Exocytosis In Mast Cells Research Articles

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP‐III at the level of endosomal membranes from rat mast cells

It is well known that the activation of mast cells due to the binding of mastoparan to the G(α) subunit of trimeric G proteins involves exocytosis regulation. However, experimental evidence in the literature indicates that mastoparan can also activate certain regulatory targets of exocytosis at the level of the mast cell endosomal membranes that have not yet been identified. Therefore, the aim of the present investigation was the proteomic identification of these targets. To achieve these objectives, mast cells were activated by the peptide Protopolybia MP-III, and the proteins of the endosomal membranes were converted to proteoliposomes using sonication. Proteins were separated from one another by affinity chromatography using proteoliposomes as analytes and Protopolybia MP III-immobilized Sepharose 4B resin as the ligand. This experimental approach, which used SDS-PAGE, in-gel trypsin digestion and proteomic analysis, permitted the identification of five endosomal proteins: Rho GTPase Cdc 42 and exocyst complex component 7 as components of the Ca(2+) -independent FcεRI-mediated exocytosis pathway, synaptosomal-associated protein 29, and GTP-binding protein Rab3D as components of the Ca(2+) -dependent FcεRI-mediated exocytosis pathway and Ras-related protein M-Ras, a protein that is related to the mediation of cell shaping and proliferation following exocytosis. The identification of the five proteins as targets of mastoparans may contribute in the near future to the use of this family of peptides as novel tools for dissecting the mechanism of exocytosis in mast cells.

Protein profiling of the secretome of FcεRI activated RBL-2H3.1 cells.

Secretory proteins of IgE receptor activated mast cells and basophils play a pivotal role in the generation of immediate and long term immune responses in allergy and type I hypersensitivity. The present study aims to generate a 2-D map and profile of proteins secreted from a high secretory variant of the rat basophilic leukemia cell line, RBL-2H3.1, which in view of the difficulty associated with gaining adequate numbers of pure primary mast cell and basophiles, represents an accepted model system for the study and standardization of the methodology to characterize the secretome of these cell types. A 2-D map of secretory proteins was generated by 2-D PAGE and a shotgun mass spectrometric approach carried out for protein identification. Study resulted into identification of 299 proteins released from resting and IgE receptor activated RBL-2H3.1 cells after 90 s, 30 min and 3 h antigen challenge. Further sequence analysis identified ~53% of total proteins as secretory proteins which could be attributed to classical and non-classical secretory pathways. Additionally, functional classification of classic secretory proteins verified the presence of proteins belonged to cytokines, receptors, membrane proteins, lysosomal proteins and proteins associated with specific sub-cellular localizations such as endoplasmic reticulum, mitochondria, nucleus, cytoplasm and ribosome. According to this data the presence of some secretory proteins such as cytokines (e.g. MCP-2, PF-4, CSF-1 and TGF-β1) are all subject to Ag challenge which may point to their importance toward pathogenesis in allergic diseases. In view of both a beneficial and adverse role of mast cell mediators in health and disease, an identification of temporal changes in the secretory pattern may form the basis for future tailor made intervention strategies that may enable us to harvest the therapeutic potential inherent in mast cell exocytosis while inhibiting/attenuating negative outcomes.

SNAP-23 and syntaxin-3 are required for chemokine release by mature human mast cells

Mast cells play a key role in allergic and non-allergic disease by releasing a broad array of mediators. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) are necessary for membrane fusion events during mast cell exocytosis. We have shown recently that the SNAREs SNAP-23, syntaxin (STX)-4, vesicle associated membrane protein (VAMP)-7, and VAMP-8 are required for release of pre-stored histamine by mast cells. Here we analyze the involvement of different SNARE isoforms in exocytosis of de novo synthesized chemokines in mast cells isolated from human intestine. Following IgE receptor cross-linking, mast cells released substantial amounts of the chemokines CXCL8, CCL2, CCL3, and CCL4. Measurement of SNARE mRNA expression revealed only a moderate up-regulation of mRNA for STX-4 after stimulation for 1.5h. Inhibition of SNAP-23 or STX-3 abolished IgE mediated release of the chemokines CXCL8, CCL2, CCL3, and CCL4. In contrast, blocking of STX-2, or VAMP-3 did not affect the chemokine release. Inhibition of STX-4 or VAMP-8 resulted in a reduced release of CXCL8, but not of CCL2, CCL3, or CCL4. Inhibition of STX-6 attenuated the release of CXCL8 and CCL2, inhibition of VAMP-7 that of CCL3. In summary, STX-3 and SNAP-23 are crucial for the release of all chemokines in mature human mast cells whereas other SNAREs affect only release of selected chemokines.

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca2+ concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca2+ dependent, the target molecule that interacts with Ca2+ is not clear. Synaptotagmin is a Ca2+ sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca2+, low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca2+, and phosphatidylserine.

