Eucalyptus oil reduces allergic reactions and suppresses mast cell degranulation by downregulating IgE-FcεRI signalling (2024)

Eucalyptus oil suppresses IgE-mediated local allergic inflammation in mice

To determine whether Eucalyptus oil can suppress mast cell degranulation, we tested its effects using passive cutaneous anaphylaxis (PCA) responses in mice. PCA is a local skin allergic reaction resulting from hyperpermeability and plasma extravasation following allergen exposure, and it has been used as an animal model for IgE-mediated allergic reactions^22,23. We evaluated whether Eucalyptus oil can suppress IgE-mediated allergic reactions by vascular hyperpermeability and local ear oedema by assessing the leakage of Evans blue dye (Fig.1).

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Figure 1

Eucalyptus oil (Eu) suppresses passive cutaneous anaphylaxis (PCA) in a mouse model. (a) Schedule of treatment and experimental procedures for the PCA and ear swelling response tests. (b) Anti-DNP-IgE was injected intravenously 24h before DNFB application. One hour before DNFB application, Eucalyptus oil or vehicle (3:1 acetone/olive oil) was applied to the inside of each ear of all animals. DNFB was applied to the outside of the ear, followed by an immediate injection of Evans blue dye. Thirty minutes later, the ears were excised, and the absorbance of the dye was measured. Data are presented as the mean ± SEM (N = 8/group). The statistical significance of the differences was assessed using Dunnett’s test (**P < 0.01 versus vehicle-DNFB). (c) The time course of ear oedema was investigated. Ear thickness was measured using a thickness gauge. Data are presented as the mean ± SEM (N = 8/group). (d) Haematoxylin and eosin staining of ear tissue 6h after DNFB application. (e,f) Toluidine blue staining of ear tissue 1 or 6h after DNFB application. (e) Tissue sections were mast cells obtained from ear tissue collected 6h after the application of DNFB. Mast cells were identified in tissue sections by their characteristic granular, deep blue-purple metachromatic appearance against blue orthochromatic background tissue. ND no degranulation, D degranulation. (f) Left panel shows the number of cutaneous mast cells at the dermal/epidermal (D/E) junction. Right panel shows the percentage of degranulated mast cells. Each bar is mean ± SEM (N = 6/group) of 12 fields of views. Statistical significance of the differences was assessed using the Tukey–Kramer test (**P < 0.01 versus vehicle, ##P < 0.01 versus vehicle-DNFB).

In addition, prior to the Eucalyptus oil test, it was verified whether the inhibitory effect can be detected, using sodium cromoglycate, which has an inhibitory effect on mast cell degranulation (Supplementary Fig.2).

Eucalyptus oil significantly suppressed the amount of dye leaking into the tissue compared with the effects of the vehicle-DNFB. Eucalyptus oil also inhibited dye leakage in a dose-dependent manner (Fig.1b). Meanwhile, oedema was induced in the ears of mice following DNFB application, peaking at 6h, and Eucalyptus oil inhibited this oedema in a dose-dependent manner (Fig.1c).

Haematoxylin and eosin (H&E) staining of the auricle was performed 6h after DNFB application. As a result, swelling of the dermal tissue, thickening of the stratum corneum and infiltration of inflammatory cells into the dermal layer via DNFB application were noticed, and these findings were suppressed by Eucalyptus oil (Fig.1d).

Toluidine blue staining was performed to confirm that Eucalyptus oil suppressed mast cell degranulation after DNFB application. The number of degranulated mast cells was quantified by counting the mast cells in the image of skin tissue^24,25.

Almost no degranulation of mast cells was observed in the vehicle. Vehicle-DNFB degranulated 50% of mast cells after 1h and 68% after 6h. Furthermore, after 6h, the number of mast cells confirmed by toluidine blue staining reduced.

In the group to which the Eucalyptus oil was applied, a significant suppression of mast cell degranulation rate was observed 1h after the application of DNFB as compared with that observed after the application of vehicle-DNFB. A significant inhibitory effect was observed even 6h after DNFB application; however, the inhibitory effect observed 1h after DNFB application was weaker than that observed 6h after DNFB application (Fig.1e,f).

