Smits E, Cifrian E, Guidry A J, Rainard P, Burvenich C, Paape M J
Immunology and Disease Resistance Laboratory, USDA, Beltsville, MD 20705, USA.
J Dairy Sci. 1996 Aug;79(8):1353-60. doi: 10.3168/jds.S0022-0302(96)76492-5.
Neutrophils are the major defense against bacterial infection in the bovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane. Confluence was determined by electrical resistance and trypan blue diffusion. Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers. Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis. Neutrophil diapedesis was greater 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 diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E. coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils. Monomeric IgG2, immune complexes, and E. coli endotoxin did not induce neutrophil diapedesis.
中性粒细胞是牛乳腺抵御细菌感染的主要防线。中性粒细胞会响应炎症刺激从血液迁移至乳腺管腔。本研究描述了一种细胞培养系统的开发，该系统可用于研究中性粒细胞通过分泌性和导管性乳腺上皮屏障的渗出过程。该培养系统由胶原蛋白层、成纤维细胞层、胶原蛋白层以及位于多孔膜上的分泌性或导管性上皮细胞汇合单层组成。通过电阻和台盼蓝扩散来确定汇合情况。中性粒细胞从单层细胞的基底表面向顶端表面渗出。纯化的补体C5a、经酵母聚糖激活的胎牛血清以及经大肠杆菌激活的胎牛血清均可诱导中性粒细胞渗出。中性粒细胞通过导管细胞单层的渗出量更大。来自五头奶牛的血液中性粒细胞在响应C5a时穿过多层培养系统的迁移能力存在差异。针对C5a的单克隆抗体可阻断纯化C5a诱导的渗出，但对经酵母聚糖激活的胎牛血清或经大肠杆菌内毒素激活的胎牛血清诱导的渗出没有影响，这表明除C5a外的其他因子对中性粒细胞具有趋化作用。单体IgG2、免疫复合物和大肠杆菌内毒素均不诱导中性粒细胞渗出。
