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. 2018 Jan 3;18(1):2.
doi: 10.1186/s12906-017-2071-y.

Shuang-Huang-Lian Prevents Basophilic Granulocyte Activation to Suppress Th2 Immunity

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Free PMC article

Shuang-Huang-Lian Prevents Basophilic Granulocyte Activation to Suppress Th2 Immunity

Qiaoling Fei et al. BMC Complement Altern Med. .
Free PMC article

Abstract

Background: Basophilic granulocytes (BGs) not only initiate the induction of Th2 cell differentiation, but also amplify the ongoing Th2 response. Shuang-Huang-Lian (SHL) is clinically used for relieving type I hypersensitivity by continuous treatment for several weeks.

Methods: ELISA, flow cytometry, magnetic activated cell sorting, isoelectric precipitation, hybridoma technique, transfection and luciferase reporter assay were used in this study. The statistical analysis was performed using a one-way ANOVA.

Results: Our recently published study demonstrated that SHL exerted a remarkable effect on mast cell stabilization. Herein, we sought to elucidate the effect of SHL on shrimp tropomyosin (ST)-induced Th2 immunity and its underlying mechanisms. The obtained data showed that continuous treatment with SHL significantly suppressed ST-stimulated Th2-cytokines release and IgE synthesis. A mechanistic study indicated that SHL not only reduced BG early IL-4 release before ST-specific IgE (sIgE) production, but also inhibited BG activation in the presence of sIgE, including suppressing CD200R surface expression and decreasing IL-4 production. Moreover, SHL markedly decreased the cytosolic Ca2+ (Ca2+[c]) level and inhibited the nuclear factor of activated T cells (NFAT) activation in RBL-2H3 cells.

Conclusions: Collectively, SHL potently reduces ST-induced Th2 immunity by inhibiting the BG Ca2+-NFAT pathway and, thus, suppressing the early IL-4 release before sIgE synthesis and inhibiting BG activation in the presence of sIgE. This study provides the pharmacological basis for the clinical use of SHL to relieve type I hypersensitivity by a successive dose regimen.

Keywords: Basophilic granulocytes (BGs); Cytosolic Ca2+ (Ca2+[c]); IL-4; NFAT; Shuang-Huang-Lian (SHL); Th2 immunity.

Conflict of interest statement

Ethics approval

All the animal experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Animals Ethics Committee of the IMPLAD of Chinese Academy of Medical Sciences.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Effects of SHL on the serum tIgE (a), sIgE (b), sIgG2a (c) and sIgG2b (d) productions in the ST-sensitized mice. Mice were intraperitoneally immunized with ST (60 μg/mouse) once a week. SHL (3 mL/kg or 6 mL/kg, i.g.) was daily administrated daily to the mice. 4 weeks later the mice were sacrificed and the sera were collected. The serum tIgE was determined by a commercial ELISA kit. The sIgE, sIgG2a and sIgG2b levels were assayed by our established ELISA. The data represent the means ± SD (n = 10). *P < 0.05 and **P < 0.01
Fig. 2
Fig. 2
Effect of SHL on the cytokines produced by splenocytes. The mice were intraperitoneally immunized with ST (60 μg/mouse) once a week. SHL (3 mL/kg or 6 mL/kg, i.g.) was administered daily to the mice. 4 weeks later the spleens were taken, and single cell suspensions were aseptically prepared. The obtained splenocytes (4 × 106 cells/well) were seeded in a 24-well plate and stimulated with ST (8 μg/mL) at 37 °C for 72 h. The levels of IL-4 (a), IL-5 (b), IL-10 (c), IL-13 (d) and IFN-γ (e), in the culture medium, were measured using the ELISA kits. The data represent the means ± SD (n = 10). ##P < 0.01 vs. the normal control group; *P < 0.05 vs. the ST group
Fig. 3
Fig. 3
a SHL decreased early IL-4 produced by BG-rich splenocytes. Splenic BGs, preliminarily purified by a MACS system, were seeded in a 96-well plate (1 × 106 cells/well) and treated with SHL (0.5–2%) and ST (100 μg/mL) or active papain (100 μg/mL) in the presence of IL-3 (1 μg/mL) for 24 h. IL-4 levels in the supernatants were analyzed by ELISA. ##P < 0.01 vs. NS; $$P < 0.01 vs. papain alone; **P < 0.01 vs. ST alone. b-c SHL inhibited BG CD200R surface expression. The whole blood from the ST-sensitized mice was collected and stained with anti-IgE FITC and anti-CD200R antibodies after an incubation with ST (5 μg/mL) for 2 h at 37 °C. BGs in the whole blood were identified by an anti-IgE FITC antibody. The surface expression of CD200R on the BGs was assayed by flow cytometry. Isotype control and unstained cells were used as the negative controls. b Representative dot plots of BG CD200R surface expression. c Effect of SHL on the mean fluorescence intensity (MFI) of CD200R in the BGs after stimulation with ST. d SHL decreased IL-4 release in the sensitized RBL-2H3 cells. The cells were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were pretreated with or without SHL at 37 °C for 30 min and were then stimulated with ST (20 ng/mL) for 6 h. IL-4 levels in the supernatants were assayed using a commercial ELISA kit. e SHL suppressed ST-induced NFAT activation in BGs. RBL-2H3 cells stably transfected with the pNFAT-luc plasmid were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were pretreated with or without SHL at 37 °C for 30 min and were then stimulated with ST (20 ng/mL) for 6 h. The cells were lysed and the luciferase activity was measured using the luciferase assay system. f SHL reduced Ca2+[c] levels in the RBL-2H3 cells. The RBL-2H3 cells were sensitized with anti-ST IgE (25 μg/mL) at 37 °C overnight. The cells were loaded with Fluo-3 AM (4 μM) at 30 °C for 30 min. The stained cells were treated with or without SHL for 30 min and were then exposed to ST (20 ng/mL). The fluorescent intensity (λex 485 nm and λem 538 nm) was recorded every 30 s. #P < 0.05 and ##P < 0.01 vs. the control; *P < 0.05, **P < 0.01 vs. ST alone
Fig. 4
Fig. 4
Schematic diagram depicting how SHL reduces ST-induced Th2 immunity

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