Mucosal Mast Cell Degranulation Research Articles

Is Wheat Glutenin Extract Intrinsically Allergenic? Evaluation Using a Novel Adjuvant-Free Mouse Model of Systemic Anaphylaxis.

Wheat is a prominent allergenic food that can trigger life-threatening anaphylaxis. Presently, it remains unclear whether wheat glutenin (WG) extract possesses inherent sensitization potential independently, without the use of adjuvants, and whether it can sensitize mice to the extent of inducing life-threatening systemic anaphylaxis. In this study, we tested the hypothesis that repeated skin exposures to WG extract without adjuvant will sensitize mice with the resultant anaphylactic reaction upon systemic WG challenge. Balb/c mice were bred and maintained on a strict plant protein-free diet and were repeatedly exposed to a WG extract or vehicle once a week for 9 weeks. WG-specific (s)IgE and total (t)IgE levels were quantified. Mice were challenged with WG extract to induce anaphylactic reactions as measured by hypothermic shock response (HSR) and mucosal mast cell degranulation response (MMCR). We also conducted proteomic analysis of 120 spleen immune markers. These skin-sensitized mice exhibited exposure-dependent IgE responses and near-fatal anaphylaxis upon challenge. Proteomic analysis identified seven dramatically elevated immune biomarkers in anaphylactic mice. These data reveal that WG is intrinsically allergenic, and that chronic skin exposure to WG extract can prime the mice for potentially fatal anaphylaxis.

Chronic application of alcohol-soluble gluten extract over undamaged skin causes clinical sensitization for life-threatening anaphylaxis via activation of systemic Th2 immune responses in mice.

Gluten allergy is a major public health problem that is growing at an alarming rate. Specific mechanisms underlying sensitization to gluten remain incompletely understood. Currently, it is unclear whether chronic exposure to alcohol-soluble gluten extract via undamaged skin has the capacity to clinically sensitize mice for life-threatening anaphylaxis. Using an adjuvant-free mouse model, here we tested the hypothesis that chronic application of alcohol-soluble durum gluten (ASDG) extract will clinically sensitize mice for life-threatening anaphylaxis. This study was conducted in a gluten-free Balb/c mouse colony that was established and maintained on a plant protein-free diet. Groups of adult female mice were exposed dermally to ASDG extract or vehicle once a week for 9-weeks. Specific (s) and total (t) IgE levels were quantified. Mice were challenged systemically with ASDG to measure symptoms of systemic anaphylaxis. Hypothermic shock response (HSR) and mucosal mast cell degranulation response (MMCR) were determined upon challenge. Spleen Th1, Th2, and other immune markers were quantified. We found that chronic exposure to ASDG elicited robust elevation of sIgE and tIgE. Systemic challenge with ASDG, but not vehicle, elicited life-threatening anaphylaxis associated with dramatic HSR and MMCR. Correlation analysis demonstrated direct positive inter-relationships among IgE, HSR, and MMCR. Anaphylaxis was associated with significant elevation of prototypic Th2 but not Th1 immune markers in the spleen. Our study collectively demonstrates that ASDG is intrinsically allergenic; and chronic exposure to ASDG via undamaged skin can clinically sensitize mice for life-threatening anaphylaxis via activating the systemic Th2 immune responses.

Comparison of sensitizing and disease-eliciting capacities of salt-soluble protein extracts from diploid (einkorn) versus tetraploid (durum) wheats

Abstract Hypersensitivity reactions to wheat have reached significant levels of global public health concern for reasons that are incompletely understood. Einkorn wheat (Triticum monococcum, genome AA) is commercially available but is rarely consumed, whereas durum wheat (Triticum durum, genome AABB) is commonly used for pasta making. At present, most mouse models of wheat allergy are adjuvant-based, which fail to reveal the intrinsic allergenic potential of different wheat products. Here we compared einkorn and durum wheats in terms of their sensitizing and disease-eliciting capacities in an adjuvant-free mouse model of anaphylaxis induced by salt-soluble protein extract (SSPE). Balb/cJ female mice were sensitized transdermally with SSPE or saline for nine weeks. Later, systemic or oral anaphylactic reactions were elicited in these mice using SSPE via intraperitoneal or oral route, respectively. We did not observe major differences in sensitizing capacity as suggested by antibody responses (i.e., sIgE and tIgE levels) in SSPE-sensitized mice. However, SSPE from durum appears to be more potent than that from einkorn in eliciting systemic but not oral anaphylaxis as shown by hypothermic shock responses as well as mucosal mast cell degranulation. This is the first report on comparing the intrinsic allergenicity between tetraploid and diploid wheats in an adjuvant-free mouse model. This research was funded by: the United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA); Hatch project MICL02486 (Accession Number: 1012322), Hatch project MICL01699; Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876; and Project GREEEN (Michigan State University); R.J., was supported by the John Harvey Kellogg Graduate Assistantship and the Academic Achievement Graduate Assistantship from the Michigan State University.

