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

doi:10.1016/0022-1759(94)90052-3

Journal of Immunological Methods

Volume 168, Issue 2, 10 February 1994, Pages 167-181

https://doi.org/10.1016/0022-1759(94)90052-3 Get rights and content

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.

References (36)

N.O. Davidson et al.
Thyroid hormone modulates the introduction of a stop codon in rat liver apolipoprotein B messenger RNA
J. Biol. Chem.
(1988)
M.A. Innis et al.
Optimization of PCRs
R.J. Wiesner et al.
Counting target molecules by exponential polymerase chain reaction: copy number of mitochondrial DNA in rat tissues
Biochem. Biophys. Res. Commun.
(1992)
H.C. Birnboim et al.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA
Nucleic Acids Res.
(1979)
C.A. Brenner et al.
Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNA's from small numbers of cells
Biotechniques
(1989)
J. Chelly et al.
Transcription of the dystrophin gene in human muscle and non-muscle tissues
Nature
(1988)
P. Chomcyzynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction
Anal. Biochem.
(1987)
C.J. Corrigan et al.
Biology of other inflamatory cells: Lymphocytes
G.F. Del Pret et al.
Purified protein derivative of Mycobacterium tuberculosis and exretory-secretory antigen(s) to Toxocara canis expand in vitro human T cells with stable and opposite (Type 1 T helper or Type 2 T helper) profile of cytokine production
J. Clin. Invest.
(1991)
P. Desreumaux et al.
Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease
J. Exp. Med.
(1992)

J. De Vries

Interleukin-4

S.R. Durham et al.

Cytokine mRNA expression for IL-3, IL-4, IL-5 and granulocyte-macrophage colony stimulating factor in the nasal mucosa after local allergen provocation: Relationship to tissue eosinophilia

J. Immunol.

(1992)

S. Ehlers et al.

Differentiation of T cell lymphokine gene expression: The in vitro acquisition of T cell memory

J. Exp. Med.

(1991)

H.A. Erlich et al.

Recent advances in the polymerase chain reaction

Science

(1991)

A.M. Feldman et al.

Selective gene expression in failing human heart: Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction

Circulation

(1991)

G. Gilliland et al.

Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction

P.W. Gray

Interferon-γ

P.W. Gray et al.

Structure of the human interferon gene

Nature

(1982)

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Background: Systemic glucocorticoids are a major therapy for the management of allergic inflammation and asthma; however, information about their effects in vivo are limited. Objective: This study was performed to examine the effects of prednisone on inflammatory mediators, cytokines, and cellular responses in the model of segmental allergen challenge (SAC) of allergic asthmatic subjects. Methods: The effects of a 3-day pretreatment with oral prednisone (30 mg twice daily) on the physiologic and inflammatory responses to SAC were studied in 10 allergic asthmatic subjects in a double-blind, placebo-controlled, crossover protocol. Results: Prednisone improved baseline FEV₁ by 10% and modestly inhibited the SAC-induced fall in FEV₁ at 30 minutes and at 6 to 8 hours. Five minutes after challenge, levels of histamine, PGD₂, 9α,11β-PGF₂, and thromboxane B₂ increased in bronchoalveolar lavage fluid (median increase, 5- to 14-fold); prednisone did not inhibit these responses. Prednisone inhibited (median decrease, 66%-97%) the total influx of inflammatory cells, specifically eosinophils, basophils, and some subsets of T lymphocytes (CD4, CD45RA, and CD45RO cells) assessed 19 hours after SAC, but it did not inhibit the influx of neutrophils. Increases in soluble E-selectin, kinins, and albumin were also inhibited by the glucocorticoid (median decrease, 36%-74%). Prednisone treatment inhibited the appearance of mRNA, protein, or both for T_H2 cytokines (IL-4 and IL-5), as well as for IL-2 and transforming growth factor α, but did not inhibit increases of immunoreactive GM-CSF in bronchoalveolar lavage fluid. Conclusion: These studies indicate that prednisone suppresses multiple components of allergic airway inflammation, including cell recruitment, adhesion molecule expression or release, airway permeability, and production of cytokines potentially involved in airway immunity or remodeling. (J Allergy Clin Immunol 2001;108:29-38.)
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¹: Pressent address: Division of Allergy and Clinical Immunology, East Tennessee State University, Johnson City, TN, USA.

²: Pressent address: Department of Otolaryngology, Asahikawa Medical School, Asahikawa, Japan.

³: Present address: Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.

View full text

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

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Nucleic Acids Res.

Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNA's from small numbers of cells

Biotechniques

Transcription of the dystrophin gene in human muscle and non-muscle tissues

Nature

Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction

Anal. Biochem.

Biology of other inflamatory cells: Lymphocytes

Purified protein derivative of Mycobacterium tuberculosis and exretory-secretory antigen(s) to Toxocara canis expand in vitro human T cells with stable and opposite (Type 1 T helper or Type 2 T helper) profile of cytokine production

J. Clin. Invest.

Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease

J. Exp. Med.

Interleukin-4

Cytokine mRNA expression for IL-3, IL-4, IL-5 and granulocyte-macrophage colony stimulating factor in the nasal mucosa after local allergen provocation: Relationship to tissue eosinophilia

J. Immunol.

Differentiation of T cell lymphokine gene expression: The in vitro acquisition of T cell memory

J. Exp. Med.

Recent advances in the polymerase chain reaction

Science

Selective gene expression in failing human heart: Quantification of steady-state levels of messenger RNA in endomyocardial biopsies using the polymerase chain reaction

Circulation

Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction

Interferon-γ

Structure of the human interferon gene

Nature