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. SAMPLE POPULATION: 12 tracheas obtained within 5 hours after euthanasia from horses free from respiratory tract disease. PROCEDURES: 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 AND CLINICAL RELEVANCE: 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.