Upregulation of heme oxygenase-1 by degranulation in rat basophilic leukemia cells

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.     

The phenoxazine derivative Phx-1 suppresses IgE-mediated degranulation in rat basophilic leukemia RBL-2H3 cells

Antigen-induced aggregation of the high affinity IgE receptor (FcepsilonRI) on mast cells induces degranulation to release chemical mediators, leading to acute allergic inflammation. We have demonstrated that the treatment of rat mast cells, RBL-2H3, with a phenoxazine derivative Phx-1 (2-amino-4,4alpha-dihydro-4alpha,7-dimethyl-3H-phenoxazine-3-one) suppresses the antigen-induced degranulation. Biochemical analysis reveals that the complementary signaling pathway through Gab2 and Akt is inhibited by this compound in mast cells. These findings suggest that phenoxazine derivatives may have a therapeutic potential for allergic diseases by inhibiting mast cell degranulation.     

Differential regulation of IL-4 expression and degranulation by anti-allergic olopatadine in rat basophilic leukemia (RBL-2H3) cells

Olopatadine hydrochloride (olopatadine) is an anti-allergic drug that functions as a histamine H(1) antagonist and inhibits both mast cell degranulation and the release of arachidonic acid metabolites in various types of cells. In this study, we examined the ability of olopatadine to inhibit the expression of cytokine genes in vitro via high-affinity receptors for immunoglobulin E in mast cells, using a rat basophilic leukemia (RBL-2H3) cell line and an in vivo mouse model. Levels of gene expression in RBL-2H3 cells were determined by semi-quantitative RT-PCR, and serum interleukin-4 (IL-4) level in mice was quantified by ELISA. Olopatadine inhibited significantly the induction of IL-4 expression by mast cells both in vivo and in vitro. Olopatadine inhibited Ca(2+) influx through receptor-operated channels (ROC) without affecting Ca(2+) release from intracellular stores. Comparative analysis of olopatadine with other anti-allergic drugs and the ROC blocker SKF-96365 demonstrated that the potency of inhibition of Ca(2+) influx correlated with the degree of suppression of degranulation and arachidonic acid release. Inhibition of Ca(2+) influx decreased phosphorylation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase, which participate in regulation of cytokine (e.g. IL-4) gene expression. However, the rank order of inhibition of Ca(2+) influx did not correspond to reduction of IL-4 expression, suggesting that an unknown mechanism(s) of action, in addition to inhibition of Ca(2+) influx, is involved in the expression of cytokines in mast cells.     

Anti-allergic properties of cyclosporin A: inhibition of mediator release from human basophils and rat basophilic leukemia cells (RBL-2H3).

In vitro the immunosuppressive drug cyclosporin A (CS-A) strongly inhibited histamine release from human basophils (HB) and the rat basophilic leukemia cell line (RBL) 2H3. It also inhibited leukotriene release from HB. In HB the IC50 values for inhibition of histamine release induced by Con A, anti-IgE, calcium ionophore A23187 and antigen (mite) were 0.03, 0.12, 0.36 and 2.0 microM, respectively. In fact, these figures underestimate the potency of CS-A, since studies with 3H-CS-A showed substantial adsorption to plastic experimental wares which was inversely proportional to drug concentration. With anti-IgE and A23187, the drug acted promptly when added at the same time as the inducers but, with antigen, inhibition increased with time of pre-incubation. Washing of HB after pre-incubation with CS-A did not remove the drug effect. Inhibition of histamine release was abolished by Ca2+ excess (5 mM). For TPA-induced release, the drug inhibited the Ca2(+)-dependent but not the Ca2(+)-independent component. In Ca2(+)-free conditions, ionophore A23187, which caused little or no histamine release on its own, was able to synergize with TPA in causing release, apparently by mobilizing intracellular Ca2+. CS-A blocked the synergism but not the original TPA effect. CS-A was compared with the calmodulin inhibitors, W7, TFP and ABCNS; all inhibited histamine release. CS-A also potently inhibited IgE-mediated histamine release from RBL-2H3 cells, without affecting their growth or viability.     

