Karen L Oslund1, Grete Adamson, Reen Wu. 1. 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
Abstract
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.
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 equineMUC5AC 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 equinetumor 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 equinepulmonary diseases.
Authors: Karine Rousseau; Jacqueline M Cardwell; Emma Humphrey; Richard Newton; David Knight; Peter Clegg; David J Thornton Journal: PLoS One Date: 2011-05-13 Impact factor: 3.240
Authors: Getu Abraham; Claudia Zizzadoro; Johannes Kacza; Christin Ellenberger; Vanessa Abs; Jana Franke; Heinz-Adolf Schoon; Johannes Seeger; Yohannes Tesfaigzi; Fritz R Ungemach Journal: BMC Vet Res Date: 2011-06-07 Impact factor: 2.741