Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity

  • Ji Hyung Kim1,
  • A-Ram Kim1,
  • Hyuk Soon Kim1,
  • Hyun Woo Kim1,
  • Young Hwan Park1,
  • Jueng Soo You1,
  • Yeong Min Park1,
  • Erk Her1,
  • Hyung Sik Kim2,
  • Young Mi Kim3 and
  • Wahn Soo Choi1Email author
Contributed equally
BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201515:80

https://doi.org/10.1186/s12906-015-0607-6

Received: 3 June 2014

Accepted: 12 March 2015

Published: 25 March 2015

Abstract

Background

Complementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo.

Methods

The effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis.

Results

LERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells.

Conclusion

This study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

Keywords

Rhamnus davurica Herbal medicine Mast cells Allergy Anaphylaxis Fyn

Background

Allergic diseases such as allergic asthma, allergic rhinitis, and atopic dermatitis have been increasing, particularly in many developed countries. Generally, around 10 to 20% of the population are reported as presenting with allergic diseases in those countries [1,2]. Usually, patients with allergy-prone genetic factors have type 2 helper T cells (Th2) reactions under a certain environment. When patients are exposed to potential allergens such as milk, egg, nuts, and shellfish, various allergic symptoms may occur. In the course of early and late allergic reactions, mast cells are solidly acknowledged as one of the major culprit effector cells [3].

Mast cells play a key role in prompting a variety of allergic symptoms [4,5]. Mast cells have FcεRI, a high-affinity receptor for IgE, which makes a complex with antigen-specific IgE. When mast cells are stimulated by an antigen, various allergic mediators including histamines, eicosanoids, and pro-inflammatory cytokines are secreted from mast cells [6,7]. Thus, mast cells play a critical role in causing allergic diseases, and therefore mast cells-stabilizing therapies have been increasingly reported [8,9].

When mast cells are stimulated by antigens, the initial activation of Lyn or other Src-family kinases is observed for the phosphorylation of an immunoreceptor tyrosine activation motif (ITAM) of FcεRI γ subunit and Syk is, then, recruited to tyrosine phosphorylated ITAMs for the full activation of Syk [10,11]. This event leads to the activation of downstream signaling molecules including a linker for the activation of T cells (LAT), SLP-76, Gab2, phospholipase (PL) Cγ, and MAP kinases. This series of activations of signaling proteins leads to the full activation of mast cells in prompting allergic responses [12,13].

The Src family kinases, including Lyn, Fyn, and Fgr, are activated by antigen stimulation in mast cells [11]. The initial activation of Lyn is essential for the activation of mast cells, and Lyn additionally also has a negative role in mitigating the excess activation of mast cells [14]. Whereas Fyn and Fgr show solely a positive role in the activation of Syk and its downstream signaling cascades for mast cell activation [11]. Therefore, a Src-family kinase-targeted study could be an efficient direction for treatment of allergic diseases.

Rhamnus davurica is widely found throughout fields of Korea, China, and other Asian countries. Extract of Rhamnus davurica has long been in use as a folk remedy in the treatment of several diseases including pruritus, dysuresia, and constipation. However, the effect of Rhamnus davurica on allergic diseases remains to be unclear.

In this study, we investigated the anti-allergic effects of the leaf extract of Rhamnus davurica (LERD) in mast cells cultures and in passive cutaneous anaphylaxis animal models. LERD suppressed the activation of mast cells and anaphylaxis responses through the inhibition of the activation of Fyn/Syk pathway in antigen-stimulated mast cells.

Methods

Reagents

Antibodies that work against the phosphorylated forms of Akt, Erk1/2, p38, JNK, Syk (Y525/526), and LAT (Y191) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The 4-Amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine (PP2) was obtained from Calbiochem (La Jolla, CA, USA). Enzyme-linked immunosorbant assay (ELISA) kits for analyzing IL-4 in media was obtained from Invitrogen-Biosource Cytokine & Signaling (Camarillo, CA, USA). An ELISA kit for TNF-α measurement was from R&D Systems, Inc. (Minneapolis, MN, USA). Reagents for cell culture media were obtained from GIBCO/Life Technologies, Inc. (Rockville, MD, USA). Most of all the other reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animals

BALB/c mice (5-weeks old) were used for the isolation of bone marrow-derived mast cells (BMMCs) and the induction of passive cutaneous anaphylaxis (PCA). All animal studies were performed according to institutional guidelines after obtaining approval from the Institutional Animal Care and Use Committee (IACUC) at Konkuk University.

