Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca(2+) mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)(2) rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca(2+) mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the gamma-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.     

High-affinity IgE receptor-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells induces early and late protein-tyrosine phosphorylations.

We reported previously that stimulation of RBL-2H3 cells through the high-affinity IgE receptor resulted in tyrosine phosphorylation of a 72-kDa protein (pp72) that was coupled to signal transduction. In the present study, although pp72 tyrosine phosphorylation was induced only by antigen triggering, stimulation of RBL-2H3 cells by either antigen or the calcium-ionophore A23187 led to increased tyrosine phosphorylation of a 110-kDa protein (pp110). This tyrosine phosphorylated protein was also observed when RBL-2H3 cells were transfected with the G protein-coupled m3 muscarinic receptor and then stimulated to secrete with carbachol. In contrast to tyrosine phosphorylation of pp72, antigen-induced pp110 tyrosine phosphorylation required extracellular calcium, was absent in cells depleted of protein kinase C, and was detected between 1 and 5 min after stimulation. The protein-tyrosine kinase inhibitor genistein blocked both histamine release and tyrosine phosphorylation induced by A23187. Altogether, the data suggest a role for pp110 in secretion. However, protein kinase C activation induced pp110 tyrosine phosphorylation but not histamine release demonstrating that pp110 tyrosine phosphorylation alone is not sufficient for degranulation. We conclude that tyrosine phosphorylation of pp72 is associated with the early steps of IgE receptor-generated signaling, whereas pp110 tyrosine phosphorylation occurs secondary to calcium influx and protein kinase C activation.     

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).     

Regulation of FcepsilonRI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells

Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma.     

Receptor-mediated release of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate in rat basophilic leukemia RBL-2H3 cells permeabilized with streptolysin O

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.     

Dexamethasone inhibits receptor-activated phosphoinositide breakdown in rat basophilic leukemia (RBL-2H3) cells

The activation of rat basophilic leukemia cells for histamine release is accompanied by Ca2+ influx and arachidonic acid release. IgE receptor but not A23187 ionophore stimulation of these cells also resulted in phosphoinositide breakdown. In these experiments, the culture of these cells with dexamethasone inhibited IgE- and ionophore-mediated histamine release. The concentration for 50% of maximal inhibition was 12 nM, and prolonged exposure to the drug was required, with maximal effect observed in 8 to 15 hr. The inhibitory effect of dexamethasone was reversible (t1/2 for recovery was 16 hr). Dexamethasone blocked the IgE-mediated 45Ca2+ influx and the release of [14C]-arachidonic acid (IC50 of 1 nM and 10 nM respectively). Dexamethasone inhibited the IgE receptor-mediated phosphoinositide breakdown (IC50 of 5 nM). It also decreased arachidonic acid release after A23187 stimulation demonstrating an effect on phospholipase A2. Therefore, exposure of the cells to dexamethasone results in the inhibition of both phospholipase A2 and phospholipase C pathways of arachidonic acid generation.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

Adherence of rat basophilic leukemia (RBL-2H3) cells to fibronectin-coated surfaces enhances secretion.

Rat basophilic leukemia (RBL-2H3) cells are a useful in vitro model for studies of mast cells and basophils. We examined the adherence of RBL-2H3 cells to different extracellular matrix proteins and the effect of such attachment on secretion. The cells bound to fibronectin-coated surfaces with maximum binding by 1 h at 37 degrees C. There was less attachment to laminin, collagen type I, and collagen type IV. There was no adherence to uncoated surfaces or in the absence of Ca2+. Binding to fibronectin was blocked by a synthetic peptide containing the sequence Arg-Gly-Asp. Therefore, the binding of RBL-2H3 cells to fibronectin may be mediated by surface molecules that belong to the integrin family. Adherence to fibronectin-coated surfaces resulted in cell spreading, a reorganization of the cytoskeletal elements, and a redistribution of the secretory granules. Attachment to fibronectin also dramatically enhanced high affinity IgE receptor-mediated histamine release. This enhancement was maximum by 1 h of adherence and lasted for at least 6 h. There was also enhanced secretion by the Ca2+ ionophore A23187. Thus, adherence to fibronectin can enhance both receptor and non-receptor-mediated release. Addition of soluble fibronectin to RBL-2H3 cells in suspension had no effect on secretion. Therefore, enhanced histamine release required cell attachment to immobilized fibronectin. These results suggest that secretion from mast cells/basophils may be modulated by their interaction with the extracellular matrix.     

IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.     

Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3).

Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL-2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells.     

