Involvement of hrs binding protein in IgE receptor-triggered exocytosis in RBL-2H3 mast cells

Hrs binding protein (Hbp) tightly associated with Hrs is thought to play a regulatory role in vesicular trafficking during endocytosis and exocytosis. In this study, we have expressed dominant-negative mutants of Hbp to evaluate their effects on the degranulation of secretory granules in RBL-2H3 mast cells. The dominant-negative mutants of Hbp significantly inhibited IgE receptor (FcepsilonRI)-triggered secretory response as tested by beta-hexosaminidase release. These results suggest that Hbp functions as a regulator in the FcepsilonRI-triggered degranulation of secretory granules in mast cells.     

Calmodulin spatial dynamics in RBL-2H3 mast cells

A line of rat basophilic leukaemia (RBL) cells, a model of mast cells, stably expressing EGFP-tagged calmodulin secreted normally in response to standard agonists. As reported for other cell types, calmodulin was concentrated in the mitotic spindle poles of dividing cells. In unstimulated interphase cells calmodulin was concentrated in the cell cortex and at a single central location. Disruption of cortical actin eliminated the concentration of calmodulin at the cortex while the central calmodulin concentration was associated with an enrichment of tubulin and is likely to represent the centrosome. Following stimulation with either an agonist that crosslinks Fc receptors or co-application of phorbol ester and a calcium ionophore the interior of the cells lost calmodulin while cortical fluorescence became more pronounced but also less uniform. After stimulation discrete bright puncta of calmodulin-EGFP (CaM-EGFP) appeared in the cell interior. Puncta colocalised with moving lysotracker-labelled granules, suggesting that calmodulin may play a role in organising their transport. Our results show that in interphase RBL cells a large fraction of the calmodulin pool is associated with targets in the actin cytoskeleton and demonstrate the utility of this model system for studying calmodulin biology.     

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).     

Calcium-dependent threonine phosphorylation of nonmuscle myosin in stimulated RBL-2H3 mast cells

Stimulation of RBL-2H3 m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by calcium/calmodulin-dependent protein kinase II (CaM kinase II), activated by increased intracellular calcium. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region, and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA carboxyl terminus was phosphorylated by CaM kinase II in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans Thr-1940 is replaced by Ala, and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. Similarly, co-transfected Ala --> Thr-1940 human NMHC-IIA was phosphorylated by activated CaM kinase II in HeLa cells, while wild type was not. In RBL-2H3 m1 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation, and inhibited release of the secretory granule marker hexosaminidase in response to carbachol but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3 m1 cell activation.     

Involvement of ELKS, an active zone protein, in exocytotic release from RBL-2H3 cells

Recent studies have indicated that SNARE proteins and their accessory proteins are involved in exocytotic release in mast cells and neurotransmitter release in neuronal cells. These data suggest that a similar molecular mechanism operates in both systems. However, mast and neuronal cells are structurally very different; an active zone is found in neuronal cells. In the present study, we examined the involvement of active zone proteins during exocytosis in mast cells. We found that several active zone proteins are expressed in RBL-2H3 cells and focused on one of those proteins called ELKS. Overexpression and knockdown of ELKS caused an increase and decrease in exocytotic activity, respectively. Immunocytochemical analysis and live imaging of the expression of YFP-conjugated ELKS showed that ELKS was translocated to the plasma membrane after antigen stimulation. These results suggest that ELKS positively regulates exocytotic release in RBL-2H3 by acting on the plasma membrane upon stimulation.     

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.     

Relationship of IgE receptor topography to secretion in RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking IgE-receptor complexes with anti-IgE antibody leads to degranulation. Receptor cross-linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti-IgE with 15 nm protein A-gold particles that are visible by back-scattered electron imaging in the scanning electron microscope. We report that anti-IgE binding stimulates the redistribution of IgE-receptor complexes at 37 degrees C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross-linked receptors. Cells incubated with 1 microgram/ml anti-IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 micrograms/ml anti-IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti-IgE concentrations, to reduce cross-linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F-actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton-membrane interaction is implicated in the formation or stabilization of the large receptor clusters.     

2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.     