These results revealed that Eucalyptus oil suppressed PCA induced by anti-DNP-IgE antibody exposure in mice. It is known that mast cell degranulation is strongly involved in the very early phase of ear oedema 1h after reaction caused by intravenous administration of anti-DNP IgE antibody and subsequent application of DNFB, and that migration of inflammatory cells is involved in the early phase of ear oedema^26,27. The ear oedema suppression effect of Eucalyptus oil has been observed from a relatively early stage and this effect gradually weakens after 3h. As a result of the inhibition of mast cell degranulation by toluidine blue staining, it was clarified that Eucalyptus oil plays a role in mast cell degranulation inhibition.

However, the inflammation caused by anti-DNP-IgE antibodies also involves inflammatory cells other than mast cells and therefore we aimed to clarify how Eucalyptus oil affects mast cell deficient mice^27,28.

The inhibitory effect of Eucalyptus oil was not observed in mice lacking mast cells

W/Wv mice are known to lack mast cells because of the lack of mast cell progenitor cells, and they are widely used to confirm the physiological roles of mast cells^28–30. We measured the ear thickness by PCA reaction and analysed the inhibitory effect of Eucalyptus oil on the increase of vascular permeability. In wild-type mice, Eucalyptus oil significantly suppressed dye leakage into the tissue compared with the effects of the vehicle. In W/Wv mice deficient in mast cells, both vehicle and Eucalyptus oil had significantly less tissue dye leakage than wild-type vehicle application. Also, there was no difference in the dye leakage by vehicle and Eucalyptus oil (Fig.2a).

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Figure 2

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Inhibitory effect of Eucalyptus oil (Eu) on passive cutaneous anaphylaxis (PCA) in mast celldeficient mice. (a) Effect of Eucalyptus oil on Evans blue dye leakage in wild-type and W/Wv mice. One hour before DNFB application, Eucalyptus oil or vehicle (3:1 acetone/olive oil) was applied to the inside of the ears of wild-type and W/Wv mice sensitised with anti-DNP-IgE. DNFB was applied to the outside of the ear, which was immediately injected intravenously with Evans blue dye. Thirty minutes later, the ears were excised, and the absorbance of the dye was measured. Data are presented as the mean ± SEM (N = 8). The statistical significance of the differences was assessed using Tukey–Kramer test (**P < 0.01, *P < 0.05 versus vehicle). (b) Time-dependent changes in the suppression of pinna oedema by Eucalyptus oil in wild-type and W/Wv mice. Ear thickness was measured using a thickness gauge. Data are presented as the mean ± SEM (N = 8). (c) Haematoxylin and eosin-stained and toluidine blue-stained images of ear tissue from Eucalyptus-oil-treated wild-type and W/Wv mice 6h after DNFB application.

DNFB application induced oedema in the ears of wild-type mice, peaking at 6h in the vehicle (Fig.2b), and these effects were suppressed by Eucalyptus oil. The vehicle- and Eu-treated W/Wv mice showed increased ear thickness with the application of DNFB, peaking at 3h, but the increase was obviously smaller in the vehicle-treated wild-type mice (Fig.2b).

H&E and toluidine blue staining of the auricle 6h after DNFB application revealed swelling of dermal tissue, thickening of the stratum corneum and infiltration of inflammatory cells into the dermal layer in vehicle-treated wild-type mice, and these effects were suppressed by Eucalyptus oil treatment (Fig.2c, Supplementary Fig.3). Vehicle-treated W/Wv mice exhibited less swelling of dermal tissue, thickening of the stratum corneum and infiltration of inflammatory cells into the dermis layer compared with the findings in vehicle-treated wild-type mice, but there was no difference in the findings between vehicle- and Eucalyptus-oil-treated W/Wv mice (Fig.2c).

Toluidine blue staining of the ear 6h after DNFB application confirmed mast cell degranulation in vehicle-treated wild-type mice. Fewer mast cells were degranulated in Eucalyptus-oil-treated wild-type mice. Meanwhile, no mast cells were identified in either vehicle- or Eucalyptus-oil-treated W/Wv mice (Fig.2c).

