title: Short communication: Differential loss of bovine mammary epithelial barrier integrity in response to lipopolysaccharide and lipoteichoic acid.
abstract: In the mammary gland, the blood-milk barrier prevents an uncontrolled intermixture of blood and milk constituents and hence maintains the osmotic gradient to draw water into the mammary secretion. During mastitis, the permeability of the blood-milk barrier is increased, which is reflected by the transfer of blood constituents into milk and vice versa. In this study, we aimed to investigate changes in the barrier function of mammary epithelial cells in vitro as induced by cell wall components of different pathogens. Primary bovine mammary epithelial cells from 3 different cows were grown separately on Transwell (Corning Inc., Corning, NY) inserts. The formation of tight junctions between adjacent epithelial cells was shown by transmission electron microscopy and by immunofluorescence staining of the tight junction protein zona occludens-1. The integrity of the epithelial barrier was assayed by means of transepithelial electrical resistance, as well as by diffusion of the fluorophore Lucifer yellow across the cell layer. The release of lactate dehydrogenase (LDH) was used as an indicator for cytotoxic effects. In response to a 24-h challenge with bacterial endotoxin, barrier integrity was reduced after 3 or 7h, respectively, in response to 0.5mg/mL lipopolysaccharide (LPS) from Escherichia coli or 20mg/mL lipoteichoic acid (LTA) from Staphylococcus aureus. No paracellular leakage was observed in response to 0.2mg/mL LPS or 2mg/mL LTA. Although LPS and LTA affected barrier permeability, most likely by opening the tight junctions, only LPS caused cell damage, reflected by increased LDH concentrations in cell culture medium. These results prove a pathogen-specific loss of blood-milk barrier integrity during mastitis, which is characterized by tight junction opening by both LPS and LTA and by additional epithelial cell destruction through LPS.
authors.affiliation: Veterinary Physiology, Vetsuisse Faculty University of Bern, CH-3012 Bern, Switzerland. Electronic address: olga.wellnitz@vetsuisse.unibe.ch.
authors.forename: Olga
authors.initials: O
authors.lastname: Wellnitz
authors.affiliation: Veterinary Physiology, Vetsuisse Faculty University of Bern, CH-3012 Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Christina
authors.initials: C
authors.lastname: Zbinden
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Xiao
authors.initials: X
authors.lastname: Huang
authors.affiliation: Veterinary Physiology, Vetsuisse Faculty University of Bern, CH-3012 Bern, Switzerland.
authors.forename: Rupert M
authors.initials: RM
authors.lastname: Bruckmaier
mesh_terms: D000818:Animals; D002417:Cattle; D004847:Epithelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007774:Lactation; D008070:Lipopolysaccharides / Q000633:toxicity*; D008321:Mammary Glands, Animal / Q000166:cytology*; D008414:Mastitis, Bovine / Q000139:chemically induced / Q000175:diagnosis; D008892:Milk / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism; D013682:Teichoic Acids / Q000633:toxicity*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D013682:Teichoic Acids; D062826:Zonula Occludens-1 Protein; C009900:lipoteichoic acid; D007770:L-Lactate Dehydrogenase
keywords: blood–milk barrier; mammary; mastitis; tight junction