Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis and in vitro cytokine-stimulated blood eosinophils

Abstract

Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha and beta chain and interleukin (IL)-3 receptor alpha chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor beta chain and IL-3 receptor alpha chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-gamma and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-gamma. The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.

Fig 1

The clinical scores of allergy in blood samples of study patients. Total IgE (UA/ml) (a) and the percentage ratio of eosinophils (b) in blood of healthy controls, mild AD, moderate AD and severe AD patient groups are shown (***P < 0·001).

Fig 2

The expression levels of various genes in peripheral blood eosinophils. The expression of CD44 (a), GM-CSF receptor α chain (b), GM-CSF receptor β chain (c), IL-3 receptor α chain (d), PAF receptor (e), bcl-2 (f) and bcl-xL (g) in eosinophil samples from healthy controls and patients with mild, moderate and severe AD are shown (*P < 0·05, **P < 0·01, ***P < 0·001). The copy numbers of each transcript per 1 ng RNA, standardized to levels of GAPDH transcript, are shown on the ordinate.

Fig 3

In vitro dose–response of the expression of various genes in peripheral blood eosinophils of healthy volunteer after cytokine stimulation. The expression of CD44 (a), GM-CSF receptor α chain (b), GM-CSF receptor β chain (c), IL-3 receptor α chain (d), PAF receptor (e), bcl-2 (f), bcl-xL (g) and IL-5 receptor α chain (h)after a 3-h stimulation with the indicated concentrations of IL-5 (red bar), IL-4 (blue bar), IFN-γ (yellow bar), GM-CSF (green bar) and eotaxin (brown bar) is shown. An arbitrary unit contrasted with the unstimulated control level (standardized to levels of GAPDH transcript) is shown on the ordinate. These experiments were performed three times. The mean ± standard error of data from three independent experiments for each stimulation condition and the statistical results of the paired t-test are shown (*P < 0·05, **P < 0·01, ***P < 0·001).

Fig 3

In vitro dose–response of the expression of various genes in peripheral blood eosinophils of healthy volunteer after cytokine stimulation. The expression of CD44 (a), GM-CSF receptor α chain (b), GM-CSF receptor β chain (c), IL-3 receptor α chain (d), PAF receptor (e), bcl-2 (f), bcl-xL (g) and IL-5 receptor α chain (h)after a 3-h stimulation with the indicated concentrations of IL-5 (red bar), IL-4 (blue bar), IFN-γ (yellow bar), GM-CSF (green bar) and eotaxin (brown bar) is shown. An arbitrary unit contrasted with the unstimulated control level (standardized to levels of GAPDH transcript) is shown on the ordinate. These experiments were performed three times. The mean ± standard error of data from three independent experiments for each stimulation condition and the statistical results of the paired t-test are shown (*P < 0·05, **P < 0·01, ***P < 0·001).

Fig 3

In vitro dose–response of the expression of various genes in peripheral blood eosinophils of healthy volunteer after cytokine stimulation. The expression of CD44 (a), GM-CSF receptor α chain (b), GM-CSF receptor β chain (c), IL-3 receptor α chain (d), PAF receptor (e), bcl-2 (f), bcl-xL (g) and IL-5 receptor α chain (h)after a 3-h stimulation with the indicated concentrations of IL-5 (red bar), IL-4 (blue bar), IFN-γ (yellow bar), GM-CSF (green bar) and eotaxin (brown bar) is shown. An arbitrary unit contrasted with the unstimulated control level (standardized to levels of GAPDH transcript) is shown on the ordinate. These experiments were performed three times. The mean ± standard error of data from three independent experiments for each stimulation condition and the statistical results of the paired t-test are shown (*P < 0·05, **P < 0·01, ***P < 0·001).

Fig 3

In vitro dose–response of the expression of various genes in peripheral blood eosinophils of healthy volunteer after cytokine stimulation. The expression of CD44 (a), GM-CSF receptor α chain (b), GM-CSF receptor β chain (c), IL-3 receptor α chain (d), PAF receptor (e), bcl-2 (f), bcl-xL (g) and IL-5 receptor α chain (h)after a 3-h stimulation with the indicated concentrations of IL-5 (red bar), IL-4 (blue bar), IFN-γ (yellow bar), GM-CSF (green bar) and eotaxin (brown bar) is shown. An arbitrary unit contrasted with the unstimulated control level (standardized to levels of GAPDH transcript) is shown on the ordinate. These experiments were performed three times. The mean ± standard error of data from three independent experiments for each stimulation condition and the statistical results of the paired t-test are shown (*P < 0·05, **P < 0·01, ***P < 0·001).