Masaru Kagami

IL-5 but not interferon-gamma (IFN-gamma) inhibits eosinophil apoptosis by up-regulation of bcl-2 expression.

In order to determine regulatory mechanisms of eosinophil apoptosis, we examined the effect of recombinant IL‐5 and interferon‐gamma (IFN‐γ) on eosinophil apoptosis and bcl‐2 expression. rhIL‐5 (2.5 ng/ml) significantly inhibited eosinophil apoptosis in 96 h in vitro culture compared with medium only‐cultured eosinophils (89.4 ± 3.6% versus 31.3 ± 12.2% (mean ± s.d.); n = 7, P < 0.05). Further, rhIL‐5 significantly increased bcl‐2 protein and mRNA expression on cultured eosinophils. A phosphorothioate antisense oligonucleotide targeted at the ATG translation initiation codon of bcl‐2 (10−5 m) could significantly block the supportive effect of rhIL‐5 (0.25 ng/ml) for eosinophil survival compared with sense cDNA of bcl‐2 on 96 h culture (inhibition rate 28.01 ± 4.56% versus 0.07 ± 1.73%; n = 4, P < 0.05). In contrast, rhIFN‐γ (100 U/ml) significantly inhibited eosinophil apoptosis on 96 h in vitro culture (72.7 ± 10.5%; n = 7, P < 0.05), but did not significantly up‐regulate bcl‐2 protein and mRNA. These results indicate that IL‐5 has inhibitory effects on eosinophil apoptosis by regulation of bcl‐2 expression.

Human peripheral eosinophils express functional interferon‐gamma receptors (IFN‐γR)

In order to determine whether or not IFN‐γR is associated with regulatory mechanisms on human eosinophil function, we examined the expression of functional IFN‐γR on human peripheral eosinophils. In this study, peripheral blood eosinophils were obtained from seven normal controls and 12 patients (bronchial asthma, n = 9, and hypereosinophilic syndrome (HES), n = 3), and the purity of eosinophils was 97.11 ± 2. 31%, n = 19. We first showed that anti‐IFN‐γR α‐chain MoAb reacted with all tested eosinophils of both normal controls and patients by flow cytometry analysis. We also showed expression of mRNA for the α‐chain of IFN‐γR in all purified eosinophils of six individuals. Further, to characterize IFN‐γR on eosinophils, we did binding experiments with 125I‐IFN‐γ on purified peripheral eosinophils. The linear Scatchard plot indicated a single type of high‐affinity binding sites (dissociation constant (Kd) = 3.89–4.95 × 10−10 M, numbers of binding sites = 183–233/cell, n = 3). To determine whether IFN‐γR on eosinophils is functional, we examined surface eosinophilic cationic protein (ECP) and CD69 induction after IFN‐γR ligation with recombinant human IFN‐γ (rhIFN‐γ) on eosinophils by flow cytometry. rhIFN‐γ stimulation significantly induced both ECP and CD69 expression on the 2–18 h‐cultured eosinophils in a dose‐dependent manner. Further, the effects of rhIFN‐γ stimulation were significantly blocked by both a neutralizing anti‐IFN‐γ MoAb and a blocking anti‐IFN‐γR MoAb. These results suggest that human peripheral eosinophils express functional IFN‐γR.

Role of JAK2 signal transductional pathway in activation and survival of human peripheral eosinophils by interferon-gamma (IFN-γ)

The purpose of this study was to determine whether the JAK pathway is involved in eosinophil activation and survival through IFN‐γ receptor signalling in human peripheral eosinophils. Eosinophils were purified from the blood of six atopic disease patients by anti‐CD16 magnetic bead‐negative selection. IFN‐γ significantly up‐regulated survival and CD69 expression in 24–48 h cultured eosinophils. Further, IFN‐γ induced tyrosine phosphorylation of JAK2 in eosinophils, as indicated by Western blot analysis. Finally, the specific JAK2 inhibitor AG‐490 inhibited the tyrosine phosphorylation of JAK2, IFN‐γ‐induced survival and CD69 expression in eosinophils. In conclusion, these results indicate that IFN‐γ induces eosinophil survival and CD69 expression through the activation of JAK2 in peripheral eosinophils, suggesting that JAK2 may play a significant role in eosinophil regulation by IFN‐γ–IFN‐γR interaction.

Heterogeneous Expression of Tumor Necrosis Factor-α Receptors I and II on Human Peripheral Eosinophils

Background: Expression of the tumor necrosis factor-α receptor by human eosinophils has not been determined. We examined the surface expression and mRNA for tumor necrosis factor-α receptors I and II (TNF-αRI and TNF-αRII) on peripheral eosinophils. Methods: Eosinophils were obtained from 17 asthma patients and 3 healthy volunteers. Flow cytometry was used to examine receptor expression, and the reverse transcriptase-polymerase chain reaction was used to examine receptor mRNA expression. Results: Flow cytometry revealed that 16 out of 20 subjects had eosinophils expressing TNF-αRI and 18 subjects had eosinophils expressing TNF-αRII. All eosinophils expressed at least one receptor. The mRNA for TNF-αRII was detected in all eosinophils examined, but the signals for TNF-αRI mRNA were only found in cells expressing this receptor. Conclusions: Human peripheral eosinophils show heterogeneous expression of TNF-αRI and TNF-αRII.

Expression of High-Affinity IgE Receptor (FcεRI) on Human Alveolar Macrophages from Atopic and Non-Atopic Patients

The expression of high-affinity IgE receptor (FcERI) on alveolar macrophages was examined in order to determine the association of alveolar macrophages with IgE-dependent inflammation. Immunostai

IN VITRO INTERLEUKIN-5 (IL-5) PRODUCTION BY PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH DRUG HYPERSENSITIVITY

It has been shown that CD4+ lymphocytes and eosinophils pathologically infiltrate the dermis in skin eruptions due to hypersensitivity to the administration of certain drugs associated with peripheral eosinophilia. However, the mechanisms involved in drug related skin eruptions with eosinophilia are largely unknown. There are several methods of diagnosing drug hypersensitivity, but no single in vitro method is available for the detection of the sensitizing drug.

Eosinophil activation and in situ interleukin-5 production by mononuclear cells in skin lesions of patients with drug hypersensitivity.

In order to determine the inflammatory mechanisms of skin lesions in patients with drug hypersentivity, we examined eosinophil activation and interleukin‐5 (IL‐5) production in infiltrating lymphocytes. First, we showed that the number of peripheral eosinophils and the level of serum IL‐5 at the eruption‐active stage were both significantly higher than those in healed skin eruptions. Histological and immunohistological examination revealed that CD4+ T cells and eosinophils significantly more densely infiltrated drug eruptions than control skin lesions. The infiltrating eosinophils were also shown to be activated by immunostaining using anti‐secreted formed eosinophilic cationic protein monoclonal antibody. The expression of mRNA for IL‐5 in the infiltrating mononuclear cells at drug eruptions was shown by in situ hybridization. These results suggest that infiltrating CD4+ T cells might regulate both peripheral and tissue eosinophils and facilitate allergic inflammation at drug eruptions by means of IL‐5 production.

Interferon-γ Receptor β-Chain Expression and Formation of α- and β-Chain Complexes after Receptor Conjugation on Human Peripheral Eosinophils

Background: It has been unclear whether or not the interferon-γ receptor (IFN-γR) on human peripheral eosinophils is completely functional. Accordingly, we examined the expression of IFN-γR α- and β-chains and the association of the two chains after binding of IFN-γ to the receptor. Methods: Peripheral blood eosinophils were obtained from 8 patients (bronchial asthma, n=6, and hypereosinophilic syndrome, n=2), and expression of the α- and β-chain was investigated by flow cytometry with specific antibodies. Expression of mRNA for the α- and β-subunits was also investigated by the reverse transcriptase-polymerase chain reaction. Results: Eosinophils from all the patients were positive for both the α- and β-chains by flow cytometry. In addition, mRNA for both chains was detected by the polymerase chain reaction. Furthermore, coprecipitation of the α- and β-chains was only found after eosinophil stimulation with IFN-γ. Conclusions: Human peripheral eosinophils apparently express both the α- and β-chains of the IFN-γR, and the two chains may only form the receptor complex after ligand binding.

Regulation of CD69 Expression on Eosinophil Precursors by Interferon-γ

The purpose of this study was to determine whether interferon-γ (IFN-γ) induced CD69 expression by eosinophil precursors. Eosinophil precursors were induced from CD34+ cord blood cells using recombinant human interleukin-3 (IL-3) and interleukin-5 (IL-5). On day 14 of culture, cells constitutively expressed CD69 and the IFN-γ receptor (IFN-γR). Stimulation with IFN-γ for 24 h did not affect IFN-γR expression by the cells. On the other hand, IFN-γ significantly upregulated CD69 expression by the precursors after 24 h of incubation. A specific JAK2 inhibitor (AG-490) caused a concentration-dependent suppression of IFN-γ-induced CD69 expression by the precursors. In conclusion, these results indicate that IFN-γ induces CD69 expression by eosinophil precursors via the activation of JAK2.