Mast cell preparation and cell culture

BMMCs were isolated from the thigh bones of 5 week-old BALB/c mice as in previous studies [15]. The BMMCs were cultured in media (RPMI 1640), containing 2 mM L-glutamine, 0.1 mM nonessential amino acids, antibiotics, and 10% fetal bovine serum (FBS) containing 10 ng/ml IL-3 in 5% CO2, 37°C incubator. Four weeks following isolation, the BMMCs were used for the following experiments. The RBL-2H3 cells from ATCC (American Type Culture Collection, VA, USA) were cultured in a minimum essential medium (MEM) with Earle’s salts, supplemented with glutamine, antibiotics, and 15% FBS.

Preparation of LERD and other plant extracts

Leaf of Rhamnus davurica was collected from Hantaek Botanical Garden (Yongin-si, Korea) and was authenticated by the Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). The methanol extracts of Rhamnus davurica leaf (LERD) and other plants were manufactured according to the institute’s standard protocol. The yield of the extraction process was approximately 15% of total dry leaf amount. The extracted and plant specimen (017-005 for LERD or as indicated in Table 1) were deposited at the Plant Extract Bank and Konkuk University. The extracts were solubilized in dimethyl sulfoxide (DMSO) for cell culture experiments and suspended in 5% Gum arabic for oral administration of extracts in the animal study.
Table 1

