Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism

Masayuki Niwa1,2,*, Yutaka Kanamori2, Koichi Hotta2, Hiroyuki Matsuno2, Osamu Kozawa2, Sadaki Fujimoto3 and Toshihiko Uematsu2

1 Medical Education Development Center, 2 Department of Pharmacology, Gifu University School of Medicine, 40-Tsukasamachi, Gifu 500-8705; 3 Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Nakauchi-cho 5, Yamashina-ku, Kyoto 607-8414, Japan

Received 1 May 2002; returned 7 June 2002; revised 16 June 2002; accepted 21 June 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.

Keywords: free radicals, fluoroquinolone antimicrobial agents, grepafloxacin, human neutrophils, p47-phox


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolone derivatives are frequently used therapeutically to treat bacterial infections, owing to their highly potent, wide-spectrum antimicrobial activity combined with good tissue penetration.13 These agents are widely acknowledged to display intracellular activity against bacteria that reside and/or multiply within phagocytes.47 Grepafloxacin is a newly developed fluoroquinolone antimicrobial agent,8,9 which exhibits higher penetration into various tissues compared with other fluoroquinolones.1014

At the site of inflammation, products released from activated cells such as mononuclear leucocytes can subsequently interact with granulocytes to result in the augmentation of their cytotoxic activities, phagocytic action and production of superoxides. These processes are generally referred to as priming.1517 It has been reported that several cytokines, such as granulocyte colony stimulating factor (GCSF) or tumour necrosis factor-{alpha} (TNF-{alpha}), may contribute to superoxide generation by neutrophils. When human blood neutrophils in suspension are pre-incubated with either GCSF or TNF-{alpha}, they exhibit greater superoxide generation in response to physiological stimuli such as chemotactic factors.1821 Interestingly, Matsumoto et al. reported that several fluoroquinolones, such as ofloxacin, fleroxacin and ciprofloxacin, but not lomefloxacin or sparfloxacin, significantly enhanced neutrophil superoxide production stimulated by a chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), or a phorbol ester, phorbol 12-myristate 13-acetate (PMA).2225 This effect is thought to facilitate the cooperation of fluoroquinolones with host defence mechanisms. However, the precise mechanism(s) underlying the priming effect of these fluoroquinolones has not yet been clarified.

In this report we evaluated whether grepafloxacin exhibits a priming effect on superoxide generation from human neutrophils. Furthermore, we also examined the contribution of p47-phox and p67-phox to the priming effect of grepafloxacin in human neutrophils.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals

fMLP, PMA, leukotriene B4 (LTB4), cytochalasin B, ferricytochrome c and Histopaque were purchased from Sigma (St Louis, MO, USA). Dextran (mol. wt 208 000) and HEPES were purchased from Nacalai (Kyoto, Japan) and DOJIN (Kumamoto, Japan), respectively. Pertussis toxin was purchased from Funakoshi Co. Ltd (Tokyo, Japan). Anti-p47-phox antibody, anti-p67-phox antibody and anti-phosphotyrosine (PY-20) were purchased from Transduction Laboratories (Lexington, KY, USA). Grepafloxacin and superoxide dismutase (SOD) were gifts from Otsuka Pharmaceutical Co. Ltd (Tokyo, Japan) and Nippon Kayaku Co. Ltd (Tokyo, Japan), respectively. Ofloxacin and ciprofloxacin were prepared from the respective marketed tablets. All reagents used were endotoxin-free, as determined by the limulus lysate assay, in which minimum detectable levels are 8 pg/L. Endotoxin-free 0.9% sodium chloride solution and distilled H2O (both injection grade, JP), buffers and salt solutions from Life Technologies Inc. (Tokyo, Japan) were used to help prevent inadvertent priming during the isolation procedure.

