Updated: Sep 25
To determine novel anti-inflammatory effects of MLK on resting and GM-CSF- stimulated eosinophils using the Cellix VenaFlux platform to mimic physiological adhesion to rhVCAM-1.
Asthma is one of the most common chronic respiratory diseases in developed countries. For most asthmatics their symptoms are satisfactorily controlled by the regular use of inhaled glucocorticoids (GC). However, these drugs are not without side effects and some asthmatic patients derive only partial and in some cases no relief of their symptoms even when using high doses of GC. Clearly, we need more effective therapy for asthma. Eosinophils are key pro-inflammatory cells in the asthmatic lung where their cytotoxic products cause damage to the airway epithelium, tissue inflammation and airflow obstruction. Eosinophil adhesion to and transmigration through the endothelial cells lining the post-capillary venules are key events in their accumulation in the asthmatic lung. Understanding these mechanisms may lead to the identification of compounds that can blunt eosinophil accumulation. Activation of the cysteinyl leukotriene cysLT1 receptor (cysLT1R) results in eosinophil migration and damage to the mucus layer in the lung. Montelukast (MLK) is part of a new class of anti-asthma drugs that are antagonists to cysLT1R reducing eosinophil migration. Evidence is accumulating that MLK may have additional anti-inflammatory effects on eosinophil function which were further investigated in this study.
Refer to Application Note R200 for eosinophil isolation and flow assay details. Concentrations of adhesion proteins, mediators and drugs used were rhVCAM-1; 10 μg/mL, BSA; 10 μg/mL, GM-CSF; 10 ng/mL, LTC4/D4; 100 nM, MLK; 0.1–100 nM, MK571; 100 nM, MK886; 100 nM, Anti- α4β1; 10 μg/mL, anti-cysLT1R; 10 μg/mL, Isotype Control; 10 μg/mL.
MLK (10 and 100 nM) gave partial (~40%) but significant (P<0.05) inhibition of unstimulated eosinophil adhesion to rhVCAM-1 at 2 dyne/cm2 (Figures 1 and 2). GM-CSF-stimulated eosinophil adhesion under flow was characterised by greater cell flattening with significant (P<0.05) inhibition of adherent cell numbers by 100 nM MLK observed (Figure 2). This effect appeared specific for MLK as the analogue MK571 had no significant effect on eosinophil adhesion to VCAM-1 (Figure 3). LTC4 released from unstimulated or GM- CSF-treated eosinophils did not contribute to their adhesion to VCAM-1 as the leukotriene biosynthesis inhibitor MK886 had no inhibitory effect (Figure 3) while exogenously added LTC4 did not enhance eosinophil adhesion (Table 1). In contrast, LTD4, enhanced eosinophil adhesion to VCAM-1; an effect blocked by MLK (Table 1). Comparable observations were also made at 1 dyne/cm2 (data not shown).
Physiologically relevant concentrations of MLK inhibited resting and GM-CSF-stimulated eosinophil adhesion to VCAM-1 in an in vitro model of the post- capillary venules.
Inhibitory effects by MLK appeared independent of cysLT1R blockade.
Our study confirms a previous report that MLK inhibited transmigration of eosinophils across human umbilical veins endothelial cells under static conditions .
These findings may provide important clues for developing novel therapy aimed at blunting eosinophil-induced inflammation in allergic-based disease.
Robinson A.J., Kashanin D, O’Dowd F, Williams V. and Walsh G.M. (2008) Montelukast inhibition of resting and GM- CSF -stimulated eosinophil adhesion to VCAM-1 under flow conditions appears independent of CysLT1 antagonism Journal of Leukocyte Biology (in press).
Virchow Jr., J.C., Faehndrich, S., Nassenstein, C., Bock, S., Matthys, H., & Luttman, W. (2001). Effect of a specific cysteinyl leukotriene receptor antagonist (montelukast) on the transmigration of eosinophils across human umbilical vein endothelial cells. Clin. Exp. Allergy, 31, 836- 844.
©2018 Cellix Limited. All rights reserved.