Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC24
Lipoxin expression in horses with recurrent airway obstruction
1. School of Veterinary Medicine and Science, Nottingham, United Kingdom.
Recurrent airway obstruction (RAO) is characterised by chronic lower airway inflammation in stabled mature horses. Moderately afflicted animals exhibit a type-1 hypersensitivity reaction accompanied by reversible airway obstruction. Advanced cases experience severe respiratory distress and irreversible remodelling of bronchial smooth muscle. RAO has serious health and welfare implications, and is estimated to affect 1 in 6 of the UK equine population. Lipoxins prevent chronic inflammation by active resolution of acute inflammation. Severe asthma in humans has similarities to RAO. Asthma is associated with deficient lipoxin signalling. Establishing dysfunctional lipoxin expression in RAO offers potential remedial therapies. Primary equine tracheal epithelial cells (ETE) were cultured following dissection and enzymatic digestion of equine respiratory mucosa. Primary ETE cells were identified using morphology and immunocytochemical staining. Isolated cells were incubated over 24 hours with LPS (Escherichia coli, serotype 0127:B8). Control groups were cultured without LPS. Both groups were cultured for time points 0, 4, 8, 12, 16, and 24 hours. A reverse transcriptase method generated cDNA using total RNA from cell samples. cDNA products corresponding to COX-2, iNOS and lipoxin receptor ALX were generated using a standard polymerase chain reaction (PCR). Quantification of COX-2 was achieved by using q-PCR. Primer pairs were from published papers with the exception of ALX. This was designed on the published mRNA sequence for equine N-formylpeptide receptor-like1 (Accession number XM_001497411). PCR products corresponding to ALX were sequenced and certified using NCI-BLAST. Cell viability was estimated to be > 85% (number of replicates = 9) and reached confluency after approximately 3 days. Isolated cells exhibited a morphology characteristic of epithelial cells with positive staining for cytokeratin (PCK-26), an intermediate filament in epithelial tissue. Isolated cells did not stain for vimentin (V9), a marker for smooth muscle. mRNA corresponding to COX-2, ALX and iNOS was expressed at all time points with no apparent difference between control groups or treated cells, at all stated time points. Preliminary findings suggest ETE cells are in a pre-inflammatory state prior to incubation with LPS. Modifications to culturing conditions and tissue sources did not decrease COX-2 expression. Future work will consider epithelial explant cultures and primary culture of airway smooth muscle. Co-culture systems between epithelial layers and airway smooth muscle will be established. Signalling pathways between the two tissue types will be examined with RNA interference. Development of successful equine primary cell culture is vital to provide an in vitro model to study the disease.
Where applicable, experiments conform with Society ethical requirements