Preparation of neutrophils

Human blood neutrophils were isolated as previously described26 with minor changes.27 Briefly, venous blood donated from healthy volunteers with informed consent was collected on sodium citrate solution (3.8%) and centrifuged (110g, 10 min), and the platelet-rich plasma was discarded. The remaining part of the blood was mixed (1:1, v/v) with a solution of 3% dextran in 0.9% sodium chloride solution in a plastic syringe and fixed vertically for 20 min at 25°C. Neutrophil-rich plasma was collected from the upper layer of the suspension and centrifuged (250g, 10 min). The pellet was subjected to hypotonic lysis to destroy the remaining erythrocytes, centrifuged and then suspended in Hanks Balanced Salt Solution (HBSS, containing 10 mM HEPES, pH 7.4). The suspension was cushioned carefully on Histopaque solution (d = 1.077) and centrifuged (420g, 30 min) at 20°C. The purified neutrophils of the bottom pellet were finally resuspended in HBSS.

The purity of neutrophils was greater than 95%. Cell number was counted by a Coulter counter model ZM (Coulter Electronics Ltd, UK), diluted in HBSS to the final required concentrations and kept on ice until examined. To correct for possible time-dependent changes in neutrophils ex vivo, the time between preparation of neutrophils and their experiment was adjusted for all experiments to be ~4 h.

Superoxide measurement

Superoxide releasing activity of neutrophils was assessed by SOD-inhibitable reduction of ferricytochrome c28 with minor changes.29 Briefly, neutrophils (1 x 106 cells/mL) were incubated with a fluoroquinolone for indicated periods at 37°C. After incubation, 100 µM ferricytochrome c (horse ferricytochrome c, type IV) and fMLP (200 nM), LTB4 (100 nM) or PMA (10 nM) were added simultaneously and the mixture was then incubated for 5 min for fMLP and LTB4 or 30 min for PMA. The reaction was terminated by adding SOD under incubation on ice. The tubes were centrifuged at 500g for 10 min and the supernatants were examined with a dual wavelength spectrophotometer (U-best-50, JASCO, Japan) at 550–540 nm. One millimolar extinction coefficient of reduced cytochrome c at 550 nm is 29.5.

Luminol-dependent chemiluminescence measurement

Luminol-dependent chemiluminescence was measured as described previously.30 Aliquots of neutrophil preparation (1 mL, 2 x 105 cells/mL), fluoroquinolones and luminol (113 µM) were incubated at 37°C for 10 min before the addition of fMLP. The development of luminol-dependent chemiluminescence was continuously monitored by a six-channel photon counter (Biolumat LB 9505, Berthold, Bad Wildbad, Germany). Maximum peak count was used for the evaluation.

Determination of concentration of grepafloxacin in neutrophils

After incubation of neutrophils with varying doses of grepafloxacin, cells were separated from extracellular solution by centrifugation (10 000g, 3 min) through a water-impermeable silicon-oil barrier (SH550:SH556/1:4, Toray Dow Corning Co. Ltd, Japan) in a microcentrifuge tube. The neutrophil pellet formed on the bottom of the microcentrifuge tube and obtained by cutting off this portion of the microcentrifuge tube was resuspended with methanol and agitated vigorously in a vortex shaker. The samples were then centrifuged at 21 600g for 10 min, and the concentration of fluoroquinolones in supernatant was determined by HPLC (Shimadzu, Japan) with a spectrofluorometer (Waters, USA). The fluorescence excitation and emission maxima of grepafloxacin in methanol were 285 and 448 nm, respectively. The velocity of grepafloxacin accumulation in neutrophils was determined by the method described previously.13

Preparation of membrane fractions and immunochemical detection of p47-phox, p67-phox and phosphotyrosine

Neutrophils (107 cells/mL) were incubated with grepafloxacin (200 mg/L) in HBSS at 37°C for the indicated periods. Then, neutrophils were separated by centrifugation (100g, 5 min at 4°C), resuspended in relaxation buffer31 and sonicated. After centrifugation (100g, 10 min, at 4°C), the supernatant was separated by centrifugation (85 000g, 30 min) and the precipitate as the plasma membrane fraction was analysed for detection of p47-phox, p67-phox and phosphotyrosine.

