Cytokine mRNA quantification in histologically normal canine duodenal mucosa by real-time RT-PCR

CD4+ T helper cells are important for the regulation of immune responses in the intestinal mucosa and they exert their effects through the secretion of pro-inflammatory and immunomodulatory cytokines. Human patients with inflammatory bowel diseases (IBD) such as Crohnʹs disease and ulcerative colitis have alterations in the normal intestinal cytokine profile. These cytokine abnormalities have been shown at both the protein and messenger RNA (mRNA) level. le that mucosal cytokines play in the pathogenesis of canine IBD has only been investigated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis of gut tissue, as cytokine antisera are not available for this species. Real-time RT-PCR has been recognised to be a more accurate and sensitive method of quantifying mRNA transcripts, so in this study TaqMan real-time RT-PCR assays for the quantification of mRNA encoding IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, IFN-γ, TNF-α and TGF-β in canine intestinal mucosa were developed. The amount of these templates was quantified in normal canine duodenal mucosa (n = 8). IL-18, TGF-β and TNF-α were found to be the most abundant transcripts, with IL-10 and IFN-γ present at levels approximately 10-fold less. IL-2, IL-4, IL-5, IL-6 and IL-12 were the least abundant templates, with some RNA samples having no detectable mRNA copies. thods developed in this study will form the basis of further work investigating the expression of mRNA encoding cytokines in mucosa from dogs with chronic enteropathies. In addition, these real-time PCR assays can also be used for the quantification of canine cytokine mRNA in other diseases.