Journal of Antimicrobial Chemotherapy (2002) 49, 953–959 DOI: 10.1093/jac/dkf042
Regulation of fluoroquinolone uptake by human neutrophils: involvement of mitogen-activated protein kinase Koichi Hotta1,2, Masayuki Niwa1,3*, Masao Hirota4, Yutaka Kanamori1, Hiroyuki Matsuno1, Osamu Kozawa1, Takanobu Otsuka2, Nobuo Matsui2 and Toshihiko Uematsu1 1Department
of Pharmacology and 3Medical Education Development Center, Gifu University School of Medicine, 40-Tsukasamachi, Gifu 500-8705; 2Department of Orthopedic Surgery, Nagoya City University Medical School, Nagoya 467-8601; 4Department of Drug Metabolism, Drug Safety Center, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd, Tokushima 771-0192, Japan Received 24 September 2001; returned 13 December 2001; revised 28 January 2002; accepted 28 February 2002
Although human neutrophils actively internalize fluoroquinolones, the precise uptake mechanism is not fully understood. In this study, we investigated the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in fluoroquinolone uptake in neutrophils. Spontaneous grepafloxacin uptake was significantly enhanced by SB203580, a p38 MAPK inhibitor, in a dose-dependent manner, but not by PD98059, a specific inhibitor of the upstream kinase that activates p44/42 MAPK. Neither inhibitor affected spontaneous ciprofloxacin or ofloxacin uptake. Phorbol myristate acetate (PMA) treatment enhanced ciprofloxacin uptake, whereas it reduced grepafloxacin uptake. These effects by PMA were significantly inhibited by the pretreatment of neutrophils with GF109203X, a specific inhibitor of PKC. PMA had no effect on ofloxacin uptake. The PMA-induced enhancement of ciprofloxacin uptake was inhibited by PD98059, but not by SB203580. On the other hand, the PMA-induced reduction of grepafloxacin uptake was not inhibited by either MAPK inhibitor. Grepafloxacin, but not ciprofloxacin or ofloxacin, strongly phosphorylated p38 MAPK. This phosphorylation of p38 MAPK was not inhibited by GF109203X pretreatment. None of these three fluoroquinolones phosphorylated p44/42 MAPK. PMA phosphorylated both p38 and p44/42 MAPK. These findings indicate that grepafloxacin negatively regulates its uptake in neutrophils, and p38 MAPK activation is involved in this down-regulation of grepafloxacin uptake. Ciprofloxacin uptake is positively regulated by the activation of PKC, and p44/42 MAPK activation is involved in this up-regulation. Neither PKC, p38 nor p44/42 MAPK is involved in the regulation of ofloxacin uptake. Keywords: fluoroquinolone, human neutrophils, MAPK, PKC
Introduction Antimicrobial agents that accumulate and remain active inside phagocytic cells are particularly useful for treating infections by intracellular bacteria. Neutrophils usually play crucial roles in host defence mechanisms, including the phagocytosis of bacteria. Many fluoroquinolones have been reported to accumulate in human neutrophils. This process enhances the killing of intracellular pathogens and could facilitate the delivery of these agents to infection sites by
migrating neutrophils. The cellular-to-extracellular concentration (C/E) ratios of several fluoroquinolones have been reported previously.1,2 Grepafloxacin is a fluoroquinolone that possesses antimicrobial activity towards a broad spectrum of bacteria, and has one of the highest C/E ratios.3,4 However, the precise mechanism determining the uptake of fluoroquinolones into phagocytic cells is not yet fully elucidated. Recently, Loo et al.5 reported that protein kinase C (PKC) activation by phorbol myristate acetate (PMA) enhances the
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*Corresponding author. Tel: +81-58-267-2233; Fax: +81-58-267-2959; E-mail:
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953 © 2002 The British Society for Antimicrobial Chemotherapy
K. Hotta et al. uptake of ciprofloxacin by human neutrophils. It may be logical to hypothesize that fluoroquinolone uptake is enhanced as a consequence of human neutrophil priming or activation of host defences.6 It has also been reported that mitogenactivated protein kinase (MAPK) is involved in human neutrophil activation by various stimuli, such as tumour necrosis factor, colony simulating factor, N-formyl-methionyl-leucylphenylalanine and PMA. Loo et al.5 also suggested that p44/42 MAPK is involved in a PMA-induced ciprofloxacin uptake enhancement effect in human neutrophils. However, the precise mechanism of this process has not yet been fully clarified, and it is not known whether other fluoroquinolones are also regulated by PKC and/or MAPK systems. In this report, to evaluate whether PKC activation is involved in fluoroquinolone uptake by human neutrophils, we tested the effect of PMA stimulation and/or GF109203X,7 a specific PKC inhibitor, pretreatment on the uptake of grepafloxacin, ciprofloxacin and ofloxacin. Furthermore, we also examined the contribution of MAPK systems to the uptake of these fluoroquinolones in human neutrophils.
