Sk. Huang et al., DETECTION OF ALLERGEN-INDUCED AND MITOGEN-INDUCED HUMAN CYTOKINE TRANSCRIPTS USING A COMPETITIVE POLYMERASE CHAIN-REACTION, Journal of immunological methods, 168(2), 1994, pp. 167-181
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.