Montelukast inhibits neutrophil pro-inflammatory activity by a cyclic AMP-dependent mechanism

Abstract

Background and purpose: The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1-2 micromol x L(-1)), on Ca(2+)-dependent pro-inflammatory activities, cytosolic Ca(2+) fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 micromol x L(-1)) and platelet-activating factor (200 nmol x L(-1)).

Experimental approach: Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B(4) and cAMP by competitive binding ELISA procedures, and Ca(2+) fluxes by fura-2/AM-based spectrofluorimetric and radiometric ((45)Ca(2+)) procedures.

Key results: Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B(4) by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 micromol x L(-1) (mean percentages of the control values of 30 +/- 1, 12 +/- 3 and 21 +/- 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca(2+) concentrations and store-operated Ca(2+) influx. These montelukast-mediated alterations in Ca(2+) handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.

Conclusions and implications: Montelukast, primarily a cysteinyl leukotriene (CysLT(1)) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.

Figure 1

Effects of montelukast (0.1–2 µmol·L⁻¹) on the lucigenin- and luminol-enhanced chemiluminescence responses of neutrophils activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 µmol·L⁻¹). The results are expressed as the mean peak chemiluminescence values in relative light units measured 30–50 s after the addition of FMLP and vertical lines show SEM. In the case of lucigenin-enhanced chemiluminescence (left graph, n = 3 with three to four replicates for each drug concentration and control system in each experiment), the absolute values for unstimulated neutrophils and for cells activated with FMLP in the absence of montelukast were 1086 ± 147 and 4729 ± 325 respectively, while the corresponding values for luminol-enhanced chemiluminescence (right graph, n = 5 with two to three replicates for each drug concentration and control system in each experiment) were 3047 ± 127 and 56 397 ± 8394 relative light units. *P < 0.01 to P < 0.001 for comparison with the FMLP-activated, montelukast-free control system.

Figure 2

Effects of montelukast (0.1–2 µmol·L⁻¹) on the release of elastase from neutrophils activated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 µmol·L⁻¹)/cytochalasin B (0.5 µmol·L⁻¹). The results (n = 4 with duplicate data sets for each experiment with 10 replicates for each drug concentration and control system in each experiment) are expressed as the mean values for total extracellular elastase (milliunits 10⁷ cells⁻¹) and vertical lines show SEM. The absolute values for the unstimulated control system and for cells activated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B in the absence of montelukast were 221 ± 44 and 892 ± 9.6 milliunits elastase 10⁷ cells⁻¹ respectively. *P < 0.001 for comparison with the drug-free control systems.

Figure 3

Effects of montelukast (0.25–2 µmol·L⁻¹) on the production of leukotriene B₄ (LTB₄) by neutrophils activated with platelet-activating factor (200 nmol·L⁻¹). The results are presented as the mean values for total extracellular LTB₄ (pg 10⁷ cells⁻¹) and vertical lines show SEM (n = 8, with two to three replicates for each drug concentration and control system in each experiment). The absolute values for the unstimulated control system and for cells activated with platelet-activating factor in the absence of montelukast were 16 ± 6 and 993 ± 107 pg LTB₄ 10⁷ cells⁻¹ respectively. *P < 0.001 for comparison with the drug-free control system.

Figure 4

Chemoattractant-activated fura-2 fluorescence responses of control and montelukast (2 µmol·L⁻¹)-treated neutrophils. Platelet-activating factor (PAF) (200 nmol·L⁻¹) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 µmol·L⁻¹) were added as indicated (↑) when a stable baseline was obtained (±1 min). The traces shown are from three different representative experiments (8 for FMLP and 12 for PAF in the series).

Figure 5

Effects of montelukast (0.25–2 µmol·L⁻¹) on the influx of ⁴⁵Ca²⁺ into the neutrophils activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 µmol·L⁻¹, upper graph) or platelet-activating factor (PAF) (200 nmol·L⁻¹, lower graph). The results are expressed as the mean percentages of the drug-free control systems and vertical lines show SEM (n = 4–8 with two to four replicates for each drug concentration and control system). The absolute values for uptake of ⁴⁵Ca²⁺ by unstimulated neutrophils and for cells activated with FMLP or PAF were 47 ± 25, 150 ± 34 and 148 ± 14 pmol ⁴⁵Ca²⁺ 10⁷ cells⁻¹ respectively. *P < 0.05 to P < 0.01 for comparison with the corresponding chemoattractant-activated montelukast-free control systems (according to the repeated measures anova, there were significant effects of montelukast at both 1 and 2 µmol·L⁻¹ for the FMLP system; on post hoc testing significance remained at 2 µmol·L⁻¹).

Figure 6

Effects of CGS21680 (CGS; 1 µmol·L⁻¹), salbutamol (Sb; 5 µmol·L⁻¹), rolipram (ROLI; 0.1 µmol·L⁻¹) and montelukast (2 µmol·L⁻¹) individually, as well as those of montelukast in combination with the other agents on neutrophil intracellular cAMP. The results are presented as the mean percentages of the drug-free control system and vertical lines show SEM (n = 6, with two to three replicates for each drug concentration and control system in each experiment). The absolute value for the drug-free control system was 4.3 ± 0.3 pmol cAMP 10⁷ cells⁻¹. *P < 0.05 for comparison with the drug-free control systems.

Figure 7

Effects of montelukast (0.5–20 µmol·L⁻¹), rolipram (20 µmol·L⁻¹) and 3-isobutyl-1-methylxanthine (50 µmol·L⁻¹) on cAMP (upper graph) and cGMP (lower graph) phosphodiesterase (PDE) activities in neutrophil cytosol. The results of four to eight and two to four experiments for cAMP and cGMP PDE activity respectively are presented as the mean percentages of the drug-free control systems and vertical lines show SEM. In the case of the cAMP/PDE experiments, the absolute values for the cytosol-free background system and for the cytosol-containing systems in the absence of the drugs were 1213 ± 53 and 14 525 ± 232 counts per minute respectively. The corresponding values for the cGMP/PDE experiments were 2031 ± 206 and 21 381 ± 501 counts per minute. *P < 0.05 for comparison with the corresponding drug-free control system.