Elsevier

Cell Calcium

Volume 67, November 2017, Pages 31-39
Cell Calcium

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

https://doi.org/10.1016/j.ceca.2017.08.002Get rights and content

Highlights

  • Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca2+ increase.

  • FcεRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs.

  • Ca2+ mobilization from the ER is both necessary and sufficient for the release of histamine.

  • Extracellular Ca2+ promotes, but is not essential for, mast cell degranulation.

  • Downregulation of SOCE reduces the intracellular Ca2+ levels and delays mast cell degranulation and histamine release.

Abstract

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca2+ levels, respectively. Histamine release and Ca2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca2+ increase. FcεRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca2+ influx enhanced the process. Furthermore, cytosolic Ca2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca2+ signaling in regulating histamine degranulation.

Introduction

The release of histamine from mast cells is a key event during an allergic response. When allergy-prone people come in contact with an allergen such as pollen or dust mite for the first time, their B cells differentiate into plasma cells, which produce large quantities of antigen-specific IgE. Following this, IgE binds to the mast cells but does not cause an allergic reaction. Upon subsequent attack by the same allergen, IgE stimulates the mast cells to release chemical mediators such as cytokines and histamine [1], [2]. These chemical mediators cause the characteristic symptoms of allergy.

The allergic responses are mediated via two signaling pathways resulting in mast cell degranulation. One is the IgE-dependent, and the other is the IgE-independent signaling pathway. The IgE-dependent signaling pathway mimics physiological antigen exposure by cross-linking the IgE-FcεRI complexes with the multivalent antigen [3], while the IgE-independent signaling pathway occurs through the activation of G protein-coupled receptors (GPCRs) [4]. The high-affinity immunoglobulin E receptor (FcεRI) expressed on the surface of mast cells [5] is a tetrameric receptor complex consisting of one α, one β, and two γ chains. While the α-chain forms the IgE-binding site, the β-chain amplifies and the γ-chain initiates the downstream signaling pathway, respectively. Cross-linking of the IgE-FcεRI complex by a multivalent antigen initiates the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) found in the cytoplasmic β and γ2 subunits of the FcεRI receptor by Lyn, a Src protein tyrosine kinase, which then leads to the recruitment and activation of another tyrosine kinase, Syk. Syk phosphorylates and activates phospholipase Cγ (PLCγ) tethered to LAT, a transmembrane adaptor molecule. Hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) by PLCγ produces two second messengers, diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). IP3-mediated Ca2+ release from the ER and the combination of Ca2+ activation of PKC are necessary and sufficient for degranulation of the mast cells [6], [7], [8].

In addition to the above, mast cell activation can also be triggered by a large number of polycationic molecules, collectively known as basic secretagogues. These include polyamines like spermine and compound 48/80 (48/80) [9], [10], venom peptides like mastoparan, and neurotransmitters such as substance P [11], which cause neurogenic inflammation [12]. The negatively charged sialic acid residues (SIA) present on the mast cell membranes, are thought to assemble the positively charged molecules, such as 48/80, at the cell surface. Following this, the polycationic molecules interact with the GPCRs that are coupled with the Gαi family of heterotrimeric G proteins [13], [14]. Previous studies have demonstrated that the basic secretagogue signaling pathway induces degranulation in the mast cells by the activation of the heterotrimeric Gα protein (Gαi2 and Gαi3), by associating with the Gβγ subunits to stimulate phospholipase Cβ (PLCβ), which results in the release of histamine [12], [15], [16]. Similar to the activation of PLCγ by the IgE-FcεRI complexes, activation of PLCβ by GPCRs also leads to the hydrolysis of PIP2 to yield DAG and IP3.

During exocytosis and release of histamine [17], [18], Ca2+ plays an important role in the fusion of the secretory vesicles with the plasma membrane [19], [20]. Elevation in the intracellular Ca2+ level after exposure to the allergens may be due to the ER-releasable fraction and extracellular Ca2+ influx. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores in non-excitable cells, like mast cells. Activation of SOCE is used to refill intracellular Ca2+ stores, regulate basal Ca2+, and excute a wide range of Ca2+-associated specialized activities [21]. Activation of both IgE-FcεRI complexes and GPCRs by allergens shares a similar signaling mechanism in inducing ER-releasable Ca2+ and SOCE. Stromal interaction molecule 1 (STIM1) is a transmembrane protein, which is localized mainly in the ER and acts as a Ca2+ sensor. When STIM1 senses a decrease in the Ca2+ concentration in the ER, it translocates to specific ER-plasma membrane junctional regions, where it activates CRACM1 (calcium release-activated calcium modulator 1 or Orai1) and TRPC1 (transient receptor potential canonical 1) channels, enabling extracellular Ca2+ influx. Microtubules are essential for translocation of STIM1 to the plasma membrane and its interaction with Orai1 for activation of SOCE [22], [23]. Microtubule-associated histone deacetylase 6 (HDAC6) induces the activation of STIM1-mediated SOCE by enhancing the translocation of STIM1 to the plasma membrane. Therefore, SOCE activated in the plasma membrane induces extracellular Ca2+ influx and promotes the events related to degranulation [24], [25], [26].