Detection of p47-phox, p67-phox and phosphotyrosine was performed according to the method described previously.32,33 Briefly, the plasma membrane fractions obtained from grepafloxacin-treated and -untreated neutrophils were mixed (1:1, v/v) with lysis buffer composed of 2% SDS, 30% glycerol, 10% 2-mercaptoethanol and 0.01% Bromophenol Blue in 0.25 M Tris–HCl (pH 6.8),34 heated at 100°C for 5 min, subjected to electrophoresis on 10% SDS–polyacrylamide gels, electrotransferred to Immuno-Blot PVDF membrane (Bio-Rad), blocked for 1 h with Tris-buffered saline containing 0.1% Tween 20 (TTBS), and then incubated with anti-p47-phox (1:250), anti-p67-phox (1:2000) or anti-phosphotyrosine (1:1000) rabbit polyclonal antibodies in TTBS containing 5% dried milk for 18 h at 4°C. Blots were washed with TTBS and incubated with horseradish peroxidase-labelled anti-rabbit IgG (Amersham, UK). Bound antibodies were revealed by enhanced chemiluminescence western blotting detection reagents (Amersham, UK) using Fuji RX medical X-ray film.

Binding of [3H]fMLP to neutrophils

fMLP binding to neutrophils was studied by a modification of the method of Snyderman et al.20,34 Neutrophil suspension containing [3H]fMLP was incubated at 0°C for 90 min. Then, bound [3H]fMLP was separated from free [3H]fMLP by rapid filtration using a Cell Harvester over G-10 glass filters (Inotech, Switzerland). The filters were washed with 15 mL of ice-cold HBSS, dried and counted in a 4 mL scintillation cocktail using a scintillation counter (Beckmann-LS6500, USA). Specific binding of [3H]fMLP was calculated from the difference between the counts in the presence and absence of 10 µM fMLP.

Statistical analysis

All results are expressed as means ± S.D. Significant differences were analysed by analysis of variance (ANOVA) with Fisher’s Protected Least Significant Difference (PLSD) test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Modulation of fMLP-, LTB4- and PMA-induced superoxide generation by fluoroquinolones in neutrophils

We examined the effect of pre-incubating neutrophils with grepafloxacin, ciprofloxacin or ofloxacin on fMLP-, LTB4- and PMA-stimulated superoxide generation. Neutrophils at 106 cells/mL were pre-incubated with 1–200 mg/L grepafloxacin or ciprofloxacin, or 1–50 mg/L ofloxacin for 10 min at 37°C. The cells were then stimulated by adding 200 nM fMLP, 100 nM LTB4 or 10 nM PMA for 5, 5 or 30 min, respectively. The capability of fMLP to stimulate superoxide production was significantly enhanced following the pre-incubation of neutrophils with grepafloxacin or ciprofloxacin, but not with ofloxacin (Figure 1). The degree of the enhancing effect by grepafloxacin reached ~300% of the control at 200 mg/L, whereas ciprofloxacin showed at most 140%. Grepafloxacin enhanced LTB4-induced superoxide generation in a similar manner to fMLP (Figure 1a). The degree of the enhancement of LTB4-induced superoxide generation by grepafloxacin reached ~150% of the control (LTB4 alone) at 100 mg/L. In contrast, none of the fluoroquinolones tested enhanced PMA-stimulated superoxide generation from neutrophils (Figure 1c). The treatment of neutrophils with fluoroquinolones alone did not stimulate superoxide production in human blood neutrophils (data not shown).



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Figure 1. The effects of fluoroquinolones on fMLP-, PMA- and LTB4-stimulated superoxide generation in human neutrophils. Neutrophils were incubated with (a) grepafloxacin, (b) ciprofloxacin or (c) ofloxacin for 10 min, then stimulated by 200 nM fMLP (open circles), 100 nM LTB4 (filled squares) or 10 nM PMA (open triangles). Each value represents the mean ± S.D. of at least four independent experiments. Control value: 2.75 ± 0.45 nmol/106 cells for fMLP-stimulated, 2.31 ± 0.33 nmol/106 cells for LTB4 and 30.4 ± 2.57 nmol/106 cells for PMA-stimulated superoxide production. *P < 0.05, compared with the value of fMLP, LTB4 or PMA alone.

