Short-term effects on pro-inflammatory cytokine, lactoferrin and CD14 mRNA expression levels in bovine immunoseparated milk and blood cells treated by LPS.

Leucocytes (WBC) are recruited from peripheral blood into milk as part of the inflammatory response, mediated through cytokines or interleukins (IL) synthesized by mammary tissue and the milk somatic cells (SC). The inflammatory response is related to the concentration of SC and the cytokines produced. To investigate and to compare the kinetics of cytokine production in SC and WBC during inflammation, cell culture models were established, where SC and WBC were cultured in parallel (n = 3). In addition, macrophages or monocytes were isolated from milk and blood with antibody-coated magnetic beads and cultivated separately. Isolated cells were pure, unaltered and viable. Cultures were activated with 10 microg/ml lipopolysaccharide (LPS). After 0, 1, 2, 3, 4 and 8 h cells were harvested for RNA isolation. Cytokine [tumour necrosis factor alpha (TNFalpha), IL-1beta, IL-6] mRNA expression responses and transcriptional activity of CD14 and lactoferrin (LF) were quantified via a one-step real-time RT-PCR. Significant cytokine mRNA increases were found in all four cell culture types and genes, with peaks after 1 and 2 h (TNFalpha > IL-6 > IL-1beta). In WBC or monocytes higher LPS responses and longer persistence could be found than in corresponding milk cells (IL-1beta > IL-6 > TNFalpha). SC and macrophages are less responsive to LPS stimulation than WBC or monocytes. The strength of the immune response in the blood system is much more prominent than in the mammary gland. This may be ascribed to the role of CD14 on the cytokine production of the investigated cells, or may be caused by the blood-to-milk diapedesis. The constitutive transcription of CD14 mRNA in WBC and monocytes was found to be 6 to 15 times higher than in adequate milk cells.