Novel aspect of amphotericin B action: accumulation in human monocytes potentiates killing of phagocytosed Candida albicans

Abstract

The influence of low doses of amphotericin B on the capacity of human monocytes to kill Candida albicans was investigated. Killing rates were quantified by a novel flow cytometric assay and were found to be 37% 3% (standard error of the mean) after 3 h. Preincubation of monocytes for 6 to 20 h with low concentrations of amphotericin B (0.2 ,ug/ml) Despite its toxicity, amphotericin B has remained the prime drug for treating life-threatening fungal infections. This agent binds with high affinity to ergosterol. Its fungicidal action is generally ascribed to an enhancement of membrane permeability as a result of the formation of ion channels (5, 16) and perhaps also because of oxidative damage of membrane constituents (4, 6). Unfortunately, amphotericin B also binds to cholesterol in mammalian cell membranes and elicits severe toxic effects. Many efforts have therefore been devoted to establishing treatment regimens that combine the merits of antifungal activity with a minimum of untoward side effects. The possibilities of local amphotericin B application are also receiving increased attention. Aerosol application, first described nearly 20 years ago (23), and low-dose intravenous application are emerging as prophylactic measures against pulmonary fungal infections in neutropenic patients (13,20,29).General belief holds that the beneficial effect of amphotericin B derives mainly from its direct fungicidal action. In addition, the drug reportedly can act on macrophages to stimulate [3H]thymidine uptake (5) and the respiratory burst (34, 35), promote tumor necrosis factor secretion (9), and enhance the tumoricidal (8, 26) and microbicidal (3,24,26,32) actions of these cells. The mechanisms underlying these processes and the relevance thereof remain unclear.We performed with a high degree of accuracy (18,19). The assays were first introduced for quantifying granulocyte phagocytic function and have now been adapted to the analysis of monocytes. In the studies described here, we showed that monocytes preincubated for hours with low doses of amphotericin B acquire a remarkably augmented capacity to kill ingested Candida albicans. This effect appears to be due to the intracellular accumulation of the drug rather than to a nonspecific stimulation of the cells. Potentiation of the fungicidal capacity of monocytes is likely to be an important novel determinant underlying the therapeutic efficacy of amphotericin B. MATERIALS AND METHODSMicroorganisms. An isolate of C. albicans from our diagnostic laboratory or an amphotericin B-resistant mutant, kindly provided by H. Dermoumi, Essen, Germany, was cultured at 37°C in tryptic soy broth for 16 to 20 h. A total of 1.5 ml of yeast cell cultures was centrifuged and the pelleted cells were resuspended in 1 ml of saline and incubated with 2 ,uM bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester (BCECF-AM; Becton Dickinson, Heidelberg, Germany) for 30 min in an Eppendorf Thermomixer 5436

Production and Characterization of Stable Amphotericin-Resistant Amastigotes and Promastigotes of Leishmania mexicana

“…However, direct experimental proof for some of these assumptions is lacking, and a number of models have been proposed to explain the biological activity of AMB (5). Other mechanisms of action have also been suggested, including lipid peroxidation and stimulation (activation) of phagocytic cells such as monocytes (25). From the current study it is clear, however, that resistance of L. mexicana amastigotes to AMB is accompanied by marked changes in membrane sterol composition.…”

Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells

“…Others have seen that Amphotericin B, which is structurally related to nystatin, increased the killing A. fumigatus conidia and C. albicans yeast cells by macrophages (Jahn et al, 1998;Martin et al, 1994). Jahn et al (Jahn et al, 1998) reported a killing rate of only 10-15% after a 12 hour incubation of conidia with monocyte-derived macrophages.…”

“…Preincubation of the cells for 16 hours with 1 µg/ml Amphotericin B increased the kill rate to 57%. Martin et al (Martin et al, 1994) observed a synergistic effect of Amphotericin B with monocyte killing of C. albicans. However, this effect was not due to amphotericin B-dependent monocyte activation as the respiratory burst and expression of human leukocyte antigen-DR was unaltered (Martin et al, 1994).…”

“…Martin et al (Martin et al, 1994) observed a synergistic effect of Amphotericin B with monocyte killing of C. albicans. However, this effect was not due to amphotericin B-dependent monocyte activation as the respiratory burst and expression of human leukocyte antigen-DR was unaltered (Martin et al, 1994). In contrast, we determined that 99% of the added conidia were washed off or killed by 12 hours.…”

Amphotericin B: A New Look at Cellular Binding

“…The absorbance difference between the trough at 398 nm and the peak at 408 nm was determined. Incubation with AmB 1.0uM resulted in intracellular concentrations, as early as 30 minutes that progressively increased over 20 hours to a maximum of ~50fg/cell or an estimated intracellular concentration of ~100pM (assuming a cell volume of 500 fL per cell) [21]. Phagocytic cells have been shown to accumulate AmB 0.8 & 3.5 ng/10 6 peritoneal exudative cell and L-AmB 4.4 & 7.9ng/10 6 cells in vivo at 6 and 24 hours after AmB administration [22].…”