Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4.
The primary objective of this study was to probe the involvement of leukotriene B(4) (LTB(4)) in itraconazole (0.1-5 microM)-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 microM 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 microM) and MK886 (0.5 microM), 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.
MRC Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
H C
HC
Steel
G R
GR
Tintinger
A J
AJ
Theron
R
R
Anderson
D000255:Adenosine Triphosphate / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D017964:Itraconazole / Q000494:pharmacology*; D020024:Leukotriene Antagonists / Q000494:pharmacology; D007975:Leukotriene B4 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013777:Tetrazoles / Q000494:pharmacology; D002722:Chlorobenzenes
D000255:Adenosine Triphosphate; D000935:Antifungal Agents; D002118:Calcium; D007211:Indoles; D017964:Itraconazole; D020024:Leukotriene Antagonists; D007975:Leukotriene B4; D016859:Lipoxygenase Inhibitors; D010972:Platelet Activating Factor; D013777:Tetrazoles; C060893:MK-886; D002722:Chlorobenzenes; C063823:LY 255283