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

Abstract

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.

Introduction

Itraconazole, an imidazole anti-mycotic, antagonizes the uptake of Ca²⁺ by activated human neutrophils, as well as by other types of mammalian cell [1–5]. In the case of neutrophils, itraconazole-mediated inhibition of Ca²⁺ influx not only interferes with the Ca²⁺-dependent proinflammatory activities of the cells, but also prevents their reactivation by preventing store-operated refilling of intracellular Ca²⁺ stores [1,2,5]. These inhibitory effects of the anti-mycotic on Ca²⁺ influx have been observed not only in chemoattractant-activated neutrophils, but also following the addition of Ca²⁺ to neutrophils with depleted intracellular Ca²⁺ stores suspended in Ca²⁺ free medium [5]. Although potentially beneficial, the molecular/biochemical basis of these secondary, anti-inflammatory interactions of itraconazole with neutrophils have not been established. Possible mechanisms include inhibition of cytochrome P450 (CYP450) enzyme systems, specifically CYP450 epoxygenase, which converts arachidonate to epoxyeicosatrienoic acid, a putative Ca²⁺ influx factor [1,2,6,7], as well as direct antagonism of Ca²⁺ channels [8,9].

Interference with the production of leukotriene B₄ (LTB₄) represents an alternative, albeit unexplored, mechanism of itraconazole-mediated interference with store-operated uptake of Ca²⁺ by activated neutrophils. In this respect it is noteworthy that itraconazole has been reported to inhibit the production of LTB₄ by intact neutrophils activated with the Ca²⁺ ionophore, A23187 [10], while endogenous-generated LTB₄ has been reported to amplify the uptake of Ca²⁺ following addition of the chemoattractant, platelet-activating factor (PAF), to neutrophils [11].

In the current study we have investigated the effects of itraconazole on the production of LTB₄ by PAF-activated neutrophils and its relationship to alterations in Ca²⁺ influx.

Materials and methods

Chemicals

Itraconazole was kindly provided by Janssen Pharmaceutica (Geel, Belgium) and dissolved in dimethylacetamide (DMA) to give a stock concentration of 10 mM. Subsequent dilutions were made in the same solvent and the final concentrations of itraconazole used in the assays described below ranged from 0·1 to 5 µM, while that of DMA was 0·1%. Unless indicated, all other chemicals and reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Neutrophils

The study was approved by the Faculty of Health Sciences Research Ethics Committee of the University of Pretoria (Protocol 43/2006), and prior informed consent was obtained from all participants.

Purified neutrophils were prepared from heparinized (5 units of preservative-free heparin/ml) venous blood of healthy, non-smoking adult human volunteers and separated from mononuclear leucocytes by centrifugation on Histopaque®-1077 (Sigma Diagnostics) cushions at 400 g for 25 min at room temperature. The resultant pellet was suspended in phosphate-buffered saline (PBS; 0·15 M, pH 7·4) and sedimented with 3% gelatine to remove most of the erythrocytes. After centrifugation, erythrocytes were removed by selective lysis with 0·84% ammonium chloride at 4°C for 10 min. The neutrophils, which were routinely of high purity (> 90%) and viability (> 95%), were resuspended at 1 × 10⁷/ml in PBS and held on ice until used.

Measurement of LTB₄

A competitive binding enzyme immunoassay procedure (Correlate-EIA™; Assay Designs Inc., Ann Arbor, MI, USA) was used to measure LTB₄ in the supernatants of neutrophils activated with PAF (200 nM) in the absence or presence of itraconazole (0·1–5 µM). Neutrophils (2 × 10⁶/ml, final) in Hank's balanced salt solution (HBSS) were preincubated for 10 min at 37°C with itraconazole after which the chemoattractant, platelet-activating factor (PAF; 200 nM, final) was added to the cells and the reactions stopped after 3 min incubation at 37°C (predetermined in preliminary time–course experiments) by the addition of an equal volume of ice-cold HBSS to the tubes which were then held in an ice-bath prior to pelletting the cells by centrifugation. The cell-free supernatants were then assayed for LTB₄ using the enzyme immunoassay (EIA) procedure. Supernatants from cells activated with PAF were diluted 1 : 4 prior to assay. These results are expressed as picograms (pg)/10⁷ cells.

