Prolactin-induced activation of nuclear factor kappaB in bovine mammary epithelial cells: role in chronic mastitis.

We sought to determine whether prolactin (PRL) could influence the neutrophilic inflammation that characterizes chronic mastitis. Most of the genes encoding inflammatory proteins depend on the nuclear factor kappaB (NF-kappaB) for their expression. We addressed the hypothesis that immunomodulatory activities of PRL might arise from an increase in NF-kappaB activity. MAC-T cells, a bovine mammary epithelial cell line, were stimulated with increasing concentrations of bovine PRL (1, 5, 25, 125, and 1,000 ng/mL). Level of NF-kappaB binding activity was measured and mRNA was evaluated for IL-1beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GMCSF), IFN-gamma, and tumor necrosis factor (TNF)-alpha, cytokines known to require NF-kappaB for their maximal transcription. Prolactin activated NF-kappaB; maximal NF-kappaB activation was weaker with PRL than with TNF-alpha at 30 or 180 min poststimulation. In addition, PRL significantly amplified, in a dose-dependent manner, mRNA expression of IL-1beta, IL-6, IL-8, GMCSF, and TNF-alpha. We measured PRL concentrations in blood and milk from healthy and chronic mastitis-infected cows, and studied the relationship between the PRL concentration and the degree of inflammation in the mammary gland as indirectly assessed by somatic cell counts (SCC). Plasma PRL did not differ significantly between healthy and chronic mastitis-affected cows (63.7 and 67.5 ng/mL, respectively). Milk PRL concentration was significantly increased in chronic mastitis-affected quarters with the highest SCC, and had a positive significant correlation between SCC, as well as between the number of neutrophils present in milk samples. The present findings show that PRL promotes an inflammatory response in bovine mammary epithelial cells via NF-kappaB activation, and suggest a role for PRL in the pathogenesis of chronic mastitis.

Delayed neutrophil apoptosis in bovine subclinical mastitis.

Bovine subclinical mastitis can be defined as a moderated inflammatory disease characterized by a persistent accumulation of neutrophils in milk. As GMCSF-mediated delay of neutrophil apoptosis contributes to the accumulation of inflammatory cells at the site of inflammation in many human diseases, we sought to determine whether subclinical mastitis in cows is also associated with a GMCSF-dependent increase in milk-neutrophil survival. We first addressed the hypothesis that GMCSF delays bovine neutrophil apoptosis by activation of the signal transducer and activator of transcription (STAT) family members STAT3 and STAT5, which are critical regulators of the expression of various Bcl-2 family proteins. Granulocyte-macrophage colony-stimulating factor significantly delayed apoptosis of blood neutrophils obtained from healthy cows. In these cells, GMCSF activated STAT5, but not STAT3, and induced an increase in the mRNA of the antiapoptotic Bcl-2 member, Bcl-xL. Granulocyte-macrophage colony-stimulating factor-dependent STAT5 activation and up-regulation of Bcl-xL mRNA were blocked by the Jak inhibitor, AG-490. This inhibition was associated with abrogation of the prosurvival effect of GMCSF, demonstrating a key role for STAT5 in delayed neutrophil apoptosis. We further found that GMCSF expression was increased in milk cells from cows affected with subclinical mastitis. Neutrophils from these cows demonstrated a significant delay of apoptosis as compared with neutrophils obtained from healthy cows and were unresponsive to GMCSF. Active STAT5 complexes were detected in these neutrophils. Finally, in the presence of AG-490, apoptosis was induced and a time-dependent down-regulation of Bcl-xL mRNA was observed in milk neutrophils from mastitis-affected cows. These results indicate that neutrophil survival is enhanced in milk of subclinical mastitis-affected cows and suggest a role for a GMCSF-activated STAT5 signaling pathway in this phenomenon. This pathway could thus represent a target for the control of persistent accumulation of neutrophils in the bovine mammary gland.