Leukotriene D4-induced activation of protein kinase C in rat basophilic leukemia cells

We studied the subcellular distribution of protein kinase C (PKC) in the particulate and cytosolic fractions of rat basophilic leukemia (RBL-1) cells treated with leukotriene D4 (LTD4) and compared these results with those of phorbol myristate acetate (PMA). Consistent with the earlier reports, treatment of RBL-1 cells with PMA resulted in a time- and dose-dependent translocation of PKC from cytosolic to the particulate fractions, sustained for at least 10 min. When RBL-1 cells were treated with LTD4, a small, transient decrease in PKC activity in cytosolic fraction was observed within 7.5 s after LTD4 treatment. This was accompanied by a significant increase of PKC in the particulate fraction. However, at 15 and 30 s, both particulate and cytosolic PKC activities were increased with LTD4 treatment. The activation induced by LTD4 was dose- and time-dependent with maximal effects occurring within 30 s, declining at the later time points. Pretreatment of the cells with 2(R)-hydroxy-3(S)-carboxyethylthio-3-[2-(8-phenyloctyl,pheny l]propanoic acid (SK&F 104353), a high affinity specific LTD4 receptor antagonist, and also with staurosporine, a potent inhibitor of PKC, completely inhibited the LTD4-induced activation of PKC both in the particulate and cytosolic fractions. These results suggest that activation of PKC by LTD4 is different from that elicited by PMA. The ability of SK&F 104353 to block LTD4-induced activation of PKC further suggests that stimulation of PKC might be an important intermediate step in the signal transduction mechanism of the LTD4 receptor in RBL-1 cells.     

Interaction of monoclonal antibodies with the IgE-receptor on rat mast cells and rat basophilic leukemia (RBL) cells

The close relation between rat mast cells and rat basophilic leukemia (RBL) cells with regard to the presence of receptors for IgE and Fc gamma led us to generate monoclonal antibodies directed against cell surface antigens. Hybridomas were obtained by the fusion of NS1 mouse myeloma cells with murine spleen and lymph node cells. The culture supernatants were assayed by two ELISA techniques: a) for the production of mouse immunoglobulin in general and b) for antibodies directed against surface antigens of RBL cells. For this purpose RBL cells were attached to polyvinyl chloride microtitre plates. Eight hybrids produced antibodies directed against surface antigens on RBL cells. Hybrids were cloned and characterized with regard to their isotype and light chains. All eight clones secreted IgM with K light chains. Immunofluorescence studies performed with RBL cells revealed that all eight antibodies were able to show a specific fluorescence. Furthermore, four of these eight antibodies also showed a specific fluorescence with purified rat mast cells. These four antibodies were analyzed as to their ability of interacting with the IgE-receptor on RBL cells and purified rat mast cells. They reduced the binding rate of radiolabelled rat IgE to RBL and rat mast cells. A mutual inhibition of the passive cutaneous anaphylaxis (PCA) reaction in the rat by either mixing mouse reaginic serum directed against 2,4-dinitrophenol bovine serum albumin (DNP-BSA) or by mixing monoclonal mouse anti-DNP IgE with the monoclonal mouse anti-cell surface (rat basophilic leukemia, rat mast cell) IgM was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspirin induces the production of the inflammatory mediator 8-epi-PGF in mast cells

Recently it has been shown that mast cells, through release of their pro-inflammatory mediators, are involved in aspirin attacks in aspirin-sensitive patients. To date, little information is available concerning 8-isoprostane (8-epi-prostaglandin F) production by mast cells. Therefore, we examined whether exposure of mast cells to aspirin can lead to isoprostane production. In this study we show that in mast cells, IgE and antigen stimulates an intracellular oxidative burst inducing H(2)O(2) and 8-epi-PGF production. Moreover, we show that exposure of mast cells to aspirin directly induces the production of 8-epi-PGF. Our study suggests that production of 8-epi-PGF by mast cells could contribute to the inflammatory response in e.g. aspirin-sensitive asthma patients.     

Inhibitory effects of an ellagic acid glucoside, okicamelliaside, on antigen-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice

Degranulation inhibitors in plants are widely used for prevention and treatment of immediate-type allergy. We previously isolated a new ellagic acid glucoside, okicamelliaside (OCS), from Camellia japonica leaves for use as a potent degranulation inhibitor. Crude extracts from leaves also suppressed allergic conjunctivitis in rats. In this study, we evaluated the in vivo effect of OCS using a pure sample and performed in vitro experiments to elucidate the mechanism underlying the extraordinary high potency of OCS and its aglycon. The IC(50) values for degranulation of rat basophilic leukemia cells (RBL-2H3) were 14 nM for OCS and 3 μM for aglycon, indicating that the two compounds were approximately 2 to 3 orders of magnitude more potent than the anti-allergic drugs ketotifen fumarate, DSCG, and tranilast (0.17, 3, and >0.3 mM, respectively). Antigen-induced calcium ion (Ca(2+)) elevation was significantly inhibited by OCS and aglycon at all concentrations tested (p<0.05). Upstream of the Ca(2+) elevation in the principle signaling pathway, phosphorylation of Syk (Tyr525/526) and PLCγ-1 (Tyr783 and Ser1248) were inhibited by OCS and aglycon. In DNA microarray-screening test, OCS inhibited expression of proinflammatory cytokines [interleukin (IL)-4 and IL-13], cytokine-producing signaling factors, and prostaglandin-endoperoxidase 2, indicating that OCS broadly inhibits allergic inflammation. During passive cutaneous anaphylaxis in mice, OCS significantly inhibited vascular hyperpermeability by two administration routes: a single intraperitoneal injection at 10 mg/kg and per os at 5 mg/kg for 7 days (p<0.05). These results suggest the potential for OCS to alleviate symptoms of immediate-type allergy.     