Predictably, treatment with vehicle and Eucalyptus oil in mast cell deficient W/Wv mice did not differ in terms of the inhibitory effect on PCA responses. However, W/Wv and wild-type mice are known to have no differences in T lymphocytes, B lymphocytes and NK cells³¹, but have low anaemia and red blood cell count due to lack of skin pigment cells²⁷. Therefore, detailed thorough examination is needed in the future.

Mast cells play an important role in IgE-mediated hypersensitivity reactions, making them an important target for alleviating allergic symptoms³². When mast cells are exposed to antigens, IgE-binding high-affinity IgE receptors (FcεRI) on the plasma membrane are cross-linked, triggering mast cell activation and degranulation. Therefore, an in vitro cell-based assay using BMMCs was performed to investigate the mechanism by which Eucalyptus oil suppresses mast cell degranulation.

Eucalyptus oil and 1,8-cineole inhibit the degranulation of BMMCs

The effects of Eucalyptus oil and its main component 1,8-cineole on BMMC degranulation were investigated. Anti-DNP-IgE antibody-sensitised BMMCs were treated with Eucalyptus oil or 1,8-cineole and stimulated with DNP-human serum albumin (HSA) as an antigen. The amount of β-hexosaminidase released from the cells was used as a marker of enzymatic activity to evaluate the effects of Eucalyptus oil and 1,8-cineole on mast cell degranulation. β-Hexosaminidase is released together with histamine during mast cell degranulation, and the amount released is generally used as an indicator of degranulation^33,34.

First, the viability of BMMCs following exposure to various concentrations of Eucalyptus oil or 1,8-cineole was examined. Both Eucalyptus oil and 1,8-cineole had no effect on cell viability at concentrations below 1µg/mL (Fig.3a). When Eucalyptus oil or 1,8-cineole was applied to BMMCs at a concentration of 0.1, 0.5 or 1µg/mL, β-hexosaminidase release was significantly suppressed. These results indicated that Eucalyptus oil and 1,8-cineole suppressed mast cell degranulation (Fig.3b).

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Figure 3

Effect of Eucalyptus oil (Eu) and 1,8-cineole on IgE-induced degranulation in bone-marrow-derived mast cells (BMMCs). (a) Effect of Eucalyptus oil and 1,8-cineole on BMMCs viability. Cells were treated with various concentrations of Eucalyptus oil or 1,8-cineole for 24h. Next, cell viability was measured using the MTT assay. Relative cell viability was calculated by comparing the absorbance in cells treated with Eucalyptus oil or 1,8-cineole with that in cells treated with vehicle (0.1% DMSO). Data are expressed as the mean ± SEM (N = 3). (b) Eucalyptus oil or 1,8-cineole was applied to anti-DNP-IgE-sensitised BMMCs immediately after stimulation with DNP-human serum albumin (HSA), and after 1h, β-hexosaminidase release was determined by comparing the enzymatic activity in BMMCs between treatment with vehicle (0.1% DMSO) and DNP-HSA. Data are expressed as the mean ± SEM (N = 4). Dunnett’s test was used to assess the statistical significance (**P < 0.01versus vehicle-DNP-HSA).

Eucalyptus oil and 1,8-cineole suppress inflammatory chemokine, cytokine and lipid mediator production by BMMCs

