nonstimulated whole blood was below the limit of detection. However, after incubation for 6 h with LPS (control), the plasma TNF-{alpha} concentration showed a significant increase (1660.3 ± 50.2 pg/mL). Three of the local anesthetics examined did not influence LPS-induced TNF-{alpha} production.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 1. The effect of local anesthetics on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-{alpha} production. After local anesthetic was added, human whole blood was stimulated by LPS (10 ng/mL) and incubated for 6 h. A, Lidocaine (n = 10); B, mepivacaine (n = 10); C, bupivacaine (n = 10). Values are expressed as mean ± SEM (n = 10). *P < 0.05 compared with control. MFI = mean fluorescence intensity.

 
The effects of local anesthetics on SEB-induced TNF-{alpha} production are shown in Figure 2. The plasma TNF-{alpha} concentration of nonstimulated whole blood was below the limit of detection. After incubation for 6 h with SEB (control), the plasma TNF-{alpha} concentration showed a significant increase (1210.3 ± 10.4 pg/mL). Consistent with LPS-induced TNF-{alpha} production, local anesthetics had no effect on that induced by SEB.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 2. The effect of local anesthetics on staphylococcal enterotoxin B (SEB)-induced tumor necrosis factor-{alpha} production. After local anesthetic was added, human whole blood was stimulated by SEB (10 µg/mL) and incubated for 6 h. A, Lidocaine (n = 10); B, mepivacaine (n = 10); C, bupivacaine (n = 10). Values are expressed as mean ± SEM (n = 10). *P < 0.05 compared with control.

 
The effects of local anesthetics on monocyte mCD14 expression are shown in Figure 3. The three different kinds of local anesthetic used in this study showed no effect on monocyte mCD14 expression.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. The effect of local anesthetics on mCD14 expression. A, Lidocaine (n = 10); B, mepivacaine (n = 10); C, bupivacaine (n = 10). Values are expressed as mean ± SEM (n = 10). *P < 0.05 compared with control. TNF = tumor necrosis factor.

 
However, local anesthetics suppressed monocyte HLA-DR expression (Fig. 4). Lidocaine, mepivacaine, and bupivacaine significantly suppressed monocyte HLA-DR expression in a dose-dependent manner, in comparison with controls (P < 0.05). Lidocaine (1–100 µg/mL), mepivacaine (1–100 µg/mL), and bupivacaine (0.25–25 µg/mL) had no effect on white blood cell viability, as determined by the exclusion of the vital stain trypan blue.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 4. The effect of local anesthetics on monocyte human leukocyte antigen-DR expression. A, Lidocaine (n = 10); B, mepivacaine (n = 10); C, bupivacaine (n = 10). Values are expressed as mean ± SEM (n = 10). *P < 0.05 compared with control. TNF = tumor necrosis factor.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of this study demonstrate that local anesthetics suppress HLA-DR expression on the surface of human monocytes but have no effect on monocyte mCD14 expression. They also have no effect on LPS- or SEB-induced TNF-{alpha} production in human whole blood.

Innate immunity is an early organism defense reaction against invading pathogenic agents. Phagocytes, such as monocytes and macrophages, play important roles in innate immunity. LPS, a body-wall component of Gram-negative bacteria, participates in innate immunity, stimulates host monocytes and macrophages, and contributes to innate immunity strengthening by the induction of bioactive substance production such as TNF-{alpha} and IL-1ß. CD14, a cell-surface molecule of macrophages/monocytes and neutrophils, has been shown to be not only the LPS receptor involved in activation of cytokine production, but also an important receptor with roles in the activation of phagocytosis of Escherichia coli (14) and in priming the respiratory burst in human neutrophils (15). In this study, we showed that local anesthetics have no effect on LPS-induced TNF-{alpha} production or monocyte mCD14 expression. In contrast to our results, Okuno et al. (6) reported that large-dose lidocaine (>2 mg/mL) inhibited phagocytosis and superoxide production by human monocytes and granulocytes. The concentration of lidocaine in human plasma reaches 2.2 µg/mL after epidural administration (16) or 1.5–1.9 µg/mL after 2 mg/mL IV administration (17), so the concentration of lidocaine used in Okuno et al.’s study was approximately 1000-fold larger than that used in a clinical setting. However, we cannot exclude the possibility that when the drugs are administered locally for nerve blockade, the concentration of local anesthetic at a local site is much larger than in plasma. Our results indicate that local anesthetics at clinically relevant concentrations do not adversely affect the innate immune system, such as TNF-{alpha} production and monocyte mCD14 expression.

