Detection of Interleukin-1 and Interleukin-6 on Cryopreserved Bovine Mammary Epithelial Cells in Vitro.

Abstract

ABSTRACT. This investigation was performed to determine whether primary cultures of mammary cells from lactating cows would sustain production of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF), and express mRNA for cytokines interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, interferon (INF)-τ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the reverse transcription-polymerase chain reaction (RT-PCR) in vitro. Cryopreserved mammary epithelial cells collected from cows at 1 week post calving were plated in collagen-coated 24-well culture plates (250,000 cells/well). IL-1 and IL-6 productions were measured using a A375 cell growth inhibition assay and a 7TD1 hybridoma proliferation assay, respectively. Production of IL-1 was demonstrated in mammary epithelial cells cultured with unsupplemented medium, but was not produced by cells cultured in medium supplemented with fetal bovine serum. IL-6 production in the conditioned medium was continued at steady level until day 14, whereas IL-6-like bioactivity was not detected in medium alone. TNF-like activity was not detectable in any experiments. This study also demonstrated the expression of mRNA for multiple cytokines including IL-1α, IL-1β, IL-6, IL-10, TNF-α, and GM-CSF by RT-PCR in mammary cell cultures. The results indicate that bovine mammary epithelial cells of lactating cows produce IL-1 and IL-6 and have gene expression for multiple cytokines. This in vitro model will be useful to investigate the function and regulation of IL-1 and IL-6 in the lactating mammary gland. -KEY WORDS: bovine, cytokine, mammary.

Effects of Lipopolysaccharide on Production of Interleukin-1 and Interleukin-6 by Bovine Mammary Epithelial Cells in vitro

“…For culture, cryopreserved cells were washed and separated by a preformed 45% Percoll density gradient (Pharmacia, Uppsala, Sweden) as described previously [8]. Morphologically, the predominant cells recovered from the Percoll density gradient centrifugation were mammary epithelial cells [8].…”

“…Morphologically, the predominant cells recovered from the Percoll density gradient centrifugation were mammary epithelial cells [8]. A suspension of cells with >70% viability usually was obtained by use of trypan blue dye exclusion test.…”

“…The medium was then replaced with serum-free M199 or M199 supplemented with various concentrations of LPS (E. coli 055:B5; 0.1, 1.0 or 10 µg/ml, Sigma) and 2% FBS. At 4,8,24, and 48 hr after treatment, the conditioned medium was collected and stored at 20°C until assayed for IL-1 and IL-6-like bioactivity.…”

“…IL-1 and IL-6-like activities in medium samples were measured by cytolytic assay using A375 human melanoma cell line and IL-6-dependent 7TD1 hybridoma proliferation assay, respectively [8]. The colored reaction products were measured at a wavelength of 570 nm with a microplate reader (BioRad, Tokyo, Japan).…”

“…Total cellular RNA (1 µg) was isolated as described previously [8]. A 456-bp fragment of the bovine IL-1 receptor antagonist (IL-1ra) and a 552-bp fragment of bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were a m p l i f i e d b y R T -P C R .…”