Evaluation of MUC5AC expression and upregulation in airway epithelial cells of horses.
OBJECTIVE
To isolate and culture primary equine airway epithelial cells in vitro and elucidate the major cytokines involved in expression of the gel-forming mucin gene MUC5AC in horses.


METHODS
12 tracheas obtained within 5 hours after euthanasia from horses free from respiratory tract disease.


METHODS
Tracheal rings were digested overnight in 0.2% protease, and dissociated airway epithelial cells were grown in a serum-free defined medium at an air-liquid interface until confluence was achieved. Differentiated airway epithelial cells were treated with a panel of recombinant equine cytokines followed by quantitative reverse transcriptase PCR assay for mRNA of equine MUC5AC and the control gene glyceraldehyde 3-phosphate dehydrogenase. Cultures were incubated in the presence of isohelenin, a nuclear factor kappaB-DNA-binding inhibitor, to investigate transcriptional regulation of MUC5AC.


RESULTS
Light and electron microscopy revealed a differentiated epithelium with ciliated cells, nonciliated mucous cells, and basal-like cells. Recombinant equine tumor necrosis factor-alpha was the major mediator in the cytokine panel that significantly increased MUC5AC mRNA by a factor of 5 in a dose- and time-dependent manner. This enhancement was attenuated by isohelenin.


CONCLUSIONS
Data suggested that a nuclear factor KB-based transcriptional mechanism is involved in induction of MUC5AC expression by tumor necrosis factor-A. Understanding the molecular mechanism of cytokine-enhanced MUC5AC expression in horses may lead to better treatment options and understanding of the pathogenesis of equine pulmonary diseases.
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine and Center for Comparative Respiratory Biology and Medicine, School of Medicine, University of California-Davis, Davis, CA 95616, USA. kloslund@ucdavis.edu
Karen L
KL
Oslund
Grete
G
Adamson
Reen
R
Wu
D000402:Airway Obstruction / Q000235:genetics / Q000662:veterinary*; D000818:Animals; D004847:Epithelial Cells; D006734:Horse Diseases / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D008855:Microscopy, Electron, Scanning / Q000662:veterinary; D055271:Mucin 5AC / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012717:Sesquiterpenes / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D055271:Mucin 5AC; D016328:NF-kappa B; D012333:RNA, Messenger; D012717:Sesquiterpenes; D014409:Tumor Necrosis Factor-alpha; C103906:isohelenin