 
Effect of grepafloxacin on the time-profile of fMLP-induced superoxide generation in neutrophils

The priming effects of grepafloxacin and ciprofloxacin on fMLP-stimulated superoxide production were examined further by changing the pre-incubation time as follows: neutrophils were incubated at 37°C in the presence of grepafloxacin (50 and 200 mg/L) or ciprofloxacin (200 mg/L) for different time periods, and stimulated with 200 nM fMLP. The enhancement of fMLP-stimulated superoxide production was manifested dependent on the time of pre-incubation with grepafloxacin, attaining a maximum at 10 min pre-incubation (Figure 2) and gradually decreasing with longer pre-incubation periods. A similar time-profile of enhancement was obtained for ciprofloxacin, although the magnitude of enhancement was much smaller.



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Figure 2. The time course of the priming effect of grepafloxacin and ciprofloxacin on fMLP-induced superoxide production in human neutrophils. Neutrophils were pre-treated with 50 (open circles) or 200 mg/L grepafloxacin (open squares), or 200 mg/L ciprofloxacin (filled circles) for indicated periods on the abscissa at 37°C, then mixtures were stimulated by 200 nM fMLP. Each value represents the mean ± S.D. of at least three independent experiments. *P < 0.05, compared with the value of the control (at 0 min).

 
Effect of fluoroquinolones on luminol-dependent fMLP-induced chemiluminescence development in neutrophils

As shown in Figure 3, not only ciprofloxacin and ofloxacin but also grepafloxacin significantly enhanced luminol-dependent fMLP-stimulated chemiluminescence development in a dose-dependent manner.



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Figure 3. The effects of fluoroquinolones on fMLP-stimulated luminol-dependent chemiluminescence in human neutrophils. Neutrophils were incubated with grepafloxacin (open circles), ciprofloxacin (filled squares) or ofloxacin (open triangles) for 10 min, then stimulated with 200 nM fMLP. Each value represents the mean ± S.D. of at least three independent experiments. Control value: 2.76 ± 0.45 x 104 count/106 cells/3 s for fMLP-stimulated chemiluminescence development. *P < 0.05, compared with the value of fMLP alone.

 
Effect of removal of extracellular grepafloxacin on the grepafloxacin priming effect on fMLP-induced superoxide generation

To determine the influence of extracellular grepafloxacin on the priming effect of grepafloxacin on fMLP-stimulated superoxide generation, grepafloxacin priming was examined after the removal of the extracellular grepafloxacin by centrifugation (washing neutrophils). Neutrophils were incubated with different concentrations of grepafloxacin at 37°C for 10 min, the cell mixture was then washed by centrifugation and the pellet was resuspended with HBSS. fMLP-stimulated superoxide generation was measured by the cytochrome c method. Control neutrophils were treated in the same manner as the washed neutrophils except that the supernatant was not discarded following centrifugation. In these control neutrophils, fMLP-stimulated superoxide generation was dose-dependently enhanced by grepafloxacin treatment. Very similar results were observed in the washed neutrophils (Figure 4); there was no statistically significant difference in superoxide generation between the washed and unwashed neutrophils.



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Figure 4. The effect of removal of extracellular grepafloxacin after pre-loading on the priming of fMLP-induced superoxide generation by grepafloxacin. After pre-incubation of neutrophils with grepafloxacin at 37°C for 10 min, extracellular grepafloxacin was washed by centrifugation, then fMLP-stimulated superoxide generation was measured. White bars, control neutrophils (resuspended without discarding the washing medium after centrifugation); black bars, washed neutrophils (supernatant was discarded after centrifugation, then suspended with new medium). Each value represents the mean ± S.D. of at least three independent experiments. aP < 0.05, compared with the value of each control (without grepafloxacin); bnot significant, compared with the value of control neutrophils.

 
Correlation of grepafloxacin priming of superoxide generation with grepafloxacin accumulation in neutrophils

The magnitude of grepafloxacin accumulation was compared with grepafloxacin priming on fMLP-induced superoxide generation. The degree of grepafloxacin priming effect was highly correlated with that of grepafloxacin accumulation (Figure 5).



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Figure 5. Correlation of grepafloxacin priming effect on fMLP-stimulated superoxide generation (open circles) and kinetics of grepafloxacin uptake (filled circles) in neutrophils. The values of grepafloxacin priming effect on fMLP-stimulated superoxide generation are from Figure 1. The kinetics of grepafloxacin uptake are shown as grepafloxacin accumulating velocity. Cellular uptake of grepafloxacin for 1 and 5 min by neutrophils at the different extracellular concentrations of grepafloxacin (1, 10, 30, 100 and 200 mg/L) were determined, then the values of velocity for cellular grepafloxacin accumulation were calculated. Velocity (pmol/min/106 cells) = (grepafloxacin accumulation at 5 min – grepafloxacin accumulation at 1 min)/4 min.

 
Effect of extracellular calcium on the priming of fMLP-induced superoxide generation by grepafloxacin

We examined the effect of chelating extracellular calcium on the priming of fMLP-stimulated superoxide generation in neutrophils by grepafloxacin. The addition of 1 mM EGTA to the incubation medium significantly enhanced the priming effect of grepafloxacin on fMLP-induced superoxide generation (Figure 6).



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Figure 6. The priming effect of grepafloxacin on fMLP-induced superoxide generation in the presence of Ca2+ (white bars) or EGTA (black bars). Neutrophils were incubated with grepafloxacin in the presence or absence of 1 mM EGTA for 10 min, then fMLP-stimulated superoxide generation was determined. Each value represents the mean ± S.D. of at least three independent experiments. aSignificant differences at P < 0.05 compared with the value of each control (without grepafloxacin); bsignificant differences at P < 0.05 compared with the values in the presence of Ca2+.

 
Effects of calphostin C and ST-638 on grepafloxacin priming of fMLP-induced superoxide generation

Neither calphostin C (20 and 200 nM), a protein kinase C inhibitor,35 nor ST-638 (5, 10 and 20 µM), a tyrosine kinase inhibitor,36 affected the priming of fMLP-induced superoxide production by grepafloxacin (data not shown).

Translocation of p47-phox and p67-phox to plasma membrane and tyrosine phosphorylation in neutrophils

Next, we investigated whether the translocation of p47-phox and/or p67-phox to the plasma membrane might be involved in the priming effect of fMLP-stimulated superoxide generation by grepafloxacin. After having added grepafloxacin to the neutrophils, translocation of p47-phox to the membranes was promoted in a time-dependent manner, and reached a maximum at 10 min, maintaining that level for a further 20 min (Figure 7a). A similar result was obtained for p67-phox translocation (Figure 7b). On the other hand, we could not detect significant p47-phox translocation by ciprofloxacin or ofloxacin treatment (data not shown). No significant tyrosine phosphorylation in the membrane fraction was observed after the treatment of grepafloxacin (Figure 7c).



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Figure 7. The effects of grepafloxacin on the translocation of p47-phox protein (a) to plasma membrane fraction and p67-phox protein (b), and on tyrosine phosphorylation (c) in neutrophils. (a and b) Neutrophils were incubated with grepafloxacin (100 mg/L) for the indicated periods at 37°C, then p47-phox and p67-phox protein in plasma membrane fractions prepared from the cells were immunodetected as described in Materials and methods. Lane 1 in (a): control of p47-phox protein. (c) Neutrophils were incubated with grepafloxacin (100 mg/L) for the indicated periods at 37°C, then tyrosine phosphorylation was immunodetected as described in Materials and methods. Lane 1 in (c): molecular marker. Similar results were obtained in another two separate experiments.

