The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maeda-Yamamoto, M.
Right arrow Articles by Kawakami, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maeda-Yamamoto, M.
Right arrow Articles by Kawakami, T.
The Journal of Immunology, 2004, 172: 4486-4492.
Copyright © 2004 by The American Association of Immunologists

O-Methylated Catechins from Tea Leaves Inhibit Multiple Protein Kinases in Mast Cells1

Mari Maeda-Yamamoto*, Naoki Inagaki{ddagger}, Jiro Kitaura||, Takao Chikumoto{ddagger}, Hiroharu Kawahara§, Yuko Kawakami||, Mitsuaki Sano{dagger}, Toshio Miyase{dagger}, Hirofumi Tachibana, Hiroichi Nagai{ddagger} and Toshiaki Kawakami2

* National Institute of Vegetable and Tea Science, National Agriculture Research Organization, and {dagger} School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan; {ddagger} Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; § Kitakyushu National College of Technology, and Division of Bioresources and Bioenvironmental Science, Kyushu University, Fukuoka, Japan; and || Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton’s tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells play a critical role in the effector phase of IgE-dependent immediate hypersensitivity and allergic diseases (1). Cross-linking of high affinity IgE receptors (Fc{epsilon}RI) with IgE and allergen initiates the activation process, leading to the release of preformed and de novo synthesized vasoactive amines, proteases, leukotrienes, cytokines, and chemokines. These chemical and polypeptide agents elicit various allergy-associated pathophysiological changes locally and systemically: for instance, amines such as histamine and serotonin enhance vascular permeability, and cytokines such as TNF-{alpha} recruit inflammatory cells to the site of allergen exposure.

Over the past two decades, we have seen a tremendous advancement in our understanding of what happens inside mast cells following Fc{epsilon}RI cross-linking (2). Fc{epsilon}RI is a heterotetrameric receptor composed of an IgE-binding {alpha} subunit, a four-transmembrane {beta} subunit, and disulfide-bonded two {gamma} subunits (3). {gamma} subunits are critical for both membrane expression of the receptor and signal transduction, while {beta} subunits amplify the {gamma} subunit-generated signals and stabilize the receptor. {beta} subunits are associated constitutively with a predominant Src family protein tyrosine kinase (PTK)3 (3), Lyn (4). Upon Fc{epsilon}RI cross-linking, Lyn becomes activated and phosphorylates tyrosine residues in the immunoreceptor tyrosine-based activation motifs in {beta} and {gamma} subunits (5, 6). Tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs of {beta} and {gamma} subunits recruit Lyn and Syk (another PTK with two tandem Src homology 2 domains), respectively, to the plasma membrane and activate these PTKs (4, 7, 8, 9). Active Lyn and Syk phosphorylate numerous signaling proteins. Another PTK, Bruton’s tyrosine kinase (Btk) (10), is phosphorylated at its critical activation-loop tyrosine by Lyn and activated (11). Active Syk and Btk in turn phosphorylate and activate phospholipase C (PLC)-{gamma} in a concerted manner (12, 13). PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular storage sites. Diacylglycerol and Ca2+ activate some isoforms of protein kinase C (PKC). Downstream of these early activation events, at least four major mitogen-activated protein kinase (MAPK) subfamilies of protein serine/threonine kinases (PS/TKs), i.e., extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) 1/2, p38, and big mitogen-activated kinase 1/ERK5 (14, 15, 16, 17, 18, 19), and a critical survival kinase, Akt/protein kinase B (Akt) (20), are also activated. Many of these kinases are shown to be involved in production and secretion of cytokines.

Tea (Camellia sinensis L.) has been consumed all over the world, particularly in large quantities in Japan and China, where it has also been used for medicinal purposes for tens of centuries. It has been reported that tea has various bioregulatory activities, such as anticarcinogenetic (21, 22) and antimetastatic activities (23, 24, 25, 26), antioxidative activity (27, 28, 29), antihypertensive activity (30), antihypercholesterolemic activity (31, 32), antidental caries activity (33), antibacterial activity (34), and intestinal flora amelioration activity (35). Major principles for these activities were shown to be catechins, a group of polyphenolic compounds. In previous studies, we have reported that O-methylated forms of (-)-epigallocatechin-3-O-gallate (EGCG), i.e., (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3''Me) and (-)-epigallocatechin-3-O-(4-O-methyl) gallate (EGCG4''Me), which were contained in some cultivars of tea such as ‘Benifuki’ or ‘Benifuji,’ had antiallergic action in in vivo type I and type IV allergies (36, 37). In this study, we present evidence that two O-methylated catechins, EGCG3''Me and EGCG4''Me, have an antiallergic activity and inhibit several protein kinases as a molecular basis for such an activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Sources of commercial Abs are as follows: anti-phosphotyrosine mAb 4G10 from Upstate Biotechnology (Charlottesville, VA); anti-Lyn (38), anti-Syk (C-20), anti-Akt (C-20), and anti-JNK (C17) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-ERK1/2, anti-phospho-JNK, and anti-phospho-p38 from Cell Signaling Technology (Beverly, MA); anti-mouse IgG F(ab')2 from BioSource International (Camarillo, CA); anti-Btk from BD PharMingen (San Diego, CA); anti-ERK1/2 from Zymed Laboratories (South San Francisco, CA); and DNP-specific IgE from Sigma-Aldrich (St. Louis, MO). Anti-Fc{epsilon}RI {beta} subunit mAb JRK was kindly provided by J. Rivera (National Institutes of Health).

Chemicals

OVA and DNP conjugates of human serum albumin (DNP-HSA) were purchased from Sigma-Aldrich; IFA and diphenhydramine hydrochloride were from Wako Pure Chemical (Osaka, Japan).