Platelet-activating factor (PAF-acether) formation in rat basophilic leukemia cells stimulated by ionophore A23187

In rat basophilic leukemia cells (2 H3-RBL) stimulated with the calcium ionophore A23187, a rapid build-up of PAF-acether was observed within 5 minutes. Thereafter, a slow and complete catabolism was observed within the next 55 minutes. Accumulation of PAF-acether required calcium in the medium and was increased in the presence of acetyl-CoA. Phenyl methyl sulfonyl fluoride, a serine hydrolase inhibitor active on 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetyl hydrolase also produced an increased accumulation of PAF-acether in ionophore-stimulated cells. Quinacrine, a non specific inhibitor of phospholipase A2 impaired PAF-acether formation in a dose-dependent manner. The time-course of PAF-acether formation was compared with the ionophore-induced release of arachidonic acid from these cells.     

Effects of inhibitors of arachidonic acid metabolism on serotonin release from rat basophilic leukemia cells

Mast cells can release arachidonic acid (AA) metabolites as well as preformed mediators with IgE mediated stimulation, and these mediators are considered to play an important role in allergic reactions. The coincident release of preformed mediators and AA metabolites suggests that AA metabolism is related to mast cell degranulation. To clarify the relationship between mast cell degranulation and AA metabolism, the effects of various AA cascade inhibitors on rat basophilic leukemia cell (RBL) mediator release induced by either anti-IgE or A23187 were examined. 5,8,11,14-eicosatetraynoic acid (ETYA) inhibited both PGD₂ and LTC₄/D₄ generation, and partially inhibited serotonin release. Nordihydroguaiaretic acid (NDGA) caused complete inhibition of LTC₄/D₄ generation, and partial inhibition of PGD₂ generation and serotonin release. The cyclooxygenase inhibitor, indomethacin, and the specific 5-lipoxygenase inhibitor, L-651,392 completely inhibited PGD₂ and LTC₄/D₄ generation, respectively, without affecting release of other mediators. Both PGD₂ and LTC₄/D₄ generation were abolished by the combination of indomethacin and L-651,392, however, serotonin release remained intact. HPLC analysis showed that no shift to other AA metabolites occurred after the treatment with these inhibitors. Mepacrine, a phospholipase A₂ inhibitor, completely inhibited PGD₂ and LTC₄/D₄ generation, as well as AArelease itself, without affecting serotonin release. Therefore, neither AA metabolism nor AA release is necessary for RBL degranulation.     

Role of galectin-3 in acetaminophen-induced hepatotoxicity and inflammatory mediator production

Galectin-3 (Gal-3) is a β-galactoside-binding lectin implicated in the regulation of macrophage activation and inflammatory mediator production. In the present studies, we analyzed the role of Gal-3 in liver inflammation and injury induced by acetaminophen (APAP). Treatment of wild-type (WT) mice with APAP (300 mg/kg, ip) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was associated with increased hepatic expression of Gal-3 messenger RNA and protein. Immunohistochemical analysis showed that Gal-3 was predominantly expressed by mononuclear cells infiltrating into necrotic areas. APAP-induced hepatotoxicity was reduced in Gal-3-deficient mice. This was most pronounced at 48-72 h post-APAP and correlated with decreases in APAP-induced expression of 24p3, a marker of inflammation and oxidative stress. These effects were not due to alterations in APAP metabolism or hepatic glutathione levels. The proinflammatory proteins, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, and MIP-3α, as well as the Gal-3 receptor (CD98), were upregulated in livers of WT mice after APAP intoxication. Loss of Gal-3 resulted in a significant reduction in expression of iNOS, MMP-9, MIP-3α, and CD98, with no effects on IL-1β. Whereas APAP-induced increases in MIP-2 were augmented at 6 h in Gal-3(-/-) mice when compared with WT mice, at 48 and 72 h, they were suppressed. Tumor necrosis factor receptor-1 (TNFR1) was also upregulated after APAP, a response dependent on Gal-3. Moreover, exaggerated APAP hepatotoxicity in mice lacking TNFR1 was associated with increased Gal-3 expression. These data demonstrate that Gal-3 is important in promoting inflammation and injury in the liver following APAP intoxication.     