The effects of LY255283 (1 µM) and MK886 (0·5 µM), an LTB₄ receptor antagonist [12] and 5′-lipoxygenase-activating protein (FLAP) inhibitor [13], respectively (both from the Cayman Chemical Co., Ann Arbor, MI, USA), on LTB₄ production by PAF-activated neutrophils were also investigated. The concentrations of MK886 and LY255283 used for these experiments (0·5 µM and 1 µM, respectively) were based on data derived from preliminary experiments. At a concentration of 0·5 µM, MK886 effectively attenuated the generation of LTB₄ by neutrophils activated with the calcium ionophore, A23187 (1 µM), while LTB₄ (50 nM)-mediated mobilization of Ca²⁺ from neutrophil intracellular stores was completely antagonized by 1 µM LY255283 (data not shown).

The effects of itraconazole (5 µM) on the production of LTB₄ by PAF-activated neutrophils suspended in Ca²⁺ replete and Ca²⁺ free HBSS were also investigated. To minimize loss of Ca²⁺ from intracellular stores, neutrophils were preincubated in Ca²⁺ replete HBSS for 10 min at 37°C, pelleted by centrifugation and resuspended in either Ca²⁺ replete HBSS (control cells) or HBSS containing 50 µM CaCl₂. The cells were then preincubated at 37°C for 9 min, after which the Ca²⁺ chelating agent, ethylene glycol tetraacetic acid (EGTA) (500 µM final) was added to cells suspended in HBSS containing 50 µM CaCl₂, followed by transfer to cuvettes and activation 1–2 min later with PAF. With this procedure we were unable to detect influx of Ca²⁺ into neutrophils treated with the pneumococcal pore-forming toxin, pneumolysin (8·37 ng/ml, final), which causes influx of extracellular Ca²⁺ without mobilizing the cation from neutrophil intracellular stores [14].

To exclude possible interference of itraconazole, LY255283, and MK886 at concentrations of 5, 1 and 0·5 µM, respectively, with LTB₄ in the EIA, we investigated the reactivity of a fixed concentration (3000 pg/ml) of the LTB₄ standard in the presence and absence of the LTB₄ receptor antagonist.

Activity of purified 5′-lipoxygenase (5-LO)

The effects of itraconazole (5–50 µM, final) or the DMA solvent (1% final in this system) on the reactivity of purified 5-LO were investigated using the Lipoxygenase Inhibitor Screening Assay Kit from the Cayman Chemical Co., which is a colorimetric assay using purified potato 5-LO (5 units/assay) and linoleic acid (100 µM, final) as enzyme and substrate, respectively (as recommended by the manufacturer). The enzyme and DMA or itraconazole in assay buffer were preincubated for 15 min at room temperature, followed by addition of substrate and incubation for an additional period of 5 min. Enzyme activity was monitored spectrophotometrically at 490 nm according to the magnitude of chromogen-reactive hydroxperoxide formation. Substrate and enzyme were omitted from negative control systems. The results of these experiments are expressed as the mean percentage activity of the itraconazole-free control systems.

Spectrofluorimetric measurement of Ca²⁺ fluxes

Fura-2/AM was used as the fluorescent Ca²⁺ sensitive indicator for these experiments [15]. Neutrophils (1 × 10⁷/ml) suspended in PBS were prewarmed for 5 min at 37°C followed by addition of fura-2/AM (2 µM, final) and reincubated for 25 min at 37°C. The fura-2-loaded neutrophils were then pelleted by centrifugation and resuspended in indicator-free, Ca²⁺ replete (1·25 mM CaCl₂) HBSS, pH 7·4. The neutrophils (2 × 10⁶/ml) were then preincubated for 10 min at 37°C with one of the following: itraconazole (0·1–5 µM); MK886 (0·5 µM) or LY255283 (1 µM); an equivalent amount of the relevant DMA (itraconazole) or dimethylsulphoxide (DMSO; MK886, LY255283) solvent control systems. The cells were then transferred to disposable reaction cuvettes which were maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths set at 340 and 500 nm, respectively. After a stable baseline was obtained (1 min), the neutrophils were activated by addition of PAF (200 nM). Alterations in fluorescence intensity were then monitored over a 5–10-min period. The final volume in each cuvette was 3 ml containing a total of 6 × 10⁶ neutrophils.

In a related series of experiments, the involvement of LTB₄ in sustaining cytosolic Ca²⁺ transients in neutrophils activated with PAF was also probed using an anti-LTB₄ antibody produced in rabbit whole anti-serum (Sigma). The antibody (60 µl of a 5-mg/ml solution) was added to the cells 1 min prior to the activators. An equivalent concentration of a control antibody to prostaglandin F_2α (PGF_2α) (rabbit whole anti-serum; Sigma) was added to control systems. The efficacy of the LTB₄ antibody was demonstrated in preliminary experiments in which it was found to antagonize the mobilization of Ca²⁺ from neutrophil intracellular stores following addition of purified LTB₄ (50 nM) to the cells.