The HLA-DR antigen on the monocyte cell membrane surface, which is necessary for antigen presentation to the T-cell receptor on T cells, is important for the activation of T cells, which are the central cells involved in adaptive immunity (10). The expression of HLA-DR, one of the major histocompatibility complex class 2 proteins, on mononuclear cells has a central role in antigen presentation to lymphocytes and initiation of immune responses (10,13). In trauma or surgical patients, monocyte HLA-DR expression is decreased, and this is associated with an increased risk of developing severe sepsis (18–21). Loss of cell-surface HLA-DR was suggested to reduce the antigen-presenting capacity of monocytes, resulting in impaired T-cell stimulation. It has been reported that neutrophils with increased expression of HLA-DR were more phagocytic than those with lower levels of expression (22). This study demonstrated that local anesthetics (lidocaine, mepivacaine, and bupivacaine) reduce monocyte HLA-DR expression, suggesting that these local anesthetics not only impair the antigen-presenting capacity and T-cell stimulation ability of monocytes, but also adversely affect phagocytic ability. Consistent with our results, Dickstein et al. (23) reported that lidocaine induced permanent changes on the surface of lymphocytes and reversible changes on the surface of macrophages. They also reported that chronic exposure to lidocaine resulted in impairment of lymphocyte function (24). The mechanism of the inhibitory effect of local anesthetics on monocyte HLA-DR expression remains unclear, but we assume that it is due to a direct effect of local anesthetics on monocytes. Further studies are required.

The concentrations of local anesthetics in human plasma reach 2.2 µg/mL (lidocaine), 2.53 µg/mL (mepivacaine), and 0.73 µg/mL (bupivacaine) after their epidural administration (16,25). In this study, the concentrations of lidocaine, mepivacaine, and bupivacaine that suppressed monocyte HLA-DR expression were 1, 1, and 0.25 µg/mL, respectively. These results suggest that local anesthetics suppress monocyte HLA-DR expression at clinically relevant concentrations.

In conclusion, we demonstrated that local anesthetics suppress HLA-DR expression on the surface of human monocytes. This suggests that local anesthetics suppress the antigen-presenting ability of monocytes at clinical concentrations. This suppressive effect might be stronger after local injection of these anesthetics, because the local concentration might be larger than that in plasma. Careful attention must be paid to the effects of local anesthetics on the host-defense mechanisms in the clinical setting.


