Multidimensional suppression of acute pulmonary inflammation by the leukotriene modifiers Montelukast and Zileuton

Abstract

The hallmark of acute lung inflammation is neutrophil infiltration in inflamed lung tissue, and leukotrienes are important mediators that drive immune cell migration. However, the effects of leukotriene modifiers on acute lung inflammation and the mechanisms of their effects are unclear. Flow cytometry revealed that the leukotriene modifiers montelukast and zileuton inhibited the migration of neutrophils and the expression of the adhesion molecules P-selectin, L-selectin and PSGL-1, as well as the integrins LFA-1, Mac-1 and VLA-4 during migration in each lung compartment. Combined ELISA and blood smear analysis confirmed that both agents reduced the release of bone marrow CXCR4+ neutrophils into the blood by maintaining bone marrow SDF-1 concentrations. The application of montelukast and zileuton resulted in a significant reduction in gene and protein expression of the lung inflammatory factors TNF-α, IL6, and CXCL2/3, but only a reduction in CXCL1 at the gene expression level, which may be related to the rapid release of CXCL1 from endothelial cell preexisting. Further studies confirmed that both agents inhibited the expression of the ERK1/2 pathway and downstream pCREB, which are involved in the transcription of inflammatory chemokines. Montelukast and zileuton also inhibited the production of oxidative bursts in the lung interstitium and the formation of neutrophil-platelet complexes in the vasculature, which promote neutrophil migration. RT-qPCR and immunofluorescence confirmed the key enzymes of the leukotriene synthesis pathway and CysLTR1, LTB4R1 expression on neutrophils, lung endothelial and epithelial cells were inhibited by both modifiers. We observed that montelukast and zileuton exhibited different functions in addition to the classical effects of CysLTR1 antagonist and inhibitor of arachidonate-5-Lipoxygenase. As has been demonstrated: both agents inhibited caspase-1 protein expression and reduced IL-1β production; They inhibited gene expression of MMP-3 and MMP-9 in lung tissue and protein expression of platelet-activated receptor P2Y12 as well. In summary, montelukast and zileuton have multitargeted, multidimensional, and potent anti-inflammatory functions during acute pulmonary inflammation.