硫酸皮肤素衍生物对大鼠肥大细胞脱颗粒的影响

目的研究不同来源不同分子量的低分子硫酸皮肤素 (LMWDS)及多硫酸化硫酸皮肤素 (PSDS)对大鼠肥大细胞 (MC)脱颗粒的影响。方法通过大鼠腹腔被动MC脱颗粒实验 ,观察LMWDS及PSDS对卵清蛋白引起MC脱颗粒的影响。结果与Hank液组比较 ,硫酸皮肤素衍生物均能明显抑制MC脱颗粒 ;与色甘酸钠组比较 ,PSDS组、牛肺来源的BLMWDS 3组和猪肠来源的PLMWDS 3组有显著的抑制MC脱颗粒作用。PSDS、BLMWDS、PLMWDS抑制MC脱颗粒作用与剂量呈正相关。结论硫酸皮肤素衍生物具有抑制MC脱颗粒作用 ,该作用与其分子量大小及结构有关     

Microglial derived nitric oxide decreases serotonin content in rat basophilic leukemia (RBL-2H3) cells

Nitric oxide (NO) and serotonin (5-hydroxytryptamine; 5-HT) are important neuromodulators that are involved in a myriad of biochemical reactions. In this work, we describe a novel model co-culture system to study the interactions between NO and 5-HT. NO derived from cytokine stimulated Bv2 microglial cells depleted 5-HT from RBL-2H3 cells. Reduction of 5-HT content by NO derived from the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was concentration-dependent, independent of intracellular Ca(2+) and inhibited by reduced glutathione (GSH). Collectively, these data indicate that this cell co-culture system is a viable model to study the mechanisms of interaction between nitrergic and serotonergic pathways.     

脱硫酸化与多硫酸化肝素衍生物对肥大细胞脱颗粒的影响

目的　研究脱硫酸化与多硫酸化肝素对大鼠肥大细胞 (MC)脱颗粒的影响。方法　采用不同方法制得不同程度硫酸化肝素 :2 O 脱硫酸化肝素 (2DeSH)、N 脱硫酸化 重乙酰化肝素 (NDeSAcH)、6 O 脱硫酸化肝素 (6DeSH)、多硫酸化肝素 (PSH) ,通过大鼠腹腔被动MC脱颗粒实验 ,观察肝素及不同程度硫酸化肝素对卵清蛋白引起MC脱颗粒的影响。结果　与Hank液组比较 ,各样品组均有显著抑制MC脱颗粒的作用 (P <0 0 1 ) ,但 6DeSH组与色苷酸钠组差异有显著性 (P <0 0 1 ) ,显示较弱的抑制活性。结论　肝素抑制MC脱颗粒的活性与肝素结构上的硫酸基位置和多少有关 ,其中6 O 硫酸基对肝素的抑制MC脱颗粒的活性影响较大。     

Calcium signals in rat basophilic leukemia (RBL-2H3) cells primed with the neuropeptide substance P

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We have recently shown that direct nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P is an important mediator of this communication. Here we study the calcium signals in rat basophilic leukemia cells (RBL-2H3; mucosal-type mast cells) primed with substance P. RBL cells responded only slightly to stimulation with compound 48/80, however they responded to the stimulation when the cells had been primed with substance P (0.5 microM) for one week. The present results provide a foundation to study the neuroimmune cross-talk in a co-culture system.     

Nonpolar lipid methylation—identification of nonpolar methylated products synthesized by rat basophilic leukemia cells,retina and parotid

Incorporation of radioactivity from [³H- or ¹⁴C-methyl]methionine into nonpolar lipids has been investigated in rat basophilic leukemia (RBL) cells, retina, and rat parotid gland. These nonpolar methylated lipids were extracted into heptane and characterized by thin-layer chromatography, high performance liquid chromatography, gas chromatography, and mass spectrometry. The major methylated nonpolar lipid product in the RBL cells themselves was ubiquinone-9, which accounted for about 90% of the nonpolar lipid and 20–30% of the total radioactive lipid formed. There was a modest increase in the methylation of nonpolar lipids upon stimulation of the RBL cells with IgE and anti-IgE, but the significance of this change is uncertain. In contrast to whole cells, RBL membrane fractions (incubated with [³H-methyl]-S-adenosylmethionine) incorporated radioactivity primarily into fatty acid methyl esters and not ubiquinone. A third product, 2-(methylthio)-benzothiazole, was formed by RBL cells, retina and minced parotid upon incubation in enriched media. This product was formed enzymatically, apparently by the known enzyme S-thiolmethyltransferase, using the thiol substrate which contaminates these media. Evidence suggests that the enzyme may reside, at least in part, on the surface of the cells.     

