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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF RESPIRATORY DISEASE SUSCEPTIBILITY Title: Characterization of two porcine macrophage cell lines for the expression of pathogen-recognition receptors, defensins, cytokines, chemokines, and surface sialic acid

Authors
item Joseph, Jocelin -
item Sreenivasan, Chithra -
item Chitko Mckown, Carol
item Kaushik, Radhey -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 20, 2010
Publication Date: October 1, 2010
Citation: Joseph, J., Sreenivasan, C., Chitko Mckown, C.G., Kaushik, R.S. 2010. Characterization of two porcine macrophage cell lines for the expression of pathogen-recognition receptors, defensins, cytokines, chemokines, and surface sialic acid [abstract]. American Society for Microbiology 70th Annual Meeting of the North Central Branch. Poster No. P-10.

Technical Abstract: Macrophages express various pathogen-recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) and activate genes responsible for host defense. The aim of this study was to characterize two porcine macrophage cell lines (Cdelta+ and Cdelta–) for the expression of PRRs, beta-defensins (BD), cytokines, and chemokines. Cdelta+ cells were cultured in RPMI-10 containing L929-conditioned media, while Cdelta– cells were cultured in RPMI-10 media. Total RNA obtained from these cells was used to prepare cDNA. Gene expressions of toll-like receptors (TLRs), retinoic acid–inducible gene 1 (RIG-1), melanoma differentiation–associated antigen 5 (MDA-5), nucleotide oligomerization domain (NOD) proteins, BD, cytokines, and chemokines were quantified using real time RT-PCR relative to cyclophilin-A. The results from a single experiment showed that TLR-5, TLR-10, IL-1beta, TNF-alpha, IL-7, IL-15, IL-18, CCL-20, and BD-1 genes were expressed at higher levels, while TLR-7, TLR-9, MD-2, NOD-1, NOD-2, IL-6, IL-10, IL-12p40, osteopontin, BD-2, RIG-1, and MDA-5 genes were expressed at lower levels in Cdelta2+ cells compared to Cdelta2- cells. TLR-1, 2, 3, 4, 6, and 8, IL-1alpha, IL-12p35, MCP-1, and MIF were expressed at similar levels by both cell types. A minimum two-fold increase or decrease in gene expression was considered as a higher or lower level of gene expression. We also tested both cell lines for the presence of surface sialic acid by flow cytometry using Maackia amurensis lectin (MAL-II) and Sambucus nigra agglutinin (SNA), as many viruses use sialic acid as receptor for cell entry. Both cell types showed approximately 3-6% MAL-II and 62-78% SNA positive cells.

Last Modified: 8/22/2014
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