Purification of EGCG3''Me and EGCG4''Me

Dried tea leaves (cultivars ‘Benihomare’ and ‘Benifuki’ or Ton-chin oolong tea) were extracted with boiling water for 30 min. The extract was passed through a porous polymer gel (Mitsubishi Diaion HP-20; Mitsubishi Oil Chemicals, Tokyo, Japan) column (9 x 40 cm). After the column had been washed with water, the absorbed fraction was eluted with water/methanol (1:1) to give a pale brown residue (31 g). The residue was chromatographed on a silica gel column (9 x 25 cm) and eluted with chloroform/methanol/water (82:16:2) and subsequently with chloroform/methanol/water (40:50:10) to yield six fractions. The peak fraction (2.657 g) that contained EGCG3''Me and EGCG4''Me was chromatographed on a reversed-phase (LOP-ODS; Nomura Chemical, Aichi, Japan) column (two columns connected in tandem; 5 x 50 cm, 2x) and eluted with water/acetonitrile (88:12) to give 241 mg of EGCG3''Me and 101 mg of EGCG4''Me. The physical properties of EGCG3''Me and EGCG4''Me (Fig. 1) were confirmed by 1H nuclear magnetic resonance spectrum and 13C nuclear magnetic resonance spectrum.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Structural formula of EGCG and O-methylated EGCGs isolated from tea leaves.

 
Cells, stimulation, and biological consequences

Bone marrow cells from femur of CBA/J mice were cultured in mouse IL-3-containing RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies, Carlsbad, CA), 2 mM glutamine, and 50 µM 2-ME in humidified 95% air/5% CO2 at 37°C (39). More than 95% pure mast cells (bone marrow-derived mast cells (BMMC)) were obtained after 4 wk of culture. These cells were incubated overnight with 0.5 µg/ml DNP-specific IgE mAb, and stimulated with a multivalent Ag, DNP-HSA, at 100 ng/ml in Tyrode solution for the indicated intervals. In some experiments, IgE-sensitized cells were precincubated with the indicated concentrations of EGCGs for 30 min and stimulated with Ag in the continued presence of EGCGs for 3 min or various periods. Cell suspensions were briefly centrifuged to separate Tyrode solution from cell pellets. Histamine secreted into the Tyrode solution during 30-min stimulation was measured by on-column HPLC. Leukotrienes C4/D4/E4 secreted into the Tyrode solution were quantified by an enzyme immunoassay (Amersham Biosciences, Piscataway, NJ) and IL-2 by an ELISA kit (Endogen, Woburn, MA).

Immunoblotting and immunoprecipitation

Cell pellets were lysed in ice-cold 1% Nonidet P-40-containing lysis buffer (20 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 25 µM p-nitrophenyl p'-guanidinobenzoate, 1 µM pepstatin, and 0.1% sodium azide) immediately after stimulation and centrifuged. Protein concentrations of cell lysates were measured using a DC protein assay reagent (Bio-Rad, Richmond, CA). For immunoprecipitation, lysates were incubated on ice with an appropriate Ab for 1.5 h, and immune complexes were recovered by brief centrifugation following another 30-min incubation with Pansorbin (Calbiochem, San Diego, CA) for rabbit polyclonal Abs or anti-mouse Ig-conjugated agarose (Sigma-Aldrich) for mouse mAbs. Immune complexes were washed in lysis buffer four times. Cleared cell lysates or immunoprecipitates were separated by SDS-PAGE and blotted electrophoretically to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Billerica, MA). Membranes were blocked and incubated with a primary Ab and then with a HRP-conjugated secondary Ab. Visualization of immunoreactive proteins was performed with ECL reagents (PerkinElmer Life Sciences, Wellesley, MA). Relative amounts of immunoreactivity were quantified by National Institutes of Health Image Analysis software (v.1.62).

Immune complex kinase assays

Immunoprecipitates were washed four times in lysis buffer and once with kinase buffer (50 mM Tris, pH 7.4, 0.1% Nonidet P-40, 10 mM MnCl2, 10 mM MgCl2) without ATP. Washed immune complexes were incubated in kinase buffer with or without exogenous substrate (Btk and Lyn without exogenous substrate; Syk with 2 µg of GST-HS1 containing the sequence from position 352 to position 486 of the human HS1 protein (40)) in the presence of [{gamma}-32P]ATP (ICN Biomedicals, Irvine, CA). Reaction products were analyzed by SDS-PAGE, followed by electroblotting onto PVDF membranes and autoradiography. Phosphorylated protein bands were quantified by densitometry.

In vitro kinase assays using purified or recombinant enzymes

Lyn and Btk were expressed in Sf9 insect cells and partially purified, as described (41). Recombinant human Syk was purchased from Upstate Biotechnology. Kinase assays were performed, as described above, for immune complex assays, except for the presence of various concentrations of EGCGs throughout the assay period. Kinase activities of PKC{alpha}, PKC{beta}I, and PKC{beta}II (all recombinant human isoforms from Panvera, Madison, WI) in the absence or presence of EGCG3''Me and EGCG4''Me were measured using the PKC pseudosubstrate peptide PKC (19, 20, 21, 22, 23, 24, 25, 26, 27) in a PKC assay kit (Invitrogen). Assays on ERK2 (recombinant mouse; New England Biolabs, Beverly, MA) and JNK1 (recombinant mouse; Upstate) in the absence or presence of O-methylated EGCGs were performed using myelin basic protein and GST-c-Jun (1–79), respectively, as substrate.

Kinetic analysis of EGCG3''Me inhibition of Lyn kinase was performed at pH 7.2 in kinase buffer, using 1 µg of purified Lyn per assay in the presence of various concentrations of [{gamma}-32P]ATP with or without 0.4 µg/ml EGCG3''Me. Reaction products were analyzed by SDS-PAGE, followed by electroblotting onto PVDF membranes and autoradiography. Phosphorylated protein bands were quantified by densitometry. Kinetic data were analyzed by Lineweaver-Burk plotting.

