DETECTION OF ALLERGEN-INDUCED AND MITOGEN-INDUCED HUMAN CYTOKINE TRANSCRIPTS USING A COMPETITIVE POLYMERASE CHAIN-REACTION

Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in t he regulation of IgE synthesis and atopic diseases. In this communicat ion, we describe the development of a quantitative assay of steady-sta te cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell s ources, including peripheral blood mononuclear cells (PBMCs) stimulate d with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analys is of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competiti ve reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonu cleotide primers. Each IS was generated from a plasmid vector containi ng 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 l inearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4 , IL-5, and IFN-gamma transcripts in PBMCs before and after stimulatio n with PHA or crude ragweed allergen. Finally, we analyzed cells isola ted from the lung lavage fluids of an atopic subject following allerge n challenge, and showed a significant increase of IL-4 and IL-5 transc ripts, but not IFN-gamma, in the allergen-challenged site when compare d to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allerg ic inflammatory diseases.