   Acknowledgments
 
This work was supported in part by Grants-in-Aid for Scientific Research 12770850 (TK) and 14370499 (MO) from the Ministry of Education, Science, Sport, and Culture of Japan.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Rosenberg PH, Renkonen OV. Antimicrobial activity of bupivacaine and morphine. Anesthesiology 1985; 62: 178–9.[Web of Science][Medline]
  2. Grimmond TR, Brownridge P. Antimicrobial activity of bupivacaine and pethidine. Anaesth Intensive Care 1986; 14: 418–20.[Web of Science][Medline]
  3. Hollmann MW, Durieux ME. Local anesthetics and the inflammatory response. Anesthesiology 2000; 93: 858–75.[Web of Science][Medline]
  4. Hyvonen PM, Kowolik MJ. Dose-dependent suppression of the neutrophil respiratory burst by lidocaine. Acta Anaesthesiol Scand 1998; 42: 565–9.[Web of Science][Medline]
  5. Mikawa K, Akamatsu H, Nishina K, et al. Inhibitory effect of local anesthetics on reactive oxygen species production by human neutrophils. Acta Anaesthesiol Scand 1997; 41: 524–8.[Web of Science][Medline]
  6. Okuno S, Noda H, Kugimiya T, et al. The influence of local anesthetics on human leukocyte functions studied by micro whole blood collection and flowcytometry. Masui 1996; 45: 317–25.[Medline]
  7. Takagi S, Kitagawa S, Oshimi K, et al. Effect of local anaesthetics on human natural killer cell activity. Clin Exp Immunol 1983; 53: 477–81.[Web of Science][Medline]
  8. Ferrero E, Jiao D, Tsuberi BZ, et al. Transgenic mice expressing human CD14 are hypersensitive to lipopolysaccharide. Proc Natl Acad Sci U S A 1993; 90: 2380–4.[Abstract/Free Full Text]
  9. Ziegler-Heitbrock HW, Ulevitch RJ. CD14: cell surface receptor and differentiation marker. Immunol Today 1993; 14: 121–5.[Web of Science][Medline]
  10. Kappler J, Kotzin B, Herron L, et al. V beta-specific stimulation of human T cells by staphylococcal toxins. Science 1989; 244: 811–3.[Abstract/Free Full Text]
  11. Kotb M. Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 1995; 8: 411–26.[Abstract]
  12. Akatsuka H, Imanishi K, Inada K, et al. Production of tumor necrosis factors by human T cells stimulated by a superantigen, toxic shock syndrome toxin-1. Clin Exp Immunol 1994; 96: 422–6.[Web of Science][Medline]
  13. Gonwa TA, Picker LJ, Raff HV, et al. Antigen-presenting capabilities of human monocytes correlates with their expression of HLA-DS, an Ia determinant distinct from HLA-DR. J Immunol 1983; 130: 706–11.[Abstract]
  14. Schiff DE, Kline L, Soldau K, et al. Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism. J Leukoc Biol 1997; 62: 786–94.[Abstract]
  15. Yan SR, Al-Hertani W, Byers D, et al. Lipopolysaccharide-binding protein- and CD14-dependent activation of mitogen-activated protein kinase p38 by lipopolysaccharide in human neutrophils is associated with priming of respiratory burst. Infect Immun 2002; 70: 4068–74.[Abstract/Free Full Text]
  16. Burm AG, van Kleef JW, Gladines MP, et al. Epidural anesthesia with lidocaine and bupivacaine: effects of epinephrine on the plasma concentration profiles. Anesth Analg 1986; 65: 1281–4.[Abstract/Free Full Text]
  17. Tsai PS, Buerkle H, Huang LT, et al. Lidocaine concentration in plasma and cerebrospinal fluid after systemic bolus administration in humans. Anesth Analg 1998; 87: 601–4.[Abstract/Free Full Text]
  18. Livingston DH, Appel SH, Wellhausen SR, et al. Depressed interferon gamma production and monocyte HLA-DR expression after severe injury. Arch Surg 1988; 123: 1309–12.[Abstract/Free Full Text]
  19. Hershman MJ, Cheadle WG, Wellhausen SR, et al. Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient. Br J Surg 1990; 77: 204–7.[Web of Science][Medline]
  20. Cheadle WG, Hershman MJ, Wellhausen SR, Polk HC Jr. HLA-DR antigen expression on peripheral blood monocytes correlates with surgical infection. Am J Surg 1991; 161: 639–45.[Web of Science][Medline]
  21. Wakefield CH, Carey PD, Foulds S, et al. Changes in major histocompatibility complex class 2 expression in monocytes and T cells of patients developing infection after surgery. Br J Surg 1993; 80: 205–9.[Web of Science][Medline]
  22. Alexis N, Soukup J, Ghio A, et al. Sputum phagocytes from healthy individuals are functional and activated: a flow cytometric comparison with cells in bronchoalveolar lavage and peripheral blood. Clin Immunol 2000; 97: 21–32.[Web of Science][Medline]
  23. Dickstein R, Kiremidjian-Schumacher L, Stotzky G. Effects of lidocaine on the function of immunocompetent cells. I. In vitro exposure of mouse spleen lymphocytes and peritoneal macrophages. Immunopharmacology 1985; 9: 117–25.[Web of Science][Medline]
  24. Dickstein R, Kiremidjian-Schumacher L, Stotzky G. Effects of lidocaine on the function of immunocompetent cells. II. Chronic in vivo exposure and its effects on mouse lymphocyte activation and expression of immunity. Immunopharmacology 1985; 9: 127–39.[Web of Science][Medline]
  25. Takasaki M. Blood concentrations of lidocaine, mepivacaine and bupivacaine during caudal analgesia in children. Acta Anaesthesiol Scand 1984; 28: 211–4.[Web of Science][Medline]
Accepted for publication October 14, 2003.




This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
J. G. Bovill
Anesthetic Pharmacology: Reflections of a Section Editor
Anesth. Analg., November 1, 2007; 105(5): 1186 - 1190.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
T. Kawasaki, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry
Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice
Am J Physiol Cell Physiol, November 1, 2006; 291(5): C1049 - C1055.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawasaki, T.
Right arrow Articles by Shigematsu, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawasaki, T.
Right arrow Articles by Shigematsu, A.
Related Collections
Right arrow Pharmacology

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2004 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press