 
Effects of pertussis toxin treatment on the grepafloxacin priming effect in fMLP-stimulated superoxide generation from neutrophils

To evaluate whether grepafloxacin interacts with GTP-binding protein-linked receptors, the effect of pertussis toxin treatment was investigated. Pertussis toxin inhibited superoxide generation in neutrophils stimulated by either fMLP alone or fMLP primed by grepafloxacin (Figure 8). In contrast, pertussis toxin did not inhibit PMA-induced superoxide generation in the presence or absence of grepafloxacin.



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Figure 8. The effect of pertussis toxin treatment on grepafloxacin-primed fMLP- (a) or PMA- (b) stimulated superoxide generation in neutrophils. Neutrophils were pre-treated with various concentrations of pertussis toxin at 37°C for 3 h. Neutrophils were incubated further with 200 mg/L grepafloxacin (black bars) or vehicle (white bars) for 10 min, then 200 nM fMLP- (a) or 10 nM PMA- (b) stimulated superoxide production was determined. Each value represents the mean ± S.D. of four independent experiments. *P < 0.05, compared with the value of each control (without pertussis toxin).

 
Effects of pertussis toxin treatment on the grepafloxacin-induced translocation of p47-phox protein to the plasma membrane fraction of neutrophils

We investigated the effect of pertussis toxin on the grepafloxacin-induced translocation of p47-phox protein to the membrane fractions. Grepafloxacin treatment of neutrophils for 10 min resulted in a translocation of p47-phox to the membranes (Figure 9, lane 3). This translocation was not affected by the pre-treatment of neutrophils with pertussis toxin for 3 h (Figure 9, lane 4). Pertussis toxin by itself also did not show any effect on the control (Figure 9, lanes 1 and 2).



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Figure 9. The effect of pertussis toxin treatment on the grepafloxacin-induced translocation of p47-phox protein to plasma membrane fraction of neutrophils. Neutrophils were incubated with pertussis toxin (100 ng/mL, lanes 2 and 4) or vehicle (lanes 1 and 3) at 37°C for 3 h, then grepafloxacin (200 mg/L, lanes 3 and 4) or vehicle (lanes 1 and 2) were treated for 10 min at 37°C. p47-phox protein in plasma membrane fractions prepared from the cells were immunodetected as described in Materials and methods. Similar results were obtained in another two independent experiments. Abbreviation: GRX, grepafloxacin.

 
Effect of grepafloxacin on the binding characteristics of [3H]fMLP to neutrophils

Typical Scatchard plots of equilibrium binding of [3H]fMLP to neutrophils were investigated. Neutrophils expressed 12 500 ± 740 receptors/cell (n = 3), with a Kd of 2.5 ± 0.5 nM. Grepafloxacin did not show any effects on these [3H]fMLP binding characteristics (Figure 10).



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Figure 10. The equilibrium binding of [3H]fMLP to neutrophils as analysed by Scatchard analysis. Neutrophils were pre-incubated with 200 mg/L grepafloxacin (closed circle) or vehicle (open circle) for 10 min at 37°C, then incubated further with increasing amounts of [3H]fMLP at 0°C for 90 min. Specific binding of [3H]fMLP to neutrophils was performed as described in Materials and methods. Similar results were obtained in another two independent experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been highlighted recently that several fluoroquinolones may play important roles in host defence by priming mature human phagocytes, such as neutrophils. For example, the ability of fMLP and/or PMA to stimulate superoxide production is significantly enhanced by pre-incubation of neutrophils with several fluoroquinolones.2225 However, the exact mechanism of this priming effect still remains unclear. In this study we have shown that the newly developed fluoroquinolone, grepafloxacin, has a remarkable priming effect on superoxide generation from human neutrophils. Furthermore, we provide evidence that grepafloxacin may act on the targets in neutrophils from inside to accelerate the translocation of p47-phox protein and p67-phox to cellular membranes. Thus, we can conclude that grepafloxacin facilitates the collaboration of its own antimicrobial activities and host defence mechanisms exhibited by neutrophils to greatly improve the antimicrobial effects as a whole.

