Quantitative analysis of immune-mediated stimulation of tumor necrosis factor-alpha in macrophages measured at the level of mRNA and protein synthesis

Abstract.

Expression of cytokines such as tumor necrosis factor-alpha (TNF-α) induced by lipopolysaccharide (LPS) has been associated with inflammatory and regulatory immune reactions. Antigen-presenting cells, especially macrophages, play a central role in directing immune responses by synthesizing different cytokines. For the analysis of cytokine synthesis, we compared quantitative changes in mRNA and protein synthesis of TNF-α in RAW 264.7 cells stimulated with 0.1 ng/ml LPS. TNF-α mRNA was quantified using the LightCycler SYBR Green technology (Idaho Technology, Inc., Salt Lake City, Utah, USA). RAW 264.7 cells showed a basal TNF-α mRNA expression which increased approximately sixfold after 2 h of stimulation with LPS. TNF-α synthesis was analyzed at the protein level using a mouse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and indicated a 56-fold increase in TNF-α protein concentration after 4 h. Thus, real-time polymerase chain reaction (PCR) is a sensitive and rapid method for quantitative determination of LPS-induced TNF-α expression. However, it requires extremely robust reaction parameters to be reliable for accurate quantification.