Background: Previous studies have shown that clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonucelar cells (PMNs) and other cells of myeloid lineage possess a similar transport system for taking up clarithromycin. In addition, we predicted that clarithromycin accumulation inside PMNs would enhance killing of Aggregatibacter actinomycetemcomitans.
Methods: To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [3H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was evaluated with PMNs loaded by brief incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37° C with control and clarithromycin-loaded PMNs and in the presence and absence of clarithromycin (5 μg/ml).
Results: Undifferentiated HL-60 cells, HL-60 cells that had been differentiated into PMN-like granulocytes, and mature human PMNs all took up clarithromycin in a saturable manner. The kinetics of uptake by all three types of cells yielded linear Lineweaver-Burk plots. PMNs and HL-60 granulocytes transported clarithromycin with a Km of approximately 250μg/ml and a Vmax that was not significantly different. When extracellular concentrations were diluted, HL-60 granulocytes loaded with clarithromycin lost approximately 20% of their antibiotic content within 3 minutes and approximately 45% of their original content within 60 minutes. HL-60 granulocytes and PMNs accumulated intracellular levels of clarithromycin that were 28- to 71-fold higher than their extracellular concentration. When assayed at a bacteria-to-PMN ratio of 100:1, clarithromycin-loaded PMNs killed significantly more bacteria and achieved shorter half times for killing than control PMNs (P < 0.04). At a ratio of 30:1, these differences were not consistently significant.
Conclusion: PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could enhance the ability of PMNs to cope with an overwhelming infection by A. actinomycetemcomitans.