Transcription assays

BMMC were transiently transfected by electroporation with a reporter construct, IL-2/luc (42). Twenty-four hours later, the cells were sensitized with IgE and stimulated by Ag in the absence or presence of O-methylated EGCGs for another 7 h. Luciferase activity was measured, as described previously (42).

Measurement of Ca2+ influx

Ca2+ influx was analyzed by spectrofluorometry, as previously described (43). Briefly, BMMC were incubated at 37°C for 30 min with 5 µM fura 2-AM (Dojindo, Osaka, Japan) in Tyrode/HEPES solution after overnight sensitization with anti-DNP IgE. After washing with HBSS, aliquots of the cell suspension were placed in a cuvette and incubated with O-methylated EGCGs at 37°C for 10 min, and then 100 ng/ml DNP-HSA was added. Fluorescence was recorded using a fluorometer (CAF-110; Spectroscopic, Tokyo, Japan) by alternating excitation at 340 and 380 nm and reading the emission at 510 nm. O-methylated EGCGs did not exhibit autofluorescence nor interfere with fura 2 fluorescence.

Passive cutaneous anaphylaxis (PCA) reactions

As a model of in vivo type I allergic reactions, we performed mast cell-dependent PCA experiments, as previously described (44). Briefly, male ddY mice (Japan SLC, Hamamatsu, Japan) were intradermally injected with 10 µl of anti-DNP IgE at both ears. Catechin samples (or distilled water as control) were administered i.v. or orally to the mice 60 min before Ag administration. Intravenous administration of a 0.25-ml mixed solution of Ag (0.1 mg/ml DNP-BSA) and Evans blue dye (0.5%) was i.v. injected 24 h after IgE sensitization. The mice were killed by cervical dislocation 30 min later, and the ears were removed. Evans blue dye leaked into ear tissues was extracted and quantified by spectrophotometry. To confirm the role of histamine released from activated mast cells in inducing PCA reactions, diphenhydramine, an inhibitor of the histamine H1 receptor, at 20 mg/kg, was i.p. injected 30 min before Ag challenge. As shown in Fig. 2, this treatment consistently inhibited PCA reactions by 70–85%.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. Effects of EGCG and O-methylated EGCGs on vascular permeability increase caused by PCA reactions. Mice were sensitized intradermally at the ear with anti-DNP IgE 24 h before Ag challenge. Catechin samples were administered i.v. (A) or orally (B) to the mice 60 min before Ag challenge. As a control, an H1 histamine receptor inhibitor, diphenhydramine (DPH), was i.p. injected 30 min before Ag challenge. The mice were challenged with Ag mixed with Evans blue dye for 30 min and sacrificed. Dye leakage into ear tissues was quantified. PCA- and PCA+ denote negative (no Ag) and positive (Ag without inhibitor) controls. Each value represents the mean ± SD of seven or eight mice. ***, p < 0.01 (vs PCA+, Student’s t test). {dagger}, {dagger}{dagger}, {dagger}{dagger}{dagger}, p < 0.05, p < 0.01, p < 0.001 (vs PCA+, Dunnett’s multiple comparison test).

 
Statistics

Statistical analysis was performed using Student’s t test or Dunnett’s multiple comparison test. Values with p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of tea catechins on PCA reactions

EGCG is known to inhibit immediate hypersensitivity reactions (38, 45). We first compared in vivo effects on mast cell-dependent PCA reactions between different tea catechins, EGCG and its O-methylated derivatives (Fig. 2). Mice were sensitized for 24 h by intradermal injection of anti-DNP IgE at the ear, and then they were i.v. injected with DNP-BSA solution containing Evans blue dye. Thirty minutes later, the mice were sacrificed, and the dye leaked into the ear was extracted and quantified. Intraperitoneal administration of EGCG 60 min before Ag/dye injection substantially inhibited allergic reactions (10, 27, and 43% inhibition by a single dose of EGCG at 5, 10, and 20 mg/kg, respectively). Both 3-O- and 4-O-methylated derivatives, EGCG3''Me and EGCG4''Me, were 2 times more potent in inhibiting PCA reactions than EGCG. Oral administration of EGCG and O-methylated derivatives 60 min before Ag/dye injection exhibited similar inhibition of PCA reactions, with slightly weaker inhibition compared with i.p. administration. Because our experiments were designed to monitor early phase reactions, these results suggest that these catechins, particularly the O-methylated EGCGs, inhibit immediate hypersensitivity reactions by suppressing activation of mast cells. Alternatively, the catechins might antagonize effects of chemical mediators or affect vascular permeability nonspecifically.

O-methylated EGCGs inhibit mast cell activation

Because mast cells are the major effector cells in the early phase of immediate hypersensitivity, we explored the effect of the O-methylated EGCGs on mast cell activation. To this end, BMMC sensitized with IgE were pretreated with these catechins for 30 min before Ag stimulation. As readouts of mast cell activation, we measured histamine release (as a surrogate marker of degranulation), leukotriene release, and cytokine secretion. As shown in Fig. 3A, both EGCG3''Me and EGCG4''Me inhibited histamine release in a dose-dependent manner. Inhibition by EGCG3''Me and EGCG4''Me at 100 µg/ml reached 70%. Leukotriene release and IL-2 production/secretion were also inhibited by these catechins (Fig. 3, B and C). It is noteworthy that more than control levels of leukotriene release and IL-2 secretion were observed at lower doses (5 or 25 µg/ml) of the O-methylated EGCGs.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 3. Effects of O-methylated EGCGs on biologic outcomes of mast cell activation. IgE-sensitized BMMC were pretreated with EGCGs and stimulated with Ag for 30 min (histamine release), 45 min (leukotrienes release), or 24 h (cytokine secretion). A, Inhibition of histamine release was expressed as a percentage of reduction compared to control values (histamine release induced by Ag without inhibitor). At least 30–70% of cellular histamine content was released upon stimulation in the absence of inhibitor. *, Significantly different from the EGCG-treated group (p < 0.05). B, Release of leukotrienes. C, IL-2 secretion. Results of histamine release are representative of four independent experiments, and those of leukotriene release and cytokine secretion of at least three independent experiments.

