Interleukin 8 response by bovine mammary epithelial cells to lipopolysaccharide stimulation

Abstract

Objective - To determine whether an established bovine mammary epithelial cell line expresses interleukin 8 (IL-8) mRNA and synthesizes antigenic IL-8 in response to lipopolysaccharide (LPS) stimulation. Sample Population - A bovine mammary epithelial cell line (MAC-T). Procedure - mRNA was isolated from cells stimulated with graded concentrations of LPS. The first strand of IL-8 cDNA was synthesized, using a reverse transcriptase (RT) reaction with a specific oligonucleotide. Amplification of IL-8 cDNA was obtained by use of polymerase chain reaction (PCR). The MAC-T-derived antigenic IL-8 was quantified by use of a commercial anti-human IL-8 kit in a sandwich ELISA. Results - RT-PCR revealed expression of MAC-T-derived mRNA within the first hour after stimulation with LPS. Expression of IL-8 mRNA was correlated to production of IL-8 protein detected in medium by use of the sandwich ELISA. Amounts of antigenic IL-8 increased in a dose-and time-dependent manner, and were maximal (57 pg/ml) at 48 hours after stimulation with 20 μg of LPS/ml. Conclusions - MAC-T cells secrete IL-8 in response to stimulation with LPS in a dose-and time-dependent manner. The results were consistent with our hypothesis that mammary gland epithelial cells can be a source of IL-8 during the early stage of mastitis. Therefore, IL-8 may have a pivotal role in resolving bacterial infections.