In an attempt to clarify the possible association between itraconazole-mediated inhibition of LTB₄ production and Ca²⁺ influx, an additional series of experiments was designed to investigate the potential of added, purified LTB₄ to reverse the inhibitory effects of itraconazole (5 µM) on the uptake of Ca²⁺ by PAF-activated neutrophils. Three strategies were used: (i) LTB₄ (75 nM) and PAF (200 nM) were added simultaneously to control or itraconazole-treated cells; (ii) LTB₄ (75 nM) was added 10 s after PAF; and (iii) LTB₄ (15 nM) and PAF were added simultaneously, followed 10 s later by addition of a second, higher dose of LTB₄ (75 nM) only.

Cellular adenosine triphosphate (ATP) levels

The cytotoxic potential of itraconazole (5 µM), LY255283 (1 µM) and MK886 (0·5 µM) was determined by measurement of cellular ATP in the lysates of neutrophils (10⁶/ml) which had been exposed to the test agents for 10 min at 37°C using a luciferin/luciferase chemiluminescence procedure [16]. The results are expressed as picomoles ATP/10⁷ cells.

Expression and statistical analysis of results

The results of each series of experiments are expressed as the mean values ± s.e.m., with the exception of several of the fura-2 experiments for which the traces are shown. Statistical analysis was performed using Student's t-test when comparing two groups or by analysis of variance with subsequent Tukey–Kramer multiple comparisons test for multiple comparisons.

Results

Effects of itraconazole on LTB₄ production by activated neutrophils

The effects of itraconazole at concentrations of 0·1–5 µM on the production of LTB₄ by neutrophils activated with PAF are shown in Fig. 1. Itraconazole caused dose-related inhibition of the production of LTB₄ by the chemoattractant, which attained statistical significance at concentrations of 0·1 µM.

Fig. 1.

Effects of itraconazole (0·1–5 µM) on the production of leukotriene B₄ (LTB₄) by platelet-activating factor (PAF) (200 nM)-activated neutrophils. The results of three experiments with five replicates for each system are expressed as the mean values ± s.e.m. The values for the unstimulated control, and for the PAF-activated control systems were 43 ± 5 and 1190±67 pg LTB₄/10⁷ cells, respectively. *P < 0·05 for comparison with the corresponding itraconazole-free control system.

A comparison of the effects of LY255283 and MK886 on PAF-activated production of LTB₄ by neutrophils is shown in Table 1. Both agents caused statistically significant inhibition of LTB₄ production. Importantly, itraconazole (5 µM), LY255283 (1 µM) and MK886 (0·5 µM) did not affect the reactivity of LTB₄ in the EIA (data not shown).

Table 1.

Effects of LY255283 (1 µM) and MK886 (0·5 µM) on the production of leukotreine B₄ (LTB₄) by platelet-activating factor (PAF)-activated neutrophils.

PAF-activated neutrophils incubated with	Leukotriene B4 (pg/107 cells)
Solvent only (control)	1054 ± 49a
1·0 µM LY255283	396 ± 42*
0·5 µM MK886	78 ± 16*

^aThe results of three different experiments with five replicates for each system are expressed as the mean values ± s.e.m. The basal values for unstimulated cells was 59 ± 10 pg LTB₄/10⁷.

^*P < 0·05.

Comparison of the effects of itraconazole on LTB₄ production by PAF-activated neutrophils suspended in Ca²⁺ replete and Ca²⁺ free HBSS

These results are shown in Fig. 2. Although markedly reduced relative to the corresponding responses in Ca²⁺ replete HBSS, the production of LTB₄ by PAF-activated neutrophils was still detectable and increased significantly above basal, unstimulated values. The production of LTB₄ by PAF-activated neutrophils was significantly (P < 0·05) inhibited by itraconazole irrespective of the Ca²⁺ content of the cell-suspending medium (Fig. 2).

Fig. 2.

Effects of itraconazole (Itra) at concentrations of 1 and 5 µM on the production of leukotriene B₄ (LTB₄) by platelet-activating factor (PAF) (200 nM)-activated, Ca²⁺ loaded neutrophils (to ensure that intracellular Ca²⁺ stores are full) suspended in Ca²⁺ free (□) or Ca²⁺ replete (▪) medium. The results of three experiments with five replicates for each system in each experiment are expressed as the mean values ± s.e.m. *P < 0·05 for comparison with the corresponding itraconazole-free, PAF-activated system.

Cytosolic Ca²⁺ fluxes of neutrophils suspended in either Ca²⁺ replete or Ca²⁺ free HBSS and treated with pneumolysin or PAF are shown in Fig. 3. The influx of Ca²⁺ which accompanies treatment of neutrophils with pneumolysin was abolished completely in the Ca²⁺ free system, while the abrupt increase in cytosolic Ca²⁺ remained intact in PAF-activated cells, but was associated with attenuation of the plateau phase and a rapid decline in fura-2 fluorescence intensity due, presumably, to absence of influx of Ca²⁺ as described in detail in the section on cytosolic Ca²⁺ fluxes below. These observations validate the experimental design used to eliminate Ca²⁺ influx during activation of the cells with PAF, in the setting of retention of mobilization of the cation from intracellular stores.

Fig. 3.

Traces showing the fura-2 fluorescence responses of Ca²⁺ loaded neutrophils (to ensure that intracellular Ca²⁺ stores are full) suspended in either Ca²⁺ free or Ca²⁺ replete Hank's balanced salt solution, followed by addition of either pneumolysin (8·37 ng/ml) or platelet-activating factor (PAF) (200 nM) as denoted by the arrow (↓).

Effects of itraconazole on purified 5-LO

Itraconazole caused dose-related inhibition of the activity of purified 5-LO in a cell-free assay system with linoleic acid as substrate. The mean percentages of the DMA-treated control systems were 83 ± 4, 77 ± 4, 71 ± 6, and 55 ± 5% for systems containing 5, 10, 20 and 50 µM itraconazole, respectively (data from six experiments; P < 0·05 for comparison of each of the itraconazole-treated systems with the control system). These experiments were complicated by the insolubility of itraconazole in aqueous, cell-free systems, and may have under-estimated the inhibitory potency of itraconazole for 5-LO.

Effects of itraconazole, LY255283, MK886 and LTB₄-antibody on cytosolic Ca²⁺ transients in activated neutrophils

The effects of itraconazole at concentrations of 0·1–5 µM on cytosolic Ca²⁺ fluxes in PAF-activated neutrophils are shown in Fig. 4 and Table 2, while the comparative effects of itraconazole, LY255283, MK886 and the LTB4-antibody are shown in Fig. 5 and Table 2. Addition of PAF to neutrophils was accompanied by the typical, abrupt increase in fluorescence intensity which accompanies G-protein-coupled receptor activation of phospholipase C and inositol triphosphate-mediated release of Ca²⁺ from intracellular stores. Peak fluorescence intensity was sustained for about 1–1·5 min, subsiding gradually thereafter, compatible with the previously described early influx of Ca²⁺ into PAF-activated neutrophils and slow clearance of cytosolic Ca²⁺ [17]. Pretreatment of neutrophils with itraconazole did not affect the magnitude of the immediate peak cytosolic Ca²⁺ response of PAF-activated neutrophils (535 ± 41 nM for the control system, rising from a basal value of 116 ± 12 nM). However, the duration of the plateau phase of peak fluorescence intensity was shortened significantly by treatment of the cells with the anti-mycotic, compatible with inhibition of Ca²⁺ influx. These effects of itraconazole were statistically significant at concentrations of ≥ 0·5 µM (Fig. 4 and Table 2).

Fig. 4.

Traces from a single representative experiment (six in the series) showing the effects of itraconazole (Itra) at concentrations of 0·25, 0·5 and 5 µM on the fura-2 fluorescence responses of platelet-activating factor (PAF) (200 nM)-activated neutrophils added as denoted by the arrow (↓).

Table 2.

Effect of itraconazole, LY255283, MK886 and the leukotreine B₄ (LTB₄) antibody on duration of the peak cytosolic response of platelet-activating factor (PAF)-activated neutrophils.

PAF-activated neutrophils incubated with	Duration of the peak cytosolic Ca2+ response (min)
Solvent only (control)	1·20 ± 0·05a
0·1 µM itraconazole	1·00 ± 0·10
0·25 µM itraconazole	0·80 ± 0·13
0·5 µM itraconazole	0·23 ± 0·08*
1·0 µM itraconazole	0·25 ± 0·03*
2·5 µM itraconazole	0·30 ± 0·03*
5·0 µM itraconazole	0·33 ± 0·02*
1 µM LY255283	0·60 ± 0·03*
0·5 µM MK886	0·13 ± 0·02*
LTB4-antibody	0·70 ± 0·03*

^aThe results of six different experiments are presented as the mean duration (min) ± s.e.m. of the peak cytosolic Ca²⁺ response. The mean peak cytosolic Ca²⁺ response of the control system was 535 ± 41 nM, rising from a basal value of 116 ± 41 nM.

