CELL-CULTURE SYSTEM FOR STUDYING BOVINE NEUTROPHIL DIAPEDESIS

Neutrophils are the major defense against bacterial infection in the b ovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes th e development of a system of cell culture that can be used to study ne utrophil diapedesis through secretory and ductal mammary epithelial ba rriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ducta l epithelial cells layered on a porous membrane. Confluence was determ ined by electrical resistance and trypan blue diffusion. Neutrophil di apedesis occurred from the basal to the apical surface of the monolaye rs. Purified complement C5a, fetal bovine serum that had been activate d by zymosan, and fetal bovine serum that had been activated by Escher ichia coli induced neutrophil diapedesis. Neutrophil diapedesis was gr eater across ductal cell monolayers. Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a. Monoclonal antibodies to C5a blocked diaped esis induced by purified C5a but had no effect on diapedesis induced b y fetal bovine serum that had been activated by zymosan or by fetal bo vine serum that had been activated by E. coli endotoxin, indicating th at factors other than C5a were chemotactic for neutrophils. Monomeric IgG(2), immune complexes, and E. coli endotoxin did not induce neutrop hil diapedesis.