 
O-methylated EGCGs inhibit PTK activity in mast cells

To gain insight into the mechanism of mast cell inhibition by O-methylated EGCGs, we investigated their effects on early signaling events. IgE-sensitized BMMC were preincubated with the catechins for 30 min and stimulated with Ag for 3 min in the continued presence of the catechins. Effects on one of the earliest signaling events, tyrosine phosphorylation of the Fc{epsilon}RI {beta} subunit, were examined by immunoprecipitation with anti-Fc{epsilon}RI {beta} Ab, followed by immunoblotting with anti-phosphotyrosine Ab. As shown in Fig. 4A, up to 50 µg/ml of either EGCG derivative did not substantially affect Fc{epsilon}RI {beta} phosphorylation. Interestingly, however, tyrosine phosphorylation of other cellular proteins, as revealed by immunoblotting of total cell lysates (Fig. 4B), was more strongly inhibited: tyrosine phosphorylation of several proteins (100-, 85-, 75-, 56-, 53-, 40-, 38-, and 30-kDa proteins) was obviously inhibited by both catechins in a dose-dependent manner.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 4. Effects of O-methylated EGCGs on early mast cell activation events. A, IgE-sensitized BMMC were left unstimulated (-) or stimulated with Ag for 3 min (+) in the presence of the indicated concentrations of O-methylated EGCGs. Fc{epsilon}RI {beta} was immunoprecipitated with anti-Fc{epsilon}RI {beta} mAb from cell lysates and immunoblotted with anti-phosphotyrosine 4G10 mAb. B, Immunoblotting of total cell lysates with 4G10 was performed with mast cells left unstimulated (-) or stimulated with Ag for 3 min (+) in the presence of the indicated concentrations of O-methylated EGCGs. C, Cells were left unstimulated (-) or stimulated with Ag for 3 min (+). In vitro kinase assays were done on immune complexes precipitated with anti-Syk using GST-HS1 as an exogenous substrate. Lyn and Btk immunoprecipitates were subjected to kinase assays without substrate. Phosphorylated GST-HS1 and autophosphorylated Lyn or Btk bands were detected by SDS-PAGE and blotting, followed by autoradiography. The amounts of the respective PTKs immunoprecipitated were measured by reprobing the same blots. Approximate IC50 values estimated from these kinase assays are also presented.

 
We next performed in vitro kinase assays on Lyn, Syk, and Btk immunoprecipitated from O-methylated EGCG-pretreated, IgE/Ag-stimulated cells (Fig. 4C). The results demonstrated that both O-methylated EGCGs inhibit catalytic activity of these critical PTKs: EGCG3''Me inhibited Lyn more preferably (IC50 of 30 µg/ml) than Syk (IC50 of 100 µg/ml). Lyn activity was inhibited almost completely by EGCG3''Me at 100 µg/ml, but substantial levels of Lyn activity remained at 100 µg/ml EGCG4''Me. In contrast, Syk activity was totally abrogated by 100 µg/ml EGCG4''Me, but substantial Syk activity remained at 100 µg/ml EGCG3''Me. Btk was also inhibited by EGCG4''Me with an IC50 of 50 µg/ml (Fig. 4C). However, Btk activity was not affected by EGCG3''Me. Treatment with 50 or 100 µg/ml O-methylated EGCGs did not change kinetics of activation of these PTKs or tyrosine phosphorylation (data not shown).

We next investigated whether inhibition of PTKs by O-methylated EGCGs is a direct action or not. To this end, we performed kinase assays on purified Lyn, Syk, and Btk proteins in the presence of various concentrations of O-methylated EGCGs (Fig. 5A). We found that the catalytic activity of all these PTKs is inhibited by both O-methylated EGCGs in a dose-dependent manner. These PTKs were much more sensitive to O-methylated EGCGs in these cell-free kinase assays than when mast cells were treated with O-methylated EGCGs: for instance, IC50 for cell-free inhibition of Lyn by EGCG3''Me was 0.2 µg/ml compared with the IC50 of 30 µg/ml when cells were pretreated with EGCG3''Me. Kinetic analysis of EGCG3''Me on Lyn inhibition showed that inhibition was noncompetitive with ATP (Fig. 5B); thus, EGCG3''Me is a nonsubstrate inhibitor. The results collectively indicate that O-methylated EGCGs directly inhibit three PTKs critical for mast cell activation.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 5. Direct inhibition of PTKs by O-methylated EGCGs. A, Kinase assays on purified Lyn, Syk, and Btk proteins were performed in the presence of various concentrations of O-methylated EGCGs. Approximate IC50 values estimated from these kinase assays are also presented. B, Double-reciprocal plot for Lyn kinase inhibition by EGCG3''Me. Kinetic analysis was performed using 1 µg of purified Lyn per assay with various concentrations of [{gamma}-32P]ATP in the presence (•) or absence ({triangleup}) of 0.4 µg/ml EGCG3''Me.

