Diphenyleneiodonium prevents reactive oxygen species generation, tyrosine phosphorylation, and histamine release in RBL-2H3 mast cells

Mast cells play a central role in immediate allergic reactions mediated by immunoglobulin E. It has recently been reported that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. However, the physiological role of ROS is poorly understood. Here we demonstrate that mast cell model rat basophilic leukemia (RBL-2H3) cells generate ROS in response to antigen and the calcium-ionophore A23187 via activation of diphenyleneiodonuim (DPI)-sensitive enzyme and that blockade of ROS generation by DPI suppresses histamine release induced by either stimulant. Increased tyrosine phosphorylation of pp125(FAK) and a 77-kDa protein coprecipitating specifically with the kinase occurred in parallel with the secretion, and blockade of ROS generation by DPI also suppressed the tyrosine phosphorylation of both proteins. These findings suggest that ROS generated by a flavoenzyme-dependent mechanism may be involved in histamine release through the pp125(FAK) pathway.     

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).     

Real-time monitoring of histamine released from rat basophilic leukemia (RBL-2H3) cells with a histamine microsensor using recombinant histamine oxidase

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.     

Positive regulation of c-Jun N-terminal kinase and TNF-alpha production but not histamine release by SHP-1 in RBL-2H3 mast cells

The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcepsilonRI aggregation-induced tyrosine phosphorylation of the beta and gamma subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcepsilonRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-alpha mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.     

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).     

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.     

Involvement of Rab27 in antigen-induced histamine release from rat basophilic leukemia 2H3 cells

The Rab family small G proteins regulate discrete steps in vesicular transport pathways. Recent studies indicate that one member of the Rab family, Rab27A, regulates the transport of lysosome-related organelles, such as melanosome distribution in melanocytes, lytic granule release in cytotoxic T cells, and dense granule release in platelets. Here, we have examined the involvement of Rab27A in the exocytic transport of another lysosome-related organelle, the basophilic secretory granule, in basophils. We have found that Rab27A locates on basophilic secretory granules containing histamine in rat basophilic leukemia (RBL) 2H3 cells. In addition, exogenous expression of dominant active Rab27A reduces antigen-induced histamine release from the cells. We have moreover identified Munc13-4 as a Rab27A target using a CytoTrap system and found that exogenous expression of Munc13-4 affects antigen-induced histamine release from RBL-2H3 cells. These results demonstrate that Rab27A plays a crucial role in antigen-induced histamine release from RBL-2H3 cells.     

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.     

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.     

Inhibitory effect of fulvic acid extracted from Canadian sphagnum peat on chemical mediator release by RBL-2H3 and KU812 cells

Fulvic acid (FA) was extracted and purified from Canadian Sphagnum peat (CP-FA) and characterized by using an element analysis meter, Fourier transform infrared (FT-IR) spectroscopy, electron spin resonance (ESR) spectroscopy, and (13)C-nuclear magnetic resonance ((13)C-NMR) spectroscopy. To investigate the antiallergic effect of CP-FA, we incubated rat basophilic leukemia (RBL-2H3) cells with 0.001-10.0 microg/ml of CP-FA and determined the beta-hexosaminidase release inhibition at different response stages. The intracellular calcium [Ca(2+)](i) level was also determined by using Fluo 3-AM, a calcium-specific fluorescent probe, and the cytotoxicity of CP-FA was determined by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The results revealed that RBL-2H3 cells incubated for 48 h with 0.001-10.0 microg/ml of CP-FA did not show any decreased viability. CP-FA inhibited the beta-hexosaminidase release by IgE-sensitized, antigen-stimulated RBL-2H3 cells at the antigen-antibody binding stage and the antibody-receptor binding stage. CP-FA also inhibited histamine release from A23187 plus PMA- or compound 48/80-stimulated KU812 cells. Furthermore, there was a decrease in the intracellular [Ca(2+)](i) level in IgE-sensitized cells incubated with CP-FA and stimulated with antigen. Our results show that CP-FA may be useful for the treatment or prevention of allergic diseases.     

Effect of interleukin-1 receptor antagonist (IL-1RA) on histamine and serotonin release by rat basophilic leukemia cells (RBL-2H3) and peritoneal mast cells

Recently, it has been appreciated that cultured mast cells are significant sources of cytokines. However, the role of interkeukin-1 (IL-1) on mast cells and/or basophil degranulation is still unclear. In this report we provide evidence that rat basophilic leukemia cells (RBLC) cultured with a natural inhibitor of IL-1, interleukin-1 receptor antagonist (IL-1RA) (500 ng/ml) for 48 h, strongly inhibited the spontaneous release of serotonin (5HT) and histamine (from 22.50 to 43.49%), compared to untreated cells (control). When IL-1RA-treated and untreated RBLC were stimulated with a secretagogue (anti-IgE), no difference was found in the percent of 5HT and histamine release. Moreover, in another set of experiments using rat peritoneal mast cells (RPMC) treated and untreated with IL-1RA, we found that IL-1RA did not affect the release of 5HT or histamine, even when the secretagogue anti-IgE or compound 48/80 (C48/80) were used. The present studies describe an additional biological activity of IL-1RA, inhibiting histamine and 5HT release from RBLC cultures.Abbreviations IL-1 interleukin-1 - RA receptor antagonist - 5HT serotonin - RBLC rat basophilic leukemia cells - RPMC rat peritoneal mast cells - IgE immunoglobulin E - Fc immunoglobulin E receptor - CPM counts per minute - BSA bovine serum albumin - C48/80 compound 48/80 - TNF tumor necrosis factor     

The mechanism of [3H]noradrenaline release by histamine and its analogs from the rat vas deferens

In this study the mechanism by which histamine and H1 and H2 agonists evoked an overflow of radioactivity from rat vasa deferentia preloaded with [3H]noradrenaline was investigated. The overflow evoked by the various agonists was unaffected by the presence of such receptor antagonists as propranolol, phentolamine, cimetidine, or scopolamine. On the other hand, the overflow evoked by all agonists except dimaprit was inhibited by mepyramine and by two well-known neuronal uptake inhibitors, cocaine and desipramine. The inhibition by mepyramine has been attributed to its effect on the neuronal uptake process. Metabolic profile studies showed that 3,4-dihydroxyphenylglycol (DOPEG) was the major constituent in the evoked overflow caused by histamine, 2-methylhistamine, 4-methylhistamine, and dimaprit and that the overflow evoked by 2-pyridylethylamine and 2-thiazolylethylamine consisted predominantly of unchanged noradrenaline. Based on these findings, it is concluded that all of the agonists tested evoke noradrenaline release intraneuronally by entering the adrenergic nerve terminals. While dimaprit might enter by passively diffusing into the adrenergic nerves, other agonists seem to use the neuronal uptake process. Noradrenaline released intraneuronally is subsequently degraded by neuronal monoamine oxidase to form DOPEG. However, there are qualitative and quantitative differences in the metabolic profile of the overflow evoked by various agonists. It is suggested that these differences could arise from their additional properties, such as their effect on the neuronal uptake process and (or) their ability to act as substrate for neuronal monoamine oxidase.     

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells

Antigen-mediated activation of mast cells results in Ca²⁺-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca²⁺ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response .     

The control of mediator release from RBL-2H3 cells: roles for Ca2+, Na+, and protein kinase C1

Antigen-stimulated rat basophilic leukemia (RBL-2H3) cells release serotonin and other inflammatory mediators by a process that requires Ca2+ influx and increased cytoplasmic Ca2+ levels, and is mimicked by Ca2+ ionophores. We report here that the Ca2+ response to antigen and to ionomycin has two components, a Ca2+ spike and a Ca2+ plateau. In nominally Ca2+-free medium, both components of the Ca2+ response are inhibited and secretion does not occur. In Na+-free medium, the initial Ca2+ spike induced by antigen or ionomycin occurs, but the plateau is again absent and secretion is inhibited by 30 to 50%. Secretion is also reduced by 10(-4) M amiloride, an inhibitor of Na+ transport pathways, and by 10(-5) M concentrations of two amiloride analogs with greater activity than amiloride, respectively, against Na+ channels and Na+/Ca2+ exchange. Phorbol esters, which stimulate protein kinase C, enhance the Ca2+ plateau and secretion caused by suboptimal amounts of both antigen and ionomycin; this enhancement depends on extracellular Na+. The Na+ ionophore, monensin, mimics the Ca2+ plateau. From these data, we infer that the Ca2+ spike and plateau reflect separate responses of RBL-2H3 cells to antigen or ionomycin. We propose that the Ca2+ plateau results at least in part from the activation of a Na+-dependent Ca2+ influx pathway. One possible mechanism is that antigen binding stimulates a protein kinase C-regulated Na+ transport system. The resulting influx of Na+ may activate a Na+/Ca2+ antiporter that supports the Ca2+ plateau and mediator release.     

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.     

Coumarin and flavone derivatives from estragon and thyme as inhibitors of chemical mediator release from RBL-2H3 Cells

Estragon and thyme extracts showed potent inhibitory activities against chemical mediator release from rat basophilic leukemia RBL-2H3 cells. 7-Methoxycoumarin was isolated from estragon, and 5,4'-dihydroxy-6,7,3'-trimethoxyflavone, 5,4'-dihydroxy-6,7,8,3'-tetramethoxyflavone, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone and luteolin were isolated from thyme as active components. Structure-activity relationship studies among the active isolates and their related compounds indicated that the oxygen-containing functional group at the 7-position of the coumarin structure was advantageous for the inhibitory activity and that methylation of the hydroxyl group at the 4'-position of the flavone structure was disadvantageous. It was also found that coumarin derivatives inhibited an earlier step than intracellular calcium release and proteinkinase C activation, while flavones inhibited a later step or both earlier and later steps.     

The mechanism of the calcium signal and correlation with histamine release in 2H3 cells

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.     

Ionophore A-23187 induced histamine release from rat mast cells and rat basophil leukemia (RBL-1) cells.

Ionophore A-23187 releases histamine from normal mast cells apparently by promoting Ca++ influx (Foreman et al, Nature 245: 249, 1973). In our hands at concentrations of greater than 0.2 mug/ml release occurs in 1 to 2 min, is blocked by metabolic inhibitors, and is unaccompanied by cytotoxicity (trypan-blue uptake, lactic dehydrogenase (LDH) release). At higher doses (0.5 mug/ml) histamine release is followed by significant cytotoxicity, but again Ca++ is required. In parallel studies, we examined cultured rat basophilic leukemia (RBL-1) cells. These cells, which apparently have normal surface receptors for IgE, contained approximately 700 ng histamine/10(6) cells but did not release histamine when IgE-mediated release was looked for. They do not respond to doses of ionophore which would be expected to give non-cytotoxic histamine release. At higher doses histamine release is preceded by progressive LDH release: LDH release is 75% complete at 5 min whereas 10 min are required for 75% maximal histamine release. This reaction requires Ca++ and is temperature dependent but is not inhibited by metabolic poisons (2-deoxyglucose, dinitrophenol, CN-). These studies suggest that either Ca++ does not enter into these cells normally or that one or more mechanisms which are ordinarily triggered by the changes in Ca++ flow are unresponsive in the RBL-1 cells. These studies also underline the importance of ruling out cytotoxicity in ionophore-induced phenomena.