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Originally published online as doi:10.1189/jlb.0305169 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1356-1365.)
© 2005 by Society for Leukocyte Biology

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms

Akio Jinnouchi*, Yoshitomi Aida*,1, Kohji Nozoe*, Katsumasa Maeda* and Michael J. Pabst{dagger}

* Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan; and
{dagger} Dental Research Center and Department of Molecular Sciences, The University of Tennessee, Memphis

1 Correspondence: Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 Japan. E-mail: yaida{at}dent.kyushu-u.ac.jp


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ABSTRACT
 
Local anesthetics have anti-inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2–) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2– in response to triggering by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma ± local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS-induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 but not LPS-induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up-regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.

Key Words: mitogen-activated protein kinase kinase • p38 mitogen-activated protein kinase • NADPH oxidase • tetracaine • fMLP • granule mobilization


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INTRODUCTION
 
Neutrophils play an important role in host defense and inflammatory responses [1 ]. In the peripheral blood of a healthy human, neutrophils are relatively inactive, but several agents, such as lipopolysaccharide (LPS), tumor necrosis factor {alpha} (TNF-{alpha}), and granulocyte macrophage-colony stimulating factor (GM-CSF), act on neutrophils to cause enhanced responses, including increased production of superoxide anion (O2–) in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) [2 3 4 5 6 ], adherence to endothelial cells [7 ], migration [5 ], lysosomal enzyme release [4 ], and adherence-triggered production of H2O2 [8 ]. This enhanced response of neutrophils is termed "priming." Primed neutrophils play a major role in protecting the host from invading microorganisms by generating several active oxygen species that kill microorganisms in vivo [1 ]. When neutrophils respond to foreign bodies, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is activated through signal transduction pathways and catalyzes the one-electron reduction of oxygen to O2–, followed by the generation of several other active oxygen species [9 ].

Human neutrophils express several isoforms of p38 mitogen-activated protein kinase (MAPK), kinases whose activation is mediated by dual phosphorylation by upstream kinases, MKK3/MKK6 [10 , 11 ]. Once activated, p38 MAPK phosphorylates a number of substrates, including activating transcription factor-2 (ATF-2), MAPK-activated protein kinase 2 (MAPKAP-K2), MAPKAP-K3, c-jun, ERK-1, and myelin basic protein [10 , 12 ]. p38 MAPK plays an important role in priming of neutrophils by LPS, TNF-{alpha}, or interleukin-8 [13 14 15 16 17 18 ], activation of NADPH oxidase, adhesion, and migration [19 20 21 ]. Priming of neutrophils with LPS via CD14 initiates a signal that results in the activation of MKK3, which in turn phosphorylates and activates p38{alpha} MAPK. After activation, p38{alpha} MAPK regulates at least three distinctly different functions: adhesion, activation of nuclear factor-{kappa}B (NF-{kappa}B), and the synthesis of TNF-{alpha} [12 ].

A diagram of part of the LPS signaling pathway is shown in Figure 1 . We indicate the sites of action of tetracaine, namely blockade of phosphorylation and activation of MKK3 and p38 MAPK, as shown by our experiments here. Dashed arrows show possible additional sites of action for tetracaine and the other local anesthetics.



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  		Figure 1. Scheme of part of the LPS signal transduction pathway in neutrophils. The sites of action of tetracaine, blockade of phosphorylation and activation of MKK3 and p38 MAPK are shown (solid arrows), along with possible additional sites of action for tetracaine and the other local anesthetics (dashed arrows). TLR4, Toll-like receptor 4; LBP, LPS-binding protein; MEKK-X, MKK kinase X.

Local anesthetics have a variety of actions, including modulation of the inflammatory response, in addition to sodium channel blockade to relieve pain [22 ]. In vivo, local anesthetics prevent or reduce inflammatory disorders such as reperfusion injury in heart, lung, and brain, as well as endotoxin- or hypoxia-induced pulmonary injury [22 ]. In vitro, local anesthetics inhibit cellular functions of neutrophils that mediate early steps of the inflammatory response [22 23 24 25 26 27 ]. However, the mechanisms behind these potentially beneficial effects of local anesthetics are largely unknown. It is clear that these actions do not result primarily from sodium channel blockade. In this study, we investigated the effects of local anesthetics on neutrophil responses to LPS, and we determined the site of action of local anesthetics on neutrophils. We used local anesthetics as tools to better understand priming of neutrophils. We did not expect that the effective concentrations or relative potencies of the local anesthetics, as anesthetics (reported in the literature), would necessarily have any relationship with their effects on neutrophils in our experiments.