 
Effects of O-methylated EGCGs on late signaling events

Subsequent to activation of the PTKs, various signaling pathways are activated upon Fc{epsilon}RI stimulation. Increased intracellular Ca2+ concentration levels following PLC-{gamma} activation by Syk and Btk are critical for Fc{epsilon}RI-induced degranulation (46). As expected from PTK inhibition (Figs. 4 and 5) and inhibition of degranulation, leukotriene release, and cytokine production (Fig. 3) by O-methylated EGCGs, both EGCG3''Me and EGCG4''Me inhibited IgE/Ag-induced Ca2+ response (Fig. 6).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of O-methylated EGCGs on Ca2+ flux. BMMC loaded with fura 2-AM dye in the absence or presence of the indicated concentrations of O-methylated EGCGs were stimulated with IgE/Ag. Intracellular Ca2+ levels were determined by spectrofluorometry.

 
In light of inhibition of IL-2 production/secretion by O-methylated EGCGs at a high concentration of 100 µg/ml, we examined their effects on the activation of MAPKs (ERKs, JNK, and p38) and Akt. Among these PS/TKs that were shown to be involved in cytokine production and secretion (17, 20, 47, 48), we previously showed that Akt regulates production of IL-2 and TNF-{alpha} (20) and JNK is believed to control the transcription of the IL-2 gene through c-Jun phosphorylation (49). BMMC pretreated with O-methylated EGCGs were stimulated with Ag, and cell lysates were analyzed by immunoblotting with Abs that react with the activated forms of these kinases. As shown in Fig. 7A, EGCG3''Me inhibited Akt and JNK1, but not ERK1, ERK2, or p38. Effects of EGCG4''Me were more complex: it inhibited Akt at 25 mg/ml; it inhibited JNK activity less potently than EGCG3''Me, but p38 activity was rather enhanced by EGCG4''Me. Potentially related to the increased IL-2 production/secretion in cells treated with low concentrations of O-methylated EGCGs (Fig. 3C), increased phosphorylations of some of these kinases were observed in cells treated with low concentrations of O-methylated EGCGs: for instance, ERK1, ERK2, and p38 in EGCG3''Me-treated cells and p38 in EGCG4''Me-treated cells (Fig. 7A).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 7. Effects of O-methylated EGCGs on Akt and MAPK activities. A, IgE-sensitized BMMC were stimulated with Ag for 3 min in the presence of the indicated concentrations of O-methylated EGCGs. Immunoblotting of total cell lysates was performed with anti-phospho-Akt, anti-Akt, anti-phospho-ERK, anti-ERK, anti-phospho-JNK1, anti-JNK1, anti-phospho-p38, or anti-p38. B, In vitro kinase assays were performed using pure preparations of ERK2, JNK1, and PKC{beta}II in the presence of myelin basic protein (MBP), GST-c-Jun (1–79), and pseudosubstrate peptide, respectively. The data shown in A and B were confirmed in another experiment. C, BMMC were transfected with the IL-2/luc reporter construct. Twenty-four hours later, the cells were sensitized with IgE and stimulated with Ag for 7 h before luciferase activities were measured. Values were normalized against those of unstimulated cells.

 
Downstream of these serine/threonine kinases, transcription factors are activated. Such activation of transcription factors was monitored by tranfecting BMMC with IL-2/luc, followed by pretreatment of the cells with O-methylated EGCGs and stimulation with IgE and Ag (Fig. 7C). Again, consistent with the inhibition of IL-2 production/secretion with O-methylated EGCGs at high concentrations, IL-2/luc activity was inhibited by both O-methylated EGCGs at 100 µg/ml. Lower concentrations of O-methylated EGCGs rather enhanced the IL-2/luc activity.

Direct effects of high concentrations of O-methylated EGCGs on PS/TK

Given the broad specificity of PTK inhibition by O-methylated EGCGs, we explored whether these catechins might directly affect the catalytic activity of serine/threonine kinases as well. For this purpose, effects of O-methylated EGCGs were measured in in vitro kinase assays using pure preparations of ERK2, JNK1, and PKC{beta}II (Fig. 7B). All of these kinases were inhibited by either EGCG in a dose-dependent manner. However, their IC50 were at least 10- to 50-fold higher than those for PTK inhibitions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that two tea-derived O-methylated catechins, EGCG3''Me and EGCG4''Me, inhibit PCA reactions with higher potency than their nonmethylated form of catechin does (Fig. 2). Consistent with such in vivo effects, these O-methylated catechins inhibited all tested aspects (i.e., degranulation, leukotriene release, and cytokine production/secretion) of mast cell activation induced by Fc{epsilon}RI cross-linking (Fig. 3). Mast cell inhibition could be explained at least in part by their activity to directly inhibit several PTKs (Figs. 4 and 5). Although the O-methylated catechins can directly inhibit several PS/TKs, their potencies against the PS/TKs are 10- to 50-fold lower than those against the PTKs. Therefore, it is likely that the inhibition of several PS/TKs observed in mast cells treated with the O-methylated catechins is due to inhibition of upstream PTKs. Effects of O-methylated EGCGs on mast cell signal transduction appear more complex than simply inhibiting PTKs: although Lyn kinase activity was inhibited by these agents, Fc{epsilon}RI {beta} phosphorylation that is mediated by Lyn was not substantially affected by them. They rather increased the activity of PS/TKs, particularly p38, at low concentrations. These results suggest that O-methylated EGCGs influence other signaling molecules, e.g., a protein phosphatase(s). These catechins are unique in their broad specificity among other known mast cell-inhibitory agents. Other PTK inhibitors that were shown to inhibit mast cell activation exhibit more stringent selectivity: for instance, ER-27319 (50) and terreic acid (51) inhibit Fc{epsilon}RI-induced mast cell activation by specifically inhibiting Syk and Btk, respectively. This broad specificity might limit the usefulness of these catechins as an antiallergic drug. However, drinking tea containing these compounds has not imposed apparent risks on human health. Therefore, our results may be a foundation for encouraging allergic patients to drink tea rich in these O-methylated catechins. Indeed, a clinical trial to treat allergic cedar pollinosis patients with tea rich in O-methylated catechins is ongoing, and promising results have been obtained by using a protocol of drinking two cups of tea daily (data not shown). Another potential use could be topical treatment of allergic diseases with these O-methylated catechins: for example, washing with these catechins or tea extracts, nasal cavity of allergic rhinitis patients and skin lesions of atopic dermatitis patients might have beneficial effects.