Regulation of exocytosis by the small GTP-binding protein rho in rat basophilic leukemia (RBL-2H3) cells

• 1.1. We investigated the effect of Clostridium botulinum C3 ADP-ribosyltransferase upon β-hexosaminidase release induced by various stimuli from streptolysin-O (0.5-1 U/ml)-permeabilized rat basophilic leukemia (RBL-2H3) cells.
• 2.2. The C3 transferase inhibited β-hexosaminidase release induced by Ca²⁺ or by guanosine-5'-(3-thio-triphosphate) (GTPyS) plus Ca²⁺.
• 3.3. The C3 transferase also inhibited β-hexosaminidase release induced by stimulating high affinity IgE and m3 muscarinic acetylcholine receptors.
• 4.4. The substrate for the C3 transferase was present in cytosol of RBL-2H3 cells, indicating the presence of rho p21. About 60% of the total cellular substrate protein remained within the cells permeabilized by 1 U/ml of streptolysin-O.
• 5.5. The protein rho p21 appears to be regulated by several pathways and it may function as an integration point for exocytosis.

     

1,3-selenazol-4-one derivatives inhibit inducible nitric oxide-mediated nitric oxide production in lipopolysaccharide-induced BV-2 cells

Activated microglia extensively produce nitric oxide (NO) by inducing expression of inducible NO synthase (iNOS). NO plays a deleterious role in brain inflammation and neuronal death. In the present study, we investigated the effects of 1,3-selenazol-4-one derivatives (Sz-A, B, C, D and E) on NO production and iNOS expression in lipopolysaccharide (LPS)-induced BV-2 cells, a murine microglia cell line. Among these compounds, Sz-B and C remarkably inhibited LPS-induced NO production relative to that of Sz-A, D, and E at 5 microM in BV-2 cells. Sz-B and C dose-dependently inhibited NO production at 1, 5, and 10 microM without toxicity to BV-2 cells. Sz-B and C also dose-dependently suppressed iNOS expression at the same concentrations in LPS-induced BV-2 cells. This result suggests that Sz-B and C inhibit iNOS-mediated NO production in LPS-induced BV-2 cells. Structurally, Sz-B and C bear an ethyl or methyl group at the 5 positions of the 4-selenazolone skeletons, which could play an important role in inhibiting iNOS-mediated NO production.     

Enhancing effect of chlorinated organic solvents on histamine release and inflammatory mediator production

We investigated the effect of several chlorinated organic solvents on antigen-induced histamine release and inflammatory mediator production. Non-purified rat peritoneal mast cells (NPMC) and rat basophilic leukemia (RBL-2H3) cells were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody, and then stimulated with DNP-conjugated bovine serum albumin (DNP-BSA) and several chlorinated organic solvents. Trichloroethylene (TCE) and tetrachloroethylene (PCE) enhanced histamine release from antigen-stimulated NPMC and RBL-2H3 in a dose-dependent manner. In addition, TCE and PCE increased IL-4 and TNF-alpha production from antigen-stimulated RBL-2H3. In an in vivo study, we investigated the effect of TCE and PCE on passive cutaneous anaphylaxis (PCA) reaction. TCE and PCE enhanced PCA reaction markedly. These results suggest that TCE and PCE increase histamine release and inflammatory mediator production from antigen-stimulated mast cells via the modulation of immune responses. In addition, exposure to TCE and PCE may lead to the augmentation of allergic diseases.     

Studies of phosphorylation in rat mast cells (third report)--isolation of calcium-activated, phospholipid, diacylglycerol-dependent protein kinase from rat basophilic leukemia cells (RBL-1 cells)

Evidence is presented that RBL-1 cells, which are similar to normal rat mast cells in morphology and contain IgE receptors and histamine, contain a calcium-activated, phospholipid, diacylglycerol-dependent protein kinase. This enzyme is very similar in its activation requirements to the calcium-dependent enzyme termed protein kinase C in other tissues. The enzyme is activated by Ca2+. Diolein, but not other di, mono or triglycerides, substantially increases the enzyme activity. Among various phospholipids, phosphatidylserine is the most reactive activator; phosphatidylinositol, phosphatidic acid and phosphatidylethanolamine are less effective; and phosphatidylcholine is practically inactive. The enzyme is inhibited by chlorpromazine and local anesthetics such as dibucaine, tetracaine and procaine.