Nile red is a fluorescent probe used for the detection of histamine [27], [28]. The measurement of histamine is based on a ligand exchange reaction between the former and the iminodiacetic acid ligands in the probe coordinated with Ni2+. Nile red is lipophilic in nature and localizes in the intracellular lipid droplets. It comprises iminodiacetic acid moieties, whose fluorescent properties are known to change upon binding to metal ions like Ni2+. Ni2+ bound to the iminodiacetic acid residues acts as a quencher, and strongly quenches the fluorescence of Nile red. When the probe is present in the histamine-containing secretory granules of the mast cells, it preferably coordinates with histamine. Ni2+ is preferably exchanged with histamine to form a histamine-Ni2+ complex, and Nile red exhibits a stronger fluorescence. Therefore, Nile red is suitable for the real-time monitoring of histamine in living cells [27].

The mast cell degranulation pathway has been popularly studied using the rat basophilic leukemia RBL-2H3 cell line, because of its reliability in the release of histamine upon initiation by IgE-FcεRI complexes and their functional similarity to the primary human basophils and rodent mast cells. IgE-FcεRI complex-mediated and GPCR-activated signaling pathways commonly require the participation of Ca2+. However, the differences between these pathways in Ca2+ mobilization and mast cell degranulation have not yet been fully understood. Regular assays for mast cell degranulation, such as β-hexosaminidase release assay, are either single point assays or endpoint assays measuring the cumulative release of mediators. Their limitations regarding single-cell level, real-time and sensitive analysis make them unsuitable for dynamic and high spatiotemporal detection. Kim et al. have demonstrated the temporal relationship between changes in intracellular Ca2+ and serotonin secretion at the single-cell level using simultaneous indo-1 photometry and constant potential amperometry, which has a much faster temporal resolution and reveals dynamic changes in living RBL-2H3 cells [29]. In this study, we examined the relationship between Ca2+ mobilization and mast cell degranulation after stimulation of two different signaling pathways using total internal reflection fluorescence microscopy (TIRFM) in real-time. We found that Ca2+ mobilization from the ER is both necessary and sufficient for the release of histamine. However, extracellular Ca2+ promotes, but is not essential for, mast cell degranulation. We also observed that downregulation of SOCE reduces the intracellular Ca2+ levels and delays mast cell degranulation and histamine release.

Section snippets

Results

The aim of this study was to understand the effect of oscillations in Ca2+ level on mast cell degranulation and histamine release through activation of FcεRI and GPCR-mediated signaling pathways in real-time. The RBL-2H3 cell line is routinely used for measuring histamine release in inflammation, allergic responses, and other immunology-related studies. Here, we used TNP-BSA and monoclonal anti-TNP IgE antibody to activate the high-affinity IgE receptor, FcεRI; and 48/80 to activate the GPCRs

Discussion

Our study demonstrated that Ca2+-mediated degranulation of mast cells was induced by both the IgE-FcεRI and GPCR signaling pathways, and was associated with Ca2+ mobilization. Previous studies have reported Ni2+ to act as a quencher of Nile red fluorescence [27], [28]. In our study also the fluorescence of Nile red was strongly quenched by Ni2+ coordinated to the iminodiacetic acid moiety. When these ions were present in the secretory granules containing histamine, they preferably coordinated

Cell culture maintenance and transfection

The rat basophilic leukemia (RBL-2H3) cell line was maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Big Cabin, OK, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Big Cabin, OK, USA), penicillin (100 IU/mL), and streptomycin (100 μg/mL) in 5% CO2 at 37 °C. For transient transfection, the CD63-GFP plasmid construct, scrambled siRNA (siCtrl) and Orai1 siRNA (siOrai1) were transfected into RBL-2H3 cells using Lipofectamine 3000 (Invitrogen, San Diego, CA, USA)

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We thank Dr. Juan S. Bonifacino (NIH, MD) for kindly providing the CD63-GFP plasmid construct and Dr. Lung-Sen Kao (National Yang-Ming University, Taiwan) for providing technical support. We also thank the “Bio-image Core Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology, Taiwan” for their technical services. We acknowledge the financial support provided by the Ministry of Science and Technology of Taiwan under Grant No. MOST 105-2633-B-006-002

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