Plant-Derived Molecule 4-Methylumbelliferone Suppresses FcεRI-Mediated Mast Cell Activation and Allergic Inflammation

Abstract

Mast cells (MCs) are an important treatment target for high-affinity IgE Fc receptor (FcεRI)-mediated allergic diseases. The plant-derived molecule 4-methylumbelliferone (4-MU) has beneficial effects in animal models of inflammation and autoimmunity diseases. The aim of this study was to examine 4-MU effects on MC activation and probe the underlying molecular mechanism(s). We sensitized rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated them with exposure to DNP-human serum albumin (HSA), and then treated stimulated cells with 4-MU. Signaling-protein expression was determined by immunoblotting. In vivo allergic responses were examined in IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) mouse models. 4-MU inhibited β-hexosaminidase activity and histamine release dose-dependently in FcεRI-activated RBLs and BMMCs. Additionally, 4-MU reduced cytomorphological elongation and F-actin reorganization while down-regulating IgE/Ag-induced phosphorylation of SYK, NF-κB p65, ERK1/2, p38, and JNK. Moreover, 4-MU attenuated the PCA allergic reaction (i.e., less ear thickening and dye extravasation). Similarly, we found that 4-MU decreased body temperature, serum histamine, and IL4 secretion in OVA-challenged ASA model mice. In conclusion, 4-MU had a suppressing effect on MC activation both in vitro and in vivo and thus may represent a new strategy for treating IgE-mediated allergic conditions.

Keywords: 4-methylumbelliferone; FcεRI signaling; SYK; mast cell.

Figure 1

Treatment with 4-MU suppresses FcεRI-mediated degranulation in RBLs. (a) Chemical structure of 4-MU. (b) Viability of RBLs incubated with 0–100 μM 4-MU for 24 h determined by CCK-8 assays. (c–g) Secretion of β-hexosaminidase (c), histamine release (d), and expression of inflammatory cytokine genes IL1β (e), IL4 (f), and TNFα (g) in RBLs sensitized with anti-DNP IgE, with or without 4-MU, for 1 h, and then challenged with DNP-HSA. The data are shown in means ± SDs (five duplicate experiments); ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. non-treated activated cells. 4-MU, 4-methylumbelliferone; DNP-HSA, dinitrophenyl-human serum albumin protein conjugate; RBL, RBL-2H3 cell.

Figure 2

4-MU suppresses FcεRI-mediated morphological changes in RBLs. Anti-DNP IgE-sensitized RBLs were pretreated (or not) with 4-MU for 1 h and then challenged with DNP-HSA (100 ng/mL) for 30 min. (a,b) Representative micrographs of toluidine blue-stained RBLs. Red arrows indicate irregular cell morphology and the release purple particles. (c,d) FITC-phalloidin stained RBLs. Blue arrow indicates cell morphology irregularity due to decomposition of F-actin cytoskeleton. Means ± SDs (of three independent experiments) are shown; * p < 0.05, ** p < 0.01 vs. activated cells without treatment. Abbreviations as in Figure 1.

Figure 3

4-MU suppresses FcεRI-mediated signaling protein activation in RBLs. IgE-sensitized RBLs were pre-incubated with 4-MU for 1 h and then stimulated with DNP-HSA for 20 min. (a–c) Western blot analysis of SYK (a), MAPK (p38, ERK, and JNK) (b), and NF-κB pathway signaling molecules (p-p65, p65, and IκBα) (c) in activated RBLs. (d,e) β-Hexosaminidase release from IgE-activated RBLs treated with 4-MU and MAPK inhibitors (ERK inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB203580) (d) or the NF-κB inhibitor BAY 11-7082 (e) for 1 h prior to the DNP-HSA challenge. Means (of three duplicate experiments) ± SDs are shown; * p < 0.05, ** p < 0.01 vs. activated cells without treatment. Abbreviations as in Figure 1.

Figure 4

4-MU inhibition of IgE/Ag stimulation of primary BMMCs. (a) Cell viability, as determined by CCK-8 assay, of BMMCs incubated with 0–100 μM of 4-MU for 24 h. Cells were sensitized with anti-DNP IgE, with or without 4-MU, for 1 h, and then challenged with DNP-HSA. (b) β-Hexosaminidase release. (c) Histamine release. (d–f) mRNA expression of IL1β (d), IL4 (e), and IL6 (f) determined by RT-qPCR. BMMCs were sensitized with anti-DNP IgE with or without 4-MU for 1 h and challenged with DNP-HSA for 4 h. Means ± SDs (of five duplicate experiments) are shown; * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations as in Figure 1.

Figure 5

4-MU attenuation of IgE-mediated allergic reactions in PCA mice [sensitized with anti-DNP-IgE (or saline control) for 12 h, 4-MU for 1 h, and injected with DNP-HSA/0.5% Evans blue]. (a) Representative photos of showing dye extravasation from ears. (b) Representative photomicrographs of sections from PCA ears. (c) Reversal of DNP-HSA-induced increases in ear thickness. (d) Formaldehyde-extracted Evans blue dye quantified at 620 nm. Means (of three independent experiments) ± SD are shown; ** p < 0.01, *** p < 0.001 vs. activated cells, not treated. Abbreviations as in Figure 1.

Figure 6

4-MU attenuates allergic reactions in ASA mice. (a) ASA protocol (ketotifen, positive control). (b) Mean (± SD) rectal temperatures taken every 10 min after initiation of an OVA challenge (three independent experiments). (c) Effects of 4-MU on histamine release into serum (determined by ELISA). (d) Effects of 4-MU on serum IL-4 levels in ASA mice (determined by ELISA). Means ± SDs (three independent experiments) are shown (N = 5/group); * p < 0.05, ** p < 0.01 vs. activated group not pretreated with 4-MU.