^*P < 0·05 for comparison with the itraconazole-free control system.

Fig. 5.

Traces from a single representative experiment (six to eight in the series) showing the effects of an anti-leukotriene B₄ (LTB₄) antibody (60 µg/ml), MK886 (0·5 µM) and LY255283 (1 µM) on the fura-2 fluorescence responses of platelet-activating factor (PAF) (200 nM)-activated neutrophils added as denoted by the arrow (↓).

As shown in Fig. 5 and Table 2, treatment of neutrophils with LY255283, MK886 or the LTB₄-antibody also (P < 0·05) shortened significantly the duration of the peak plateau phase of fura-2 fluorescence intensity in PAF-activated cells, without affecting the magnitude of the immediate peak increase in cytosolic Ca²⁺. The duration of the plateau phase was not affected by treatment of the cells with the control, PGF_2α-antibody (not shown).

As shown in Table 3, pulsed addition of purified LTB₄ (i.e. PAF + 15 nM LTB₄, followed 10 s later by 75 nM LTB₄) substantially restored Ca²⁺ influx to itraconazole (5 µM)-treated neutrophils, without affecting the fura-2 responses of itraconazole-free control cells. On the other hand, simultaneous addition of LTB₄ (75 nM) with PAF only, or addition of LTB₄ (75 nM) 10 s after PAF, was less effective in counteracting the inhibitory effects of itraconazole on Ca²⁺ influx (data not shown).

Table 3.

Effects of pulsed addition of leukotriene B₄ (LTB₄) on itraconazole (5 µM)-mediated attenuation of the sustained, peak cytosolic Ca²⁺ transient in platelet-activating factor (PAF) (200 nM)-activated neutrophils.

System	Duration of the peak cytosolic Ca2+ response (min)
Neutrophils + PAF	1·05 ± 0·03a
Neutrophils + itraconazole + PAF	0·25 ± 0·02
Neutrophils + PAF + LTB4	1·09 ± 0·03
Neutrophils + itraconazole + PAF + LTB4	0·82 ± 0·03*

^aResults are expressed as the mean values ± s.e.m. for the duration of the abruptly occurring peak Ca²⁺ transients in neutrophils activated with PAF only, or with PAF + LTB₄ (15 nM) added simultaneously followed 10 s later by 75 nM LTB₄ in the absence or presence of 5 µM itraconazole (data from 10 determinations).

^*P < 0·05 for comparison between the itraconazole-treated systems activated with PAF only or with PAF + LTB₄.

Although not shown, we also observed that treatment of neutrophils with itraconazole (5 µM) did not affect the fura-2 fluorescence responses of cells activated with purified LTB₄ (50 nM), excluding possible LTB₄ receptor blockading effects of the anti-mycotic.

ATP levels

At the maximum concentrations used, ATP was unaffected by treatment with itraconazole (5 µM), LY255283 (1 µM) or MK886 (0·5 µM). In the case of itraconazole the values for the solvent control (0·1% DMA) and drug-treated systems were 29·4 ± 2·7 and 30·0 ± 2·1 pmoles ATP/10⁷ cells, respectively, while the corresponding values for the DMSO (0·1%) and LY255283- and MK886-treated systems were 25·5 ± 2·4, 25·2 ± 1·8 and 27·0 ± 2·1 pmoles ATP/10⁷ cells. At the concentrations used (0·1%), neither of the solvents affected neutrophil ATP levels (not shown).

Discussion

In the current study, we have observed that itraconazole attenuates both LTB₄ production and Ca²⁺ influx following activation of human neutrophils with the chemoattractant, PAF. Assuming a mechanistic relationship between these events, two possibilities were evident. Firstly, the anti-mycotic acts primarily as an antagonist of Ca²⁺ influx leading to secondary inhibition of LTB₄ production; alternatively, the inhibitory effects of the anti-mycotic on Ca²⁺ influx are indirect, resulting from interference with production of LTB₄.

Taken together, the following observations clearly supported a primary inhibitory effect of itraconazole on LTB₄ production, with consequent secondary inhibition of Ca²⁺ influx in PAF-activated neutrophils; (i) although the magnitude of the LTB₄ response was dependent upon the Ca²⁺ content of the cell-suspending medium, the inhibitory effects of the anti-mycotic were evident irrespective of the presence or absence of the cation in the medium; (ii) the inhibitory effects of itraconazole on Ca²⁺ uptake were mimicked by treatment of the cells with an antibody to LTB₄, as well as by non-cytotoxic concentrations of LY255283 and MK886, antagonists of LTB₄ receptors and FLAP, respectively; (iii) the inhibitory effects of itraconazole on Ca²⁺ influx were negated largely by addition of LTB₄, while the responses of itraconazole-free control cells were unaffected by the eicosanoid; and (iv) itraconazole antagonized the activity of purified 5′-lipoxygenase (5-LO) in a cell-free system.

In addition to 5-LO, other possible targets of itraconazole which would result in decreased production of LTB₄ by activated neutrophils include cytosolic phospholipase A₂ and FLAP. Cytosolic phospholipase A₂, however, appears to be an improbable target of the anti-mycotic. This contention is based on a previous report that the production of prostaglandin E₂ by human neutrophils activated with the calcium ionophore, A23187 is unaffected by itraconazole [10], and our own unpublished observations that the generation of arachidonic acid by A23187 (1 µM)-activated human neutrophils is insensitive to the anti-mycotic. Although involvement of FLAP cannot be excluded, the direct inhibitory effects of itraconazole on purified 5-LO suggest that this is the primary target of the anti-mycotic, which is in keeping with the well-recognized inhibitory interactions of pharmacological agents which possess an imidazole moiety with this enzyme [18,19]. It is noteworthy, however, that the inhibitory effects of itraconazole on purified 5-LO were of lesser magnitude than those observed with LTB₄ production by PAF-activated neutrophils. This may simply reflect the strongly lipophilic properties of itraconazole, resulting in intracellular accumulation of the anti-mycotic. Alternatively, other intermediates of arachidonic acid metabolism, such as 5-hydroperoxyeicosatetraenoic acid, may also contribute to Ca²⁺ uptake by PAF-activated neutrophils.

The inhibitory effects of LY255283 on the production of LTB₄ by PAF-activated neutrophils are compatible with a sequence of events which result in a protracted, autocrine activation of the cells. Exposure of neutrophils to the chemoattractant results in mobilization of Ca²⁺ from both intracellular and extracellular reservoirs and consequent production of LTB₄; the eicosanoid in turn interacts with LTB₄ receptors and initiates a secondary, autocrine amplification cascade resulting in sustained Ca²⁺ influx and prolonged activation of the cells.

Unlike the well-recognized involvement of cysteinyl leukotrienes in the pathogenesis of bronchial asthma, the clinical importance of LTB₄-mediated autocrine amplification cascades remains to be established. Very recently, however, LTB₄ has been reported to be involved critically in both the initiation and perpetuation of neutrophil influx and synovial damage in experimental inflammatory arthritis [20,21], while membrane and nuclear receptors for LTB₄ are up-regulated in the lungs of smokers who are susceptible to the development of chronic obstructive pulmonary disease [22]. These reports, together with recent studies which have identified an important role for LTB₄ in the pathogenesis of asthma [23,24], clearly underscore the validity of pharmacological anti-inflammatory strategies which target LTB₄ and its receptors, although no such agents are currently available for widespread clinical use. In this regard it is noteworthy that the fenamate subgroup of non-steroidal anti-inflammatory agents has been reported to inhibit both leukotriene B₄ production and Ca²⁺ uptake by activated human neutrophils; however, the relationship between these two events was not investigated in these studies [25,26].

Notwithstanding conventional anti-fungal activity, the inhibitory effects of itraconazole on LTB₄ production by activated neutrophils described in the current study, together with a possible small contribution from contaminating cells, may also contribute to the usefulness of this and other imidazole anti-mycotics in the prophylaxis and/or therapy of both acute and chronic inflammatory disorders, including septic shock [27], chronic granulomatous disease [5,28] and allergic bronchopulmonary aspergillosis [29]. Moreover, PAF, and by implication LTB₄, is considered to be a key proinflammatory mediator of both hyperacute and chronic inflammatory disorders such as septic shock and bronchial asthma [30,31].

In conclusion, itraconazole at therapeutically attainable concentrations of 0·5 µM [32], antagonizes the activity of 5-LO in PAF-activated neutrophils, preventing the initiation of a secondary amplification mechanism with the potential to intensify and prolong harmful inflammatory responses.