The Distribution of Synaptotagmin Ⅱ in RBL-2H3 and Its Regulation on Exocytosis of Lysosomes in RBL-2H3

Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin II （Syt2） in RBL-2H3 （RBL） and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL. Cellular ＆ Molecular Immunology. 2005;2（3）：205-209.     

反义RNA抑制synaptotagminⅡ在RBL-2H3的表达及对胞吐的作用

目的：研究SynaptotagminⅡ（简称syt2）在RBL-2H3（简称RBL）中的表达及其在胞吐中的作用。方法：通过Westernblot检测Syt2在RBL中的表达情况。用高保真酶扩增Syt2，与pEGFP-N1构建全长反义基因表达载体，电穿孔转染RBL，G418筛选获得稳定转染细胞。通过钙离子载体和抗原刺激，应用Westernblot检测稳定转染细胞和对照细胞分泌的组织蛋白酶D，分析Syl2对胞吐的影响。结果：在RBL中检测到Syt2表达。构建了pEGFP-N1-Syt2-AS质粒，插入片段测序结果与GenBank登录号NM012665（ratsyt2）序列完全一致。经转染和G418筛选，获得了稳定转染细胞RBL-Syt2-AS，Westernblot结果显示，两株RBL-Syt2-AS表达的syt2均明显减少，分别只有对照的8％和10％。经刺激后，RBL-Syt2．AS分泌的组织蛋白酶D较对照明显增加。结论：Syt在RBL中表达，其对RBL溶酶体胞吐起负调控作用。     

Toll-like Receptors and RBL-2H3 Mast Cells

Inflammation Research -     

Ubiquitination of Lyn-kinase in rat basophilic leukemia RBL-2H3 cells

Receptor-mediated signal transduction pathways of cells involved in allergy and inflammations are extremely significant. Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FcepsilonRI). Lyn is necessary for FcepsilonRI-mediated mast cell activation. To investigate how the level of Lyn is maintained in mast cell activation, it was studied whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation in cells. In the yeast two hybrid system, Lyn specifically interacted with ubiquitin in vivo. Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. Pulse-chase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin. This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin demonstrating the ubiquitination Lyn. Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by a proteasome specific pathway.     

Casein kinase II-like ectokinase activity on RBL-2H3 cells.

We studied the properties of the ectokinase activity on the outer cell surfaces of RBL-2H3 cells and examined the phosphorylation of exogenous substrates to clarify the substrate specificity of the ectokinases on RBL-2H3 cells. Among the several protein substrates tested, casein was the most strongly phosphorylated with [gamma-32P]ATP, and the net incorporation of 32P into casein was 0.65 pmol P/50 microg/10(6) cells. Casein kinase II peptide was also phosphorylated with [gamma-32P]ATP. The phosphorylation of casein and casein kinase II peptide was almost completely inhibited by the addition of 3 microg/ml of cell-impermeable K252b. Phosphorylation of casein and casein kinase II peptide was also observed by [gamma-32P]GTP. Western blot analysis using anti-casein kinase II antibody revealed a 44-kDa casein kinase band in the membrane fraction and Fc epsilonRI complexes. The immunofluorescence microscopic analysis using anti-casein kinase II antibody showed the existence of casein kinase II on the surface of the cells. This is the first report about the existence of ectokinase on mast cells.     

Effects of endogenous substance P expression on degranulation in RBL-2H3 cells

Objective and design  

To determine whether RBL-2H3 cells have endogenous substance P (SP) expression under immunoglobulin E (IgE)-activated and inactivated conditions, and to ascertain the function of endogenous SP in the antigen-induced degranulation of RBL-2H3 cells.     

青藤碱对RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响

目的 通过研究青藤碱对肥大细胞增殖凋亡以及活化脱颗粒的影响,深入探讨青藤碱的抗炎作用机理.方法 应用细胞培养,流式细胞术等方法,观察青藤碱等药物对体外培养的RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响.结果 青藤碱对肥大细胞增殖有明显的抑制作用,其IG50约为1 mmol/L,并能促进肥大细胞凋亡,且对RBL-2H3肥大细胞活化脱颗粒有较强的抑制作用.结论青藤碱可能部分通过下调肥大细胞功能发挥抗炎抗风湿作用.     

Inhibition of the Antigen-Induced Activation of RBL-2H3 Cells by Gab2 siRNA

Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-associated binder 2） siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2- mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl （DNP）-specific IgE overnight and challenged with dinitrophenyl-human serum albumin （DNP-HSA） to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of β-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcεRI-mediated mast cell release of β-hexosaminidase and histamine, reduced the production of IL-4 and TNF-α and inhibited the phosphorylation of Akt, PKC8 and p38 mitogen-activated protein kinase （MAPK）. Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the 2＋ FcεRI-mediated Ca -independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated. Cellular ＆ Molecular Immunology. 2008; 5（6）：433-438.     

