Regulation Of Mast Cell Degranulation Research Articles

Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74.

Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC β-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

Inhibition effect of PPAR-γ signaling on mast cell-mediated allergic inflammation through down-regulation of PAK1/ NF-κB activation

As a ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPAR-γ) plays a crucial role in allergic inflammation. Recently, the nuclear factor kappa B (NF-κB) pathway and PAK1 have been indicated to be associated with allergic diseases. However, the effect of PPAR-γ on NF-κB and PAK1 production in food allergies is not known. This study aims to 1) systematically validate that the activation of PPAR-γ attenuates allergic reactions and 2) elucidate the mechanism by which PPAR-γ regulates mast cell degranulation. Brown Norway rats were separated into control, ovalbumin, ovalbumin + rosiglitazone, and ovalbumin + GW9662 groups. In vivo experiments demonstrated that rosiglitazone administration markedly inhibited the clinical symptoms and the serum levels of immunoglobulins E and G1. In addition, cytokine release was regulated by activated PPAR-γ and characterized by increased levels of IFN-γ and decreased levels of IL-4, IL-5, and TNF-α. Our data showed that activated PPAR-γ has the potential to alleviate food allergies by enhancing intestinal mucosal integrity and tight junctions. Moreover, we found that PPAR-γ activation inhibited mast cell degranulation both in vivo and in vitro. Our in vitro findings also showed that the activated PPAR-γ signal could inhibit PAK1 phosphorylation and the expression of p65. Furthermore, the interaction between p65 and p-PAK1 during ovalbumin treatment was attenuated after PPAR-γ activation. Collectively, these results demonstrate that PPAR-γ is an important regulator of mast cell degranulation and the Th2-type response, which sheds new light on the importance of PPAR-γ in food allergies.

Autonomic nervous system receptor-mediated regulation of mast cell degranulation modulates the inflammation after corneal epithelial abrasion

Mast cells (MCs) regulate wound healing and are influenced by the autonomic nervous system (ANS). However, the underlying mechanisms affecting wound healing outcomes remain elusive. Here, we explored the specific role of the ANS by regulating MC degranulation following corneal epithelium abrasion. A mouse model of corneal abrasion was established by mechanically removing a 2-mm central epithelium. Wound closure, neutrophil infiltration, and transcription of injured corneas were investigated using whole-mount immunostaining, flow cytometry, and RNA-sequencing analysis, respectively. Inhibition of MC degranulation by the MC stabilizers cromolyn sodium and lodoxamide tromethamine increased the infiltration of neutrophils and delayed healing of abraded corneas. Moreover, transcriptomic profiling analysis showed that purified MCs from the limbus expressed adrenergic and cholinergic receptors. Pharmacological manipulation and sympathectomy with 6-hydroxydopamine confirmed that sympathetic nervous system signaling inhibited MC degranulation after corneal abrasion, whereas parasympathetic nervous system signaling enhanced MC degranulation. We conclude that normal degranulation of MCs in the corneal limbus and crosstalk between the ANS and MCs are crucial for the appropriate control of inflammation and the repair progress of wounded corneas. This suggests a potential approach for improving defective corneal wound healing by the administration of clinically available autonomic activity-modulating agents.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

GPER‐mediated, oestrogen‐dependent visceral hypersensitivity in stressed rats is associated with mast cell tryptase and histamine expression

