Entry of antibiotics into phagocytes is necessary for activity against intracellular organisms. Therefore, we examined the uptake of five of the newer antibiotics-roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim, and metronidazole-by human polymorphonuclear leukocytes (PMN). Antibiotic uptake by PMN was determined by a velocity gradient centrifugation technique and expressed as the ratio of the cellular concentration of antibiotic to the extracellular concentration (C/E). Cefotaxime, like other I-lactam antibiotics, was taken up poorly by phagocytes (C/E ' 0.3). The metronidazole concentration within PMN was similar to the extracellular level. Imipenem bound rapidly to phagocytes (C/E = 3), but cell-associated drug progressively declined during the incubation period. Trimethoprim was well concentrated by PMN (C/E = 9 to 13), and uptake was unexpectedly greater at 25°C than at 37°C. The most striking finding was that roxithromycin was more avidly concentrated by PMN (C/E = 34) than any other antibiotic we studied. Entry of roxithromycin into phagocytes was an active process and displayed saturation kinetics characteristic of a carrier-mediated membrane transport system. Ingestion of microbial particles by PMN slightly decreased the ability of these cells to accumulate roxithromycin (C/E = 24 to 31). These studies identified two antibiotics, trimethoprim and especially roxithromycin, which are markedly concentrated within human PMN and may prove useful in treatment of infections caused by susceptible intracellular organisms.
Vol. 29, no. 2, p. 214: In paragraph 2 of the Discussion, the second sentence should begin "An ampicillin-resistant strain of Pasteurella multocida.. .".
Recently we found that certain antibiotics which are markedly concentrated by human polymorphonuclear leukocytes (PMN) failed to kill susceptible, intraphagocytic Staphylococcus aureus, even though cellular drug levels were quite high. The possibility that specific antibiotics might adversely affect phagocyte antibacterial function was considered. Thus, we studied the effects of multiple antibiotics and adenosine, a known modulator of the PMN respiratory burst response, on neutrophil antibacterial function. At nontoxic concentrations, these drugs had no effect on degranulation in stimulated PMN. Adenosine was a potent inhibitor of formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated superoxide and hydrogen peroxide generation in PMN but produced less inhibition of microbial particle-induced respiratory burst activity. Three of the tested antibiotics, all of which reach high concentrations in phagocytic cels, had a marked modulatory effect on the PMN respiratory burst. Clindamycin, which enters phagocytes by the ceUl membrane adenosine (nucleoside) transport system, had only a modest effect on FMLP-mediated superoxide production but inhibited the microbial particle-induced response by -50%. Roxithromycin and trimethoprim were efficient inhibitors of PMN superoxide generation stimulated by FMLP and concanavalin A (also inhibited by erythromycin) but had less effect on zymosan-mediated respiratory burst activity. Antibiotics which entered phagocytes less readily had no effect on the respiratory burst response in PMN. These results, as well as those of experiments with inhibitors of cell membrane nucleoside receptors, indicated that the antibiotic effect is mediated through intraphagocytic pathways. The possibility that antibiotic-associated inhibition of the PMN respiratory burst response might alter leukocyte antimicrobial and inflammatory function deserves further evaluation.The interaction of antimicrobial agents with leukocytes, and especially any influence on the fate of bacteria ingested by these phagocytic cells, may be of therapeutic importance. Entry of antibiotics into phagocytes is obviously a prerequisite for activity against intracellular organisms, but we recently demonstrated a discrepancy between uptake of certain antibiotics by human neutrophilic polymorphonuclear leukocytes (PMN) and the subsequent effect of these drugs on intraphagocytic bactericidal activity (18). Thus, clindamycin and erythromycin, which were markedly concentrated by PMN, exhibited poor activity against intraphagocytic Staphylococcus aureus, even though the intracellular drug levels exceeded the MBCs for this organism (18). The possibility that certain antibiotics might adversely influence phagocyte antibacterial function was considered. In the case of cindamycin, a potential modulatory role has been identified. We have shown that clindamycin enters PMN and macrophages by means of the cell membrane nucleoside (adenosine) transport system (16, 36). Since adenosine (by binding external cell membrane nucleoside receptors) regula...
The use of antibiotics which can penetrate phagocytic cells and kill intracellular organisms is desirable in the treatment of chronic facultative bacterial infections. Recently, we reported that several antibiotics were selectively concentrated by rabbit alveolar macrophages. Clindamycin accumulation was especially marked. In the present study we evaluated the plasma membrane transport (initial uptake) of clindamycin in alveolar macrophages. The transport of clindamycin is an active process, as documented by requirements for cellular viability, elevated environmental temperature, metabolic energy, and establishment of the 40- to 50-fold cellular/extracellular gradient. Energy for membrane transport of the drug depended at least in part upon mitochondrial oxidative respiration and cell membrane Na-K pump activity. Kinetic analysis of active clindamycin transport revealed it to be saturable, with a high binding affinity (Km = 1 mM) and a high velocity of uptake (Vmax = 15.8 nmol/45 s per 10(6) cells). Clindamycin uptake was not influenced by the presence of hexose or amino acids, but was inhibited by nucleosides (adenosine, puromycin). Decreased clindamycin transport in the presence of puromycin was typical of competitive inhibition (increased Km, unchanged Vmax). Conversely, competitive inhibition of adenosine transport by clindamycin was documented. Thus, clindamycin is transported into alveolar macrophages via the nucleoside system. The potential biological consequences of this unique antibiotic transport mechanism are of interest.
Optimal therapy of infections due to organisms capable of surviving within phagocytes would include use of antimicrobials that penetrate phagocytic cells and inactivate intracellular organisms. To establish those characteristics of drug and cell that mediate the antibiotic-phagocyte interaction, we have studied the uptake of radiolabelled antibiotics by rabbit alveolar macrophages (AM), human AM from smokers and non-smokers, and human polymorphonuclear leukocytes (PMN). Relative entries of drug groups into the three types of phagocytic cells were similar. Penicillin G and cephalosporin antibiotics were taken up poorly by phagocytes. Lipid-soluble antibiotics, such as rifampicin and chloramphenicol, were concentrated several-fold (2-5) by phagocytes. Ethambutol, erythromycin and clindamycin were concentrated many-fold (5-50) by phagocytic cells. Human AM of smokers accumulated certain antibiotics more avidly than AM of non-smokers. Clindamycin entry into phagocytes was shown to be an active, energy-requiring process, mediated by the nucleoside transport system. Ingestion of microbial particles by PMN stimulated transport of both clindamycin and nucleoside (adenosine) into the cell.
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