Uptake and Intracellular Activity of Moxifloxacin in Human Neutrophils and Tissue-Cultured Epithelial Cells

Isolation of PMN.PMN were recovered from heparinized venous blood of healthy donors by using dextran sedimentation and a Ficoll-Hypaque gradient and purified by previously described methods (16). PMN preparations were 97% pure. Final cell suspensions were adjusted to 5 × 10⁶ PMN per ml in Hanks balanced salt solution (HBSS) containing 1% gelatin. PMN were 95% viable as determined by trypan blue exclusion.

Tissue culture cells.McCoy cells (Flow Laboratories, Irvine, United Kingdom) were grown in minimal essential medium (Flow) supplemented with 1 mM HEPES (Flow) and containing 10% fetal calf serum (Flow) without antibiotics. For each experiment, the cells were detached from tissue culture bottles with trypsin-EDTA (Flow), washed once with minimal essential medium containing fetal calf serum (10%), and suspended in HBSS at a concentration of 5 × 10⁶per ml.

Moxifloxacin uptake by cells.A previously described fluorometric assay was used to measure quinolone uptake by human PMN and epithelial cells (11). Moxifloxacin was kindly supplied by Bayer AG, Leverkusen, Germany. In these experiments, PMN or tissue cells were incubated in HBSS containing different concentrations of antimicrobial agent (1 to 50 mg/liter). After different incubation times at 37°C, cells were separated from the extracellular solution by centrifugation through a water-impermeable silicone-oil barrier (density, 1,029 g/cm³) in a microcentrifuge tube. The entire cell pellet, obtained by cutting off the portion of the microcentrifuge tube containing the pellet, was placed in 2 ml of 0.1 M glycine-HCl buffer (pH 3.0) and agitated vigorously in a vortex shaker. Incubation for 2 h at room temperature was sufficient to release the intracellular antimicrobial agent fully (11). Samples were centrifuged for 5 min at 5,600 × g , and the amount of antimicrobial agent was determined by fluorescence emission of supernatants with an F 2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The fluorescence excitation and emission maxima in 0.1 M glycine-HCl (pH 3.0) were 295 and 498 nm, respectively. Controls without antimicrobial agents were always used to determine the background fluorescence.

Intracellular water space was measured by using tritiated water and the extracellular marker [¹⁴C]polyethylene glycol (1.4 mCi/g; New England Nuclear Corp., Boston, Mass.). Cells were incubated with these radiolabeled compounds for 2 min at 37°C, separated from extracellular fluid by velocity gradient centrifugation as described above, and counted in a liquid scintillation counter. Total water content of the cell pellet was corrected for trapped extracellular water, i.e., polyethylene glycol space, to obtain the intracellular water space. From the values obtained by this procedure, cell-associated antimicrobial agent concentrations were calculated and expressed as ratios of the cellular concentration to extracellular concentration (C/E ratios) (8).

The efflux of reversibility of the binding of PMN or tissue cell-associated moxifloxacin was also studied. Cells were incubated for 20 min at 37°C with moxifloxacin (extracellular concentration, 5 mg/liter), collected by centrifugation, and rapidly suspended in quinolone-free medium. Cell-associated moxifloxacin was quantitated at various intervals (1, 5, 10, 20, and 30 min) after removal of the extracellular antimicrobial agent. All assays were performed in duplicate with PMN from five donors.

Characterization of moxifloxacin uptake.Further studies to elucidate the mechanism of moxifloxacin uptake by PMN were performed as described previously (11). The importance of cell viability was studied by using PMN killed by exposure to 10% formalin for 30 min. These cells were washed and then suspended in fresh medium. Moreover, the influences of environmental temperature, pH, and metabolic inhibitors were evaluated. The influence of temperature was examined by comparing antimicrobial uptake at 4 and 37°C. The pH profiles of moxifloxacin uptake in media preadjusted to different external pHs (pH 5, 6, 7, and 8) by the addition of 10 N HCl or 10 N NaOH were measured. An inhibitor of glycolysis (sodium fluoride, 1.5 × 10⁻³ M) (Sigma Chemical Co., St. Louis, Mo.), an inhibitor of mitochondrial oxidative metabolism (sodium cyanide, 1.5 × 10⁻³ M) (Sigma), a blocker of the proton gradient (carbonyl cyanide m -chlorophenylhydrazone [CCCP]; 1.5 × 10⁻⁵ M) (Sigma), and an uncoupler of oxidative phosphorylation (2,4-dinitrophenol; 1 × 10⁻⁴ M) (Sigma) were used as metabolic inhibitors. PMN in HBSS with and without metabolic inhibitors were incubated for 30 min at 37°C. Moxifloxacin (final concentration, 5 mg/liter) was then added, and the uptake was measured as described above.

In a series of experiments, moxifloxacin (extracellular concentration, 5 mg/liter) uptake by human PMN was measured after the stimulation of cells with 200 nM phorbol myristate acetate (PMA; Sigma) and after the phagocytosis of S. aureus ATCC 25923 opsonized in 5% pooled human serum (15 min, 37°C) at a 10/1 ratio of bacteria to PMN. PMA or opsonized bacteria were added to PMN suspensions at the same time as the antimicrobial agent, and the uptake was measured as described above. Controls were always used to evaluate the effects of inhibitors and substrates on the fluorescence of moxifloxacin in cell-free systems. All assays were performed in duplicate with PMN from five different donors.

Organisms and susceptibility testing. S. aureus ATCC 25923 was used for the killing assays. Susceptibility studies were determined by dilution assay. The MICs and minimum bactericidal concentrations of ciprofloxacin (Bayer AG), ofloxacin (Hoechst AG), and moxifloxacin for this strain were 0.25, 0.25, and 0.06 mg/liter, respectively.

Intracellular activity of antimicrobial agents.To evaluate the intracellular activity of antimicrobial agents, a previously described method was used (15). Briefly, 0.1 ml of bacterial suspension preopsonized in 5% pooled human serum (5 × 10⁷ CFU/ml) and 0.1 ml of PMN (5 × 10⁶per ml) were combined in a series of polypropylene biovials (Beckman) and the vials were incubated in a shaker (50 rpm) for 60 min at 37°C. After incubation, the mixtures were washed three times with 2.5 ml of ice-cold phosphate-buffered saline (pH 7.2) by using differential centrifugation (160 × g ; 5 min at 4°C) to remove the extracellular bacteria. The cells were then suspended in 0.2 ml of RPMI medium (Sigma). At this time (designated time zero) different concentrations of the different fluoroquinolones (0.125 to 5 mg/liter) were added, and the vials were reincubated in a shaker (50 rpm) at 37°C. The vials were removed at time zero (control without antimicrobial agents) and after 3 h of incubation (control without antimicrobial agents and samples with antimicrobial agents). Cells were lysed in distilled water, and samples were diluted and pour plated in agar. Colonies were counted after 24 h of incubation at 37°C. The data were expressed as percentages of staphylococci surviving, compared with the levels in the controls (without antimicrobial agents) at 3 h. In addition to determining bacterial survival, morphologic studies were also routinely performed at time zero and after 3 h of incubation to evaluate the disposition of bacteria (cell associated or extracellular). Samples (50 μl) were removed from biovials and were deposited on glass slides. After being stained with Wright stain, the samples were examined by light microscopy. All assays were performed in duplicate with PMN from five different donors.

Statistical analysis of data.Data were expressed as means ± standard deviations. Differences among groups were compared by analysis of variance, used to assess statistical significance at a P value of ≤0.05.