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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Genetics, Breeding, and Animal Health Research » Research » Publications at this Location » Publication #344846

Research Project: Genomic Intervention Strategies to Prevent and/or Treat Respiratory Diseases of Ruminants

Location: Genetics, Breeding, and Animal Health Research

Title: Development and characterization of a bovine monocyte-derived macrophage cell line

Author
item Chitko Mckown, Carol
item Bartens, Marie - Royal Veterinary College
item Gibson, Amanda - Royal Veterinary College
item Holder, Angela - Royal Veterinary College
item Vats, Ashutosh - Royal Veterinary College
item Brown, Emma - Royal Veterinary College
item Kolakowski, Jeannine - Royal Veterinary College
item Workman, Aspen
item Heaton, Michael - Mike
item Kalbfleisch, Theodore - University Of Louisville
item Werling, Dirk - Royal Veterinary College

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 9/19/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Monocytes circulate in the blood, and later differentiate into macrophages in the tissues. They are components of the innate arm of the immune response and are one of the first lines of defense again invading pathogens. However, they also serve as host cells for intracellular pathogens such as Mycobacteria, Brucella, and Salmonella. Because monocytes represent only a small percentage of circulating leukocytes, harvesting sufficient quantities of them for in vitro experiments can be costly, time consuming, and can vary among cattle. Therefore, it was our objective to develop a monocyte-derived macrophage cell line that could be utilized for studies involving macrophages. Whole blood from a cross-bred steer was collected into syringes containing EDTA as an anticoagulant, and monocytes obtained using density gradient centrifugation. The monocytes were purified from the peripheral blood mononuclear cell fraction by adherence. After extended culture in RPMI 1640 media supplemented with 10% FBS, a population emerged spontaneously that proliferated in culture and could be easily detached from the tissue culture vessels using tryspin-EDTA. These proliferating cells were tested for cell surface determinants indicative of monocyte/macrophage lineage, as well as bactericidal and phagocytic activity. Genomes of the cell line and whole blood from the donor steer were sequenced and aligned to the bovine reference assembly to identify any large deletions that may have occurred in passage during development, and to identify any cell line gene variants that may affect function. Genome analysis is ongoing. This bovine monocyte-derived macrophage cell line has been passaged over 25 times, morphologically resembles macrophages in culture, expresses the CD markers CD14/16, C172a, CD11b amongst others, is phagocytic and bactericidal (by ROS and NO production), produces IFN in response to TLR/RLR ligands, and appears to fall into the M2 macrophage category. There is a dearth of bovine macrophage cell lines available to veterinary researchers, and the relative ease of culture should render this cell line a useful tool for the in vitro study of numerous macrophage-trophic pathogens.