Role of Ca(2+) on TNF-alpha and IL-6 secretion from RBL-2H3 mast cells

Ca2+ acts as an important second messenger in mast cells. However, the mechanisms involved in the secretion of inflammatory cytokines from activated mast cells are unknown. In this study, we examined the signaling pathway involved in calcium-related cytokine secretion in a mast cell line, RBL-2H3 cells. We report that treatment with 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a chelator of intracellular calcium, can inhibit IgE-stimulated TNF-alpha and IL-6 secretion in a concentration-dependent manner with IC50 values of 0.41 and 0.014 microM, respectively. Maximal inhibition of TNFalpha- and IL-6 secretion was 58.5 +/- 3% and 87 +/- 8% in BAPTA-AM, respectively. BAPTA-AM also completely inhibited the IgE-induced TNF-alpha and IL-6 mRNA levels. In activated RBL-2H3 cells, the expression level of NF-kappaB/Rel A protein increased in the nucleus. However, the level of NF-kappaB/Rel A in nucleus was decreased by treatment of BAPTA-AM. In addition, BAPTA-AM completely inhibited the IgE-induced IkappaB kinase beta (IKKbeta) activation and IkappaBalpha phosphorylation. These observations demonstrate that the intracellular Ca2+ may play an important role in IgE-induced TNF-alpha and IL-6 secretion from mast cells via IKKbeta activation.     

Impaired secretion and increased insolubilization of IgE-receptor complexes in mycophenolic acid-treated (guanine nucleotide-depleted) RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking anti-DNP IgE-receptor complexes with multivalent antigen (DNP-BSA) activates a signal transduction pathway leading to Ca2+ influx and secretion. Cross-linking IgE-receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors; this pathway becomes important at high concentrations of cross-linking antigen. Recent evidence that antigen-induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP-binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross-linked receptors to a detergent-insoluble (cytoskeleton-associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 micrograms/ml DNP-BSA) is only inhibited by about 25% in guanine nucleotide-depleted cells, whereas secretion elicited by 5 micrograms/ml DNP-BSA, a concentration in the range that causes the high-dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE-receptor complexes are insolubilized in response to 5 but not 0.05 micrograms/ml DNP-BSA in both control and guanine nucleotide-depleted cells. Importantly, the extent of insolubilization elicited by 5 micrograms/ml DNP-BSA is increased by more than 60% in the guanine nucleotide-depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein-coupled signaling pathway and by increasing the availability of receptors to the pathway leading to receptor insolubilization and inactivation.     

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     

Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells

We have previously shown that store-associated microdomains of high Ca(2+) are not essential for exocytosis in RBL-2H3 mucosal mast cells. We have now examined whether Ca(2+) microdomains near the plasma membrane are required, by comparing the secretory responses seen when Ca(2+) influx was elicited by two very different mechanisms. In the first, antigen was used to activate the Ca(2+) release-activated Ca(2+) (CRAC) current (I(CRAC)) through CRAC channels. In the second, a Ca(2+) ionophore was used to transport Ca(2+) randomly across the plasma membrane. Since store depletion by Ca(2+) ionophore will also activate I(CRAC), different means of inhibiting I(CRAC) before ionophore addition were used. Ca(2+) responses and secretion in individual cells were compared using simultaneous indo-1 microfluorometry and constant potential amperometry. Secretion still takes place when the increase in intracellular Ca(2+) occurs diffusely via the Ca(2+) ionophore, and at an average intracellular Ca(2)+ concentration that is no greater than that observed when Ca(2+) entry via CRAC channels triggers secretion. Our results suggest that microdomains of high Ca(2+) near the plasma membrane, or associated with mitochondria or Ca(2+) stores, are not required for secretion. Therefore, we conclude that modest global increases in intracellular Ca(2+) are sufficient for exocytosis in these nonexcitable cells.     

In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells

A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.     

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.     

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.     

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.     

Rat basophilic leukemia cells (RBL-2H3) generate prostaglandin D2 (PGD2) after regulated upon activation, normal T-cell expressed and secreted (RANTES) activation

Increasing evidence indicates that local neurogenic inflammation, possibly in response to different stimuli, may be involved in sensory nerve sensitization, migraine generation and some other precipitating events leading to neuronal dysfunction in the brain. In addition, mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. Regulated upon activation, normal T-cell expressed and secreted (RANTES) is a small protein and a prototype member of the CC chemokine-beta subfamily with chemoattractant and inflammatory properties. In this study we used the RBL-2H3 cell line to determine whether or not these cells generate prostaglandin D2 (PGD2) after treatment with RANTES. After 4 hours of incubation, RBL-2H3 cells cultured with RANTES at 20 ng/mL released large amounts of PGD2 in a dose-response manner compared to control. Moreover, RBL-treated RANTES generated a large quantity of histamine. Our study confirms once again the proinflammatory action of RANTES, in this case acting on the stimulation of the arachidonic acid cascade product PGD2.     

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcRI, LAT and α-galactosyl derivatives of ganglioside GD_1b mobilized to lipid raft domains following FcRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of β-hexosaminidase activity after FcRI activation. The two mutant cell lines have a reduced release of β-hexosaminidase activity after FcRI stimulation, but not after exposure to calcium ionophore. These results indicate that the α-galactosyl derivatives of ganglioside GD_1b are important in the initial events of FcRI signaling upstream of Ca²⁺ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcRI.