BAYu9773

Methods

Cultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D4 (LTD4) and E4 (LTE4) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.

Results

LTD4 and LTE4 significantly augmented the expression of Tn, whereas LTD4, distinctly from LTE4, was able to increase also the Ln β2 chain. Although the expression of CysLT2 prevailed over that of CysLT1, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT1-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.

Cell culture

Normal human bronchial epithelial cells (line BEAS-2B) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 using serum-free epithelial growth medium (BEGM; Cambrex, Walkersville, MD, USA) supplemented with Bullet Kit (Cambrex) to contain 0.5 ng/ml human recombinant epidermal growth factor, 50 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 μg/ml gentamicin and 50 ng/ml amphotericin B. After reaching 50% subconfluency, the cells were challenged with 0 to 100 nM concentrations of either LTD4, the most potent or LTE4, the weakest though the most stable of the CysLTs (both from Cayman Chemical, Ann Arbor, MI, USA) during 24, 48, and 72 hours. TGF-β 1 (1 ng/ml) (Chemicon, Hampshire, UK), known to stimulate Tn expression in BEAS-2B cells [], served as a positive control. Montelukast sodium (1 μM, Merck-Frosst, Pointe Clarie, Quebec, Canada) or BAY u9773 (1 μM, Cayman Chemical) was added to the cell culture for 30 min before challenging with LTD4 or LTE4 to block the CysLT effect mediated via CysLT1 or both CysLT receptors, respectively. In settings, where LTD4-stimulation was involved, parallel experiments were performed with the presence of 5 mM L-cysteine (Sigma, St. Louis, MO, USA.) in the media to inhibit catabolism of LTD4 into LTE4.

Effect of antileukotrienes on CysLT-induced up-regulation of ECM proteins

Both CysLT receptor antagonists montelukast and BAY u9773 abolished the significant CysLT-induced increase in Tn production at both protein (Figure ) and mRNA level (Figure ). This inhibition was complete as the levels of Tn expression after receptor blockade did not differ from that in non-stimulated cells. Similarly, the significant up-regulation of expression of Ln β2 chain mRNA by 100 nM LTD4 was fully prevented by both montelukast and BAY u9773 (Figure ). There was no statistical difference between the inhibitory effects of the CysLT1-specific antagonist montelukast and the dual antagonist BAY u9773 in any setting. In experiments, where LTD4 was combined with 1 ng/ml TGF-β, both montelukast and BAY u9773 reduced the expression of Tn protein and mRNA down to the level not statistically differing from that what was seen after stimulation with TGF-β alone (Figures and ). Neither of the antileukotrienes alone significantly affected the baseline Tn and Ln β2 chain expression. Neither montelukast nor BAY u9773 significantly affected the TGF-β-induced Tn-expression (Figures and ).