Salicylaldehyde Suppresses IgE-Mediated Activation of Mast Cells and Ameliorates Anaphylaxis in Mice

Abstract

Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis.

Keywords: FcεRI; IgE; anaphylaxis; mast cells; salicylaldehyde.

Figure 1

Suppression of the degranulation of MCs by candidate compounds selected from an aroma library. (A) Compounds passed through a screening using 0.001% v/v (left) and 0.01% v/v (right) (Table 1) were added to culture media of BMMCs at the indicated concentrations; left: a = 0.001%, b = 0.002%; right: a’ = 0.0004%, b = 0.002%, c = 0.01%; S, salicylaldehyde; D, diacetyl; A, ambrettolide; M, mintlactone; J, cis-jasmone; I, isoamyl salicylate; B, benzyl salicylate. Forty-eight hours after a preincubation with each compound, BMMCs were stimulated with anti-TNP IgE and TNP-BSA as described in the Materials and Methods Section. The relative degranulation to that of control BMMCs without compounds (=100%) is shown. (B) Degranulation assay data obtained from 3 independent experiments, in which different independently prepared lots of BMMCs were used; left: a = 0.001%, b = 0.002%; center and right: b = 0.002%, c = 0.01%; S, salicylaldehyde; D, diacetyl; A, ambrettolide; M, mintlactone; J, cis-jasmone; I, isoamyl salicylate; B, benzyl salicylate. Dunnett’s multiple comparison test was used for statistical analyses. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant.

Figure 2

Salicylaldehyde inhibited the degranulation of and cytokine production by IgE-stimulated MCs. (A) Effects of salicylaldehyde on the IgE-mediated degranulation of BMMCs. BMMCs were incubated in the presence or absence of the indicated concentrations of salicylaldehyde (SA) for 48 h. After sensitization with IgE, BMMCs were incubated in Tyrode’s buffer containing TNP-BSA (IgE/Ag) or without Ag (IgE), and the supernatant was collected for β-hexosaminidase assay. (B) Effects of salicylaldehyde on the viability of BMMCs. BMMCs incubated with the indicated concentrations of salicylaldehyde (SA) for 48 h were treated with DAPI. DAPI-stained cells were judged to be dead cells. (C) Inhibition of cytokine release from BMMCs by salicylaldehyde. BMMCs incubated in the presence or absence of the indicated concentrations of salicylaldehyde (SA) for 48 h were stimulated with TNP-BSA (Ag) after sensitization with IgE. Data shown in (A,C) represent the mean ± SD of 4 and 3 independent experiments, respectively. Dunnett’s multiple comparison test (A,C) was used for statistical analyses. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Figure 3

Salicylaldehyde reduced cell surface expression levels of FcεRI on MCs and IgE-mediated signal transduction in MCs. (A) Cell surface expression levels of FcεRI and c-kit. Typical dot plot profiles (left) and the mean fluorescence intensity (MFI) of FcεRI obtained in 3 independent experiments. BMMCs incubated in the presence or absence of the indicated concentrations of salicylaldehyde (SA) for 48 h were stained with anti-FcεRI Ab and anti-c-kit Ab. (B) mRNA expression levels of FcεRIα (Fcer1a), β (Ms4a2), and γ (Fcer1g) subunits. BMMCs pretreated with 500 μM salicylaldehyde (SA+) or its control (SA−) were harvested to assess mRNA levels by qPCR. (C) Western blot analysis of tyrosine-phosphorylated proteins. BMMCs treated with 500 μM salicylaldehyde for 48 h (SA+) and its control (SA−) were stimulated with IgE plus TNP-BSA (Ag) for 10 min. The expected molecular weights of Lyn, Fyn, and Syk were marked on the left. A typical result is shown, and similar results were obtained in 2 other independent experiments. Data shown in (A (right) and B) represent the mean ± SD of 3 independent experiments. Tukey’s multiple comparison test (A right) and the t-test (B) were used for statistical analyses. n.s., not significant; ****, p < 0.0001.

Figure 4

The administration of salicylaldehyde alleviated the IgE-induced anaphylactic reaction. Effects of the administration of salicylaldehyde on PSA (A) and PCA (B). Mice were intraperitonially (i.p.) administered 50 mg/kg/day salicylaldehyde (SA) or saline (Ctrl) once a day for 6 days prior to the anaphylactic reaction (A,B). (A) Changes in the body temperature of control and salicylaldehyde-treated mice following PSA. Open square, control (n = 8); open circle, administration of salicylaldehyde (n = 9). The t-test was used for statistical analyses. *, p < 0.05 vs. control. (B) Thicknesses of the ear (left panel) and footpad (center panel) of saline-treated (SA−; n = 8) or salicylaldehyde-treated (SA+; n = 6) mice 1 h after the injection with TNP-BSA. Each individual was s.c. injected with IgE (IgE+) on the right ear or right footpad and s.c. injected with saline (IgE−) on the left ear, or left footpad. The amount of Evans blue in the back skin of control (SA−; n = 4) or salicylaldehyde-treated (SA+; n = 4) Balb/c mice 1 h after the injection of TNP-BSA (right panel). Dunnett’s multiple comparison test (ear thickness and footpad thickness) and the t-test (Evans blue) were used. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.