We observed that local anesthetics inhibited neutrophil responses to LPS, including priming for enhanced release of O2– in response to fMLP and mobilization of granules. Up-regulation of cytochrome b558, a component of NADPH oxidase, was inhibited by local anesthetics, but translocation of p47phox was not. Inhibitors of p38 MAPK, such as SB203580, also inhibited LPS-induced priming. Among the four local anesthetics, only tetracaine, at concentrations that inhibited priming, inhibited activation of MKK3/p38 MAPK, although tetracaine did not inhibit the enzyme activity of p38 MAPK. Nevertheless, LPS priming was inhibited by all the local anesthetics through impaired expression of cytochrome b558, consequently impairing activation of NADPH oxidase in response to fMLP.


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MATERIALS AND METHODS
 
Reagents
LPS from Escherichia coli K235 [28 ] was a gift from Dr. Floyd C. McIntire (University of Colorado, Denver). Bupivacaine hydrochloride, cytochrome c, Histopaque 1077, protease inhibitor cocktail, and phosphatase inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO). fMLP was purchased from Vega Biochemical (Tucson, AZ). Dextran (molecular weight 200,000–300,000, LPS-free) was purchased from ICN Biomedicals Inc. (Aurora, OH). Tetracaine hydrochloride, procaine hydrochloride, Triton X-100, p-nitrophenol phosphate, and 2-amino-2-methyl-1-propanol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Lidocaine hydrochloride was purchased from ICN Biomedicals, Inc. (Irvine, CA). SB203580 and PD169316 were purchased from Calbiochem-Novabiochem (San Diego, CA). TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). Peptidoglycan from Staphylococcus aureus was purchased from Fluka (Buchs, Switzerland). Limulus amebocyte lysate reagent (E-Toxate) was purchased from Sigma Chemical Co.

Antibodies
Fluorescein isothiocyanate (FITC)-labeled anti-CD11b (CR3, Bear-1) monoclonal antibody (mAb) was purchased from Sanbio (Udan, Netherlands). FITC-labeled anti-CD14 (MY4) mAb was purchased from Coulter Immunology (Hialeah, FL). FITC-labeled anti-CD66b (80H3) mAb was purchased from Serotec (Oxford, UK). Mouse anticytochrome b558 mAb (7D5) was a gift from Dr. Michio Nakamura (University of Nagasaki, Japan) [29 ]. Rabbit polyclonal antibodies against MKK3, Ser189/207-phosphorylated MKK3/6, and Thr180/Tyr182-phosphorylated p38 MAPK were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Rabbit anti-p38 MAPK polyclonal antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG), and HRP-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (CA). Mouse anti-p47phox mAb was purchased from Becton Dickinson (San Jose, CA). FITC-labeled goat anti-mouse IgG was purchased from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD).

Preparation of neutrophils
Neutrophils were prepared from freshly drawn venous blood by standard methods, involving dextran sedimentation, followed by centrifugation on Histopaque [30 ]. Neutrophils were washed by centrifugation through 20% Histopaque in phosphate-buffered saline (PBS) and finally resuspended in PBS at 5 x 106 cells/ml. Neutrophil preparations contained >96% neutrophils and only 0.1% monocytes.

Incubation of neutrophils with LPS
Neutrophils (1.25x105 cells in 50 µl) were incubated in PBS in the presence or absence of various reagents indicated in the text with 10 ng/ml LPS plus 1% plasma for indicated times at 37°C. Controls containing vehicle alone were treated in an identical manner. We were careful to maintain the neutrophils at 37°C consistently, as temperature fluctuations are known to affect neutrophil priming and degranulation.

O2– release
Generation of O2– was measured by O2– dismutase-inhibitable reduction of cytochrome c. Neutrophils (1.25x105 cells) were incubated in PBS with 1 µM fMLP in 20 µM cytochrome c at 37°C for 7 min. After the incubation, cells were centrifuged for 5 min at 300 g. The absorbance of the supernatant was measured with a spectrophotometer. The height of the peak at 550 nm as a result of reduced cytochrome c was determined with reference to the isosbestic points at 542 nm and 556 nm. The extinction coefficient 0.021 µM–1 was used to calculate the total amount of O2– released.

