engleski

Macrolide uptake and release by HL-60 cells and human polymorphonuclear neutrophils (PMN)

Introduction: Macrolides encompass several classes of drugs. All macrolides concentrate within phagocytic and other cells, with a mean intracellular to extracellular (I/E) concentration ratio higher than 10 whatever the type of phagocyte (Labro, 1993). Among macrolide antibiotics there are considerable differences in uptake and release kinetics (Gladue, 1989, Miossec-Bartoli 1999). Azithromycin has one of the highest I/E ratios in all cell types reaching up to 120-200 after 1-hour incubation (Bonnet et al. 1992, Vazifeh et al. 1997). Azithromycin not only concentrates in phagocyte but also remains in the cell for a relatively long period of time (Vazifeh, 1997). HL-60 cells (human promyelocytic leukemia cell line) can be induced to differentiate to granulocytes by retinoic acid (Breitman, 1980). HL-60 cells and PMNs were compared for their macrolide uptake and release kinetics. Materials and Methods: Uptake: PMNs were isolated from human blood by erythrocyte sedimentation on 3% dextran followed by Ficoll-Paque centrifugation. Differentiated HL-60 cells were obtained by 4-day incubation with 1uM all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (RA). Cells were incubated with azithromycin, erythromycin or HMR 3647 (10 ug/mL) in RPMI 1640 at 37°C for up to 3h. PMNs were removed from the drug-containing medium by centrifugation through silicone oil. HL-60 cells were instead washed twice in ice-cold PBS. In both cases, cell pellet was resuspended in 0.5% TritonX-100, sonicated for 1 min, filtered (10kDa MWCO) and analysed by microbiological bioassay. Release: Cells were loaded with macrolides as previously described. Afterwards, they were washed in ice-cold PBS and incubated for up to 180 min in RPMI 1640 at 37°C. PMNs and HL-60 lysates were prepared and analysed as described in procedure for uptake. Results: Compared to undifferentiated HL-60 cells and cell differentiated with RA, HL-60 cells differentiated with ATRA showed significantly higher accumulation of azithromycin during 3h incubation. Accordingly, ATRA differentiated HL-60 cells were further tested to esteblish a model for macrolide uptake and release kinetics similar to PMNs. Azithromycin and erythromycin display the same uptake kinetics in PMNs and differentiated HL-60 cells. While erythromycin's intracellular concentration remains almost unchanged after 1h incubation, azithromycin exhibits no saturation even after 3h incubation. On the other hand HMR 3647 displays saturable kinetics in differentiated HL-60 cells but not in PMNs. The results on azithromycin and erythromycin release from differentiated HL-60 cells correlate with the date obtained on PMNs (our data and Miossec-Bartoli, 1999). However while in PMNs 80% of accumulated HMR 3647 stays trapped in the cells even after 3h incubation in drug-free medium, in differentiated HL-60 cells HMR-3647 stays trapped in the cells even after 3h incubation in drug-free medium, in differentiated HL-60 cells HMR 3647 egresses rapidly from the cells during the first hour of incubation with only 5% of the initial intracellular concentration remaining in the cells after 3h incubation. Conclusion:PMNs and ATRA-differentiated HL-60 cells display similar uptake and release kinetics for azithromycin and erythromycin. Interestingly, uptake and release kinetics of HMR 3647 differ significantly between these two models. The observed discrepancies could indicate the existance of an additional release mechanism present in differentiated HL-60 cells but not in PMNs.

macrolide; uptake; release; HL-60; polymorphonuclear neutrophils

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