Mast cells activated locally by inflammation produce histamine as well as cytokines such as IL-4 and IL-13 and lipid mediators such as prostaglandin and leukotriene C4, thereby promoting allergic inflammation^32,35. The effects of Eucalyptus oil and 1,8-cineole on the production of histamine, IL-4, IL-13, prostaglandin and leukotriene C4 were investigated via an in vitro assay using BMMCs. When BMMCs sensitised with anti-DNP-IgE were stimulated with the antigen, histamine, IL-4, IL-13, prostaglandin and leukotriene C4 were detected in the supernatant (Fig. ​(Fig.4a–e).4a–e). When Eucalyptus oil or 1,8-cineole was applied at 0.1, 0.5 or 1µg/mL, histamine production was significantly inhibited (Fig.4a), and IL-4 and IL-13 production was also significantly suppressed (Fig. ​(Fig.4b,c).4b,c). Neither Eucalyptus oil nor 1,8-cineole inhibited the production of prostaglandin and leukotriene C4 at 0.1g/mL. At a concentration 0.5µg/mL, their production was suppressed, albeit without significance. At 1µg/mL, significant suppression was observed (Fig. ​(Fig.4d,e).4d,e). There was no difference between the inhibitory effects of Eucalyptus oil and 1,8-cineole. Regarding mast cell degranulation, the effects of Eucalyptus oil could be attributable to 1,8-cineole. The inhibitory effects on prostaglandin D2 and leukotriene C4 were weaker than those on histamine, IL-4 and IL-13.

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Figure 4

Effects of Eucalyptus oil (Eu) and 1,8-cineole on inflammatory chemokine and cytokine production by bone-marrow-derived mast cells (BMMCs). (a) Histamine release was determined after treating anti-DNP-IgE-sensitised BMMCs with Eucalyptus oil and 1,8-cineole. Histamine release in the supernatant was specifically after 30min of stimulation with DNP-human serum albumin (HSA) via ELISA. Histamine release was calculated relative to that in cells treated with vehicle (0.1% DMSO) and stimulated with DNP-HSA. Data are expressed as the mean ± SEM (N = 4). Dunnett’s test was used to assess the statistical significance (**P < 0.01 versus vehicle-DNP-HSA). (b,c) Production of IL-4 and IL-13 by anti-DNP-IgE-sensitised BMMCs was determined after treatment with Eucalyptus oil or 1,8-cineole. ELISA was performed after culturing cells with DNP-HSA for 3h. IL-4 and IL-13 production was calculated relative to that in cells treated with vehicle (0.1% DMSO) and stimulated with DNP-HSA. Data are expressed as the mean ± SEM (N = 4). Dunnett’s test was used to assess the statistical significance (**P < 0.01 versus vehicle-DNP-HSA). (d,e) Production of prostaglandin D2 and leukotriene C4 by anti-DNP-IgE-sensitised BMMCs was determined after treatment with Eucalyptus oil or 1,8-cineole. ELISA was performed after culturing cells with DNP-HSA for 1h. Prostaglandin D2 and leukotriene C4 production was calculated relative to that in cells treated with vehicle (0.1% DMSO) and stimulated with DNP-HSA. Data are expressed as the mean ± SEM (N = 4). Dunnett’s test was used to assess the statistical significance (**P < 0.01) of differences between vehicle- and DNP-HSA-treated cells. Dunnett’s test was used to assess the statistical significance (*P < 0.05, **P < 0.01 versus vehicle-DNP-HSA).

Eucalyptus oil and 1,8-cineole suppress the increase in intracellular Ca²⁺ concentration in BMMCs

When IgE is cross-linked by the antigen, FcεRI aggregation initiates signalling cascades, leading to the increase in intracellular Ca²⁺  concentration is one of the major signalling pathways for mast cell degranulation³⁶. We next investigated the effect of 1,8-cineole on the intracellular Ca²⁺ increase in BMMCs.

Stimulation with DNP-HSA was performed for 60s after the start of Ca²⁺ measurement. As depicted in Fig.5a, the fluorescence intensity of Fluo 4 increased immediately after the DNP-HSA stimulation of vehicle-treated cells, whereas the fluorescence intensity of Fluo 4 remained unchanged without DNP-HSA. Fluo 4 fluorescence intensity was significantly suppressed when cells treated with 1,8-cineole were stimulated with DNP-HSA.

