Real-time PCR method for the detection of cytokine expression in human proinflammatory T cells

Background and Aims: Immune-mediated diseases such as allergies and autoimmune diseases have increased during the past decades in developed countries. The incidence of type 1 diabetes is very high in Finland and both genetic and environmental factors, such as HLA genes and enteroviruses, are probably contributing to this phenomenon. Immunopathology of immune-mediated diseases is mediated by proinflammatory T cells, which can secrete several cytokines to contribute the tissue damage. Accordingly, imbalances in differentiation and function of the T cells are commonly observed in patients. The aim of this study was to create a panel of real-time PCR methods, which can detect cytokine expression of human proinflammatory T cells. Another aim was to optimize RNA extraction methods for stored buffy-coat samples, which have been collected in large clinical studies. The final goal of this study was to develop methods which can be used for the analyses of cytokine expression in different clinical conditions like acute virus infections and immune-mediated diseases. Methods: Human blood samples were obtained from healthy volunteers or from the children participating in the prospective DIPP study. White blood cells were purified from blood samples either as mononuclear cells or as buffy-coat. RNA was isolated from fresh and stored white blood cells using different commercial assays and RNA was transcripted into cDNA with RT-reaction. cDNA was then used as a template in real-time PCR to measure interferon-gamma, interleukin-4 and interleukin-17 mRNA expression. The relative gene expression was calculated with Pfaffl equation and the results were confirmed by agarose gel electrophoresis. Results: The real-time PCR was optimized successfully for three proinflammatory cytokines. RNA isolation from frozen buffy-coat samples worked best with modified protocols of Qiagen RNeasy® Mini Kit. Optimized assays were then used to analyze the effect of enteroviruses on cytokine expression. The in vitro white blood cell stimulations revealed that some enterovirus serotypes induced clear increases in the cytokines which are associated with autoimmune diseases. Conclusions: New real-time PCR assays were developed for the detection of three important proinflammatory cytokines. These real-time PCR methods can be reliably used for the detection of both basal and stimulated expression of proinflammatory cytokines in human white blood cells