Itraconazole‐mediated inhibition of calcium entry into platelet‐activating factor‐stimulated human neutrophils is due to interference with production of leukotriene B 4

Summary The primary objective of this study was to probe the involvement of leukotriene B 4 (LTB 4 ) in itraconazole (0·1–5 µM)‐mediated inhibition of Ca 2+ uptake by chemoattractant‐activated human neutrophils. Following exposure of the cells to platelet‐activating factor (PAF, 200 nM), LTB 4 was measured by immunoassay, while neutrophil cytosolic Ca 2+ concentrations were determined by a fura‐2/AM‐based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca 2+ which was associated with increased generation of LTB 4 , both of which were attenuated significantly by itraconazole at 0·5 µM and higher. The inhibitory effect of the anti‐mycotic on Ca 2+ uptake by PAF‐activated cells was mimicked by an LTB 4 antibody, as well as by LY255283 (1 µM) and MK886 (0·5 µM), an antagonist of LTB 4 receptors and an inhibitor of 5′‐lipoxygenase‐activating protein, respectively, while addition of itraconazole to purified 5′‐lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole‐mediated inhibition of LTB 4 production and Ca 2+ influx was also supported by the observation that pulsed addition of purified LTB 4 to PAF‐activated neutrophils caused substantial restoration of Ca 2+ uptake by cells treated with the anti‐mycotic. Taken together, these observations suggest that the potentially beneficial anti‐inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB 4 , with consequent attenuation of a secondary LTB 4 ‐mediated wave of Ca 2+ uptake by the cells.