Suppressing Mechanism of Mucosal Mast Cell Degranulation by Oral Immunotherapy in A Murine Food Allergy Model

Suppressing Mechanism of Mucosal Mast Cell Degranulation by Oral Immunotherapy in A Murine Food Allergy Model

The role of the stress‐activated transcription factor Nrf2 in OVA‐induced food allergy

Immune-mediated adverse reactions to food allergens are rising at a striking rate, but the underlying cause is unknown. The purpose of the present studies was to determine the effect of the food preservative tert-butylhydroquinone (tBHQ) on anaphylaxis in a mouse model of food allergy. Our preliminary studies demonstrate that skin-sensitization with OVA induced a markedly higher OVA-specific IgE response in mice on a tBHQ diet. Likewise, in response to OVA challenge by the oral route, a greater decrease in body temperature during anaphylaxis was observed in the tBHQ-treated group. Previously published in vitro studies suggest that tBHQ promotes Th2 polarization, a key step in the development of allergy, through activation of the stress-activated transcription factor, Nrf2. Therefore, we proposed the hypothesis that tBHQ exacerbates allergic response in ovalbumin (OVA) sensitized mice through activation of Nrf2. We implemented adoptive transfers of CD4 T cells from wild-type or Nrf2-null mice into immunodeficient SCID mice (all mice also received wild-type B cells). The study revealed marked genotype differences. Notably, dietary tBHQ had little effect on food allergy in recipients of Nrf2-deficient CD4 T cells. These mice showed markedly attenuated sensitization as compared to mice who received wild-type CD4 T cells. Strikingly, mice receiving Nrf2-deficient T cells did not become anaphylactic following OVA challenge. In contrast, tBHQ caused an increase in OVA-IgE plasma levels and a greater drop in body temperature in mice receiving wild-type CD4 T cells, suggesting these effects are Nrf2 dependent. In addition, recipients of Nrf2-deficient CD4 T cells showed a greatly attenuated percentage of Th2 cells and mucosal mast cell degranulation response, following OVA challenge. Taken together, these data suggest a critical role for Nrf2 during food allergen-induced sensitization and anaphylaxis and demonstrate that tBHQ exacerbates food allergy in a CD4 T cell- Nrf2 dependent manner in mice.

Protective effect of bergapten in acetic acid-induced colitis in rats

Bergapten (5-methoxysporalen) is a furanocoumarin extracted from several species of citrus and bergamot oil. Bergamot essential oil is used traditionally in the management of inflammatory conditions. Previous studies on bergapten have explored mainly its in vitro anti-inflammatory activities which include suppression of the expression and release of pro-inflammatory cytokines such as TNF-α and interleukins as well as prostaglandins. Bergapten enhances the clearance of neutrophils and macrophages from the site of inflammation and reduces oxidative stress by inhibition of reactive oxygen species (ROS). Bergapten was assessed for its anti-inflammatory properties in acetic acid-induced colitis. Animals were obtained and randomly placed in six (6) groups (n = 5) after acclimatization. Colitis was induced by rectal administration using 4% v/v acetic acid in Sprague Dawley rats after pre-treatment for 5 days. Bergapten was administered at doses of 3, 10, and 30 mg kg−1p.o. while the control group received saline 5 mL kg−1p.o. and the standard drug employed was sulphasalazine at a dose of 500 mg kg−1. Assessments made for colon-weight-to-length ratio, colonic injury, and mucosal mast cell degranulation. There were reduced colon-weight-to-length ratios in animals treated with bergapten which was significant (p < 0.5) for doses 10 and 30 mg kg−1 compared to the disease control group Both macroscopic and microscopic damage were reduced as well, with a lesser percentage of degranulated mast cells. Macroscopic damage was reduced for bergapten at doses 10 and 30 mg kg−1 significantly at p < 0.5 and p < 0.001, respectively. Similarly, microscopic damage was reduced at p < 0.01 and p < 0.001 respectively for bergapten 10 and 30 mg kg−1. The reduction of degranulation by bergapten was significant at p < 0.001. There was generally reduced damage at inflammatory sites as well as decreased infiltration of inflammatory cells. Overall, bergapten reduces inflammation in acetic acid-induced colitis.

Interleukin-9 promotes early mast cell-mediated expulsion of Strongyloides ratti but is dispensable for generation of protective memory

IL-9 is a cytokine with pleiotropic function that mediates allergic inflammation and immunity to intestinal helminth parasites. Accumulating evidence suggests that IL-9 acts via both, initiation and regulation of adaptive immune responses and direct activation of intestinal effector pathways. Here we use IL-9 receptor deficient mice on BALB/c and C57BL/6 genetic background to dissect effector and regulatory functions of IL-9 during infection with the parasitic nematode Strongyloides ratti. IL-9 receptor-deficient mice displayed increased intestinal parasite burden and prolonged infection irrespective of the genetic background of the mice. Increased parasite burden was correlated to a reciprocally reduced early degranulation of mucosal mast cells, reduced intestinal IL-13 expression and caused by IL-9 receptor deficiency on hematopoietic cells. We observed additional significant changes in the adaptive immune response to S. ratti infection in the absence of the IL-9 receptor that depended on the mouse strain. However, the generation of protective memory to a second infection was intact in IL-9 receptor-deficient mice, irrespective of the genetic background. In summary, our results support a central role for IL-9 as an early mast cell activating effector cytokine during intestinal helminth infection while non-redundant functions in the initiation and amplification of adaptive immune responses were not apparent.

Inhibition of IgE/antigen- and ionophore-induced mucosal mast cells degranulation by Alpinia galangal and acetoxychavicol acetate

Inhibition of IgE/antigen- and ionophore-induced mucosal mast cells degranulation by Alpinia galangal and acetoxychavicol acetate

A Mouse Model of Anaphylaxis and Atopic Dermatitis to Salt-Soluble Wheat Protein Extract

Background: Wheat allergy and other immune-mediated disorders triggered by wheat proteins are growing at an alarming rate for reasons not well understood. A mouse model to study hypersensitivity responses to salt-soluble wheat protein (SSWP) extract is currently unavailable. Here we tested the hypothesis that SSWP extract from wheat will induce sensitization as well as allergic disease in mice. Methods: Female BALB/cJ mice were weaned onto a plant protein-free diet. The mice were injected a total of 4 times with an SSWP (0.01 mg/mouse) fraction extracted from durum wheat along with alum as an adjuvant. Blood was collected biweekly and SSWP-specific IgE (SIgE) and total IgE (TIgE) levels were measured using ELISA. Systemic anaphylaxis upon intraperitoneal injection with SSWP was quantified by hypothermia shock response (HSR). Mucosal mast cell degranulation was measured by the elevation of mMCP-1 in the blood. The mice were monitored for dermatitis. Skin tissues were used in histopathology and for measuring cytokine/chemokine/adhesion molecule levels using a protein microarray system. Results: Injection with SSWP resulted in time-dependent SIgE antibody responses associated with the elevation of TIgE concentration. Challenge with SSWP elicited severe HSR that correlated with a significant elevation of plasma mMCP-1 levels. Sensitized mice developed facial dermatitis associated with mast cell degranulation. Lesions expressed significant elevation of Th2/Th17/Th1 cytokines and chemokines and E-selectin adhesion molecule. Conclusion: Here we report a mouse model of anaphylaxis and atopic dermatitis to SSWP extract that may be used for further basic and applied research on wheat allergy.

Eppikajutsuto Protects against Food Allergy Induced by Ovalbumin in a Murine Model

Background: Currently, there are no efficient medications available for the prevention and treatment of food allergy (FA). Herbal medicines, including traditional Japanese Kampo medicines (TJKMs), are promising therapeutic drugs. Methods: We screened 18 TJKMs for treatment of FA symptoms in a mouse FA model induced by ovalbumin (OVA). BALB/c mice were sensitized intraperitoneally by an OVA/aluminum hydroxide gel mixture followed by 4 booster doses of oral OVA and FA symptom induction by 50 mg of OVA. TJKMs were orally administered for 28 days from the day of sensitization to the day before FA symptom induction. Evaluated FA symptoms included a decrease in body temperature and allergic diarrhea. Allergic sensitization was determined by plasma OVA-specific IgE levels. Cytokine mRNA levels in mesenteric lymph nodes, plasma mouse mast cell protease-1, and the number of mast cells in the small and large intestines were analyzed. Additionally, the therapeutic effect of the TJKM eppikajutsuto (EJT) on mast cell degranulation was determined in active anaphylaxis and passive cutaneous anaphylaxis models. Results: EJT effectively prevented FA symptoms. Although OVA-specific IgE levels and the intestinal mast cell numbers were not different between the EJT-treated and untreated FA mice, plasma mMcpt1 and IL-4 levels were lower in EJT-treated FA mice than untreated FA mice. EJT could alleviate symptoms in both active and passive anaphylaxis models. Conclusion: EJT prevented OVA-induced FA symptoms in a mouse model, suggesting that EJT might exert its therapeutic activity via IL-4 suppression and the inhibition of mucosal mast cell degranulation.

