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Polymerase chain reaction quantification of cytokine messenger RNA expression in peripheral blood mononuclear cells of patients with acute exacerbations of asthma: effect of glucocorticoid therapy
Author(s) -
DOI S.,
GEMOUENGESAETH V.,
KAY A. B.,
CORRIGAN C. J.
Publication year - 1994
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.1994.tb01808.x
Subject(s) - peripheral blood mononuclear cell , glucocorticoid , messenger rna , immunology , eosinophil , cytokine , real time polymerase chain reaction , medicine , polymerase chain reaction , microbiology and biotechnology , asthma , biology , in vitro , gene , biochemistry
Summary We have measured the expression of messenger ribonucleic acid (RNA) (mRNA) encoding inlerleukin‐5 (IL‐5), IL‐4. IL‐2 and interteron‐γ (IFN‐γ) in peripheral blood mononuclear cells (PBMC) from 10 patients with acute exacerbations of asthma and nine non‐asthmatic controls. Measurements were repealed in seven of the asthmatics following 7 days of oral glucocorticoid therapy. Total RNA was extracted from the PBMC, reverse transcribed using oligo‐(dT) primers and aliquots of the resulting complementary DNA (cDNA) amplified using the polymerase chain reaction (PCR) in the presence of cytokine‐specific primers under non‐saturating conditions. PCR products were quantified on a relative basis after Southern blotting and probing with radiolabelled internal oligonucleotide probes by computer assisted densitometry of blot autoradiographs. The relative amounts of IL‐5 mRNA in PBMC from the asthmatic patients prior to glucocorticoid therapy were greater ( P < 0.01) than those in PBMC from non‐asthmatic controls. In contrast, there were no differences in the relative amounts of IL‐4. IL‐2 and IFN‐γ mRNA. In the asthmatics, the relative amounts of IL‐5 mRNA correlated with the peripheral blood eosinophil counts ( P = 0.02). After oral glucocorticoid therapy of the asthmatics, lung function improved and the relative amounts of PBMC IL‐5 mRNA were reduced ( P = 0.04) and no longer differed from those in PBMC from non‐asthmatic controls. Glucocorticoid therapy was not associated with significant changes in the relative amounts of PBMC IL‐4. IL‐2 and IFN‐γ mRNA. PBMC from alopic subjects contained significantly greater quantities of IL‐4 mRNA ( P ‐ 0.04) but not IL‐5, IL‐2 and IFN‐γ mRNA compared with non‐atopic subjects regardless of their asthmatic status. We conclude that PBMC of patients with acute exacerbations of asthma demonstrate elevated expression of mRNA encoding IL‐5, but not IL‐2, IL‐4 and IFNγ and that the clinical improvement associated with glucocorticoid therapy is associated with a reduction of IL‐5 mRNA expression. We further conclude that elevated expression in PBMC of mRNA encoding IL‐4 is a feature of atopy but not of asthma. These observations suggest that IL‐5 synthesis by activated T‐lymphocytes may be relevant to the pathogenesis of asthma, and that inhibition of this release by glucocorticoids may at least partly explain their therapeutic effect in this disease.