Cytokine production by cultured bovine mammary epithelial cells (MAC-T) upon stimulation with lipopolysaccharide

Cytokines are known to be involved in mastitis, yet their production by bovine epithelial cells has so far not been reported. In this research a bovine mammary alveolar cell line (MAC-T) was used as a simplified model for mammary glands. Cultured MAC-T cells were stimulated with 1, 10 and 20 mug/ml of a bacterial cell wall component (lipopolysaccharide) to verify the production of epithelially derived bovine cytokines. Cytokine mRNA production was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA samples isolated during the first 24 hrs of lipopolysaccharide stimulation. Interleukin-1alpha/beta, interleukin-6 and interleukin-8 mRNA production seemed to be time and dose dependent. Restriction enzyme analysis of PCR products confirmed the identity of the cytokines. Enzyme linked immunosorbent assays for interleukin-1 and interleukin-8 showed that the respective mRNA's were translated into proteins. Interleukin-8 protein was detectable 6 hrs after lipopolysaccharide stimulation; maximal levels of approximately 55 pg/ml were reached at 48 hrs post stimulation. Interleukin-1 was detected 1hr after stimulation; concentrations peaked between 500 and 600 pg/ml at 2, 5 and 12 hrs for 20, 10 and 1 mug/ml of lipopolysaccharide respectively. The amount of protein produced by the MAC-T cell line was relatively low and required high concentrations of lipopolysaccharide. Nevertheless, this model demonstrated a time and dose dependent cytokine production. These results suggest that bovine epithelial cells could be a source of cytokine production during mammary infection