Proinflammatory Responses of a hTERT-Transformed, Immortalized Line of Cultured Bovine Mammary Epithelial cells (BME)

Monday, July 21, 2014
Exhibit Hall AB (Kansas City Convention Center)
Theodore H. Elsasser , USDA, Agricultural Research Service, Beltsville, MD
Stanislaw Kahl , USDA, Agricultural Research Service, Beltsville, MD
David E Kerr , University of Vermont, Burlington, VT
Enrique Zudaire , NIH-NCI, Bethesda, MD
Frank Cuttitta , NIH-NCI, Bethesda, MD
Abstract Text:

Cell Characterization:  Primary cultures of BME were generated from healthy mammary glands as described (Vet Immunol Immunopath 101(3-4):191-202, 2004).  Towards immortalization, BME from four cows were pooled and transfected with pCI neo-hEST2-HA , a human telomerase segment containing a neomycin/Geneticin  resistance selection cassette (Cell 90:785-95, 1997).  Cells were grown in DMEM +10%FBS and Geneticin (800 μg/ml) and followed through the ensuing selection growth lag to full proliferation capacity.  Following 50+ passages, cells were further subcloned to increase epithelial and decrease myoepithelial cell content; the resulting culture was called ELS-321-Clone2B.   For function studies and to achieve hormone and cytokine receptor access by the apical-lumenal polarized cells, cultures experiments were conducted on porous (0.4 micron) hanging well inserts coated with laminin-111.  At confluence, cells had the following characteristics:  tight junctions (electron microscopic confirmation of desmosomes, EpCAM-1 and E-cadherin immunostaining), expression (immunohistochemical localization) of prolactin receptor PRLr, xanthine oxidase (XO), inducible nitric oxide synthase (iNOS), and cytokeratin-18 (<10% cells displayed myoepithelial smooth muscle actin). 

Proinflammatory Modeling:  Confluent cells were challenged with 20 ng/ml recombinant bovine TNF-α and 200 ng/ml IL-17a.  Media was collected at 0, 1, 4 and 24 h relative to challenge with respective cells on inserts fixed in paraformaldehyde for immunofluorescence analysis of nitrated proteins (pNT) and PRLr .  Media content of lactate dehydrogenase progressively accumulated past time 0.  The first noticeable cell response to challenge was the redistribution of PRLr followed by a reduction in numbers to<10% T0 values. Cell pNT content was increased at T4, and progressively increased through T24.  The data suggest that ELS-321-2B are well suited to serve as an in vitro model to characterize BME responses to proinflammatory conditions.

Keywords: mammary, cell, proinflammatory