Although it has been reported that ofloxacin shows a priming effect on both PMA- and fMLP-stimulated superoxide generation,22,23 we were unable to show it in the present study. This discrepancy may be due to the difference in superoxide detection systems: we employed the cytochrome c reduction method, whereas superoxide anion production was measured by using a luminol-dependent chemiluminescence technique in all of the previously published literature. In the present study, not only grepafloxacin but also ofloxacin showed a priming effect on luminol-dependent fMLP-induced superoxide generation in neutrophils. Since luminol detects mainly OH-radicals rather than superoxides, it is possible that ofloxacin may activate primarily an OH-radical generation system rather than a superoxide-generating system. In contrast, grepafloxacin may activate both superoxide- and OH-radical-generating systems.

It has been reported that several fluoroquinolones produce convulsive seizures when co-administered with specified non-steroidal anti-inflammatory drugs, such as fenbufen. It is recognized that free hydrogen in the 4N position in a piperazine ring, as exists in enoxacin, is thought to be essential for this convulsive activity. When this hydrogen is substituted with a bigger molecule, such as a methyl residue as in ofloxacin, convulsive properties are significantly decreased. In view of the grepafloxacin structure, the methyl residue in the 3 position in a piperazine ring may have some relation to the priming effect, because neither ofloxacin nor ciprofloxacin has such a residue in their molecules, and both showed only a weak priming effect. Further investigation will be required to elucidate the precise mechanism.

Grepafloxacin produced a greater enhancing effect for fMLP than for PMA. Grepafloxacin enhanced fMLP-induced superoxide generation by ~300% of control, whereas the priming effect of grepafloxacin on PMA-induced superoxide generation was very small. Similar observations have been reported for the priming effect produced by several cytokines, such as GCSF and TNF-{alpha}.1821 These cytokines bind to their respective specific receptors on the cell membranes of neutrophils to prime superoxide generation.20 The present observations, that despite the removal of extracellular grepafloxacin neutrophils still maintained their primed state and that a lack of intracellular grepafloxacin led to the absence of priming, strongly suggest the action of grepafloxacin on neutrophils from inside to exert the priming effect. In fact, the degree of grepafloxacin priming effect was highly correlated with intracellular grepafloxacin accumulation.

It has been reported20 that pre-treatment of neutrophils with cytochalasin B produces priming effects on both fMLP- and PMA-stimulated superoxide generation. Priming effects of cytochalasin B are considered to result from phospholipase D activation, and Ca2+ is essential for this activation.37 The priming effect of grepafloxacin on fMLP-induced superoxide generation was not inhibited by chelating extracellular Ca2+, whereas that of cytochalasin B was significantly inhibited. This suggests that the underlying mechanisms for priming neutrophil superoxide production are different. Interestingly, EGTA treatment of neutrophils to remove extracellular Ca2+ enhanced the priming effect of grepafloxacin on fMLP-stimulated superoxide generation. Since EGTA treatment of neutrophils enhanced levofloxacin accumulation,38 it is possible that increased intracellular grepafloxacin enhanced the priming effect, if EGTA increased grepafloxacin accumulation. However, as shown by our preliminary results, since EGTA did not affect grepafloxacin uptake in neutrophils, enhancement by EGTA of grepafloxacin priming is not due to the increase in penetration of grepafloxacin. EGTA alone did not show any effect on fMLP-stimulated superoxide generation. EGTA affects the grepafloxacin priming systems by an as yet unknown mechanism.

