title: Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production.
abstract: Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated the induction of IFN-α, IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam(3)Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24 h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN-α and IL-12 p40, and PGN, LPS and Pam(3)Cys inducing varying amounts of IL-12 p40, IL-1β, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1β only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1β and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results provide guidelines concerning the timing and use of protein and mRNA determinations for the characterization of porcine cytokine responses to PAMPs, although given the low number of animals used here results are preliminary and need confirmation in a larger study.
authors.affiliation: Innate Immunology Group, Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, DK-1790 Copenhagen V, Denmark. nsor@vet.dtu.dk
authors.forename: Nanna S
authors.initials: NS
authors.lastname: Sorensen
authors.forename: Kerstin
authors.initials: K
authors.lastname: Skovgaard
authors.forename: Peter M H
authors.initials: PM
authors.lastname: Heegaard
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology; D009841:Oligonucleotides / Q000276:immunology*; D010455:Peptides / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013997:Time Factors
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D009841:Oligonucleotides; D010455:Peptides; D012333:RNA, Messenger