Liprin-α is involved in exocytosis and cell spreading in mast cells

The active zone is a specialized region of the presynaptic plasma membrane where the neurotransmitter release occurs by exocytosis. Mast cells also release inflammatory mediators by exocytosis resulting in induction of allergic responses. In our previous reports, we found that active zone proteins, Munc13-1 and ELKS regulates exocytosis of mast cell positively. In this study, we investigated the involvement of liprin-α, another active zone protein, in exocytosis in mast cells.We found that three isoforms of liprin-α, liprin-α1, -α2 and -α3 were expressed. Immunocytochemical experiments revealed that liprin-α1 resided both in the cytoplasm and on the plasma membrane. Upon stimulation with antigen, the area of a cell increased remarkably due to cell spreading and the distribution of liprin-α1 became punctuated. Interestingly, knockdown of liprin-α1 caused decrease in exocytotic release and cell spreading. These results suggest that liprin-α1 facilitates exocytosis and cell spreading, and these events might have correlated each other in mast cells.

2K1624 マスト細胞の開口放出におけるアクティブゾーンタンパク質と細胞接着の関与(細胞生物的課題2,第49回日本生物物理学会年会)

2K1624 マスト細胞の開口放出におけるアクティブゾーンタンパク質と細胞接着の関与(細胞生物的課題2,第49回日本生物物理学会年会)

Advanced glycation end products (AGEs) activate mast cells.

Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca(2+) increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.

Latrotoxin-induced exocytosis in mast cells transfected with latrophilin

α-Latrotoxin (α-LTX) is known to cause massive exocytosis from presynaptic nerve terminals. We investigated the effects of α-LTX on exocytotic release from mast cells, typical non-neuronal secretory cells. When we transfected mast cells with latrophilin, a specific receptor for α-LTX, α-LTX caused intracellular Ca 2+ to increase and led to exocytosis in the presence of extracellular Ca 2+. On the other hand, neither Ca 2+ increase nor exocytosis was observed in the absence of extracellular Ca 2+. These results indicate that α-LTX, together with latrophilin, works as a Ca 2+ ionophore. However, α-LTX had additional effects on signal transduction in mast cells. We found that inhibitors of protein kinase C (PKC) partially suppressed exocytosis. Furthermore, several soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including SNAP-23, were phosphorylated by α-LTX. These results suggest that exocytosis induced by α-LTX can be explained by (1) elevation of intracellular Ca 2+, (2) phosphorylation of SNARE proteins including SNAP-23, syntaxin-4 and VAMP-8 through PKC-dependent and -independent pathways. Our study may provide a new system to investigate the action of α-LTX and the mechanism of exocytosis in mast cells.

3P227 リポソーム系を用いたシナプトタグミンによるマスト細胞開口放出に関与するSNAREの膜融合制御(生体膜・人工膜-ダイナミクス,第48回日本生物物理学会年会)

3P227 リポソーム系を用いたシナプトタグミンによるマスト細胞開口放出に関与するSNAREの膜融合制御(生体膜・人工膜-ダイナミクス,第48回日本生物物理学会年会)

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells

Ca(2+) mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca(2+) sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca(2+) waves that originate most frequently from the tips of extended cell protrusions, as well as Ca(2+) oscillations throughout the cell that usually follow the initiating Ca(2+) wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca(2+) puffs at the region of cell contact. Initiating Ca(2+) waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca(2+) release from endoplasmic reticulum stores and to extracellular Ca(2+), but they do not depend on store-operated Ca(2+) entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca(2+) signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca(2+) response.

Synaptotagmin-2 Controls Regulated Exocytosis but Not Other Secretory Responses of Mast Cells

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

Image analysis of mast cell degranulation in a concentration gradient of stimuli formed in the channel between a glass plate and a silicon substrate

Measurement of released granule components, popularly used to quantify mast cell exocytosis, does not deliver real-time information about degranulation at the single-cell level nor the ratio of responding/non-responding cells. Rather it provides, only end-point, bulk-population data. Here we studied degranulation of rat peritoneal mast cells dispersed in a narrow horizontal channel between a silicon substrate and a glass plate. Upon exposure to a concentration gradient of a soluble stimulus, degranulation started from those cells facing towards the highest concentration of stimulus. We captured images of exocytosing cells without the need for phase-contrast or differential interference-contrast microscopy. This was achieved using the reflection caused by the silicon substrate. The time-lapse images of cells in the channel were segmented into multiple concentration belts to identify the proportion of degranulated cells in each belt region. Maximum ratios of degranulated cells in the belt regions determined by time-course curve fitting calculations were then plotted against the distance from the stimulus injection site, resulting in a sigmoidal response curve. This method provides a powerful means for real-time analysis of concentration- and stimulus-dependent degranulation of mast cells and allows comparison of cell responses under different conditions. To show its effectiveness, we evaluated the effect of a protein kinase C (PKC) inhibitor, Gö6976, on degranulation induced by various stimuli. In contrast to stimulation with concanavalin A+lysophosphatidylserine (lysoPS) or nerve growth factor+lysoPS (completely inhibited by Gö6976 over the whole range of stimulus concentrations used) or compound 48/80 and mastoparan (no inhibition by Gö6976), stimulation with ionomycin, a known Ca 2+ ionophore, showed a concentration-dependent inhibition by Gö6976, with a major inhibition at low stimulus concentrations and a diminished one at higher ionomycin concentrations. The results indicate that ionomycin-induced degranulation is mainly induced via a PKC-independent signal cascade at high stimulus concentrations, whereas below a certain concentration, degranulation is completely dependent on PKC.