Effects of plant extracts on the Ag-induced degranulation in RBL-2H3 mast cells

Plant name

Part extracted

Voucher specimen number

Percent inhibition of degranulationa

Acer okamotoanum

Stem-bark

016-081

0.00

Acer ukurunduense

Leaf

016-050

40.78

Acer ukurunduense

Stem

016-051

30.32

ActinoStemma lobatum

Whole plant

017-038

18.48

Ardisia pusilla

Whole plant

016-061

0.00

Aster incisa

Whole plant

016-001

0.00

Berberis amurensis var. quelpaertensis

Stem

016-069

16.96

Betula chinensis

Leaf

017-013

39.17

Betula chinensis

Stem

017-014

27.76

Betula davurica

Stem

017-045

3.78

Betula davurica

Stem-bark

017-046

0.00

Betula ermani var. saitoana

Leaf

016-089

10.80

Betula ermani var. saitoana

Stem

016-090

1.78

Boehmeria pannosa

Whole plant

016-080

0.43

Bupleurum longeradiatum

Whole plant

017-016

29.22

Cacalia auriculata var. matsumurana

Whole plant

016-047

12.05

Campylotropis macrocarpa

Aboveground part

017-023

0.00

Celosia argentea

Whole plant

017-012

38.14

Chaenomeles sinensis

Leaf

017-040

3.85

Cinnamomum loureirii

Leaf

016-082

5.92

Clerodendrum trichotomum

Leaf

016-026

2.27

Clintonia udensis

Whole plant

016-019

51.37

Corydalis heterocarpa

Whole plant

016-079

11.79

Crataegus scabrida

fruit

017-047

0.00

Cyrtomium falcatum

Aboveground part

016-028

4.84

Daphniphyllum macropodum

Leaf

017-019

6.46

Daphniphyllum macropodum

Stem

017-020

0.29

Deutzia prunifolia

Stem

017-051

44.69

Dicentra spectabilis

Aboveground part

016-010

8.22

Diospyros kaki

Leaf

017-007

17.98

Diospyros kaki

Stem

017-008

17.68

Diospyros kaki

Stem-bark

017-009

17.25

Diospyros kaki

Root

017-011

11.64

Dryopteris championi

Whole plant

016-088

6.97

Elaeagnus macrophylla

Leaf

017-059

23.52

Forsythia nakaii

Leaf

016-085

1.93

Fraxinus sieboldiana

Leaf

017-073

9.30

Geranium wilfordii

Whole plant

017-070

42.07

Houttuynia cordata

Whole plant

016-077

7.45

Hydrangea serrata for. acuminata

Aboveground part

016-058

67.16

Hypericum erectum

Whole plant

017-018

6.53

Kalopanax pictus

Stem-heartwood

016-083

14.84

Lilium lancifolium

Aboveground part

016-091

8.82

Lindera erythrocarpa

Leaf

016-054

18.09

Lindera erythrocarpa

Stem

016-055

14.20

Lindera obtusiloba

Stem

016-063

19.80

Lycopodium clavatum var. nipponicum

Whole plant

017-069

23.56

Lycoris aurea

Underground part

017-015

52.03

Melandryum seoulense

Whole plant

017-003

62.76

Monochoria vaginalis var. plantaginea

Whole plant

017-043

2.84

Mosla punctulata

Whole plant

017-037

6.83

Nandina domestica

Leaf

016-023

6.46

Nuphar japonicum

Whole plant

016-006

9.22

Oenothera laciniata

Whole plant

016-075

14.50

Pedicularis resupinata

Whole plant

017-072

35.15

Perilla frutescens var. acuta

Whole plant

017-071

14.14

Persicaria fauriei

Whole plant

018-003

28.89

Persicaria thunbergii

Whole plant

017-017

17.79

Phlomis koraiensis

Aboveground part

017-064

19.72

Phlomis koraiensis

Underground part

017-065

1.94

Phtheirospermum japonicum

Whole plant

017-024

27.81

Picris hieracioides var. glabrescens

Whole plant

017-074

16.13

Polystichum polyblepharum

Whole plant

016-020

0.99

Potentilla dickinsii

Whole plant

017-036

7.18

Prunus sargentii

Leaf

017-062

24.50

Prunus takesimensis

Stem-bark

016-070

52.74

Pyrus pyrifolia

Leaf

017-034

3.85

Pyrus pyrifolia

Stem-bark

017-035

11.10

Rhamnus davurica

Leaf

017-005

92.35

Rhododendron tschonoskii

Leaf

016-099

9.43

Rhododendron tschonoskii

Stem

016-100

25.70

Rhododendron yedoense var. poukhanense

Stem

016-060

16.80

Rhodotypos scandens

Leaf

017-056

14.20

Rodgersia tabularis

Whole plant

016-005

3.83

Salix hallaisanensis

Stem

016-036

31.12

Sanguisorba hakusanensis

Whole plant

017-068

10.22

Sanguisorba tenuifolia var. alba

Whole plant

017-002

29.37

Sorbus commixta

Stem-bark

016-037

22.84

Sorbus commixta

Leaf

016-038

10.86

Sorbus commixta

Stem

016-039

14.46

Sorbus commixta

fruit

017-039

10.32

Spiraea salicifolia

Leaf

016-016

0.00

Stipa sibirica

Whole plant

017-025

46.66

Suaeda asparagoides

Whole plant

017-026

27.48

Symplocos coreana

Stem

016-067

3.90

Syringa velutina

Leaf

016-093

0.55

Syringa velutina

Stem

016-094

0.35

Tilia insularis

Stem-bark

016-071

10.69

Tripterygium regelii

Stem

017-048

31.59

Ulmus laciniata

Stem-bark

016-022

0.00

Veronica kiusiana

Whole plant

017-028

3.07

Veronica longifolia

Aboveground part

017-022

4.14

Viburnum sargentii

Leaf

017-052

4.31

Vicia amoena

Whole plant

017-006

27.44

Vicia angustifolia var. minor

Whole plant

017-001

17.50

Vicia unijuga

Whole plant

017-027

28.89

Vitex rotundifolia

Leaf

017-075

0.63

Vitis thunbergii var. sinuata

Whole plant

016-014

0.11

Zizyphus jujuba

fruit

017-042

0.00

aThe degranulation was assessed through the measurement of the release of the granule marker β-hexosaminidase from RBL-2H3 mast cells as described in the “Methods” section. The percent inhibition of degranulation is presented as the mean values from three independent experiments.

Degranulation assay in mast cells

Mast cells (1.8 × 105/well) were primed in 50 ng/ml anti-dinitrophenol (DNP) IgE on 24-well plates for 12 h. The cells were then washed twice with 1,4-piperazinediethanesulfonic acid (PIPES)-buffered medium [25 mM PIPES (pH 7.2), 159 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 1 mM CaCl2, 5.6 mM glucose, and 0.1% fatty acid-free fraction V from bovine serum] for RBL-2H3 cells or with Tyrode buffer [20 mM HEPES (pH 7.4), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.05% bovine serum albumin (BSA)] for BMMCs and then pre-incubated in the buffer for 1 h with or without each plant extract. The mast cells were stimulated by the antigen (DNP-BSA) for 10 min and the stimulation was terminated using ice. The cultured media were transferred to new tubes and cells were disrupted with 0.1% triton X-100. For β-hexosaminidase assay, the culture media and cell lysates were mixed with 1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide on 96 well plates and incubated at 37°C for 1 h. Next, 0.1 M carbonate was used to stop the reaction. The density of the color was measured at 405 nm by a microplate reader. Degranulation of mast cells was determined by calculating the ratio of β-hexosaminidase activity released into the culture medium to the total activity of β-hexosaminidase from the cell lysate plus the culture medium [16].

Assay of cell viability

BMMCs (5 × 104/well) were plated on 96-well plates in serum-free RPMI-1640 medium with or without LERD for 8 h. Then, cell viability was determined by using a cell counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's protocol. CCK-8 solution was added to each well of the plate at 1:10 ratio to volume of medium, and the plates were incubated for 1 h in a CO2 incubator at 37°C. The absorbance of color density was measured at 450 nm.

Measurement of TNF-α and IL-4 expression by reverse transcriptase-polymerase chain reaction (RT-PCR)

To obtain the total RNA from cells, an Easy-spinTM Total RNA Extraction Kit (iNtRON Biotechnology, Inc., Sungnam, Korea) was used. PCR was performed at 94°C for 20 sec, at 62°C for 10 sec, and at 72°C for 20 sec for 30 cycles. The primers were used as follows: rat TNF-α forward 5′-ACCACGCTCTTCTGTCTACTGAAC-3′; rat TNF-α reverse: 5′-CCGGACTCCGTGATGTCTAAGTACT-3′; rat IL-4 forward 5′-ACCTTGCTGTCACCCTGTTC-3′; rat IL-4 reverse 5′-TTGTGAGCGT GGACTCATTC-3′; rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5′-GTGGAGTCTACTGGCGTCTTC-3′; rat GAPDH reverse: 5′-CCAAGGC TGTGGGC AAGGTCA-3′.

Measurement of TNF-α and IL-4 in culture media by ELISA

RBL-2H3 cells (5.0 × 105/well/12 well-clustered plate) were plated with 50 ng/ml DNP-specific IgE overnight. Cells were stimulated by antigen with or without LERD for 8 h at 37°C and then the culture media were analyzed via the ELISA kit according to manufacturer’s protocol.

Immunoblotting analysis

The IgE-primed RBL-2H3 cells were stimulated with 25 ng/ml antigen for 7 min or as indicated. The cells were lysed with ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet p-40, 10% glycerol, 60 mM octyl β-glucoside, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 μg/ml pepstatin and protease inhibitor cocktail tablet). Lysates were kept on ice for 30 min and then centrifuged at 13,000 × g for 10 min at 4°C. After centrifuging, the supernatant proteins were denatured at 95°C for 5 min in a 3× Laemmli buffer [17]. The denatured proteins were separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to a nitrocellulose membrane. The transferred protein membrane was blocked in tris-buffered saline-0.05% Tween 20 (TBS-T) buffer containing 5% BSA. The membrane was incubated overnight with the specific antibody for the target protein. After washing the membrane with TBS-T buffer, it was incubated with a labeled secondary antibody directed against the primary antibody. The protein bands for immunoreactive proteins were detected with horseradish peroxidase (HRP)-coupled secondary antibodies and enhanced chemiluminescence according to manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ, USA).

Passive cutaneous anaphylaxis (PCA) and histological analysis

PCA was generated in mice according to a previous study [18]. DNP-specific IgE (0.5 μg per mouse) was intradermally injected into the right ear of a BALB/c mouse (male, 5 weeks old). After 24 h, LERD (0, 100, 300, and 1,000 mg/kg) or cetirizine (20 mg/kg) was orally administered to the mice. After 60 min, the mice were intravenously injected with 250 μg of antigen in Evans blue solution (5 mg/ml PBS). The ears were collected after the mice were euthanized 1 h later. The dye of ear tissue was extracted overnight in 700 μl of formamide at 63°C and then the absorbance was analyzed at 620 nm. For histological analysis, ear tissues were fixed in 4% paraformaldehyde in PBS for 24 h. 5-μm paraffin sections were stained with 0.1% toluidine blue and examined with an optical microscope (Olympus DP 70, Center Valley, PA, USA) at × 100 magnification. Degranulated mast cells in the ear tissue were counted as previously described [19].

Measurement of tyrosine kinase activity in vitro

After stimulating IgE-primed mast cells with 25 ng/ml antigen for 7 min, Lyn or Fyn were immunoprecipitated from whole cell lysates by using specific antibodies. The activity of tyrosine kinase was measured using a Universal Tyrosine Kinase Assay Kit (Gen Way, San Diego, USA) according to manufacturer’s instruction.

Presentation of results

The data were presented as the means ± SEM from three or more independent experiments. Statistical analysis was performed by using one-way ANOVA and the Dunnett test. All statistical calculations (*P < 0.05 and **P < 0.01) were performed with SigmaStat software (Systat Software Inc., Point Richmond, CA, USA).

