Bovine neutrophil migration, CD11a/CD18 expression, and cytokine response to co-culture with mammary gland endothelial cells and epithelial cells

Abstract Movement of leukocytes from the circulatory system into a site of inflammation is a highly complex process. The migration of neutrophils into the lumen of the mammary gland during mastitis is no exception. There is information about the involvement of bacterial produced products during this process, but less is known regarding the role of host products. Two bovine cell lines, a primary mammary gland endothelial cell line and an immortalized bovine mammary gland epithelial cell line (Mac-T), along with freshly isolated bovine neutrophils, were used to study this further. The cell lines were grown on inserts and in wells of tissue-culture plates. In the initial set of experiments, neutrophils were added to the inserts and then their migration into the tissue-culture plate wells was monitored using a hemocytometer or a flow cytometer. Lipopolysaccharide was added to some of the wells to induce migration. This was then followed by a similar series of experiments that were initialized by the addition of inhibitors to interleukin-8 (IL-8), platelet-activating factor (PAF), tumor-necrosis factor-α(TNF-α), or lipoxygenase (LOX) prior to the addition of the neutrophils and their enumeration. In addition, integrin expression (CD11a/18) by the neutrophils was measured using flow cytometry. In our insert/tissue culture plate well system, neutrophils readily migrated towards the epithelial cells when they were separated from them either by the insert alone or insert plus a layer of endothelial cells. The presence of LPS in the system allowed this migration to occur without the involvement of epithelial cells. The inhibition of PAF or TNF alone did not alter migration while the inhibition of either IL-8 or LOS did significantly reduce the movement of neutrophils. Only the migrating neutrophils had upregulated levels of CD11a/18 on their surface. From a host perspective, it appears that products of the LOX enzymes system and IL-8 were the primary inducers of neutrophil migration, and that mammary gland epithelial cells were capable of driving this process on their own. Understanding the role of host-produced chemotactic agents that are involved in mammary gland inflammation may allow better regulation of this activity.