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Figure 5

1,8-cineole suppresses the elevation of intracellular Ca²⁺ concentration. BMMCs sensitised with anti-DNP IgE were incubated in medium containing Fluo 4-AM. BMMCs were treated with various concentrations of 1,8-cineole and stimulated with DNP-HSA 60s after initiating the monitoring of fluorescence intensity. (a) Time course of intracellular Ca^{2 +} concentration in BMMCs. Data are displayed as mean ± SEM (N = 5). (b) Relative fluorescence intensity 60s after DNP-HSA stimulation. Data are displayed as mean ± SEM (N = 5). Statistical significance was assessed using Dunnett’s test (** P < 0.01 vs vehicle-DNP-HSA).

Figure5b shows the results 60s after DNP-HSA stimulation. Compared with the fluorescence intensity of DNP-HSA-stimulated cells treated with vehicle, there was significant suppression at 0.1, 0.5 and 1.0μg/mL every concentration of 1,8-cineole, and although no significant difference was detected, a concentration-dependent suppression was observed.

It was found that 1,8-cineole suppressed the intracellular Ca²⁺ increase stimulated by DNP-HSA. Therefore, we next examined the effect of 1,8-cineole on the intracellular signalling cascades of BMMCs other than Ca²⁺.

1,8-cineole does not affect Lyn and Syk phosphorylation

Mast cells express FcεRI on their surface, and when IgE is cross-linked by an antigen, the Src family tyrosine kinase Lyn, which is related to FcεRI, is phosphorylated, followed by a series of signal transductions. Then, signs of mast cell activation such as degranulation, cytokine production and lipid mediator production are observed^29,32,37,38. Therefore, the effects of 1,8-cineole on the phosphorylation status of intracellular signalling proteins were examined via immunoblot analysis.

Anti-DNP-IgE antibody-sensitised BMMCs were treated with 0.5µg/mL 1,8-cineole, stimulated with DNP-HSA as an antigen, lysed and immunoblotted. As a result, the phosphorylation of Syk and Lyn was not suppressed by 1,8-cineole, even when the concentration was increased to as much as 2.0µg/mL (Fig.6a,b).

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Figure 6

Effect of 1,8-cineole on the phosphorylation of Syk and Lyn. (a,b) Eucalyptus oil or 1,8-cineole was applied to BMMCs sensitised with anti-DNP-IgE 24h before stimulation with DNP-human serum albumin, and then after 10min, cells were lysed, separated by SDS-PAGE and immunoblotted to analyse p-Syk, Syk, p-Lyn and Lyn expression. The left side presents representative examples of blots for each protein, and the right bar graphs present the phosphorylation relative ratios for Syk and Lyn.Phosphorylation ratios were calculated relative to Syk and Lyn expression (each set as 1), and data are presented as the mean ± SEM of 4–6 independent experiments. Dunnett’s test was used to assess the statistical significance (**P < 0.01 versus DNP-HSA). The original blots are shown in the supplementary Fig.5.

1,8-cineole suppresses PLCγ and p38 phosphorylation

Lyn and Syk phosphorylation was not inhibited by 1,8-cineole. Therefore, we investigated the intracellular signalling cascade downstream of Lyn and Syk. Anti-DNP-IgE antibody-sensitised BMMCs were treated with Eucalyptus oil or 1,8-cineole, stimulated with DNP-HSA as an antigen, lysed and immunoblotted. As a result, PLCγ phosphorylation was significantly suppressed by 0.5μg/mL 1,8-cineole (Fig.7a), as was p38 phosphorylation (Fig.7b). Phosphorylation of phospholipase A2 (PLA2) was not suppressed by 0.5µg/mL 1,8-cineole(Fig.7c). The results confirm that 1,8-cineole suppressed PLCγ and p38 phosphorylation.

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Figure 7

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Effect of 1,8-cineole on the phosphorylation of PLCγ, p38 and phospholipase A2 (PLA2). (a–c) 1,8-cineole was applied to bone-marrow-derived mast cells (BMMCs) after sensitisation with anti-DNP-IgE for 24h, and cells were subsequently stimulated with DNP-human serum albumin (HSA). After 10min, the cells were lysed, and SDS-PAGE was performed. Proteins were separated, and p-PLCγ, PLCγ, p-p38, p38, p-PLA2 and PLA2 expression was examined via immunoblotting. The left side presents representative blots for each protein, and the right bar graphs show the relative ratios of PLCγ, p38 and PLA2 phosphorylation. Phosphorylation ratios were calculated relative to PLCγ, p38 and PLA2 expression (each set as 1), and data are presented as the mean ± SEM of 4–6 independent experiments. Dunnett’s test was used to assess the statistical significance (**P < 0.01 versus DNP-HSA). The original blots are shown in the supplementary Figs.6, 7.

