Analysis of cytokine mRNA levels in interleukin‐4‐transgenic mice by quantitative polymerase chain reaction

Abstract

Interleukin (IL)-4-transgenic mice were used as a model system to study the consequences of low levels of IL-4 expression for the expression of other cytokines examined by quantitative polymerase chain reaction (PCR). For this purpose, a plasmid was constructed which contains, in tandem array, 5′ and 3′ primer sequences specific for the cytokine genes IL-1 to IL-6, tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN)-γ and β-actin. During co-amplification, target and control DNA complete for the primers and the amount of PCR product is proportional to the amount of input DNA. Competitive PCR was performed first to adjust the cDNA to be compared to identical concentrations of β-actin cDNA and subsequently to determine cytokine mRNA levels from spleen cells of normal and IL-4-transgenic animals. The sensitivity of this approach was demonstrated by the capability to detect a twofold difference in IL-4 mRNA levels between IL-4-transgenic heterozygous and homozygous animals. Upon lipopolysaccharide activation, the IL-4 transgene which is expressed essentially in B lymphocytes was induced approximately 50-fold. Several cytokine mRNA such as those coding for IL-5, IL-6, IFN-γ and also the IL-4 receptor were found to be up-regulated in IL-4-transgenic mice, whereas IL-1, IL-2, IL-3, TNF and LT mRNA levels did not seemed to be influenced by IL-4. A possible functional significance of the elevated IFN-γ mRNA was demonstrated by showing that (a) CD23 expression was not increased, and (b) Mac-1⁺ cells were markedly increased in the spleen of transgenic mice.