Visceral hypersensitivity (VH) is common in irritable bowel syndrome (IBS), and female patients are more likely to seek healthcare services for IBS-related abdominal pain. Oestrogen has been reported to mediate pain modulation via its receptor, and mast cells are known to participate in the development of visceral hypersensitivity. Our previous studies showed that the G-protein-coupled oestrogen receptor (GPER, also known as GPR30) was expressed by mast cells in human colonic tissues and was associated with IBS type and severity of visceral pain. However, whether GPER is involved in oestrogen-dependent visceral hypersensitivity via mast cell degranulation is still unknown. Rats were subjected to wrap partial restraint stress to induce visceral hypersensitivity and were ovariectomized (OVX) to eliminate the effects of oestrogen on visceral hypersensitivity. OVX rats were treated with oestrogen, an oestrogen receptor α and β antagonist (ICI 182.780), a GPER antagonist (G15) or a GPER agonist (G1), to evaluate the effects of oestrogen via its receptor. The colorectal distention test was performed to assess visceral sensitivity. Immunofluorescence studies were performed to evaluate GPER and mast cell tryptase co-expression. Mast cell number with degranulation was detected by specific staining. Mast cell tryptase expression in rat colon was also investigated by Western blot and immunohistochemistry. Substance P and histamine expression were examined by ELISA. GPER was expressed by the majority of tryptase-positive mast cells in the colonic mucosa. Stressed rats showed increased visceral sensitivity, increased mast cell degranulation, mast cell tryptase expression, and increased colon histamine levels. Ovariectomy reduced stress-induced VH in female rats and decreased mast cell degranulation, mast cell tryptase expression, and histamine levels, whereas oestrogen replacement reversed these effects. In OVX rats, the GPER antagonist G15 counteracted the enhancing effects of oestrogen on stress-induced VH, mast cell degranulation, mast cell tryptase, and histamine expression, whereas VH was preserved after treatment with ICI 182.780. On the other hand, pretreatment with the selective GPER agonist G1 at doses between 1 and 20μg/kg significantly increased VH, mast cell tryptase, and histamine expression in OVX-stressed rats, mimicking the effects of oestrogen. GPER plays a pivotal role in the regulation of mast cell degranulation, mast cell tryptase expression, and histamine levels and contributes to the development of colonic hypersensitivity in a female rat model of IBS.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

PKC-dependent phosphorylation of Munc18a at Ser313 in activated RBL-2H3 cells.

Protein Kinase C (PKC) regulates the release of pro-inflammatory compounds from IgE/antigen-activated mast cells by unknown mechanisms. In this study, we show for the first time that PKC inhibitor Ro-03-0432, which inhibits RBL-2H3 exocytosis/degranulation in a concentration-dependent fashion, prevents the phosphorylation of membrane fusion factor Munc18a at Ser 313. Our study provides fresh evidence that PKC-dependent protein phosphorylation may contribute to the intricate regulation of mast cell degranulation by directly targeting the fusion factors.

Inhibitory effects of 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate on allergic inflammatory responses in rat basophilic leukemia cells

Mast cells play crucial roles in the initiation of allergic inflammatory responses by releasing various mediators such as histamines, cytokines, and leukotrienes. In addition, signaling cascade pathways, such as the mitogen-activated protein kinase (MAPK) pathway, contribute to the regulation of mast cell degranulation. Accordingly, different research strategies have been pursued to develop anti-inflammatory and anti-allergic drugs by regulating these signaling pathways. The development of new drugs that inhibit mast cell degranulation may help in the treatment of allergies. In this study, we investigated the effects of coumarin derivatives on mast cell degranulation. The effect of coumarin derivatives on degranulation in rat basophilic leukemia (RBL)-2H3 cells was determined by a β-hexosaminidase assay and histamine assay. A coumarin derivative 1 (C1), 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate, inhibited degranulation in a dose-dependent manner and demonstrated maximum therapeutic effect when used at 25μM. Additionally, these compounds inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. Taken together, these results indicate that 2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate inhibits mast cell degranulation by suppressing the activation of the ERK pathway and this inhibitory effect suggests potential therapeutic strategies towards the prevention of allergic disorders.

New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling.

Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

The role of the Annexin-A1/FPR2 system in the regulation of mast cell degranulation provoked by compound 48/80 and in the inhibitory action of nedocromil

1.We investigated the role of Annexin (ANX)-A1 and its receptor, ALX/FPR2, in the regulation of mast cell degranulation produced by compound 48/80.2.Both human cord-blood derived mast cells (CBDMCs) and murine bone marrow derived mast cells (BMDMCs) release phosphorylated ANX-A1 during treatment with glucocorticoids or the mast cell ‘stabilising’ drugs ketotifen and nedocromil.3.Compound 48/80 also stimulated ANX-A1 phosphorylation and release and this was also potentiated by nedocromil. Anti-ANX-A1 neutralising monoclonal antibodies (Mabs) enhanced the release of pro-inflammatory mediators in response to compound 48/80.4.Nedocromil and ketotifen potently inhibited the release of histamine, PGD2, tryptase and β-hexosaminidase from mast cells challenged with compound 48/80. Anti-ANX-A1 neutralising Mabs prevented the inhibitory effect of these drugs.5.BMDMCs derived from Anx-A1−/− mice were insensitive to the inhibitory effects of nedocromil or ketotifen but cells retained their sensitivity to the inhibitory action of hu-r-ANX-A1.6.The fpr2/3 antagonist WRW4 blocked the action of nedocromil on PGD2, but not histamine, release. BMDMCs derived from fpr2/3−/− mice were insensitive to the inhibitory effects of nedocromil on PGD2, but not histamine release.7.Compound 48/80 stimulated both p38 and JNK phosphorylation in CBDMCs and this was inhibited by nedocromil. Inhibition of p38 phosphorylation was ANX-A1 dependent.8.We conclude that ANX-A1 is an important regulator of mast cell reactivity to compound 48/80 exerting a negative feedback effect through a mechanism that depends at least partly on the FPR receptor.

Diacylglycerol kinase γ regulates antigen-induced mast cell degranulation by mediating Ca2+ influxes

Diacylglycerol (DAG) is an important lipid that acts as a signaling messenger during mast cell degranulation after allergen cross-linking of immunoglobulin (Ig) E-bound FcεRI receptors. In this study, we determined the role of diacylglycerol kinase (DGK), which negatively regulates DAG-dependent signaling by converting DAG to phosphatidic acid (PA), in the regulation of mast cell degranulation. Treating RBL (rat basophilic leukemia)-2H3 mast cells with a type I DGK inhibitor significantly reduced antigen-induced degranulation and PA production. Among type I DGK isoforms, we observed that DGKα and DGKγ mRNAs were expressed in RBL-2H3 mast cells using reverse transcription polymerase chain reaction. DGKγ knockdown, but not DGKα, by isoform-specific short hairpin RNAs reduced mast cell degranulation and Ca2+ influxes from the extracellular environment. These results suggest that DGKγ regulates mast cell degranulation after FcεRI cross-linking through mobilization of intracellular Ca2+ through Ca2+ influxes.

Dual regulation of mast cell degranulation through IgE receptor-mediated modulation of M2-type pyruvate kinase

It was reported that mast cell degranulation is inversely related to the enzymatic activity of M₂-type pyruvate kinase (M₂PK). This study shows that activation of high-affinity IgE receptor (FcεRI) evokes a sequential dual regulation of M₂PK, i.e., an immediate decrement followed by slow phase increment of enzymatic activities. Changes in the activities of M₂PK and mast cell degranulation showed similar time course after antigenic stimulation of FcεRI. The immediate inhibition of M₂PK involved tyrosine phosphorylation, and subsequently led to a cellular accumulation of glycolytic intermediates, including fructose 1,6-biphosphate (FBP), a feedforward activator of M₂PK. As the cellular levels of FBP were increased, both the enzymatic acitivity of M₂PK and mast cell degranulation slowly returned to near basal levels. A-Raf, when exogenously introduced into RBL-2H3 cells, phosphorylated M₂PK on the serine residues, elevated enzyme activities of M₂PK, and resulted in the inhibition of degranulation. These results suggest that dual regulation of M₂PK which involves the phosphorylation of M₂PK and accumulation of a feedforward activator of M₂PK plays important roles in the control of mast cell degranulation.