Mice

Female BALB/c mice (Japan SLC, Shizuoka, Japan) and male WBB6F1 + / + (wild-type) and WBB6Ft/J-W/Wv (W/Wv) mice (Japan SLC, Shizuoka, Japan) were used at 6–12weeks old. The mice were housed in a room under controlled temperature (22°C ± 2°C), humidity (50% ± 10%) and light (lights on from 07:00 to 19:00). Food and water were freely available. The study was approved by the Animal Research Committee of Ikedamohando Research Center in accordance with the National Research Council Guidelines.

Reagents

DNCB, anti-DNP-IgE and DNP-HSA were purchased from Sigma-Aldrich (St. Louis, MO, USA), Eucalyptus oil, procured from Nippon Terpene Chemicals (Tokyo, Japan), conforms with the Japanese Pharmacopoeia, and its main component, 1,8-cineole, accounts for 82.5% of the oil. 1,8-cineole was obtained from Nacalai Tesque (Kyoto, Japan), and dimethyl sulfoxide (DMSO) was acquired from Wako (Osaka, Japan). The following antibodies for immunoblotting were purchased from Cell Signalling Technology (Beverly, MA, USA): anti-Syk, anti-phospho-Syk, anti-Lyn, anti-phospho-Lyn, anti-PLA2, anti-phospho-PLA2, anti-PLCγ, anti-phospho-PLCγ, anti-p38 and anti-phospho-p38. Goat anti-rabbit IgG horseradish peroxidase was purchased from Cell Signaling Technology (Danvers, MA, USA).

Passive cutaneous anaphylaxis (PCA)

The mice were sensitised via the intravenous administration of 20μg of anti-DNP-IgE. In the increased vascular permeability test, after 24h, 0.6% DNFB was applied to the outside ear of each mouse, and 1% Evans blue (Sigma-Aldrich, St. Louis, MO, USA) was injected into the tail vein. One hour before DNFB application, 0.5%, 2% or 8% Eucalyptus oil was applied to the inside of the ear. After 30min, the mice were sacrificed, and their ears were collected. Skin was excised and incubated with KOH at 55°C for 24h, and then, the extravasated Evans blue dye was extracted. The absorbance of the dye at 620nm was measured using a multi-well spectrophotometer (Bio-Rad, Hercules, CA, USA).

In the ear swelling response test, no dye was administered, and Eucalyptus oil was applied to the inside of the ear. After 1h, DNFB was applied to the outside of the ear, and the thickness of the ear was measured with a digital thickness gauge (Mitutoyo, Kanagawa, Japan).

Preparation and culture of BMMCs

Bone marrow was collected from the femurs and tibias of wild-type BALB/c mice (female, 6–10weeks old; Japan SLC, Shizuoka, Japan) after sacrifice via cervical dislocation. Their bone marrow cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, 0.1mg/mL streptomycin, 50µM 2-ME and 5ng/mL IL-3 (PeproTech, Cranbury, NJ, USA). The medium was changed every 3days during culture. Mature BMMCs were obtained after 4weeks of culture and stained with toluidine blue. Most (> 90%) of these cells expressed both c-Kit and FcεRI. All experiments were performed with BMMCs cultured for 4–7weeks.

β-Hexosaminidase release assay

BMMCs (1 × 10⁵ cells/mL) were incubated for 24h with 1μg/mL anti-DNP-IgE. Surface-bound IgE was cross-linked for 30min at 37°C using DNP-HSA. Reactions were stopped by placing the cells on ice. β-Hexosaminidase in the supernatant and cell lysate was incubated with 26mM citrate buffer (pH 4.5) and 2.3mg/mL p-nitrophenyl-N-acetyl-α-d-glucosaminide for 60min at 37°C. The reaction was developed by adding 0.4M glycine (pH 10.7), and the absorbance was measured at 570nm using a multi-plate reader (Bio-Rad). The percentage of β-hexosaminidase release was calculated relative to the total β-hexosaminidase content^22,40.