To activate NADPH (nicotinamide adenine dinucleotide phosphate) oxidase, at least four different protein components are required: membrane-bound cytochrome b558 and three cytosolic proteins, p47-phox, p67-phox and low molecular weight GTP-binding protein.39 The catalytic activity of the oxidase is located in the plasma membrane, but at the resting stage of cells the oxidase components are distributed between the plasma membrane and cytosol. When activated, at least some of the cytosolic components such as p47-phox and p67-phox are translocated to the membrane.40,41 Stimulation of neutrophils with certain agents, such as fMLP or PMA, promotes phosphorylation of p47-phox and/or p67-phox to promote their distribution to the membrane.42,43 Based on the recent finding that granulocyte/macrophage colony-stimulating factor (GMCSF) induces partial phosphorylation of p47-phox, but not p67-phox, in neutrophils,44 it has been suggested that p47-phox protein serves as an important factor in the priming effect of GMCSF on the respiratory burst. In the present study we observed that grepafloxacin induced translocation of both p47-phox and p67-phox protein to neutrophil plasma membrane in parallel with priming of superoxide production by neutrophils in response to fMLP. This suggests that both p47-phox and p67-phox protein translocation are involved in the priming effect of neutrophils by grepafloxacin. DeLeo et al.45 recently reported that priming of the respiratory burst by lipopolysaccharide resulted in translocation of cytochrome b and p47-phox but not p67-phox or rac2. On the other hand, Green et al.46 reported that priming of NADPH oxidase by interleukin-8, another proinflammatory cytokine, does not induce translocation of cytosolic oxidase component. As GMCSF did not affect the translocation of either p47-phox or p67-phox,34 the mechanism underlying grepafloxacin priming could be different from those underlying GMCSF and interleukin-8 priming.

It has been reported that the effect of GCSF or TNF-{alpha} on neutrophil respiratory burst is regulated through tyrosine phosphorylation by tyrosine kinase,47,48 since it is inhibited by pre-treatment with tyrosine kinase inhibitors, such as genistein and ST-638.3,49 The present study indicated that grepafloxacin priming of the neutrophil respiratory burst does not involve tyrosine phosphorylation, since grepafloxacin priming of fMLP-induced superoxide generation was not inhibited by the pre-treatment with ST-638, and grepafloxacin produced no significant tyrosine phosphorylation. These findings also indicate that grepafloxacin differs from cytokines such as GCSF and TNF-{alpha} in priming neutrophils.

fMLP and LTB4 receptors are coupled to pertussis toxin-sensitive GTP-binding proteins and their agonists induce superoxide generation, which is inhibited by treatment with pertussis toxin.37 In the present study, we confirmed an inhibitory effect on fMLP-stimulated superoxide generation by pertussis toxin treatment. Superoxide generation in grepafloxacin-primed neutrophils triggered by fMLP was also significantly inhibited by the pertussis toxin treatment to almost the same degree seen in the absence of grepafloxacin. To confirm the pertussis toxin effect, we also investigated the effect of pertussis toxin on the translocation of p47-phox protein to the membrane fractions by grepafloxacin. Pertussis toxin did not inhibit translocation of p47-phox protein. These results suggest that the priming effect of grepafloxacin is unrelated to pertussis toxin-sensitive GTP-binding proteins. In addition, receptor binding analysis showed that grepafloxacin has no effect on fMLP receptor binding. This result indicates that the target site of grepafloxacin to produce priming is not the agonist–receptor interaction mechanism and that another mechanism is involved in the activation of p47-phox and p67-phox translocations.

Recently, grepafloxacin has been withdrawn from clinical use in the USA. However, this drug has unique properties affecting neutrophil function, such as high penetrating ability and in showing a priming effect. By elucidating the underlying mechanisms of these effects, grepafloxacin could provide a model for the development of new types of antimicrobial agents having activation properties in the host defence system.

In conclusion, the data represented here show that grepafloxacin primes the neutrophil respiratory burst, and that translocation of p47-phox to plasma membrane plays a key role, although further investigation will be required to elucidate precise mechanism(s).


    Acknowledgements
 
We thank Dr M. Hirota of Otsuka Pharmaceutical Company for measuring the fluoroquinolone antibiotics and for valuable discussion. This work was supported in part by a grant-in-aid for Scientific Research (Nos 12470015 and 13670084) from the Ministry of Education, Science, Sports and Culture of Japan.


    Footnotes
 
* Corresponding author: Tel: +81-58-267-2624; Fax: +81-58-267-2935; E-mail: mniwa{at}cc.gifu-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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