Once Phosphorylated, Tyrosines in Carboxyl Terminus of Protein-tyrosine Kinase Syk Interact with Signaling Proteins, Including TULA-2, a Negative Regulator of Mast Cell Degranulation

Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.

Differential regulation of mast cell degranulation versus cytokine secretion by the actin regulatory proteins Coronin1a and Coronin1b

Mast cell (MC) activation via aggregation of the high affinity IgE receptor (FcεRI) causes degranulation and release of proinflammatory mediators in a process that involves the reorganization of the actin cytoskeleton. However, the regulatory pathways and the molecular links between cytoskeletal changes and MC function are incompletely understood. In this study, we provide genetic evidence for a critical role of the actin-regulatory proteins Coronin1a (Coro1a) and Coro1b on exocytic pathways in MCs: Coro1a(-/-) bone marrow-derived MCs exhibit increased FcεRI-mediated degranulation of secretory lysosomes but significantly reduced secretion of cytokines. Hyperdegranulation of Coro1a(-/-) MCs is further augmented by the additional loss of Coro1b. In vivo, Coro1a(-/-)Coro1b(-/-) mice displayed enhanced passive cutaneous anaphylaxis. Functional reconstitution assays revealed that the inhibitory effect of Coro1a on MC degranulation strictly correlates with cortical localization of Coro1a, requires its filamentous actin-binding activity, and is regulated by phosphorylation of Ser2 of Coro1a. Thus, coronin proteins, and in turn the actin cytoskeleton, exhibit a functional dichotomy as differential regulators of degranulation versus cytokine secretion in MC biology.

Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis

Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. Human umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Protein phosphatase 2A carboxymethylation and regulatory B subunits differentially regulate mast cell degranulation

Asthma is characterised by antigen-mediated mast cell degranulation resulting in secretion of inflammatory mediators. Protein phosphatase 2A (PP2A) is a serine/threonine protein phosphatase composed of a catalytic (PP2A-C) subunit together with a core scaffold (PP2A-A) subunit and a variable, regulatory (PP2A-B) subunit. Previous studies utilising pharmacological inhibition of protein phosphatases have suggested a positive regulatory role for PP2A in mast cell degranulation. In support of this we find that a high okadaic acid concentration (1 μM) inhibits mast cell degranulation. Strikingly, we now show that a low concentration of okadaic acid (0.1 μM) has the opposite effect, resulting in enhanced degranulation. Selective downregulation of the PP2A-Cα subunit by short hairpin RNA also enhanced degranulation of RBL-2H3 mast cells, suggesting that the primary role of PP2A is to negatively regulate degranulation. PP2A-B subunits are responsible for substrate specificity, and carboxymethylation of the PP2A-C subunit alters B subunit binding. We show here that carboxymethylation of PP2A-C is dynamically altered during degranulation and inhibition of methylation decreases degranulation. Moreover downregulation of the PP2A-Bα subunit resulted in decreased MK2 phosphorylation and degranulation, whilst downregulation of the PP2A-B′δ subunit enhanced p38 MAPK phosphorylation and degranulation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A-B subunit binding.

Licochalcones suppress degranulation by decreasing the intracellular Ca 2+ level and tyrosine phosphorylation of ERK in RBL-2H3 cells

Mast cells play a key role in allergic inflammation by releasing various mediators, such as histamine, serotonin, leukotrienes and cytokines. A signaling cascade of events activated by stimulation with antigens contributes to the regulation of mast cell degranulation. While various anti-inflammatory and anti-allergic drugs have been developed that inhibit degranulation of mast cells, the inhibitory mechanism has been poorly understood. Licochalcone A (Lico A) is a retrochalcone isolated from the root of Xinjiang liquorice and has been reported to exhibit various biological activities such as anti-inflammatory activity. We examined the effects of Lico A and related chalcones on degranulation in a rat basophilic leukemia cell line, RBL-2H3. Whereas Lico A and licochalcone C (Lico C) exhibited inhibitory activity with cytotoxicity, licochalcone D (Lico D) significantly inhibited the degranulation in RBL-2H3 cells with low cytotoxicity. Moreover, Lico D significantly inhibited the Ca 2+ influx and phosphorylation of extracellular signal regulated kinase (ERK) and MEK. These results suggest that Lico D inhibits mast cell degranulation via the inhibition of both extracellular Ca 2+ influx and activation of the MEK-ERK pathway.