Flow cytometric analysis of granule mobilization
CD14 was used as a marker for secretory vesicles, CD66b for specific granules, and CD11b for secretory vesicles, specific granules, and gelatinase granules [31 ]. For determination of surface expression of CD35, CD14, CD66b, and CD11b, neutrophils were incubated with FITC-conjugated mAb, anti-CD35 (1/50), anti-CD14 (0.5 µg/ml), anti-CD66b (1/40), or anti-CD11b (1 µg/ml) for 30 min on an ice bath. Cytochrome b558 was determined by using mAb 7D5 [29 ]. Neutrophils were incubated on ice for 30 min with 7D5 (1/1000 of concentrated ascites). The cells were then washed with ice-cold PBS and incubated for a further 30 min with FITC-conjugated goat anti-mouse Ig (5 µg/ml). Antibody bindings were analyzed for fluorescence intensity with a flow cytometer (Ortho Cytoron, Ortho Diagnostic Systems, Westwood, MA). A total of 5000 gated cells was analyzed, and the results were expressed as mean channel of fluorescence intensity (MFI).

Up-regulation of alkaline phosphatase
Alkaline phosphatase activity of neutrophils was determined in the presence or absence of 0.5% Triton X-100 to compare the total activity with that present on the cell surface [32 ]. Alkaline phosphatase activity was assayed with 1 mg/ml p-nitrophenol phosphate in saline containing 0.1 M 2-amino-2-methyl-1-propanol (pH 10.0) and 10 mM MgCl2 by incubating for 60 min at 37°C. Each assay contained only 0.125 million neutrophils so that the substrate was present in excess and was not exhausted under these experimental conditions. After the incubation, tubes were treated with 0.02 N NaOH and centrifuged at 2000 g for 5 min. The absorbance of the supernatant at 410 nm was measured. Results are given as the percentage of alkaline phosphatase activity expressed on the cell surface.

Western blotting
Neutrophils (2.5x105 cells) were incubated with 10 ng/ml LPS plus 1% plasma in the presence or absence of agents indicated in the text for 20 min at 37°C. After the incubation, cells were centrifuged at 300 g for 5 min at 4°C and lysed in 10 µl solubilizing buffer [PBS, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1/100 vol protease inhibitor cocktail, 1/100 vol phosphatase inhibitor cocktail, 1% Triton X-100] for 30 min on an ice bath. After centrifugation, the supernatant was mixed 1:1 with 2x sample buffer [4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and a trace amount of bromophenol blue in 125 mM Tris-HCl, pH 6.8], heated at 100° for 5 min, and then frozen at –80° until use. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 12.5% gels. After electrophoresis, proteins were transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in blocking buffer (PBS, pH 7.4, containing 0.1% Tween 20 and 5% nonfat milk) for 2 h at room temperature and then with antiphosphorylated p38 MAPK (1 µg/ml in blocking buffer) or with antiphosphorylated MKK3/6 rabbit polyclonal antibody (1 µg/ml in blocking buffer) overnight at 4°C. After washing with 0.1% Tween 20, the membranes were incubated with HRP-conjugated goat anti-rabbit Ig (0.2 µg/ml in blocking buffer) for 1 h at room temperature. After washing, phosphorylated bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Life Science, Ltd., Buckinghamshire, UK). Immunoreactive bands were quantified by the National Institutes of Health (NIH) Image program on a Macintosh computer. For the reblotting of membrane, it was treated with Restore Western blot stripping buffer (Pierce, Rockford, IL), washed, and incubated with new antibodies.

p38 MAPK assay
p38 MAPK assays were carried out with a commercially available kit (Cell Signaling Technology, Inc.). Briefly, neutrophils were incubated with 10 ng/ml LPS and 1% plasma in PBS for 20 min at 37°C. After the incubation, cell pellets were lysed on ice with cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) for 30 min. Then, supernatants were incubated with antiphospho-p38 MAPK antibody in the presence of Protein G plus/Protein A-agarose suspension at 4°C overnight. The cell pellet was then rinsed and incubated in kinase buffer with 2 µg ATF-2 fusion protein and 200 µM adenosine 5'-triphosphate in the presence or absence of various agents at 30°C for 30 min. The reaction was terminated with threefold concentrated sample buffer for SDS-PAGE. The samples were subjected to Western blot analysis using rabbit polyclonal phospho-specific ATF-2 antibody and HRP-conjugated goat anti-rabbit Ig. Immunoreactive bands were visualized with the ECL system and quantified by the NIH Image program.