RBL-2H3细胞诊断过敏性休克的可行性初探

目的 :探索诊断过敏性休克的体外检测方法。方法 :建立豚鼠过敏性休克模型 ,培养RBL 2H3细胞株 ,用致敏动物血清在体外诱导该细胞脱颗粒 ,观察其形态改变及测定组胺释放量与细胞表面AnnexinV标记阳性率。结果 :RBL 2H3细胞在致敏血清刺激下活化 ,在抗原再次攻击后可以脱颗粒 ,AnnexinV阳性率与组胺释放量呈正比 ,二者与致敏血清的滴度呈剂量依赖关系。结论 :RBL 2H3可望用于过敏性疾病诊断及变应原筛选 ,组胺测定及AnnexinV阳性率均为较好的测定指标     

Involvement of Heme Oxygenase-1 in Kaempferol-Induced Anti-Allergic Actions in RBL-2H3 Cells

Kaempferol is one of the most commonly found dietary flavonoids. The exposure to kaempferol is known to inhibit degranulation from mast cells, but the inhibitory mechanism of degranulation has not been clarified yet. In this study, we investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of kaempferol against degranulation in rat basophilic leukemia (RBL-2H3) cells. Our results demonstrate upregulation of HO enzymatic activity after short (15 min) exposure to kaempferol, followed by the induction of HO-1 expression in protein. The involvement of HO-1 in the kaempferol-induced inhibition of degranulation was confirmed using tin protoporphyrin IX (SnPP), a HO-1 inhibitor. These findings strongly suggest that kaempferol exerts anti-allergic actions via activation of the HO-1. Etsuko Hirose and Miyoko Matsushima have contributed equally to this work.     

Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells

Objective

RBL-2H3 cells express Toll-like receptors, including TLR4. This study aims to assess various aspects of the TLR4 pathway.

Methods

RBL-2H3 cells were indirectly stained for cell surface TLR4, 25 CD14 and intracellular MyD88 proteins and analysed through flow cytometry for single-colour staining.

Results

While TLR4-receptors are expressed in RBL-2H3 cells, associated elements involved in the signaling pathway, CD14 and MyD88, are not.

Conclusion

Care should be taken if RBL-2H3 cells are used to study aspects of the innate immune system in mast cells.

     

Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells

Objective

RBL-2H3 cells express Toll-like receptors, including TLR4. This study aims to assess various aspects of the TLR4 pathway.

Methods

RBL-2H3 cells were indirectly stained for cell surface TLR4, 25 CD14 and intracellular MyD88 proteins and analysed through flow cytometry for single-colour staining.

Results

While TLR4-receptors are expressed in RBL-2H3 cells, associated elements involved in the signaling pathway, CD14 and MyD88, are not.

Conclusion

Care should be taken if RBL-2H3 cells are used to study aspects of the innate immune system in mast cells.

     

Nicorandil inhibits degranulation and TNF-α release from RBL-2H3 cells

OBJECTIVE: Nicorandil is a potassium channel opener and nitric oxide (NO) donor, and the hypothesis was tested that these modes of action may inhibit cellular degranulation and release of tumour necrosis factor-alpha (TNF-alpha). MATERIALS AND METHODS: TNF-alpha and beta-hexosaminidase secretion were measured from rat basophilic leukemia cells (RBL-2H3) activated via the high affinity IgE receptor with dinitrophenyl-albumin (DNP-A) challenge in the presence of nicorandil. Inhibitors of K+ openers and NO were pre-incubated with the RBL-2H3 cells to determine the principal mode of action. RESULTS: Nicorandil significantly inhibited the release of TNF-alpha in a dose-dependent manner (p < 0.001, ANOVA) reaching a maximum inhibition of DNP-A 74.1% at 10(-3) M, (p < 0.001). Similarly it inhibited beta-hexosaminidase release (p < 0.001, ANOVA) with maximal inhibition at 10(-3) M (p < 0.001). Other K+ openers did not show this effect. Neither the potassium channel blocker glibenclamide nor the guanylyl cyclase inhibitor, ODQ, could reverse this inhibition, but when added in combination reduced the effect by 47%. CONCLUSIONS: Nicorandil is able to inhibit degranulation and TNF-alpha release of RBL cells stimulated through the IgE receptor, and requires both the K+ opening and nitric oxide donor activity, which may represent a novel method for inhibiting cytokine release.     