A current understanding of FcɛRI-dependent mast cell activation

Mast cell activation via the high-affinity immunoglobulin (Ig) E receptor Fc epsilon RI is a topic of extensive investigation with therapeutic potential in allergic disease. The protein tyrosine kinases Fyn, Lyn, and Syk are intimately linked with the early events initiated by allergen-mediated aggregation of IgE-occupied Fc epsilon RI. Fyn and Lyn initiate signaling events that are organized by adaptor molecules, which compartmentalize and coordinate the activity of activated protein and lipid kinases and phospholipases to generate lipid products essential for signal amplification and mast cell function. Fyn and Lyn counter-regulate phosphatidylinositol 3-OH kinase (PI3K), controlling the produced amount of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a key regulator of mast cell degranulation. Fyn and Lyn also activate sphingosine kinases (SphK), which generate sphingosine-1-phosphate (S1P), thus contributing to mast cell chemotaxis and degranulation. Here, we summarize the current knowledge and future challenges and directions.

Histamine modulates mast cell degranulation through an indirect mechanism in a model IgE‐mediated reaction

Histamine is released in inflammatory reactions and exerts an immunoregulatory function on cells present in the microenvironment. In this study, we compared the effect of histamine on degranulation of mast cells derived from animals bearing a parasitic infection with those from uninfected animals. Peritoneal mast cells (PMC) were obtained 24 days after infection of Wistar rats with Toxocara canis. The degree of degranulation was assessed either morphologically or by measuring the release of beta-hexosaminidase and TNF-alpha. Non-purified PMC or mast cells immunomagnetically purified with mAb AA4 were used. An increase in degranulation of non-purified mast cells from infected animals was observed after incubation with histamine in vitro or when histamine was injected into the peritoneal cavity. When a purified mast cell population was used, this effect was no longer observed. Supernatants from spleen cells stimulated with histamine induced degranulation of purified mast cells, and again, this was potentiated with PMC from infected animals. However, when supernatants from peritoneal macrophages similarly stimulated were used, a reduction in the degranulation of PMC from infected animals was observed. Our results suggest that histamine may act as a regulator of mast cell degranulation, thus modulating inflammatory responses due to infection with certain parasites.

Regulation of mast cell degranulation in allergic eye disease by IL-10

Rationale Initial studies showed a panel of commonly used mouse strains, including A/J and C57Bl/6, to have significant variability in the development of allergic eye responses to a mast cell secretagogue, c48/80. As these mouse strains also showed significant differences in production of the immunomodulatory cytokine IL-10, the goal of these studies was to use C48/80 to assess a role for IL-10 on mast cell degranulation and allergic responses in vivo. Methods To ascertain a role for IL-10 on mast cell stability in the allergic response, we challenged either WT C57bl/6 or IL-10 KO mice with C48/80. Mice were then examined for changes in clinical symptoms of allergic disease. Eyes and attached conjunctiva were removed 1 hour after challenge, and were examined histologically for evidence of mast cell degranulation. Results While challenge with C48/80 caused mild symptoms of allergic eye disease including chemosis and edema, these symptoms were significantly increased in mice genetically depleted of IL-10. Histological examination of conjunctival tissues correlated well with disease patterns with enhanced degranulation in IL-10 KO mice. We have begun performing “add-back” experiments with rIL-10 to support a role for IL-10 in mast cell stability in vivo, and are in the process of establishing mast cell cultures to ascertain a role for IL-10 induced alterations in G-protein coupled signal transduction pathways. Conclusions IL-10 appears to increase mast cell stability and prevention of degranulation, suggesting an additional role for IL-10 in protection from the allergic response.