Materials and methods Chemicals and reagents Grepafloxacin and 1-cyclopropyl-6,8-difluoro-1,4-dihydro5-ethyl-7-(4-methyl-1-piperazinyl)-4-oxo-3-quinoline carboxylic acid (OPC-17203, used as an internal standard for the measurement of fluoroquinolones) were provided by Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan). Ciprofloxacin was provided by Bayer Japan Pharmaceutical Co., Ltd (Tokyo, Japan). Ofloxacin and PMA 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. SB203580 was obtained from SmithKline Beecham Pharmaceuticals (King of Prussia, PA, USA). PD98059 was obtained from Calbiochem-Novabiochem (La Jolla, CA, USA). GF109203X was purchased from BioMol (Plymouth Meeting, PA, USA). Rabbit polyclonal antibodies against Thr-180/Tyr-182-phosphorylated p38 MAPK, p38, Thr-202/Tyr-204-phosphorylated p44/42 and p44/42 were purchased from Cell Signalling Technology (Beverly, MA, USA). The enhanced chemiluminescence (ECL) western blotting system was purchased from Amersham Pharmacia Biotech (Tokyo, Japan). SB203580, PD98059 and GF109203X were dissolved in dimethylsulphoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the measurement of p38 or p44/42 MAPK activity, or the assay for fluoroquinolone uptake. All reagents used were endotoxin free, as determined by the limulus lysate assay, in which minimum detectable levels were 0.03 enzyme unit/mL.
Preparation of human neutrophils Human neutrophils from healthy donors were isolated as previously described8 with minor changes.9 Briefly, venous blood was collected in sodium citrate solution (3.8%), centrifuged (110g, 10 min), and 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 HBSS (Hanks Balanced Salt Solution 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 neutrophil pellet was finally resuspended in HBSS. Cell number was counted by a Coulter counter model Z1 (Coulter Electronics Ltd, Bedfordshire, UK), and diluted in HBSS to the final required concentrations and kept on ice until examined. The purity of neutrophils was >95%. The viability of neutrophils used was >95%, as evaluated by the trypan blue exclusion test. Informed consent was obtained from all donors.
Determination of fluoroquinolones in human neutrophils The previously described high performance liquid chromatography (HPLC)-fluorometric assay10 was used to measure fluoroquinolone uptake by human neutrophils. Human neutrophils (5 × 10 6 cells/mL) were pretreated with or without various inhibitors in HBSS for 10 min and were then incubated for 1–60 min with different concentrations of fluoroquinolones in the presence or absence of PMA (0–100 nM). After incubation, cells were separated from extracellular solution by centrifugation (10 000g, 3 min) through a waterimpermeable silicon–oil barrier (SH550:SH556/1:4; Toray Dow Corning Co. Ltd, Tokyo, Japan) in a microcentrifuge tube. The human neutrophil pellet formed on the bottom of the microcentrifuge tube was obtained by cutting off this portion of the microcentrifuge tube and resuspending it with methanol by agitating vigorously on a vortex shaker. These samples were then centrifuged at 21 600g for 10 min, and the concentration of fluoroquinolones in the supernatant was determined by HPLC with a spectrofluorimeter (Shimadzu, Kyoto, Japan). The fluorescence excitation and emission maxima of fluoroquinolones in methanol were 285 and 448 nm, respectively. The intracellular concentration of fluoroquinolones was expressed as pmol per 106 neutrophils. A previously determined intracellular volume of 3.3 × 10–13 L was used to determine the C/E ratios of fluoroquinolones.11
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Fluoroquinolone uptake by human neutrophils
Western blotting Human neutrophils (5 × 106 cells/mL) suspended in HBSS were pretreated with or without various inhibitors for 10 min at 37°C and were then stimulated with grepafloxacin (0– 200 mg/L), ciprofloxacin (0–200 mg/L), ofloxacin (0–50 mg/L) or PMA (0–100 nM) for the indicated period up to 20 min. The reactions were terminated by 1:10 dilution with cold HBSS and centrifugation at 250g for 10 min at 4°C. The cell pellets were treated as previously described.12 In short, they were resuspended in ice-cold solution containing 50 mM HEPES (pH 7.4), 1% (v/v) Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride, 100 mg/L aprotinin and 10 mg/L leupeptin, and were lysed for 60 min at 4°C. After centrifugation, the supernatant was mixed 1:1 (v/v) with 2× sample buffer [4% (w/v) sodium dodecyl sulphate (SDS), 20% (v/v) glycerol, 10% (v/v) mercaptoethanol and a trace amount of bromophenol blue dye in 125 mM Tris–HCl, pH 6.8], boiled for 5 min and then frozen at –30°C until used. Samples were subjected to SDS–PAGE [10% (w/v)] at 20 mA for 90 min. After electrophoresis, proteins were electrophoretically transferred from the gel on to 0.2 µm polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) at 10 V for 30 min. Blots were blocked with 5% (w/v) skimmed milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST) overnight at 4°C. The next day, blots were washed three times with TBST for 10 min, and were then incubated with primary antibodies against phospho-p38, p38, phosphop44/42 and p44/42 overnight at 4°C. Antibodies were used at a dilution of 1:1000 in 5% (w/v) bovine serum albumin (fraction V) in TBST. Blots were washed with TBST three times and were incubated for 1 h with the secondary antibody goat anti-rabbit IgG-peroxidase (Chemicon International Inc., Temecula, CA, USA) at a dilution of 1:1000 in 5% (v/v) skimmed milk in TBST. Blots were subsequently washed three times with TBST and were visualized by the ECL detection system, as directed by the manufacturer.
Results Uptake of fluoroquinolones by human neutrophils The time profiles of the uptake of the fluoroquinolones grepafloxacin (50 and 200 mg/L), ciprofloxacin (50 and 200 mg/L) and ofloxacin (50 mg/L) by human neutrophils were investigated. We selected these concentrations based on the results of a previous report.4 Grepafloxacin was rapidly taken up by human neutrophils, reaching a maximum at c. 5 min (Figure 1a). We have previously reported4 that the Km value of grepafloxacin uptake by human neutrophils was c. 170 µM, calculated using the Eadie–Hofstee plot. Similar to grepafloxacin, ciprofloxacin (50 and 200 mg/L) was also rapidly
Figure 1. Time profiles of uptake of fluoroquinolones by human neutrophils. (a) Grepafloxacin (filled circles, 50 mg/L; empty circles, 200 mg/L) and (b) ciprofloxacin (filled squares, 50 mg/L; empty squares, 200 mg/L). Uptake abilities are shown as the C/E ratio of fluoroquinolones, measured as described in Materials and methods. Results are expressed as means ± S.D. of three to five experiments run in duplicate.
taken up by human neutrophils, and reached a maximum at c. 20 min (Figure 1b). Ofloxacin was also rapidly taken up by human neutrophils, and reached a maximum at c. 5 min (data not shown). The absolute amounts and C/E ratios of ciprofloxacin and ofloxacin were
