Influence of fluconazole on phagocytosis, oxidative burst and killing activity of human phagocytes. Using a flow cytometric method with whole blood.

摘要

OBJECTIVES: The aim of this study was to investigate the influence of fluconazole, an antimycotic on phagocytosis, oxidative burst and killing activity of phagocytes in human whole blood with Candida albicans as a test strain using a flow cytometric method. METHODS: Candida albicans was stained with Calcein AM, a greenfluorescent dye from Bioprobes (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA). To measure phagocytosis and burst activity diluted monoclonal antibody (CD-13-R-PE, Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) attaching at the surface of granulocytes and monocytes was added as well as Dihydroethidium solution (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) which changes into red fluorescent Ethidium by oxidation when killing activity takes place. With Ethidium-Homodimer-1-solution (Molecular Probes, Inc., P.O. Box 22010, Eugene, OR 97402-0469, USA) killing activity can be observed. Three different tests, one incubating the Candida for 1- 4 hrs in advance, another incubating whole blood for 1 h, and the third incubating neither yeast nor blood, and a combined main test were carried out. Measurement of phagocytosis, burst- and killing activtiy was performed with a flow cytometric method (Coulter Company, type: Epics-Profile II). RESULTS: Three different concentrations of fluconazole (5, 20 and 100 microg/ml) show neither decreasing nor increasing influence on phagocytosis and burst activity, irrespective of whether yeasts or phagocytes had been incubated with fluconazole in advance or not. Also after incubating the drug with phagocytes for 1 h, neither an increase nor a decrease of killing activity was observed. A significant increase was, however, found with increasing incubation time of yeasts and fluconazole. - The minimum concentration of fluconazole, just enough to show a significant increase of the killing rate was 1 microg/ml after 3hrs of incubation. No further significant increase was detected when the concentration exceeded 5 microg/ml. CONCLUSION: 1 h incubation of human phagocytes with fluconazole does not have any significant influence on cellular activities. After advanced incubation of Candida a corresponding increase of the intracellular killing rate in phagocytes occurs, probably due to changes of the cytomorphology of yeasts.