Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B₄

Summary

The primary objective of this study was to probe the involvement of leukotriene B₄ (LTB₄) in itraconazole (0·1–5 µM)-mediated inhibition of Ca²⁺ uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB₄ was measured by immunoassay, while neutrophil cytosolic Ca²⁺ 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 Ca²⁺ which was associated with increased generation of LTB₄, both of which were attenuated significantly by itraconazole at 0·5 µM and higher. The inhibitory effect of the anti-mycotic on Ca²⁺ uptake by PAF-activated cells was mimicked by an LTB₄ antibody, as well as by LY255283 (1 µM) and MK886 (0·5 µM), an antagonist of LTB₄ 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 LTB₄ production and Ca²⁺ influx was also supported by the observation that pulsed addition of purified LTB₄ to PAF-activated neutrophils caused substantial restoration of Ca²⁺ 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 LTB₄, with consequent attenuation of a secondary LTB₄-mediated wave of Ca²⁺ uptake by the cells.