Results

The effects of LERD and other herbal extracts on antigen-stimulated degranulation in mast cells

Mast cells have secretary granules containing allergic mediators such as histamine and various proteases that cause allergic symptoms [20]. Thus, the effects of approximately 100 herbal extracts on the degranulation of mast cells were firstly measured in RBL-2H3 mast cells. The extracts from Vitex rotundifolia, Prunus sargentii, Lycoris aurea, Hydrangea serrata for. acuminata, Prunus takesimensis, Clintonia udensis, and Rhamnus davurica significantly inhibited degranulation (>50%) at a concentration of 100 μg/ml in RBL-2H3 cells (Table 1). Among them, LERD most potently suppressed degranulation, release of β-hexosaminidase, in antigen-stimulated RBL-2H3 mast cells or BMMCs in a dose dependent manner (Figure 1A and B). However, LERD did not inhibit the activity of β-hexosaminidase released from mast cells (data not shown). When mast cells were washed twice with the incubation buffer after pretreating with LERD for 1 h, the degranulation of mast cells was almost completely recovered (Figure 1C), indicating that the effect of LERD on mast cell activation was reversible. Notably, no effect on the viability of mast cells was observed by LERD at the experimental doses (Figure 1D).
Figure 1

LERD reversibly inhibits antigen-stimulated degranulation in mast cells. RBL-2H3 mast cells (A) and BMMCs (B) were incubated overnight in 24-well cluster plates with 50 ng/ml of IgE in a complete growth medium. The IgE-primed mast cells were stimulated by antigen (25 ng/ml) with or without LERD. The activity of β-hexosaminidase was measured as described in the “Methods” section. (C) RBL-2H3 cells were pre-incubated for 1 h with 0.1% DMSO or 100 μg/ml LERD. After LERD-treated cells were washed five times with PIPES buffer, degranulation of mast cells was measured as for panel A. (D) BMMCs were incubated with LERD for 8 h and the cell viability, then, was measured as described in the “Methods” section. The mean ± SEM from three independent experiments are shown here. Asterisks indicate significant differences from the controls, *P < 0.05 and **P < 0.01. PP2 is a general Src family kinase inhibitor.

Effect of LERD on the expression and secretion of inflammatory cytokines

In addition to degranulation in mast cells, mast cells also secrete various inflammatory cytokines. Among them, TNF-α and IL-4 are secreted in antigen- stimulated cells. Therefore, we measured the expression levels of TNF-α and IL-4 by RT-PCR. As shown in Figure 2A, the expression of TNF-α and IL-4 was dose-dependently suppressed in antigen-stimulated mast cells. The secretion levels of TNF-α and IL-4 were also analyzed by ELISA assay. The secretion of TNF-α and IL-4 was consistently inhibited in a dose dependent manner (Figure 2B).
Figure 2

The effect of LERD on the expression and secretion of TNF-α and IL-4. (A) The IgE-primed RBL-2H3 cells (1 × 106 cells/well) were stimulated by the antigen for 15 min for the assay of TNF-α and IL-4 mRNA by RT-PCR. The representative images (upper panel) and densitometric data (lower panel) from three independent experiments are shown. (B) The IgE-primed RBL-2H3 cells (1 × 106 cells/well) were stimulated by antigen for 8 h and the secretion of TNF-α and IL-4 was then measured by ELISA. The values are expressed as the mean ± SEM from three independent experiments. Asterisks indicate significant differences from the controls, *P < 0.05 and **P < 0.01. PP2 is a general Src family kinase inhibitor.

Effect of LERD on activations of signaling molecules by antigen

Next, to ascertain the mechanism of the LERD action, we investigated which intracellular signaling molecules were affected by LERD treatment. It is well known that Syk and its direct substrate protein LAT pathway is a pivotal signaling cascade for mast cell activation [12]. LERD suppressed the activation of Syk and LAT in RBL-2H3 cells and BMMCs (Figure 3). At a dose of 30 μg/ml, the inhibition was obvious and was nearly complete at 100 μg/ml (Figure 3). Next, we also studied MAP kinase signaling pathway which is generally accepted that three typical MAP kinases are critical for the production of inflammatory cytokines from mast cells [21,22]. In this experiment, LERD significantly reduced the activation of ERK1/2, p-38 and JNK in a dose dependent manner (Figure 3).
Figure 3

LERD inhibits the activation of Syk and Syk-mediated downstream signaling molecules. The RBL-2H3 cells (upper panel) or BMMCs (lower panel) were incubated overnight with 50 ng/ml IgE and cells were then stimulated with 25 ng/ml antigen with or without LERD for 7 min. Cell lysates were subjected to immunoblot analysis with specific antibodies to detect phosphorylated or total proteins. Representative immunoblotting images are shown from three independent experiments. PP2 is a general Src family kinase inhibitor.