Enhancement of the release of inflammatory mediators by substance P in rat basophilic leukemia RBL-2H3 cells

Summary Substance P (SP), a neurotransmitter, may play an important role in neurogenic inflammation. Ginseng has been used extensively in traditional medicine; however, few studies were focused on their anti-allergic effect. Therefore, the effect and mechanism of ginsenoside Rb1 on the SP enhancement of allergic mediators were explored. In this study, SP and dinitrophenyl-bovine serum albumin (DNP-BSA) were used to activate rat basophilic leukemia (RBL)-2H3 cells. The cultured supernatants were assayed for histamine, leukotriene C₄(LTC₄) and interleulin-4 (IL-4) production. The mitogen-activated protein kinases (MAPKs) signaling pathway was determined by Western blotting analysis. We found that IgE/DNP-BSA, SP, ginsenoside Rb1, or MAPK specific inhibitors had no effect on cell viability and cytotoxicity. SP (30 μM) alone, did not induce histamine and LTC₄ release, but it enhanced allergen-induced histamine and LTC₄ release. In␣addition, SP significantly induced and enhanced allergen-activated IL-4. Ginsenoside Rb1 dose-dependently inhibited these effects. SP enhanced the allergen-activated ERK pathway in RBL-2H3 cells, and Rb1 effectively inhibited the ERK pathway activation. Although MAPK specific inhibitors suppressed LTC₄ and IL-4, only U0126 inhibited the SP enhanced histamine release. These results demonstrate that Rb1 dose-dependently inhibited SP enhanced allergen-induced mediator release and its mechanism was through the inhibition of the ERK pathway.     

Simultaneous determination of intracellular calcium concentration and histamine secretion in rat basophilic leukemia cells (RBL-2H3).

In rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mast cells, the aggregation of IgE receptors initiates increase in the intracellular concentration of calcium ([Ca2+]i), monitored with the fluorescent Ca probe fura-2, and finally results in histamine secretion. In cell suspensions, however, the fluorescence gradually increases due to leakage and exocytosis of the dye. A superfusion system was developed to overcome these problems and [Ca2+]i was calculated from the ratio of fluorescence intensities at 505 nm of fura-2 excited at 340 and 380 nm. Histamine and beta-N-acetylglucosaminidase in granules are released during exocytosis, and both substances in the superfusates were determined simultaneously. This system is useful for studies on the relationships of cell stimulation, changes in second messengers, and final responses.     

Depletion of guanine nucleotides with mycophenolic acid suppresses IgE receptor-mediated degranulation in rat basophilic leukemia cells

In RBL-2H3 rat basophilic leukemia cells, Ag that crosslink IgE-receptor complexes stimulate the turnover of inositol phospholipids, the mobilization of Ca2+ from intra- and extracellular sources, the release of serotonin and other substances from granules and the transformation of the cell surface from a microvillous to a lamellar architecture. This study explores the role of GTP-binding proteins (G proteins) in the control of these biochemical and functional responses. We report that incubating RBL-2H3 cells for 4 h with 10 microM mycophenolic acid (MPA), an inhibitor of de novo GTP synthesis, reduces GTP levels by over 60% and causes an average reduction of 50% in Ag-stimulated serotonin release. This inhibition of secretion is associated with a 50% decrease in the rate of 45Ca2+ influx in MPA-treated cells. In contrast, Ag-stimulated inositol trisphosphate production is only slightly reduced, indicating that the phosphatidylinositol-specific phospholipase C can be activated by Ag in GTP-depleted cells. The membrane responses to IgE receptor cross-linking are unaffected by incubating cells with MPA. Exogenous guanine or guanosine protects the GTP pools in MPA-treated cells and permits normal ion transport and secretory responses to Ag; adenine does not. These results implicate a guanine nucleotide-binding protein in the control of IgE receptor-dependent signal transduction in RBL-2H3 cells. This protein may particularly control the Ca2+ influx pathway that is essential for secretion.     

Fc epsilon RI-induced protein tyrosine phosphorylation of pp72 in rat basophilic leukemia cells (RBL-2H3). Evidence for a novel signal transduction pathway unrelated to G protein activation and phosphatidylinositol hydrolysis.

