Background and purpose:The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1-2 mmol·L ).Experimental approach: Generation of reactive oxygen species was measured by lucigenin-and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B4 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 B4 by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 mmol·L -1 (mean percentages of the control values of 30 Ϯ 1, 12 Ϯ 3 and 21 Ϯ 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 (CysLT1) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 mM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca 2+ fluxes?Isolated human blood neutrophils were activated with the chemoattractant N-formyl-Lmethionyl-L-leucyl-L-phenylalanine (fMLP) (1 mM) in combination with cytochalasin B (CB; 3 mM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca 2+ fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B 4 were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca 2+, as well as release of elastase and production of superoxide and LTB 4 , and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP.The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

These previously undocumented sensitizing interactions of LTs C(4) and D(4) with neutrophils may contribute to the activation of these cells in acute and chronic inflammation of both atopic and non-atopic aetiology, while identifying a role for montelukast in regulating neutrophil reactivity.