Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C10 and PC10
Effect of lipoxin A4 on acute inflammation in primary equine airway smooth muscle cells
C. Beynon1, R. Hammond1, S. Dunham1
1. School of Veterinary Medicine and Science, Nottingham University, Leicestershire, United Kingdom.
INTRODUCTION Recurrent airway obstruction (RAO) is a condition that may occur in mature horses and has similarities to human asthma. Permanent airway remodelling occurs in long term cases. Airway remodelling features an increase in airway smooth muscle (ASM) mass. Chronic respiratory inflammation has been postulated as a factor that contributed to such remodelling. Airway inflammation in both RAO and human asthma features an increase in the expression of Toll-like receptor 4, the inducible enzymes inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2), and the release of mediators including tumour necrosis factor alpha (TNF-α) and interleukin-1 β (IL-1β).Lipoxin A4 (LXA4) is one of a group of arachidonic acid metabolites that modulate acute inflammation through action on specific lipoxin receptor (ALX) in a number of species. LXA4 has been shown to have anti-inflammatory properties in a number of models of acute inflammation. Dysfunction of the mechanisms that result in LXA4 synthesis and expression of the ALX receptor may contribute to the chronic inflammatory response seen in asthma. Abnormal LXA4 and/or ALX expression may also be present in horses with RAO thereby promoting ASM remodelling. AIMS OF THE STUDY To investigate the effect of endogenous LXA4 on acute LPS-induced inflammation in primary equine ASM cells. METHODS Primary ASM cells were cultured from explants of equine trachealis muscle. Primary cells were identified by positive staining for α-smooth muscle actin. Near-confluent ASM cells were treated with:100µM/ml endogenous LXA4, 0.1µg/ml lipolysaccharide (LPS) or a combination of LXA4/LPS. Cells were harvested after 24 and 72 hrs. Real-time PCR quantified mRNA expression of β-actin (reference gene),ALX, TLR-4, TNF- α, IL-1β, iNOS and COX-2. Protein expression of COX-2 and ALX were additionally examined using Western Blot.RESULTS After 24hrs, expression of mRNA coding for ALX peaked in the LXA4 treated cells but was decreased in other treatment groups. Expression of mRNA coding for iNOS mRNA was also upregulated in the LXA4/LPS treated cells at 24hrs. At 72hrs post treatment, mRNA for TLR-4, TNF-α, IL-1β and COX-2 was increased in LPS-treated cells. Incubation with LXA4 decreased mRNA expression for TLR-4, TNF-α, IL-1β, iNOS and COX-2 at 72hrs. Similarly, reduced expression of mRNA for TNF-α, IL-1β, iNOS and COX-2 was evident in LXA4/LPS treated cells at 72hrs. Western Blotting showed that protein for ALX was present at 24 and 72hrs in control and treated cells. COX-2 was not expressed at 72hrs in the LXA4 and LXA4/LPS treated cells. CONCLUSION These findings suggest that treatment of equine ASM cells with endogenous LXA4 causes a decrease in LPS-induced release of inflammatory mediators after 72hrs of incubation. However, an increase in expression of TLR-4 mRNA at 72hrs indicates that this effect may not be sustained at later time points
Where applicable, experiments conform with Society ethical requirements