LERD inhibited activity of Fyn, but not Lyn, in antigen-stimulated mast cells

Several lines of evidence suggest that Lyn is the initially activated tyrosine kinase in antigen-stimulated mast cells. The aggregation of IgE-high affinity receptor (FcεRI) by antigen initially leads to the activation of Lyn for downstream signaling molecules [12]. Another tyrosine kinase, Fyn, is also activated in mast cells by antigen for a complementary pathway to stimulate full activation of mast cells [23]. LERD did not show any suppressive effect on the expressions of FcεRI subunits (Figure 4A). Next, we tested whether LERD suppressed the upstream tyrosine kinases of Syk, Lyn, or Fyn in the cells. As shown in Figure 4B, LERD inhibited activation of Fyn, but not Lyn, in a dose dependent manner.
Figure 4

LERD inhibits the activity of Fyn, but not Lyn. RBL-2H3 cells (1 × 107 cells/dish) were incubated with 50 ng/ml IgE for overnight and then stimulated with 25 ng/ml antigen for 7 min after pre-incubating with or without LERD for 1 h. (A) Cell lysates were subjected to immunoblot analysis with specific antibodies to detect each FcεRI subunit. Representative immunoblotting images are shown from three independent experiments. (B) Fyn or Lyn was immunoprecipitated from the cell lysates and the immunoprecipitates were incubated in the kinase assay buffer at room temperature for 40 min and the activity was measured as described in the “Methods” section. The values are expressed as the mean ± SEM from three independent experiments. Asterisks indicate significant differences from the controls, *P < 0.05 and **P < 0.01. PP2 is a general Src family kinase inhibitor.

Effect of LERD on passive cutaneous anaphylaxis (PCA) in mice

To determine the anti-allergic effect of LERD in vivo, a PCA model was used in BALB/c mice. Histological analysis was further performed to check whether LERD inhibited mast cell degranulation in mouse ear tissues. The PCA responses were successfully induced by the injection of IgE/antigen in mice. LERD significantly inhibited the response in a dose dependent manner (Figure 5A). In the histological analysis with ear tissues, LERD suppressed the degranulation of tissue mast cells by antigen (Figure 5B). These results suggested that LERD has an anti-allergic effect in vivo through the inhibition of mast cells.
Figure 5

LERD suppresses passive cutaneous anaphylaxis in vivo. An IgE (0.5 μg) was intradermally injected into the mouse ear. After 24 h, an injection of 250 μg antigen in 4% Evans blue PBS solution was administered into the mouse tail vein. The LERD was orally administered 1 h before the treatment of the antigen. The mouse was euthanized 1 h after the antigen challenge, and the ear was then removed for measurement of the amount of dye extravasated (A) or histological changes (B) by antigen treatment. (A) Representative photographs of ears after PCA (upper panel) and quantitative data for ear-tissue content of Evans blue (lower panel). (B) The ear tissues were prepared and stained with toluidin blue for histological examination as described in the “Methods” section. Representative images (upper panel) and the percentage of degranulated mast cells in ear skin sections (lower panel) are shown: arrows indicate mast cells. bar, 50 μm. The values are expressed as the mean ± SEM from three independent experiments. *P < 0.05 and **P < 0.01.; C.Z., cetirizine.

Discussion

Allergic diseases such as asthma, allergic rhinitis, and atopic dermatitis are on the rise, particularly, in developed countries. It has been generally accepted that when people who are skewed to Th2 intake or come into physical contact with a specific allergen, they suffer from allergic symptoms. A large body of evidence suggests that mast cell is a key player of acute and chronic allergic inflammation in allergic diseases [20]. In this context, stimulation of mast cells by antigen results in the release of a variety of mediators such as histamine and an array of inflammatory cytokines that cause allergic symptoms [6,12]. Therefore, the regulation of mast cells is one of the potential therapeutic approaches for the development of allergy medication. In this study, we demonstrated for the first time that LERD suppresses mast cell-mediated PCA in mice by inhibiting the activation of the Fyn/Syk signaling pathway in mast cells.

Mast cells play a critical role in immune response when a foreign antigen infiltrates a human body. When mast cells are stimulated by antigen, they release preformed granule-associated mediators, such as histamine, serotonin, and β-hexosaminidase, eicosanoids, inflammatory cytokines, and chemokines [24,25]. These mediators lead to certain pathophysiological changes and tissue remodeling for various allergic symptoms.

The effects of drugs are generally reversible in a human body. After a certain period following the administration of a drug, the function of drug-target molecule returns to normal functioning status. If the action of a drug, on the other hand, is irreversible, its effect persists until the body generates additional protein. Therefore, it is critical to check at the early stage of the drug development whether a candidate is reversible. The inhibition of LERD on mast cell activation was reversible (Figure 1C), suggesting that LERD achieves its effect by reversibly suppressing the activations of signaling molecules in antigen-stimulated mast cells.

