Concentration-dependent noncysteinyl leukotriene type 1 receptor-mediated inhibitory activity of leukotriene receptor antagonists

Abstract

The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 receptor (CysLT(1))-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and -independent anti-inflammatory activity of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast). Using receptor-desensitization experiments in monocytes and CysLT(1)-transfected HEK293 cells and IL-10- and CysLT(1) small interfering RNA-induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists leukotriene D(4) and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also inhibited calcium ionophore-induced leukotriene (leukotriene B(4) and leukotriene C(4)) production, indicating 5-lipoxygenase inhibitory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non-CysLT(1)-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma.

Figure 1

LTD₄ and UDP induce calcium mobilization in monocytes. Monocytes were prepared and stimulated with different concentrations of LTD₄ (open squares) or UDP (open diamonds) and calcium release was measured, as indicated in Methods. Data are presented as means ± SD from 3 separate experiments each performed in triplicate.

Figure 2

Montelukast and zafirlukast inhibit UDP and LTD₄ induced calcium mobilization in monocytes. Monocytes were stimulated with UDP (100 μmol/L) (A, C) or LTD₄ (100 nmol/L) (B, D) in the presence of increasing concentrations of montelukast (A, B), zafirlukast (C, D) or vehicle control (10 min. pretreatment). Results are shown as the percentage of peak fluorescence obtained in cells stimulated without inhibitors. The means ± SD from three independent experiments are presented.

Figure 3

Desensitization effects in LTD₄ and UDP stimulated monocytes and CysLT₁transfected HEK293 cells. Representative traces of calcium flux induced by repeated exposure to LTD₄ (100 nmol/L) and UDP (100 μmol/L) in monocytes (A) and CysLT1 transfected HEK293 cells (B). Data from one of three separate experiments, each with similar results, are shown.

Figure 4

The effect of P2Y₆ inhibitor, MRS2578 on UDP- and LTD₄-induced calcium flux in monocytes. Representative traces of calcium flux induced by exposure to UDP (100 μmol/L) and LTD₄ (100 nmol/L) in the presence of increasing concentrations of MRS2578 (MRS) or vehicle control. Data from one of three separate experiments, each with similar results, are shown.

Figure 5

Down-regulation of CysLT₁ did not affect UDP induced calcium mobilization in monocytes. (A) Monocytes were incubated with or without IL-10 (20 ng/ml) overnight and calcium release was measured in response to different concentrations of LTD₄ and UDP as described in the Methods section. (B) Monocytes were nucleofected with CysLT₁ siRNA or negative control oligonucleotides (Con) as described in the Methods section, cultured for 24 hours and calcium flux was measured in response to LTD₄ and UDP. Data are presented as means ± SD from 3 separate experiments performed in triplicate.

Figure 6

Knock down of P2Y₆ did not affect LTD₄ induced signaling in monocytes. Monocytes were nucleofected with P2Y₆ siRNA or negative control oligonucleotides (Control) as described in the Methods section, cultured for 24 hours and calcium flux was measured in response to different concentrations of UDP (A, B) and LTD₄ (C, D). Representative traces of calcium flux from one of three separate experiments, each with similar results, are shown.

Figure 7

Montelukast and zafirlukast inhibit UDP induced IL-8 production in monocytes. (A) Monocytes were preincubated with MRS2578 or vehicle control for 30 min. and stimulated with LTD₄(100 nmol/L) or UDP (100 μmol/L). Supernatants were collected after 2 h and 6 h and IL-8 expression was measured. (B) Monocytes were preincubated with different concentrations of LTRAs, stimulated with UDP and IL-8 expression was measured after 6 hours. Data are presented as percentage of UDP induced IL-8 production from cells pretreated with vehicle control. Means ± SD from 3 separate experiments are presented. * p < 0.001; Student's t test.

Figure 8

Montelukast and zafirlukast inhibit leukotriene synthesis in monocytes. Monocytes were preicubated with different concentrations of montelukast (A) and zafirlukast (B) (10 min) or vehicle control, stimulated with calcium ionophore A23187 (1 μmol/L) and levels of LTB₄, LTC₄ and PGE₂ (D) released were measured. (C) Cells were preincubated with montelukast (Mon) and zafirlukast (Zaf) (5 μmol/L) and intracellular levels of LTB₄ and LTC₄ were measured as described in Methods. The means ± SD from 3 donors, performed in triplicate are shown. * p < 0.01 or ** p < 0.001 for A23187 stimulated cells with versus without inhibitor.