Histology

Ear specimens were fixed with 10% neutral buffered formalin and embedded in paraffin. Four-micrometre-thick sections cut from each paraffin block were stained routinely with H&E and toluidine blue. Extrusion of toluidine blue-stained granules was the evidence indicating mast cell degranulation. The number of mast cells and the percentage of degranulated mast cells at the dermal/epidermal junction were analysed in tissue sections, as described previously^24,25.

Cell viability assay

BMMCs (1 × 10⁴ cells/mL) were grown in 96-well micro-titre plates for 24h. Eucalyptus oil or 1,8-cineole were added to the cells at a concentration of 0.5, 1.0, 5.0, 10.0 or 50.0μg/mL, followed by incubation for 24h. The cytotoxic effects of Eucalyptus oil and 1,8-cineole were evaluated using the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Specifically, 20 μL of MTT (5mg/mL) was added to each well, followed by incubation at 37°C for 4h. The medium was removed, and 200 μL of DMSO was added to each well. The optical density was measured at 490nm using a multi-well spectrophotometer (Bio-Rad).

Measurement of intracellular calcium levels

Intracellular calcium levels were measured using a Calcium kit Fluo 4 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions^45,46. BMMCs were seeded at 4.0 × 10⁴ cells/well in a 96-well black culture plate and sensitised with anti-DNP IgE (50ng/mL) at 37°C overnight. After washing with PBS, the cells were incubated with Fluo 4-AM for 1h at 37°C. Cells were again washed with PBS, treated with the recording buffer containing Eucalyptus oil and 1,8-cineole, and then incubated at 37°C for 30min. Next, the cells were stimulated with DNP-HSA (50ng/mL), and the fluorescence intensity was immediately monitored with an excitation wavelength of 485nm and an emission wavelength of 535nm using a microplate reader (BioTeh, Winooski, St,USA).

ELISA for histamine, IL-4, IL-13, prostaglandin D2 and leukotriene C4 production

BMMCs were stimulated as described previously. The cell culture supernatants were harvested 3h after stimulation, and IL-6 and IL-13 concentrations were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA). The prostaglandin D2 and leukotriene C4 concentrations in cell culture supernatants were measured at 1h after stimulation using ELISA kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Histamine concentrations in cell culture supernatants were measured at 30min after stimulation using an ELISA kit (Cayman Chemical) according to the manufacturer’s instructions.

Immunoblot analysis

BMMCs were washed and lysed with iced lysis buffer (1% Triton X-100, 50mM Tris–HCl [pH 7.5], 150mM NaCl, protease inhibitor co*cktail and phosphatase inhibitor co*cktail; Nacalai Tesque)⁴⁷. The lysates were subjected to SDS-PAGE and immunoblotting. The reactive bands were visualised using a 5-bromo-4-chloro-3-indolyl phosphate/NBT colour development substrate (Promega, Madison, WI, USA) or an ECL chemiluminescence detection kit. Densitometric analysis of the data obtained from at least three independent experiments was performed using a cooled CCD camera system EZ-Capture II and CS analyser ver. 3.00 software (ATTO, Tokyo, Japan).

Statistical analysis

All data are expressed as the mean ± SEM. Each experiment was performed independently at least three times. For statistical significance, two-tailed Student’s t-test was performed when the results were between two groups. Comparisons between other groups were performed by one-way analysis of variance using Tukey–Kramer or Dunnett’s multiple comparison test.

Ethical approval

All animal testing protocols were approved by the Animal Experiment Committee of Ikeda Mohando Co., Ltd. The Animal Experiment Committee of Ikeda Mohando Co., Ltd., has been certified by the Japan Health Science Foundation, including on-site surveys.

Publisher's note

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

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