Cellular Accumulation and Activity of Quinolones in Ciprofloxacin-Resistant J774 Macrophages

Abstract

Ciprofloxacin is the substrate for a multidrug resistance-related protein (MRP)-like multidrug transporter in J774 mouse macrophages, which also modestly affects levofloxacin but only marginally affects garenoxacin and moxifloxacin (J. Active efflux is a general means developed by cells for protection against invasion by amphiphilic, potentially harmful molecules (18). In this context, overexpression of multidrug efflux pumps is now recognized as a common and widespread mechanism of resistance to anticancer agents in eukaryotic cells (for a review, see reference 3). These pumps often display broad substrate specificities (7,15). It is therefore not surprising that they also transport other amphipathic drugs like antibiotics (for a review, see reference 19). In this context, we showed that the fluoroquinolone ciprofloxacin is subject to active efflux by a multidrug resistance-related protein (MRP)-like transporter in J774 mouse macrophages (11). The activity of this transporter significantly reduces the accumulation of ciprofloxacin in comparison with other drugs of the same class (10) and, as a consequence, impairs its activity against intracellular bacteria such as Listeria monocytogenes (13).-In an attempt to better characterize the transporter of ciprofloxacin, we have generated J774 macrophages with increased efflux capabilities toward this drug. We applied a procedure commonly used to select resistance to anticancer drugs (6), namely, the continuous exposure to progressively increasing concentrations of the drug under study. This methodology, which can select multifactorial resistance, is intended to mimic to some extent what may develop in vivo upon chronic exposure to the corresponding drug (6). We obtained stable cell lines resistant to 68 mg/liter (0.2 mM) ciprofloxacin. The present paper deals with a description of the quinolone pharmacokinetics and pharmacodynamics in these cells in comparison with the wild-type, parent cell line. MATERIALS AND METHODSCell culture and selection of ciprofloxacin-resistant J774 macrophages. All experiments were performed with J774 mouse macrophages. Wild-type cells were maintained exactly as reported previously (11). To select ciprofloxacinresistant cells, we used a stepwise approach similar to that described previously for obtaining probenecid-resistant J774 macrophages (1, 11). Based on preliminary experiments evaluating ciprofloxacin cytotoxicity in wild-type cells, a concentration of 34 mg/liter (0.1 mM) was used as a first selection step. After 4 weeks (i.e., up to passage 6), the ciprofloxacin concentration was increased to 51 mg/liter (0.15 mM) for 3 weeks (from passage 7 to 9) and then further increased to 68 mg/liter (0.2 mM) for 6 months (up to passage 30). At each increase in ciprofloxacin concentration, cells showed a marked but transient (1 to 2 passages) decrease in their multiplication rates, after which, however, they resumed at almost normal growth. Cells were then used for experiments up to the 120th passage, while being maintained in the continuous pr...

Modulation of the Cellular Accumulation and Intracellular Activity of Daptomycin towards Phagocytized Staphylococcus aureus by the P-Glycoprotein (MDR1) Efflux Transporter in Human THP-1 Macrophages and Madin-Darby Canine Kidney Cells

“…The present data extend our previous observations underlining the role of the ATP-energized efflux transporters for modulation of the accumulation and activity of antibiotics in macrophages (33,34,46,47). Specifically, we show here that the activity of P-gp in THP-1 human macrophages decreases the relative potency of daptomycin towards phagocytized S. aureus by reducing its cellular concentration.…”

Identification of the Efflux Transporter of the Fluoroquinolone Antibiotic Ciprofloxacin in Murine Macrophages: Studies with Ciprofloxacin-Resistant Cells

“…This was achieved by longterm culture in the presence of progressively increasing drug concentrations, a procedure successfully used to select cells with efflux-mediated resistance to anticancer agents (11) (while fluoroquinolones act as antibiotics by impairing bacterial topoisomerases at low concentrations, they inhibit the eukaryotic topoisomerases II and kill mammalian cells at large concentrations). The phenotype of these cells has been described in a previous publication (24). They display a markedly reduced accumulation of ciprofloxacin, which could be brought almost to control levels by ATP depletion or addition of typical inhibitors of MRP efflux transporters, such as probenecid or MK571.…”

“…They display a markedly reduced accumulation of ciprofloxacin, which could be brought almost to control levels by ATP depletion or addition of typical inhibitors of MRP efflux transporters, such as probenecid or MK571. Additional studies comparing other quinolones in both wild-type and ciprofloxacin-resistant cells showed that moxifloxacin was not affected by this efflux transporter in both cell types, while levofloxacin and garenoxacin showed an intermediate behavior (23,24). Beyond displaying a markedly reduced accumulation of ciprofloxacin, these ciprofloxacinresistant cells provide a protected environment against ciprofloxacin for Listeria monocytogenes (24).…”

“…Additional studies comparing other quinolones in both wild-type and ciprofloxacin-resistant cells showed that moxifloxacin was not affected by this efflux transporter in both cell types, while levofloxacin and garenoxacin showed an intermediate behavior (23,24). Beyond displaying a markedly reduced accumulation of ciprofloxacin, these ciprofloxacinresistant cells provide a protected environment against ciprofloxacin for Listeria monocytogenes (24). Moreover, we recently showed that this eukaryotic transporter can cooperate with a prokaryotic ciprofloxacin transporter to make intracellular L. monocytogenes still more resistant to the drug (20).…”

Comparison of the In Vitro Efficacies of Moxifloxacin and Amoxicillin against Listeria monocytogenes

“…In our model, moxifloxacin diffused and accumulated quickly in cellular compartments, killing the intracytoplasmic forms of L. monocytogenes within the first 3 h (2, 22, 30). Despite the overexpression and/or increased activity of the MRP-like ciprofloxacin transporters, reported in ciprofloxacinresistant J744 macrophages, the intracellular accumulation of moxifloxacin would not be significantly altered, as moxifloxacin is only partially effluxed (21,22).…”