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Title: CELL CULTURE MODEL FOR STUDYING BOVINE NEUTROPHIL DIAPEDESIS

Author
item SMITS, E
item CIFRIAN, E.
item Guidry, Albert
item RAINARD, P
item BURVENICH, C
item Paape, Max

Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: White blood cells (neutrophils) are the cow's primary defense against bacteria invading the mammary gland. In the normal healthy gland, neutrophils are in low numbers, thus making the gland susceptible to bacterial infection. However, once bacteria enters the gland they release toxins and other substances that attract neutrophils from blood into the milk in an effort to ward off the invading pathogen. There is some evidence that cows differ in the ability of their neutrophils to respond to these signals from the bacteria, but testing these differences is complicated by the complexity of the mammary gland. This report describes a method for creating a laboratory model of the lining of the mammary gland that can be used to test migration of neutrophils across this lining in response to various stimuli. It will also allow for the determination of cow differences in the ability of their neutrophils to respond to these stimuli. The latter will allow for the selection of cows that are more likely to ward off bacterial invasion.

Technical Abstract: Polymorphonuclear neutrophils (PMN) are the major host defense against bacterial infection in the bovine mammary gland. PMN migrate from peripheral blood into the milk in response to inflammatory stimuli. We report here the development of a cell culture model to study PMN diapedesis through he secretory and ductal mammary epithelial barriers. The model consists of a porous membrane, a layer of collagen, a non- confluent layer of fibroblasts, another layer of collagen and confluent monolayer of secretory or ductal epithelial cells. Confluency of epithelial cell monolayers was determined by electrical resistance and trypan blue diffusion. Diapedesis of PMN occurred from the basal to the apical side of the epithelial cell monolayers. Purified complement component C5a, zymosan activated serum (ZAS) and E. coli endotoxin activated serum (EAS) induced diapedesis of PMN across secretory and ductal cell monolayers. PMN isolated from the blood of five cows differed in their ability to migrate through the model in response to C5a. In all instances PMN migration occurred during the first 3 hours. Diapedesis of PMN in response to C5a was greater across ductal than across secretory cell monolayers. Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on PMN diapedesis induced by ZAS and EAS, indicating that factors other than C5a are chemotactic for PMN. Monomeric IgG2, immune complexes and E. coli endotoxin did not induce PMN diapedesis.