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

• Published in: Clinical and experimental immunology Vol. 150; no. 1; pp. 144 - 150
• Main Authors: Steel, H. C., Tintinger, G. R., Theron, A. J., Anderson, R.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-10-2007; Blackwell; Blackwell Science Inc
• Subjects: 5′‐lipoxygenase; Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Antifungal Agents - pharmacology; Basic Immunology; Biological and medical sciences; calcium; Calcium - metabolism; Cells, Cultured; Cytosol - metabolism; Dose-Response Relationship, Drug; Fundamental and applied biological sciences. Psychology; Humans; Indoles - pharmacology; itraconazole; Itraconazole - pharmacology; Leukotriene Antagonists - pharmacology; leukotriene B4; Leukotriene B4 - antagonists & inhibitors; Leukotriene B4 - biosynthesis; Leukotriene B4 - immunology; Lipoxygenase Inhibitors - pharmacology; Molecular and cellular biology; Molecular biophysics; Neutrophil Activation - drug effects; neutrophils; Neutrophils - drug effects; Neutrophils - metabolism; Platelet Activating Factor - pharmacology; platelet‐activating factor; Spectrometry, Fluorescence - methods; Tetrazoles - pharmacology; Calcium; Granulocyte; Biochemistry; Biosynthesis; platelet-activating factor; 5'-lipoxygenase; Inorganic element; Antifungal agent; Eicosanoid; Inhibition; neutrophils; Itraconazole; Human; Enzyme; Arachidonic acid derivatives; Biophysics; Platelet activating factor; Leukotriene B4; Triazole derivatives; Inhibitor; Oxidoreductases; Neutrophil; Molecular biology; Lipoxygenase; leukotriene Β4
• ISSN: 0009-9104; 1365-2249
• DOI: 10.1111/j.1365-2249.2007.03470.x

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.