Differences of one to two orders of magnitude in effective concentrations were found with the O-methylated catechins in PTK-inhibitory potency between when mast cells were incubated with the catechins (Fig. 4C) and when the purified PTKs were directly incubated with them (Fig. 5A). Thus, cell-free IC50 of 0.2–5 µg/ml to inhibit PTKs were reflected with in vitro IC50 of 30–100 µg/ml to inhibit histamine release, leukotriene release, and IL-2 production/secretion. This strongly suggests that these O-methylated catechins penetrate the mast cell plasma membrane with very poor efficiency. Alternatively, these catechins might lose their mast cell-inhibiting activity by being modified or degraded to inert molecules within or even outside mast cells. Indeed, O-methylated EGCGs can be epimerized, methylated, and conjugated with glucuronic acid and sulfate (M. Maeda-Yamamoto, unpublished data). Bioactivities of these EGCG derivatives, including effects on mast cell activation, remain to be explored. Importantly, in vivo IC50 of 50 mg/kg EGCG3''Me by i.v. administration to inhibit PCA reactions correlate reasonably well with the in vitro IC50 of 30 µg/ml to inhibit Lyn kinase activity. Thus, in our preliminary experiments, serum concentrations of 1–25 µg/ml were achieved by i.v. injection of 50 mg/kg EGCG3''Me into mice (data not shown). Therefore, the major target of the O-methylated catechins in the acute phase of in vivo allergic reactions was considered to be mast cells, although other cell types should be affected in slower aspects of allergic reactions.


   Acknowledgments
 
We thank Dr. Masaniko Ikeda for performing Ca influx experiments, Dr. Keiko Kawamoto for technical advice, and Nahomi Matsuda, Naoko Suzuki, Nobufumi Tahara, Masayoshi Yoshimura, and Makiko Suzuki for technical assistance. This is Publication 576 from the La Jolla Institute for Allergy and Immunology.


   Footnotes
 
1 This work was supported in part by a grant from the Program for the Promotion of Basic Research Activities for Innovative Biosciences, Bio-oriented Technology Research Advancement Institution, Japan (to M.M.-Y.) and National Institutes of Health Grant AI/GM38348 to T.K. Back

2 Address correspondence and reprint requests to Dr. Toshiaki Kawakami, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: toshi{at}liai.org Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; BMMC, bone marrow-derived mast cell; Btk, Bruton’s tyrosine kinase; EGCG, (-)-epigallocatechin-3-O-gallate; EGCG3''Me, (-)-epigallocatechin-3-O-(3-O-methyl) gallate; EGCG4''Me, (-)-epigallocatechin-3-O-(4-O-methyl) gallate; ERK, extracellular signal-regulated kinase; HSA, human serum albumin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; PCA, passive cutaneous anaphylaxis; PKC, protein kinase C; PLC, phospholipase C; PS/TK, protein serine/threonine kinase; PVDF, polyvinylidene difluoride. Back