Accumulating evidence from mice and humans has argued that Th2 cytokines such as IL-4, IL-5, and IL-13 are contributors to allergic responses [26]. Mast cells secrete the cytokines. Apart from these Th2 cytokines, TNF-α, one of the critical inflammatory cytokines, is also secreted from mast cells [12]. In this study, LERD suppressed the expression and secretion of IL-4 and TNF-α in antigen-stimulated mast cells (Figure 2). The results further suggested that LERD may have a therapeutic potential in cytokine-associated allergic symptoms in allergic patients.

In the atopic environment, mast cells are activated by binding antigen to the IgE that is bound to its multimeric receptor, FcɛRI. The aggregation of FcɛRIs leads to the phosphorylation of tyrosine residues of the ITAMs of FcɛRI by Lyn and, subsequently, activation of Syk, a pivotal signaling molecule for mast cell activation by the antigen. Syk is essential for the activation of many downstream signaling molecules including LAT adaptor protein. The activation of Syk is also critical for the activation of Akt and three typical MAP kinases (ERK1/2, p38, and JNK). In such a manner, mast cells are activated to release an array of allergic mediators [12]. In this study, LERD inhibited activation of Syk and LAT in antigen-stimulated mast cells (Figure 3), suggesting that its inhibition of Syk activation was a key mechanism of LERD.

Fyn is an essential signaling molecule for full activation of antigen-stimulated mast cells. The activation of Fyn is critical for the activation of the Gab2/PI3-K/Akt pathway [23]. In other reports, Fyn is also important for the activation of Syk [11,27]. Of note, our study found that LERD inhibited only the activity of Fyn, not Lyn, in antigen-stimulated mast cells (Figure 4B), suggesting that LERD suppressed the activation of Fyn and Fyn/Syk-mediated downstream signaling molecules.

Anaphylaxis is an allergic response that is remarkably dangerous and acute as it can cause death due to asphyxiation. Systemic or local anaphylaxis may occur when the exterior allergens such as insect venom, food, and pollen infiltrate the allergic patient’s body [28]. The reactions cause the release of inflammatory mediators and cytokines from mast cells and basophils [29]. To confirm the anti-allergic effect of LERD in animal model, we utilized IgE-mediated PCA mice. In the mice, LERD remarkably suppressed the anaphylactic responses (Figure 5A) and degranulation of mast cells in ear (Figure 5B). Taken together, the results demonstrated that LERD has an anti-allergic effect in vivo.

Conclusions

Our results demonstrated for the first time that LERD suppresses degranulation and cytokine production in antigen-stimulated mast cells and, furthermore, inhibits IgE-mediated allergic responses in mice. Mechanically, LERD suppresses the activation of Fyn/Syk pathway and its downstream signaling molecules to supress secretion of allergic mediators in mast cells (Figure 6). Although further comprehensive studies for bioavailability, toxicity, and active components of LERD are required for the development of drug, our finding suggested that LERD may be a useful herbal extract for human allergic diseases.
Figure 6

LERD inhibits the activation of Fyn for the antigen-mediated activation of mast cells. LERD suppresses the activation of Fyn and Fyn/Syk-mediated downstream signaling molecules for the release of various allergic mediators in antigen-stimulated mast cells.

Notes

Abbreviations

BMMC: 

Bone marrow–derived mast cell

DNP: 

2,4-Dinitrophenyl

LAT: 

Linker for activation of T cells

MAP: 

Mitogen-activated protein

PCA: 

Passive cutaneous anaphylaxis

Declarations

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP, No. 2012R1A2A1A03670516) and in part by a National Research Foundation of Korea (NRF) grant (MSIP, NRF-2013R1A4A1069575).

Authors’ Affiliations

(1)
KU open Innovation Center, School of Medicine, Konkuk University
(2)
College of Pharmacy, Sungkyunkwan University
(3)
College of Pharmacy, Duksung Women’s University