Vacuolin-1-modulated exocytosis and cell resealing in mast cells

The small chemical vacuolin-1 induces rapid formation of large vacuoles in various cell types. In epithelial cells, vacuolin-1 has been shown to inhibit Ca 2+ ionophore-induced exocytosis depending on experimental conditions used but had no effect on repair of damaged membranes. However, it is not known whether vacuolin-1 could inhibit exocytosis induced by immunoreceptor triggering in professional secretory cells and whether there is any correlation between effect of vacuolin-1 on exocytosis and membrane repair in such cells. Here we show that in rat basophilic leukemia (RBL-2H3) cells activated by the high-affinity IgE receptor (FcεRI) triggering vacuolin-1 enhanced exocytosis. Under identical conditions of activation, vacuolin-1 inhibited exocytosis in mouse bone marrow-derived mast cells (BMMCs). This inhibition was not reflected by decreased phosphorylation of the FcεRI α and β subunits, linker for activation of T cells, non-T cell activation linker, Akt and MAP kinase Erk, and uptake of extracellular Ca 2+, indicating that early activation events are not affected. In both cell types vacuolin-1 led to formation of numerous vacuoles, a process which was inhibited by bafilomycin A1, an inhibitor of vacuolar H +-ATPase. Thapsigargin- or Ca 2+ ionophore A23187-induced exocytosis also showed different sensitivity to the inhibitory effect of vacuolin-1. Pretreatment of the cells with vacuolin-1 followed by permeabilization with bacterial toxin streptolysin O enhanced Ca 2+-dependent repair of plasma membrane lesions in RBL-2H3 cells but inhibited it in BMMCs. Our data indicate that lysosomal exocytosis exhibits different sensitivity to vacuolin-1 depending on the cell type analyzed and mode of activation. Furthermore, our results support the concept that lysosomal exocytosis is involved in the repair of injured plasma membranes.

Activation of CD47 receptors causes histamine secretion from mast cells.

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcepsilonRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the betagamma dimer of a G(i) protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G(i) protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G(i) protein complex.

Characterization of an early signaling defect following FcɛRI activation in the canine mastocytoma cell line, C2

A comparison of IgE recognition by cognate receptors expressed on the C2 canine mastocytoma cell line with analogous events in a rat basophilic leukemia cell line transfected with the α-chain subunit of the canine high-affinity IgE receptor using flow cytometry show that canine IgE recognizes the α-chain of its cognate receptor on both cell lines. Our study confirms the expression of functional IgE receptors in both cell lines, but receptor-mediated signaling in the C2 line only supports the early stages of downstream signaling as shown by the phosphorylation of the γ-chain and the failure to effect the phosphorylation of Syk. In contrast RBL-2H3 cells respond to sensitization with IgE and challenge with cognate antigen with tyrosine phosphorylation of the γ-subunits of the receptor complex followed by downstream phosphorylation of Syk and Ca2+ mobilization, culminating in β-hexosaminidase release. We propose that the identification of the precise signaling defect in C2 cells will yield useful information regarding the pathway leading to mast cell exocytosis and facilitate the restoration of the complete signaling cascade through complementation of the missing/defective signal transducer since signaling events downstream of Ca2+ mobilization are intact as demonstrated by β-hexosaminidase release following non-immunologic stimulation with the calcium ionophore, A23187.

3TA4-04 マスト細胞のヒスタミン分泌の研究(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

3TA4-04 マスト細胞のヒスタミン分泌の研究(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

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.

2P-177 リポソーム再構成SNARE系を用いたマスト細胞脱顆粒機構の解析(生体膜・人工膜-ダイナミクス,第47回日本生物物理学会年会)

2P-177 リポソーム再構成SNARE系を用いたマスト細胞脱顆粒機構の解析(生体膜・人工膜-ダイナミクス,第47回日本生物物理学会年会)

Membrane fusion between liposomes containing SNARE proteins involved in mast cell exocytosis

We monitored the membrane fusion of liposomes to determine if the minimal components of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE), which is involved in mast cell exocytosis, have fusogenic activity. Three core components of SNARE were reconstituted into liposomes. Membrane fusion between liposomes containing vesicle associated membrane protein (VAMP) -7 or -8 and liposomes containing synaptosomal-associated protein 23 kDa (SNAP23) and syntaxin-3 or -4 was monitored by fluorescence resonance energy transfer. The combination of SNAP23/syntaxin-3/VAMP-8 showed the most efficient liposome-liposome fusogenic activity. Liposomes with VAMP-7 exhibited poor fusogenic activity regardless of the syntaxin isoform. The core components of SNAP23, syntaxin-3, and VAMP-8 appear to be minimal machinery to induce membrane fusion, while VAMP-7 appears to be unessential for membrane fusion.

Munc13-1, an active zone protein, regulates exocytosis in mast cells

Munc13-1, an active zone protein, regulates exocytosis in mast cells