Received for publication June 23, 2003. Accepted for publication January 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Galli, S. J., C. S. Lantz. 1999. Allergy. W. E. Paul, ed. Fundamental Immunology 1137. Lippincott-Raven Press, Philadelphia.
  2. Kinet, J. P.. 1999. The high-affinity IgE receptor (Fc{epsilon}RI): from physiology to pathology. Annu. Rev. Immunol. 17:931.[Medline]
  3. Blank, U., C. Ra, L. Miller, K. White, H. Metzger, J. P. Kinet. 1989. Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 337:187.[Medline]
  4. Eiseman, E., J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355:78.[Medline]
  5. Reth, M.. 1989. Antigen receptor tail clue. Nature 338:383.[Medline]
  6. Cambier, J. C.. 1995. Antigen and Fc receptor signaling: the awesome power of the immunoreceptor tyrosine-based activation motif (ITAM). J. Immunol. 155:3281.[Medline]
  7. Jouvin, M. H., M. Adamczewski, R. Numerof, O. Letourneur, A. Valle, J. P. Kinet. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269:5918.[Abstract/Free Full Text]
  8. Kihara, H., R. P. Siraganian. 1994. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. Chem. 269:22427.[Abstract/Free Full Text]
  9. Wilson, B. S., N. Kapp, R. J. Lee, J. R. Pfeiffer, A. M. Martinez, Y. Platt, F. Letourneur, J. M. Oliver. 1995. Distinct functions of the Fc{epsilon}R1 {gamma} and {beta} subunits in the control of Fc{epsilon}R1-mediated tyrosine kinase activation and signaling responses in RBL-2H3 mast cells. J. Biol. Chem. 270:4013.[Abstract/Free Full Text]
  10. Kawakami, Y., L. Yao, T. Miura, S. Tsukada, O. N. Witte, T. Kawakami. 1994. Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc{epsilon}RI cross-linking. Mol. Cell. Biol. 14:5108.[Abstract/Free Full Text]
  11. Rawlings, D. J., A. M. Scharenberg, H. Park, M. I. Wahl, S. Lin, R. M. Kato, A. C. Fluckiger, O. N. Witte, J. P. Kinet. 1996. Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science 271:822.[Abstract]
  12. Fluckiger, A. C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J. P. Kinet, O. N. Witte, A. M. Scharenberg, D. J. Rawlings. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J. 17:1973.[Medline]
  13. Scharenberg, A. M., O. El-Hillal, D. A. Fruman, L. O. Beitz, Z. Li, S. Lin, I. Gout, L. C. Cantley, D. J. Rawlings, J. P. Kinet. 1998. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals. EMBO J. 17:1961.[Medline]
  14. Tsai, M., R. H. Chen, S. Y. Tam, J. Blenis, S. J. Galli. 1993. Activation of MAP kinases, pp90rsk and pp70–S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or Fc{epsilon}RI: rapamycin inhibits activation of pp70–S6 kinase and proliferation in mouse mast cells. Eur. J. Immunol. 23:3286.[Medline]
  15. Offermanns, S., S. V. Jones, E. Bombien, G. Schultz. 1994. Stimulation of mitogen-activated protein kinase activity by different secretory stimuli in rat basophilic leukemia cells. J. Immunol. 152:250.[Abstract]
  16. Hirasawa, N., F. Santini, M. A. Beaven. 1995. Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line: indications of different pathways for release of arachidonic acid and secretory granules. J. Immunol. 154:5391.[Abstract]
  17. Kawakami, Y., T. Miura, R. Bissonnette, D. Hata, W. N. Khan, T. Kitamura, M. Maeda-Yamamoto, S. E. Hartman, L. Yao, F. W. Alt, T. Kawakami. 1997. Bruton’s tyrosine kinase regulates apoptosis and JNK/SAPK kinase activity. Proc. Natl. Acad. Sci. USA 94:3938.[Abstract/Free Full Text]
  18. Ishizuka, T., A. Oshiba, N. Sakata, N. Terada, G. L. Johnson, E. W. Gelfand. 1996. Aggregation of the Fc{epsilon}RI on mast cells stimulates c-Jun amino-terminal kinase activity: a response inhibited by wortmannin. J. Biol. Chem. 271:12762.[Abstract/Free Full Text]
  19. Kawakami, Y., J. Kitaura, S. E. Hartman, C. A. Lowell, R. P. Siraganian, T. Kawakami. 2000. Regulation of protein kinase C{beta}I by two protein-tyrosine kinases, Btk and Syk. Proc. Natl. Acad. Sci. USA 97:7423.[Abstract/Free Full Text]
  20. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, T. Kawakami. 2000. Akt-dependent cytokine production in mast cells. J. Exp. Med. 192:729.[Abstract/Free Full Text]
  21. Lambert, J. D., C. S. Yang. 2003. Cancer chemopreventive activity and bioavailability of tea and tea polyphenols. Mutat. Res. 523:201.
  22. Cao, Y., R. Cao. 1999. Angiogenesis inhibited by drinking tea. Nature 398:381.[Medline]
  23. Maeda-Yamamoto, M., H. Kawahara, N. Tahara, K. Tsuji, Y. Hara, M. Isemura. 1999. Effects of tea polyphenols on the invasion and matrix metalloproteinases activities of human fibrosarcoma HT1080 cells. J. Agric. Food Chem. 47:2350.[Medline]
  24. Maeda-Yamamoto, M., N. Suzuki, Y. Sawai, T. Miyase, M. Sano, A. Hashimoto-Ohta, M. Isemura. 2003. Association of suppression of extracellular signal-regulated kinase phosphorylation by epigallocatechin gallate with the reduction of matrix metalloproteinase activities in human fibrosarcoma HT1080 cells. J. Agric. Food Chem. 51:1858.[Medline]
  25. Isemura, M., Y. Suzuki, K. Satoh, K. Narumi, M. Motomiya. 1993. Effects of catechins on the mouse lung carcinoma cell adhesion to the endothelial cells. Cell Biol. Int. 17:559.[Medline]
  26. Sazuka, M., S. Murakami, M. Isemura, K. Satoh, T. Nukiwa. 1995. Inhibitory effects of green tea infusion on in vitro invasion and in vivo metastasis of mouse lung carcinoma cells. Cancer Lett. 98:27.[Medline]
  27. Okuda, T., Y. Kimura, T. Yoshida, T. Hatano, H. Okuda, S. Arichi. 1983. Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver. Chem. Pharm. Bull. 31:1625.
  28. Bors, W., M. Saran. 1987. Radical scavenging by flavonoid antioxidants. Free Radical Res. Commun. 2:289.[Medline]
  29. Hashimoto, F., M. Ono, C. Masuoka, Y. Ito, Y. Sakata, K. Shimizu, G. Nonaka, I. Nishioka, T. Nohara. 2003. Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols. Biosci. Biotechnol. Biochem. 67:396.[Medline]
