Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction

Abstract

Human cytokines, IL-4, IL-5, and IFN-γ play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-γ) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-γ transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS)_cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector contining the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the ‘competitive’ PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-γ transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finaly, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-γ, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.