羧胺三唑对RBL-2H3细胞活化脱颗粒的影响

目的研究羧胺三唑(CAI)对RBL-2H3肥大细胞增殖、凋亡及活化脱颗粒的影响,探索CAI的抗感染作用机制。方法以C48/80诱导RBL-2H3细胞活化脱颗粒模型,中性红染色法观察细胞脱颗粒的形态学,分别用ELISA法和底物显色法检测细胞培养上清中组胺和β-氨基己糖苷酶的释放水平,CCK-8法测定细胞活力,Hoechst 33342荧光染色法检测细胞凋亡。结果与对照组相比,10、20和40μmol/L CAI能够不同程度抑制C48/80诱导的RBL-2H3细胞脱颗粒反应,20和40μmol/L CAI能够降低C48/80诱导的组胺释放(P0.01),40μmol/L CAI能够降低β-氨基己糖苷酶的释放(P0.01)。另外,所用各浓度的CAI对细胞增殖和凋亡均无明显影响。结论 CAI能有效抑制RBL-2H3肥大细胞的活化脱颗粒,此作用并不是通过细胞毒发挥作用的。CAI可能部分通过下调肥大细胞的功能活化,发挥其抗感染作用。     

Stable knockdown of MAFA expression in RBL-2H3 cells by siRNA retrovirus-delivery system

The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein first discovered on the surface of the rat mucosal-type mast cells of the RBL-2H3 line. A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering by its specific monoclonal antibody G63, has been previously shown to cause a dose-dependent inhibition of the secretory response of these cells to the FcRI stimulus. More recently, human and mouse homologues of MAFA have also been discovered. However, they are expressed also or only by NK- and T cells, suggesting that MAFA may play additional functional role(s). To further pursue MAFA's functional significance in mast cells, we have employed the recently developed siRNA (small inhibitory RNA)-dependent gene-specific silencing protocol. We have first incorporated the key elements of the well-defined and commercially available siRNA vector (pSilencer1.0-U6) together with anti-MAFA hairpin into a retroviral vector (pLXSNneo). This enabled generating retroviruses that induced a stable and efficient downregulation (knockdown) of MAFA's expression. RBL-2H3 cells with either normal or knocked-down MAFA expression levels are currently examined for their biological responses e.g. FcRI mediated secretion, cell proliferation and cell adhesion. So far however, no significant changes were resolved in the above biological functions.     

Munc18-2 regulates exocytotic membrane fusion positively interacting with syntaxin-3 in RBL-2H3 cells

Recent studies have revealed that SNARE proteins are involved in exocytotic granular content release in mast cells as well as in neurotransmitter release in neural cells. However, the proteins that regulate the structure and activity of SNARE proteins in mast cells are not well understood. Munc18 is one such regulatory protein that plays a crucial role in neurotransmitter release. In this study, we investigated the role of Munc18 and its mechanism for regulating exocytotic release (degranulation) in rat basophilic leukemia cells (RBL-2H3). We obtained RBL-2H3 cells that express a low level of Munc18-2 and found that degranulation was remarkably inhibited in knockdown cells without any change in the expression level of syntaxins or Ca(2+) mobilization. We also observed the behavior of secretory granules in a single cell, and found no significant changes in their number and distribution in Munc18-2 knockdown cells. Using chimera proteins fused with fluorescent proteins, we demonstrated that Munc18-2 interacted with syntaxin-3, but not with syntaxin-4, in vivo. Interestingly, this interaction occurred not only on plasma membrane but also on secretory granules, suggesting that Munc18-2 may regulate granule-granule fusion as well as granule-plasma membrane fusion. These observations suggest that Munc18-2 together with syntaxin-3 regulate degranulation positively during the process of membrane fusion between secretory granules and plasma membrane, rather than during processes that regulate the number or behavior of secretory granules.     

Magnoliae flos induces apoptosis of RBL-2H3 cells via mitochondria and caspase

BACKGROUND: Magnoliae flores (MF), the buds of Magnolia denudata Desrousseaux, have been successfully used for the management of allergic diseases in Korea. The purpose of the present study was to determine their causal role in inducing apoptosis of mast cells and to verify the underlying mechanism. METHODS: The viability of mast cells was assessed by the trypan blue exclusion test. Induction of apoptosis was confirmed by DNA fragmentation, nuclear staining and DNA hypoploidy. Western blotting and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Mitochondrial membrane potential (MMP) change and cytochrome C release were assayed. RESULTS: We present several lines of evidence indicating that MF induce apoptosis. Changes in cell morphology, generation of DNA fragmentation, cell cycle arrest, activation of caspase-3, and PARP and DFF degradations were demonstrated. The reduction of MMP and the release of cytochrome C to cytosol were also shown. Either PTP blockers, bongkrekic acid and cyclosporin A, or pancaspase inhibitors, Boc.D-fmk and zVAD-fmk, did not prevent the release of cytochrome C. Bax protein content was increased, and Bax was translocated from cytosol into mitochondria at early time points after MF treatment. CONCLUSIONS: We have demonstrated that MF induce mitochondria- and caspase-dependent mast cell apoptosis. Our observations contribute new insights to the role of MF and support the view that the clinical effect of MF may depend on their pharmacological efficacy in regulating mast cell apoptosis.