  30. Yokozawa, T., H. Okura, S. Sakanaka, S. Ishigaki, M. Kim. 1994. Depressor effect of tannin in green tea on rats with renal hypertension. Biosci. Biotechnol. Biochem. 58:855.
  31. Chisaka, T., H. Matsuda, Y. Kubomura, M. Mochizuki, J. Yamahara, H. Fujimura. 1988. The effect of crude drugs on experimental hypercholesteremia: mode of action of (-)-epigallocatechin gallate in tea leaves. Chem. Pharm. Bull. 36:227.
  32. Murase, T., A. Nagasawa, J. Suzuki, T. Hase, I. Tokimitsu. 2002. Beneficial effects of tea catechins on diet-induced obesity: stimulation of lipid catabolism in the liver. Int. J. Obes. Relat. Metab. Disord. 26:1459.[Medline]
  33. Sakanaka, S., N. Shiumua, M. Masumi, M. M. Kim, T. Yamamoto. 1992. Preventive effect of green tea polyphenols against dental caries in conventional rats. Biosci. Biotechnol. Biochem. 56:592.
  34. Fukai, K., T. Ishigami, Y. Hara. 1991. Antibacterial activity of tea polyphenols against phytopathogenic bacteria. Agric. Biol. Chem. 55:1895.
  35. Okubo, T., N. Ishihara, A. Okura, M. Serit, M. M. Kim, T. Yamamoto, T. Mitsuoka. 1992. In vitro effects of tea polyphenols intake on human intestinal microflora and metabolism. Biosci. Biotechnol. Biochem. 56:588.
  36. Sano, M., M. Suzuki, T. Miyase, K. Yoshino, M. Maeda-Yamamoto. 1999. Novel antiallergic catechin derivatives isolated from oolong tea. J. Agric. Food Chem. 47:1906.[Medline]
  37. Suzuki, M., K. Yoshino, M. Maeda-Yamamoto, T. Miyase, M. Sano. 2000. Inhibitory effects of tea catechins and O-methylated derivatives of (-)-epigallocatechin-3-O-gallate on mouse type IV allergy. J. Agric. Food Chem. 48:5649.[Medline]
  38. Ohmori, Y., M. Ito, M. Kishi, H. Mizutani, T. Katada, H. Konishi. 1995. Antiallergic constituents from oolong tea stem. Biol. Pharm. Bull. 18:683.[Medline]
  39. Maeda-Yamamoto, M., H. Kawahara, N. Matsuda, K. Nesumi, M. Sano, K. Tsuji, Y. Kawakami, T. Kawakami. 1998. Effects of tea infusions of various varieties or different manufacturing types on inhibition of mouse mast cell activation. Biosci. Biotechnol. Biochem. 62:2277.[Medline]
  40. Fukamachi, H., N. Yamada, T. Miura, T. Kato, M. Ishikawa, E. Gulbins, A. Altman, Y. Kawakami, T. Kawakami. 1994. Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by Fc{epsilon}RI cross-linking. J. Immunol. 152:642.[Abstract]
  41. Han, W., L. Yao, T. Kawakami. 1998. Expression of Bruton’s tyrosine kinase in Sf9 insect cells. Chin. J. Biochem. Mol. Biol. 14:269.
  42. Hata, D., J. Kitaura, S. E. Hartman, Y. Kawakami, T. Yokota, T. Kawakami. 1998. Bruton’s tyrosine kinase-mediated interleukin-2 gene activation in mast cells: dependence on the c-Jun N-terminal kinase activation pathway. J. Biol. Chem. 273:10979.[Abstract/Free Full Text]
  43. Baus, E., J. Urbain, O. Leo, F. Andris. 1994. Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor. J. Immunol. Methods 173:41.[Medline]
  44. Hata, D., Y. Kawakami, N. Inagaki, C. S. Lantz, T. Kitamura, W. N. Khan, M. Maeda-Yamamoto, T. Miura, W. Han, S. E. Hartman, et al 1998. Involvement of Bruton’s tyrosine kinase in Fc{epsilon}RI-dependent mast cell degranulation and cytokine production. J. Exp. Med. 187:1235.[Abstract/Free Full Text]
  45. Matsuo, N., K. Yamada, K. Shoji, M. Mori, M. Sugano. 1997. Effect of tea polyphenols on histamine release from rat basophilic leukemia (RBL-2H3) cells: the structure-inhibitory activity relationship. Allergy 52:58.[Medline]
  46. Ozawa, K., Z. Szallasi, M. G. Kazanietz, P. M. Blumberg, H. Mischak, J. F. Mushinski, M. A. Beaven. 1993. Ca2+-dependent and Ca2+-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. 268:1749.[Abstract/Free Full Text]
  47. Ishizuka, T., N. Terada, P. Gerwins, E. Hamelmann, A. Oshiba, G. R. Fanger, G. L. Johnson, E. W. Gelfand. 1997. Mast cell tumor necrosis factor {alpha} production is regulated by MEK kinases. Proc. Natl. Acad. Sci. USA 94:6358.[Abstract/Free Full Text]
  48. Garrington, T. P., T. Ishizuka, P. J. Papst, K. Chayama, S. Webb, T. Yujiri, W. Sun, S. Sather, D. M. Russell, S. B. Gibson, et al 2000. MEKK2 gene disruption causes loss of cytokine production in response to IgE and c-Kit ligand stimulation of ES cell-derived mast cells. EMBO J. 19:5387.[Medline]
  49. Karin, M.. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 270:16483.[Free Full Text]
  50. Moriya, K., J. Rivera, S. Odom, Y. Sakuma, K. Muramato, T. Yoshiuchi, M. Miyamoto, K. Yamada. 1997. ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fc{epsilon} receptor I-mediated activation of Syk. Proc. Natl. Acad. Sci. USA 94:12539.[Abstract/Free Full Text]
  51. Kawakami, Y., S. E. Hartman, E. Kinoshita, H. Suzuki, J. Kitaura, L. Yao, N. Inagaki, A. Franco, D. Hata, M. Maeda-Yamamoto, et al 1999. Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase. Proc. Natl. Acad. Sci. USA 96:2227.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J Med MicrobiolHome page
I. Ishida, C. Kohda, Y. Yanagawa, H. Miyaoka, and T. Shimamura
Epigallocatechin gallate suppresses expression of receptor activator of NF-{kappa}B ligand (RANKL) in Staphylococcus aureus infection in osteoblast-like NRG cells
J. Med. Microbiol., August 1, 2007; 56(8): 1042 - 1046.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Nishimoto, S.-W. Lee, H. Hong, K. G. Potter, M. Maeda-Yamamoto, T. Kinoshita, Y. Kawakami, R. S. Mittler, B. S. Kwon, C. F. Ware, et al.
Costimulation of mast cells by 4-1BB, a member of the tumor necrosis factor receptor superfamily, with the high-affinity IgE receptor
Blood, December 15, 2005; 106(13): 4241 - 4248.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maeda-Yamamoto, M.
Right arrow Articles by Kawakami, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maeda-Yamamoto, M.
Right arrow Articles by Kawakami, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS