APPLICATION NOTE R100 Respiratory: EOSINOPHIL - ADHESION ASSAY - ASTHMA
To determine novel anti-inflammatory effects of MLK on
MLK (10 nM and 100 nM) gave partial (~40%) but significant
resting and GM-CSF-stimulated eosinophils using the Cellix
(P<0.05) inhibition of unstimulated eosinophil adhesion to
VenaFluxTM platform to mimic physiological adhesion to
rhVCAM-1 at 2 dyne cm-2 (Figure 1 and Figure 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
Asthma is one of the most common chronic respiratory
(Figure 3). LTC4 released from unstimulated or GM-CSF-
diseases in developed countries. For the majority of asthmatics
treated eosinophils did not contribute to their adhesion to
their symptoms are satisfactorily controlled by the regular use
VCAM-1 as the leukotriene biosynthesis inhibitor MK886 had
of inhaled glucocorticoids (GC). However, these drugs are not
no inhibitory effect (Figure 3) while exogenously added LTC4
without side effects and some asthmatic patients derive only
did not enhance eosinophil adhesion (Table 1). In contrast,
partial and in some cases no relief of their symptoms even
LTD4, enhanced eosinophil adhesion to VCAM-1; an effect
when using high doses of GC. Clearly we need more effective
blocked by MLK (Table 1). Comparable observations were also
made at 1 dyne cm-2 (data not shown).
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 theendothelial 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 accumulatingthat MLK may have additional anti-inflammatory effectson eosinophil function which were further investigated inthis 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-1, BSA; 10 µg mL-1, GM-CSF; 10 ng mL-, LTC4/D4; 100 nM, MLK; 0.1 nM – 100 nM, MK571; 100 nM, MK886; 100
Figure 1: Representative experiment illustrating unstimulated eosinophil
nM, Anti-α4β1; 10 µg ml-1, anti-cysLT1R; 10 µg ml-1,
adhesion at 2 dyne cm-2 to rhVCAM-1 together with the inhibitory effects of
APPLICATION NOTE R100 Respiratory: EOSINOPHIL - ADHESION ASSAY - ASTHMA
Non-& GM-CSF-Stimulated Eosinophil Adhesion to VCAM-1 at 2 dyne cm-2
* denotes significance (p<0.005), compared to Nil ** denotes significance (p<0.005), compared to LTD4
Table 1: The effect of MLK and an anti-CysLT1R antibody on non-stimulated
and LTC4/D4-stimulated eosinophil adhesion to rhVCAM-1 at dyne cm-2.
Figure 2: Dose response of the effect of MLK on non-stimulated and GM- CSF-stimulated eosinophil adhesion to rhVCAM-1 under flow conditions of2 dyne cm-2 (n=4, *P<0.05).
Physiological y 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.
Non-& GM-CSF-Stimulated Eosinophil Adhesion to VCAM-1 at 2 dyne cm-2
Inhibitory effects by MLK appeared independent of cysLT1R
Our study confirms a previous report that MLK inhibited
transmigration of eosinophils across human umbilical vein
endothelial cells under static conditions2.
These findings may provide important clues for developing novel
therapy aimed at blunting eosinophil-induced inflammation
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).
2 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
Figure 3: Effect of MLK, the montelukast an MK571 and the leukotriene
human umbilical vein endothelial cells. Clin. Exp. Allergy, 31, 836-844.
biosynthesis inhibitor MK886, on unstimulated or GM-CSF-stimulated eosinophil adhesion to rhVCAM-1. Data expressed as a percentage of non-stimulated eosinophil adhesion at 2 dyne cm-2 (n=4, *P<0.05).
2008 Cellix Limited. All rights reserved.
God loads the purim for His glory All passages from the New American Standard Bible unless otherwise noted. The Book of Esther is set in the mid-fifth century in Persia before Messiah’s earthly ministry. That’s basically modern-day Iran. The events occurred just after a federation of Medians and Persians defeated Babylon, which had conquered the Kingdom of Yehudah (Judah) with the fall o
OZURDEX™ OZURDEX™ HIGHLIGHTS OF PRESCRIBING INFORMATION –––––– WARNINGS AND PRECAUTIONS ––––– 2.2 Administration These highlights do not include all the • Intravitreal injections have been associated The intravitreal injection procedure should be carried out under controlled aseptic conditions which information needed to use OZURDEX™ include
APPLICATION NOTE R100