References

  1. Erb KJ. Can helminths or helminth-derived products be used in humans to prevent or treat allergic diseases? Trends Immunol. 2009;30:75–82.View ArticlePubMedGoogle Scholar
  2. Flohr C, Quinnell RJ, Britton J. Do helminth parasites protect against atopy and allergic disease? Clin Exp Allergy. 2009;39:20–32.View ArticlePubMedGoogle Scholar
  3. Voehringer D. Protective and pathological roles of mast cells and basophils. Nat Rev Immunol. 2013;13:362–75.View ArticlePubMedGoogle Scholar
  4. Bischoff SC. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol. 2007;7:93–104.View ArticlePubMedGoogle Scholar
  5. Gregory GD, Brown MA. Mast cells in allergy and autoimmunity: implications for adaptive immunity. Methods Mol Biol. 2006;315:35–50.PubMedGoogle Scholar
  6. Beaven MA. Our perception of the mast cell from Paul Ehrlich to now. Eur J Immunol. 2009;39:11–25.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Rivera J, Gilfillan AM. Molecular regulation of mast cell activation. J Allergy Clin Immunol. 2006;117:1214–25.View ArticlePubMedGoogle Scholar
  8. Galli SJ, Tsai M, Piliponsky AM. The development of allergic inflammation. Nature. 2008;454:445–54.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Kinet JP. The essential role of mast cells in orchestrating inflammation. Immunol Rev. 2007;217:5–7.View ArticlePubMedGoogle Scholar
  10. Siraganian RP, Zhang J, Suzuki K, Sada K. Protein tyrosine kinase Syk in mast cell signaling. Mol Immunol. 2002;38:1229–33.View ArticlePubMedGoogle Scholar
  11. Lee JH, Kim JW, Kim DK, Kim HS, Park HJ, Park DK, et al. The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice. J Immunol. 2011;187:1807–15.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Gilfillan AM, Beaven MA. Regulation of mast cell responses in health and disease. Crit Rev Immunol. 2011;31:475–529.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Alvarez-Errico D, Lessmann E, Rivera J. Adapters in the organization of mast cell signaling. Immunol Rev. 2009;232:195–217.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Odom S, Gomez G, Kovarova M, Furumoto Y, Ryan JJ, Wright HV, et al. Negative regulation of immunoglobulin E-dependent allergic responses by Lyn kinase. J Exp Med. 2004;199:1491–502.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Jensen BM, Swindle EJ, Iwaki S, Gilfillan AM. Generation, isolation, and maintenance of rodent mast cells and mast cell lines. Curr Protoc Immunol 2006;3.23.1-3.23.13. doi:10.1002/0471142735.im0323s74Google Scholar
  16. Ozawa K, Szallasi Z, Kazanietz MG, Blumberg PM, Mischak H, Mushinski JF, et al. Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J Biol Chem. 1993;268:1749–56.PubMedGoogle Scholar
  17. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophages t4. Nature. 1970;227:680–5.View ArticlePubMedGoogle Scholar
  18. Kim DK, Kim HS, Kim AR, Jang GH, Kim HW, Park YH, et al. The scaffold protein prohibitin is required for antigen-stimulated signaling in mast cells. Sci Signal. 2013;6:ra80.View ArticlePubMedGoogle Scholar
  19. Kim DK, Kim HS, Kim AR, Kim JH, Kim B, Noh G, et al. DJ-1 regulates mast cell activation and IgE-mediated allergic responses. J Allergy Clin Immunol. 2013;131:53–62.Google Scholar
  20. Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nat Med. 2012;18:693–704.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Law CL, Chandran KA, Sidorenko SP, Clark EA. Phospholipase Cgamma1 interacts with conserved phosphotyrosyl residues in the linker region of Syk and is a substrate for Syk. Mol Cell Biol. 1996;16:1305–15.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Saitoh S, Odom S, Gomez G, Sommers CL, Young HA, Rivera J, et al. The four distal tyrosines are required for LAT-dependent signaling in FcepsilonRI-mediated mast cell activation. J Exp Med. 2003;198:831–43.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Parravicini V, Gadina M, Kovarova M, Odom S, Gonzalez-Espinosa C, Furumoto Y, et al. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol. 2002;3:741–8.PubMedGoogle Scholar
  24. Theoharides TC, Cochrane DE. Critical role of mast cells in inflammatory diseases and the effect of acute stress. J Neuroimmunol. 2004;146:1–12.View ArticlePubMedGoogle Scholar
  25. Theoharides TC, Kalogeromitros D. The critical role of mast cells in allergy and inflammation. Ann N Y Acad Sci. 2006;1088:78–99.View ArticlePubMedGoogle Scholar
  26. Licona-Limón P, Kim LK, Palm NW, Flavell RA. TH2, allergy and group 2 innate lymphoid cells. Nat Immunol. 2013;14:536–42.View ArticlePubMedGoogle Scholar
  27. Yu M, Luo J, Yang W, Wang Y, Mizuki M, Kanakura Y, et al. The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast cell proliferation by activating the Rac/JNK pathway. J Biol Chem. 2006;281:28615–26.View ArticlePubMedGoogle Scholar
  28. Simons FE. World Allergy Organization: World Allergy Organization survey on global availability of essentials for the assessment and management of anaphylaxis by allergy-immunology specialists in health care settings. Ann Allergy Asthma Immunol. 2010;104:405–12.View ArticlePubMedGoogle Scholar
  29. Khan BQ, Kemp SF. Pathophysiology of anaphylaxis. Curr Opin Allergy Clin Immunol. 2011;11:319–25.View ArticlePubMedGoogle Scholar

Copyright

© Kim et al.; licensee BioMed Central. 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement