Identification of the efflux transporter of the fluoroquinolone antibiotic ciprofloxacin in murine macrophages: Studies with ciprofloxacin-resistant cells

Ciprofloxacin, the most widely used totally synthetic antibiotic, is subject to active efflux mediated by a MRP-like transporter in wild-type murine J774 macrophages. To identify the transporter among the seven potential Mrps, we used cells made resistant to ciprofloxacin obtained by long-term exposure to increasing drug concentrations (these cells show a lower ciprofloxacin accumulation, and provide a protected niche for ciprofloxacin-sensitive intracellular Listeria monocytogenes). In the present paper, we first show that ciprofloxacin-resistant cells display a faster efflux of ciprofloxacin, which is inhibited by gemfibrozil (an unspecific MRP inhibitor). Elacridar, at a concentration known to inhibit P-glycoprotein and BCRP, only slightly increased ciprofloxacin accumulation with no difference between resistant and wild-type cells. Analysis at the mRNA (real-time PCR) and protein (Western blot) levels revealed an overexpression of Mrp2 and Mrp4. Mrp4 transcripts, however, were overwhelmingly predominant (45 [wild-type cells] to 95 % [ciprofloxacin-resistant cells] of all Mrp's transcripts tested [Mrp1 to Mrp7]). Silencing experiments of Mrp2 and Mrp4 with specific siRNAs showed that only Mrp4 is involved in ciprofloxacin transport in both ciprofloxacin-resistant and wild-type cells. The study, therefore, identifies Mrp4 as the most likely transporter of ciprofloxacin in murine macrophages, but leaves open a possible common upregulation mechanism for both Mrp4 and Mrp2 upon chronic exposure of eucaryotic cells to this widely used antibiotic