Recently, we demonstrated that aggregation of the high affinity IgE receptor in rat basophilic leukemia (RBL-2H3) cells results in rapid tyrosine phosphorylation of a 72-kDa protein (pp72). Here we investigated the relationship of pp72 phosphorylation to guanine nucleotide-binding protein (G protein) activation and phosphatidylinositol hydrolysis. The activation of G proteins by NaF in intact cells or by guanosine 5'-O-(3-thiotriphosphate) in streptolysin O-permeabilized cells induced both phosphatidylinositol hydrolysis and histamine release without tyrosine phosphorylation of pp72. Similarly, in RBL-2H3 cells expressing the G protein-coupled muscarinic acetylcholine receptor, carbachol activated phospholipase C and induced secretion without concomitant pp72 phosphorylation. Therefore, pp72 phosphorylation was not induced by G protein activation or as a consequence of phosphatidylinositol hydrolysis. To investigate whether pp72 tyrosine phosphorylation precedes the activation of phospholipase C, we studied the effect of the tyrosine kinase inhibitor genistein. Preincubation of cells with genistein decreased, in parallel, antigen-induced tyrosine phosphorylation of pp72 (IC50 = 34 micrograms/ml) and histamine release (IC50 = 31 micrograms/ml). However, genistein at concentrations of up to 60 micrograms/ml did not inhibit phosphatidylinositol hydrolysis nor did it change the amount of the secondary messenger inositol (1,4,5)-triphosphate. Previous observations showed that there was no pp72 tyrosine phosphorylation after activation of protein kinase C or after an increase in intracellular calcium. Taken together, these results suggest that pp72 tyrosine phosphorylation represents a distinct, independent signaling pathway induced specifically by aggregation of the Fc epsilon RI.     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells.

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.     

IgE alone-induced actin assembly modifies calcium signaling and degranulation in RBL-2H3 mast cells

In the mast cell signaling pathways, the binding of immunoglobulin E (IgE) to FcRI, its high-affinity receptor, is generally thought to be a passive step. In this study, we examined the effect of IgE alone, that is, without antigen stimulation, on the degranulation in mast cells. Monomeric IgE (500–5,000 ng/ml) alone increased cytosolic Ca²⁺ level ([Ca²⁺]_i) and induced degranulation in rat basophilic leukemia (RBL)-2H3 mast cells. Monomeric IgE (5,000 ng/ml) alone also increased [Ca²⁺]_i and induced degranulation in bone marrow-derived mast cells. Interestingly, monomeric IgE (5–50 ng/ml) alone, in concentrations too low to induce degranulation, increased filamentous actin content in RBL-2H3 mast cells. We next examined whether actin dynamics affect the IgE alone-induced RBL-2H3 mast cell activation pathways. Cytochalasin D inhibited the ability of IgE alone (50 ng/ml) to induce de novo actin assembly. In cytochalasin D-treated cells, IgE (50 ng/ml) alone increased [Ca²⁺]_i and induced degranulation. We have summarized the current findings into two points. First, IgE alone increases [Ca²⁺]_i and induces degranulation in mast cells. Second, IgE, at concentrations too low to increase either [Ca²⁺]_i or degranulation, significantly induces actin assembly, which serves as a negative feedback control in the mast cell Ca²⁺ signaling and degranulation. mast cell; immunoglobulin E; cytochalasin D; Y-27632; wortmannin     

Arachidonic acid metabolism in rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells metabolized arachidonic acid through more than one enzymatic pathway. The major cyclooxygenase product was prostaglandin (PG) D₂ as established by chromatographic and chemical behavior and the effect on platelet aggregation. PGD₂ formation from exogenous arachidonic acid was inhibited by indomethacin, 1 μg/ml. RBL-1 incubated with exogenous arachidonic acid also formed SRS-A the synthesis of which was not inhibited by indomethacin. However, the SRS-A activity was blocked by the specific receptor antagonist FPL 55712. [¹⁴C]arachidonic acid was effectively incorporated into the phospholipids of RBL-1 cells. Challenge of such prelabelled cells or unlabelled cells with A 23187 caused release of PGD₂, SRS-A and another presently unidentified product. However, with A 23187 as a stimulus, the RBL-1 cyclo-oxygenase could not be blocked by low concentrations of indomethacin. This work further substantiates our earlier findings that SRS-A formed from arachidontic acid is not a cyclooxegenase product.     

Inhibitory effects of flavonoids isolated from Fragaria ananassa Duch on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3

We isolated the 4 kinds of flavonoids from strawberry ‘Nohime’ and examined the effect of these flavonoids on the degranulation in RBL-2H3 cells. The flavonoids were found to suppress the degranulation from Ag-stimulated RBL-2H3 cells to different extents. To disclose the inhibitory mechanism of degranulation by flavonoids, we examined their effects on the intracellular free Ca²⁺ concentration ([Ca²⁺]i) and the intracellular signaling pathway such as Lyn, Syk, and PLCγs. The intracellular free Ca²⁺ concentration ([Ca²⁺]i) was elevated by FcεRI activation, but these flavonoid treatments reduced the elevation of [Ca²⁺]i by suppressing Ca²⁺ influx. Kaempferol strongly suppressed the activation of Syk and PLCγs. It was thus suggested that suppression of Ag-stimulated degranulation by the flavonoids is mainly due to suppression of [Ca²⁺]i elevation and Syk activation. These results suggested that strawberry would be of some ameliorative benefit for the allergic symptoms.