Isolation of plasma membrane fraction
Neutrophils (1x106 cells) were incubated in PBS with 10 ng/ml LPS plus 1% plasma in the presence or absence of test agents indicated in the text for 20 min at 37°C. After the incubation, cells were sonicated for 20 s with an Astracon ultrasonic processor W-225 and centrifuged for 5 min at 800 g. Unbroken cells and nuclei were discarded, and the cytosol was separated from the membranes by centrifuging at 250,000 g for 60 min using an Hitachi 70P-72 ultracentrifuge. Membranes were resuspended in 1x sample buffer for SDS-PAGE, boiled for 5 min, and subjected to SDS-PAGE in 12.5% gels. Western blotting detected translocation of p47phox to the membrane fraction as described above.

Statistics
All experiments shown were performed at least three times. Results are presented throughout as means ± SE from independent cell samples. Statistical significance was determined by ANOVA. Scheffé’s F-test was used for the post hoc comparison of specific groups.


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RESULTS
 
Local anesthetics inhibited neutrophil responses to LPS
LPS primes neutrophils for enhanced release of O2– in response to fMLP [33 ]. In addition, LPS induces modulation of the expression of surface proteins in association with granule mobilization [34 ]. We examined local anesthetics for their effects on the responses of neutrophils to LPS. Neutrophils were incubated with 10 ng/ml LPS plus 1% plasma in the presence of increasing concentrations of local anesthetics. As local anesthetics or other agents used in this study were found to affect fMLP-triggered O2– release directly, cells were washed before triggering with fMLP. As shown in Figure 2 , all four local anesthetics inhibited LPS priming for enhanced release of O2– in response to fMLP but to different extents. Half-maximal inhibition was observed with 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine.



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  		Figure 2. Inhibition by local anesthetics of LPS priming for enhanced release of O2– in response to fMLP. Neutrophils (2.5 million cells/ml) were incubated with 10 ng/ml LPS plus 1% plasma in the presence of increasing concentrations of local anesthetics at 37°C for 30 min. After the incubation, the cells were washed and triggered with 1 µM fMLP for 7 min, and released O2– was measured. A control without LPS showed 1.43 ± 0.06 nmol O2–. Results are means ± SE from six experiments. *, Significantly different from the response of neutrophils without local anesthetics by ANOVA at the 95% confidence level.

We have shown previously that neutrophils interact with LPS during the first 1–2 min and that the priming develops during subsequent incubation for 20–30 min at 37°C [35 ]. So, we examined whether the LPS-neutrophil interaction in the first 2 min was affected by local anesthetics. When neutrophils were incubated with LPS plus plasma in the presence of local anesthetics for 2 min, followed by washing and then incubating for 28 min, local anesthetics did not inhibit the priming response (data not shown). Thus, local anesthetics did not appear to interfere with the initial interaction between neutrophils and LPS.

Next, local anesthetics were tested for their ability to modulate the expression of surface proteins that are associated with mobilization of granules. As shown in Figure 3 , in a dose-dependent manner, local anesthetics inhibited LPS-induced up-regulation of CD66b, a marker for specific granules. Local anesthetics also inhibited LPS-induced up-regulation of CD14 and alkaline phosphatase, which are markers for secretory vesicles, and local anesthetics inhibited surface expression of CD11b, a marker for specific granules, gelatinase granules, and secretory vesicles (not shown).



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  		Figure 3. Inhibition by local anesthetics of up-regulation of the expression of CD66b. Neutrophils were incubated with LPS plus plasma as in Figure 2 . Expression of CD66b, a marker for specific granules, was measured by flow cytometry as described in Materials and Methods. A control without LPS was 77.6 ± 2.2. Results are means ± SE from four experiments. *, Significantly different from the response of neutrophils without local anesthetics by ANOVA at the 95% confidence level.

When local anesthetics were added later to neutrophils that had already been primed with LPS, tetracaine alone caused enhanced fMLP-stimulated O2– release (Fig. 4 ). The other local anesthetics had no effect. Local anesthetics had no effect on O2– production by unprimed neutrophils. (Except in the experiments of Fig. 4 , local anesthetics were washed away before the O2– assay or other assays were performed, so the local anesthetics could not interfere with the assays.)



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  		Figure 4. Enhancement of O2– release by tetracaine added after LPS. Neutrophils were primed with 10 ng/ml LPS plus 1% plasma at 37° for 30 min. Local anesthetics were added to LPS-primed cells, and the cells were triggered with fMLP for 7 min. No O2– was detected in the absence of fMLP. Local anesthetics added to resting neutrophils did not cause priming. Results are means ± SE from three experiments. *, Significantly different from the response of neutrophils without local anesthetics by ANOVA at the 95% confidence level.

We investigated whether the inhibition of LPS effects by local anesthetics was reversible. Neutrophils were incubated with LPS plus plasma in the presence of local anesthetics at 37°C for 30 min, washed, and then incubated for a further 5 min before adding fMLP. As shown in Figure 5 , after the incubation for 30 min with local anesthetics, priming was inhibited, but after washing off the local anesthetics, priming reappeared after 5 min of incubation. Reversal of priming by brief incubation was complete for procaine, lidocaine, or bupivacaine but partial for tetracaine. Flow cytometry experiments showed that inhibition by local anesthetics of LPS-induced movement of granule contents (cytochrome b558 and CD66b) to the plasma membrane was also reversed by brief incubation after washing (data not shown).



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  		Figure 5. Inhibition by local anesthetics of LPS priming was reversed by washing, followed by a 5-min incubation before triggering with fMLP. Neutrophils treated with LPS plus plasma in the presence of local anesthetics were washed and assayed for fMLP-stimulated O2– release (–) or were washed, incubated for 5 min, and then triggered with fMLP (+). Results are means ± SE from four experiments. *, Significantly different from the response of neutrophils without incubation for 5 min by ANOVA at the 95% confidence level.

We examined whether LPS might merely be binding to local anesthetics and lowering their effective concentration in solution. That is, local anesthetics might not be affecting signal transduction at all but only removing LPS. We noted that the effects of local anesthetics were reversible upon washing, but if the local anesthetic was washed away, and the LPS was not washed away, then the LPS could work after the washing. However, if local anesthetic and LPS formed a two-way complex in solution, then after washing, the LPS and local anesthetics should have disappeared together, as the anesthetics were present in great excess, and therefore, the cells should not have been activated. However, the cells were activated; so we concluded that LPS and local anesthetics by themselves probably did not form a significant complex in solution. We also considered a possibility in which anesthetic-LPS-TLR4 (and CD14 and LPS-binding protein and other proteins) might form a three-way complex on the cell surface, which did not activate as a result of binding of local anesthetic but that the local anesthetic could be washed away, and then the LPS could activate the cells. To address the possibility of a two- or three-way complex between local anesthetics and LPS, we tested whether local anesthetics interfered with gelation of the Limulus lysate by LPS. (Limulus reagent is a lysate of macrophage-like cells from the blue blood of the primitive horseshoe crab, which forms a turbid gel on exposure to LPS.) We prepared stock solutions of LPS (10 ng/ml) in the presence of saline, tetracaine (0.5 mM), procaine (4 mM), lidocaine (4 mM), or bupivacaine (1 mM). Serial dilutions of the LPS solutions were tested for their ability to gel the Limulus lysate. All solutions showed the same endpoint of gelation at a 1000-fold dilution (10 pg/ml LPS). Therefore, local anesthetics did not interfere with the Limulus assay for LPS, which provided some evidence that local anesthetics and LPS did not interact in a significant way.

We also tested whether local anesthetics inhibited activation of neutrophils by TNF-{alpha} or by peptidoglycan from S. aureus, in place of LPS. We found that local anesthetics inhibited activation by TNF-{alpha} or by peptidoglycan. For example, neutrophils primed with TNF-{alpha} (20 ng/ml) released 7.68 ± 0.03 nmol O2– per 0.25 million neutrophils, but after treatment with 0.5 mM tetracaine, the O2– response was reduced to 4.04 ± 0.13; after 4 mM procaine, O2– was reduced to 4.08 ± 0.08; after 4 mM lidocaine, 4.55 ± 0.14; and after 4 mM bupivacaine, 4.00 ± 0.09 (effect of all anesthetics significant at P<0.05). Similarly, neutrophils activated with peptidoglycan (20 µg/ml) produced 5.48 ± 0.14, but with tetracaine, produced 2.14 ± 0.16; with procaine, 2.15 ± 0.06; with lidocaine, 2.73 ± 0.11; and with bupivacaine, 2.07 ± 0.07 (P<0.05). As these other activators use different receptors and different initial pathways from those used by LPS, we concluded that local anesthetics affected signal transduction and that local anesthetics did not bind LPS directly or alter the interaction between LPS and its receptor directly.

These results indicated that local anesthetics inhibited neutrophil responses to LPS by interfering with LPS signal transduction but that local anesthetics did not interfere with the initial neutrophil-LPS interaction nor did local anesthetics cause irreversible damage to the neutrophils. However, Figures 4 and 5 suggested that tetracaine might inhibit LPS by a different mechanism, compared with the other local anesthetics.

Effect of local anesthetics on LPS-induced up-regulation of cytochrome b558 and translocation of p47phox
Priming for enhanced release of O2– requires the up-regulation of cytochrome b558, a component of NADPH oxidase. Up-regulation involves fusion of pre-existing cytochrome b558 in intracellular granules with the plasma membrane [34 , 36 ]. Some fraction of the cytochrome b558 might move to internal membranes such as those of phagocytic vacuoles, but we used a soluble stimulus fMLP to minimize formation of phagocytic vacuoles, so we were able to observe up-regulation of cytochrome b558 on the plasma membrane by flow cytometry. The NADPH oxidase system of neutrophils is composed of multiple membrane-associated components, such as cytochrome b558, and many cytosolic components, such as Rac, p67phox, p47phox, and p40phox. When neutrophils are activated by inflammatory mediators, the cytosolic oxidase component p47phox becomes phosphorylated and translocated to the membrane, where it binds with cytochrome b558 to form an active enzyme complex [37 , 38 ].

We investigated the effect of local anesthetics on the formation of the active NADPH oxidase system. First, we measured surface membrane expression of cytochrome b558. As shown in Figure 6 , local anesthetics inhibited LPS-induced up-regulation of cytochrome b558 in a dose-dependent manner. Next, we tested the effect of local anesthetics on the translocation of p47phox from cytosol to membrane. As shown in Figure 7 , a concentration of tetracaine (500 µM), which inhibited up-regulation of cytochrome b558, did not affect the translocation of p47phox to the membrane fraction. Other local anesthetics also did not affect LPS-induced translocation of p47phox. These results indicated that local anesthetics inhibited LPS priming by inhibiting the assembly of active NADPH oxidase, specifically by inhibiting the up-regulation of cytochrome b558 but not by blocking translocation of p47phox.



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  		Figure 6. LPS-induced up-regulation and membrane expression of cytochrome b558. Neutrophils (2.5 million cells/ml) were incubated with 10 ng/ml LPS plus 1% plasma in the presence of increasing concentrations of local anesthetics at 37°C for 30 min. After the incubation, expression of cytochrome b558 was measured by flow cytometry as described in Materials and Methods. Fluorescence intensity for No LPS control was 39.0 ± 0.5. Results are means ± SE from five experiments. *, Significantly different from the response of neutrophils without local anesthetics by ANOVA at the 95% confidence level.



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  		Figure 7. LPS failed to induce translocation of p47phox to the membrane fraction. Neutrophils (2.5 million cells/ml) were incubated with 10 ng/ml LPS plus 1% plasma in the presence and absence of tetracaine (TC; 500 µM) for 20 min at 37°C. Resting control sample was from untreated neutrophils. Membrane fractions were prepared as described in Materials and Methods. Membrane fractions from 0.5 million cells were analyzed for the amount of p47phox by Western blotting as described in Materials and Methods. Results are representative of four independent experiments.

Specific inhibitors of p38 MAPK inhibited neutrophil responses to LPS
p38 MAPK is a key enzyme in the LPS signaling pathway [12 , 14 , 15 , 34 , 39 ]. We investigated the effect of SB203580, a specific inhibitor of p38 MAPK, on the responses of neutrophils to LPS. As shown in Figure 8 , SB203580 (10 µM) inhibited LPS-induced priming for enhanced release of O2– in response to fMLP, and it inhibited up-regulation of CD66b and cytochrome b558. Another inhibitor of p38 MAPK, PD169316, showed a similar inhibitory effect on LPS responses (not shown). These results confirmed that p38 MAPK is involved in neutrophil responses to LPS, including priming and the mobilization of intracellular granules. Thus, the possibility arose that local anesthetics inhibited the responses to LPS through inhibition of the p38 MAPK pathway.



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  		Figure 8. Inhibitors of p38 MAPK inhibited neutrophil responses to LPS. Neutrophils (2.5 million cells/ml) were incubated with 10 ng/ml LPS plus 1% plasma in the presence or absence of 10 µM SB203580 at 37°C for 30 min. After the incubation, cells were washed, and fMLP-triggered O2– release was measured. Expression of CD66b and up-regulation of cytochrome b558 were measured without washing as described in Materials and Methods. Results are means ± SE from four experiments. *, Significantly different from the response of neutrophils without SB203580 by ANOVA at the 95% confidence level.

Effects of local anesthetics on the LPS-induced activation of the p38 MAPK pathway
We investigated the effects of local anesthetics on the LPS-induced phosphorylation and activation of p38 MAPK. The phosphorylation was significant at 5 min and maximal at 20 min (not shown). As shown in Figure 9 , tetracaine inhibited LPS-induced phosphorylation of p38 MAPK at the same concentrations that inhibited priming. However, the other local anesthetics did not affect LPS-induced phosphorylation of p38 MAPK (Fig. 10 ). The inhibitory effect of tetracaine on the phosphorylation was reversed by brief incubation at 37°C after washing the cells (Fig. 11 ).



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  		Figure 9. Tetracaine inhibited phosphorylation of p38 MAPK. Neutrophils (2.5 million cells/ml) were incubated with 10 ng/ml LPS plus 1% plasma in the presence of increasing concentrations of tetracaine for 20 min at 37°C. Western blotting was performed with antibodies directed to phosphorylated (phospho; top panel) or nonphosphorylated (middle panel) forms of p38 MAPK. The results of densitometric analysis of the top panel are shown in the bottom panel. Results are representative of five experiments.



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  		Figure 10. Procaine, lidocaine, and bupivacaine did not inhibit phosphorylation of p38 MAPK. Neutrophils were treated with these other local anesthetics as in Figure 9 . Band intensities did not differ significantly from the results with no local anesthetics.



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  		Figure 11. Effect of tetracaine on the LPS-induced phosphorylation of p38 MAPK was reversed by washing and incubation. Neutrophils were incubated with LPS plus plasma in the presence of increasing concentrations of tetracaine and were then washed and incubated at 0°C or at 37°C for 2.5 min. After the incubation, cells were analyzed for the phosphorylation of p38 MAPK. The intensity of the phosphorylation was restored by washing away the tetracaine and incubating the neutrophils at 37°C for 2.5 min. Results are representative of two independent experiments.

In the p38 MAPK pathway, an enzyme that phosphorylates p38 MAPK is MKK3 [12 ]. We next investigated the effect of tetracaine on LPS-induced phosphorylation of MKK3. As shown in Figure 12 , tetracaine also inhibited LPS-induced phosphorylation of MKK3. In contrast, SB203580 had no effect on phosphorylation of p38 MAPK and MKK3 (not shown).



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  		Figure 12. Effect of tetracaine on the LPS-induced phosphorylation of MKK3. Neutrophils were incubated with LPS plus plasma in the presence of increasing concentrations of tetracaine. After the incubation, phosphorylation of MKK3 was examined by Western blotting with antibody directed against the phosphorylated (top panel) or the nonphosphorylated (middle panel) forms of MKK3. The results of densitometric analysis of the top panel are shown in the bottom panel. Results are representative of four independent experiments.

Another possibility that local anesthetics affected the catalytic activity of activated p38 MAPK was studied. p38 MAPK was immunoprecipitated and assayed for its kinase activity in the presence of local anesthetics or SB203580 using ATF-2 as a substrate. As shown in Figure 13 , tetracaine (500 µM) did not inhibit phosphorylation of ATF-2, but SB203580 did. These results indicated that tetracaine and SB203580 had similar effects on neutrophil responses to LPS but that these two agents acted at different points. SB203580 inhibited the enzyme activity of activated p38 MAPK, whereas tetracaine inhibited activation of p38 MAPK and MKK3 and subsequent LPS signal transduction.



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  		Figure 13. No effect of tetracaine on p38 MAPK kinase activity. Activated p38 MAPK was isolated from lysates of neutrophils that had been incubated with 10 ng/ml LPS plus 1% plasma at 37°C for 20 min. Kinase activity was assayed in the presence of 500 µM tetracaine (TC) or 10 µM SB203580 (SB) using ATF-2 as substrate as described in Materials and Methods. Unlike SB203580, tetracaine did not inhibit, but rather enhanced, the enzyme activity of p38 MAPK. Results are representative of three experiments.

Collectively, these results suggested that neutrophil responses to LPS, especially priming, were inhibited by local anesthetics through inhibition of the mobilization of cytochrome b558. Among the four local anesthetics, tetracaine alone inhibited the p38 MAPK pathway, and the other local anesthetics inhibited by different mechanisms.


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DISCUSSION
 
Local anesthetics have been reported to have inhibitory effects on neutrophil functions, but the mechanism behind the effects is not clear [22 ]. Many cellular functions of neutrophils, such as phagocytosis or bactericidal activity, depend on priming, which is induced by bacterial components or cytokines [40 ]. We show in this report that four local anesthetics, tetracaine, procaine, lidocaine, and bupivacaine, inhibited LPS priming for enhanced release of O2– in response to fMLP. Tetracaine was the most effective of the four. The inhibitory effects were a result of interference with LPS-induced intracellular signal transduction events required for priming. Local anesthetics did not interfere with the response to fMLP (after the local anesthetic was washed away), local anesthetics did not interfere with the initial interaction between LPS and neutrophils in the first 2 min, and local anesthetics did not cause irreversible damage to the cells. LPS-induced up-regulation of CD66b, CD14, alkaline phosphatase, and CD11b was also inhibited by local anesthetics, suggesting that mobilization of specific granules and secretory vesicles was inhibited by local anesthetics. Another important finding was that local anesthetics inhibited the LPS-induced up-regulation of cytochrome b558, a component of the NADPH oxidase system. Up-regulation of cytochrome b558 is essential for priming for enhanced O2– release [34 , 36 ]. Translocation of p47phox from cytosol to the plasma membrane occurs in primed neutrophils as well as in cells triggered for O2– release [37 ]. Our results showed that LPS-induced translocation of p47phox was not affected by local anesthetics. Thus, the primary cause of inhibition of priming by local anesthetics is the inhibition of the up-regulation of cytochrome b558.

p38 MAPK is a key enzyme in LPS signal transduction [12 , 39 ]. We found that two inhibitors of p38 MAPK, SB203580 and PD169316, inhibited LPS-induced priming for O2– and inhibited up-regulation of cytochrome b558. Nick et al. [39 ] have reported that p38 MAPK is involved in the LPS signaling leading to adherence and NF-{kappa}B activation. They demonstrated that LPS causes the activation of MKK3, resulting in activation of p38 MAPK, followed by further activation of MAPKAP-K2. Our results showed that, among the four local anesthetics, only tetracaine inhibited LPS-induced phosphorylation of p38 MAPK. Tetracaine also inhibited the LPS-induced phosphorylation of MKK3. The results that local anesthetics other than tetracaine did not affect the p38 MAPK pathway suggest that tetracaine and other local anesthetics inhibit neutrophil responses to LPS by disparate mechanisms. The other local anesthetics, which were less effective inhibitors of priming than tetracaine, appeared to work by different mechanisms, as the other local anesthetics did not affect the phosphorylation and activation of p38 MAPK or MKK3. As the other local anesthetics did block up-regulation of cytochrome b558, they must affect a step in signal transduction that occurs after p38 MAPK, or they must affect some independent pathway that is also required for up-regulation of cytochrome b558 and priming.

To examine the possibility that tetracaine inhibits p38 MAPK directly, we examined the effect of tetracaine on the catalytic activity of p38 MAPK and found that tetracaine did not inhibit the kinase activity, in contrast to the known inhibitor SB203580. We do not know at this time whether tetracaine might directly inhibit a kinase up-stream from MKK3 in the LPS pathway.

In regard to potential target molecules for local anesthetics, it has been reported that local anesthetics inhibit fMLP-induced or PMA-induced phospholipase D (PLD) activation in differentiated HL60 cells [41 ]. However, priming agents such as GM-CSF or TNF-{alpha} have been shown not to activate PLD by themselves [42 ] nor to activate p38 MAPK in a PLD-dependent manner [7 ] but rather, to cause enhanced activation of PLD in response to fMLP. In our preliminary experiments, 1-butanol (an inhibitor of PLD) inhibited LPS-induced priming, up-regulation of cytochrome b558, and phosphorylation of p38 MAPK (unpublished observations). However, involvement of PLD in LPS signal transduction remains to be proven.

Our results showing that local anesthetics other than tetracaine did not inhibit LPS-induced phosphorylation of p38 MAPK suggest that the other local anesthetics may act on signal transduction components outside the p38 MAPK pathway. However, tetracaine was the most effective inhibitor of priming among the local anesthetics, which might suggest that the MKK-p38 MAPK pathway inhibited by tetracaine is the pathway most directly responsible for LPS priming.


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ACKNOWLEDGEMENTS
 
This work was supported by Grant-in-Aid 16591889 from the Ministry of Health of Japan and by Grant DE05494 to M. J. P. from the United States Public Health Service. We thank Michio Nakamura for providing anticytochrome b558 antibody and Floyd C. McIntire for providing E. coli LPS.

Received March 29, 2005; revised July 13, 2005; accepted July 19, 2005.


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