Distinct immune and clinical responses to alcohol-soluble vs. salt-soluble wheat proteins in mice

Abstract Food hypersensitivity is a growing global public health problem. Wheat has been linked to distinct types of human hypersensitivities with incompletely understood mechanisms. Although wheat proteins (WP) differ in solubility, it is unknown whether alcohol-soluble (AS) and salt-soluble (SS) WP differ in their immune and disease activation properties. Here we tested the hypothesis that AS and SSWP extracted from soft-wheat will elicit distinct types of immune and clinical responses in inbred mice. Groups of BALB/cJ mice weaned onto a plant-protein free diet received IP injections with AS and SSWP (0.01 mg/mouse) along with alum. The WP-specific (S) IgE, IgG1, IgG2a, IgA and total (T) IgE responses were analyzed. Systemic anaphylaxis and mucosal mast cell degranulation response (MMCR) upon IP challenge were quantified by hypothermia shock response (HSR) and elevation of plasma mMCP-1, respectively. Mice were monitored for dermatitis and alopecia. Spleen tissue extracts were used in measuring immune-activation biomarkers. Whereas the SSWP elicited higher titers of SIgA, the ASWP elicited higher titers of SIgE, SIgG1, SIgG2a and higher TIgE levels. The ASWP elicited significantly higher HSR and MMCR. Only ASWP but not SSWP elicited dorsal neck dermatitis lesions in 44% of mice. Alopecia lesions, without inflammation, were noted in 22% of ASWP- and 33% of SSWP-injected mice. Furthermore, ASWP and SSWP elicited distinct patterns of activation of acute-phase response proteins, chemokines, cytokines, growth factors, matrix proteins and signaling proteins. These data provide the first direct evidence that WP fractions that differ in solubility elicit qualitatively distinct immune and clinical reactions in genetically identical mice.

CRH promotes mucosal type mast cells degranulation and increases their number in situ

CRH promotes mucosal type mast cells degranulation and increases their number in situ

Effect of extrusion processing on immune activation properties of hazelnut protein in a mouse model

Although food processing can alter food allergenicity, the impact of extrusion processing on in vivo hazelnut allergenicity is unknown. Here, we tested the hypothesis that extrusion processing will alter the immune activation properties of hazelnut protein (HNP) in mice. Soluble extrusion-processed HNP (EHNP) was prepared and evaluated for immune response using an established transdermal sensitization mouse model. Mice were sensitized with identical amounts of EHNP versus raw HNP. After confirming systemic IgE, IgG1 and IgG2a antibody responses, oral hypersensitivity reaction was quantified by hypothermia shock response (HSR). Mechanism was studied by measuring mucosal mast cell (MMC) degranulation. Compared to raw HNP, the EHNP elicited slower but similar IgE antibody (Ab) response, lower IgG1 but higher IgG2a Ab response. The EHNP exhibited significantly lower oral HSR as well as MMC degranulation capacity. These results demonstrate that the extrusion technology can be used to produce soluble HNP with altered immune activation properties.

Role of colonic mast cell infiltration and nerve growth factor in visceral hypersensitivity of irritable bowel syndrome

Objective To investigate the role of mucosal mast cells infiltration and degranulation with nerve growth factor (NGF) in development of visceral hypersensitivity in Sprague-Dawley (SD) rats. Methods The model of visceral hypersensitivity of irritable bowel syndrome (IBS) was established in 19 neonate SD rats with intestinal stimulation (rectalballon distention) on 8th, 10th and 12th postnatal days. The other 19 neonate SD rats without colonic distention were assigned to the control group. After rats grew up (six to eight weeks old), the visceral sensitivity was tested by abdominal withdrawal reflex (AWR) in 10 rats of each group. Mast cell infiltration and degranulation were observed with toluidine blue staining in colon tissue slides. The NGF level of intestinal tissues was detected by enzyme-linked immunosorbent assay (ELISA) methods in the left nine rats of each group. The culture system of dorsal root ganglias (DRG) from the neonatal rats was set up. The changes of electrophysilogical characters of DRG stimulated with NGF (100 ng/mL) for four days were recorded with patch-clamp.Paired t test was performed for comparison between groups. Results The results of AWR indicated that neonatal colonic stimulation could significantly increase visceral sensitivity after growing up. Under 20, 40 and 60 mmHg (1 mmHg=0.133 kPa) distention pressure, visceral sensitivity scores of visceral hypersensitivity rats and rats of control group were 1.00±0.50 vs 1.67±0.50, 1.89±0.31 vs 2.89±0.34 and 2.89±0.33 vs 3.89±0.33, the differences were statistically significant (t=-2.83, -6.00 and -6.00, all P<0.05). The results of master cells staining in tissue slides showed colonic master cells infiltration was obvious in rats with visceral hypersensitivity, and part of mast cells were degranulation. The result of ELISA demonstrated that NGF level of visceral hypersensitivity rats was significantly higher than that of control group ((11.07±3.06) pg/mg vs (2.38±1.88) pg/mg, t=-6.93, P<0.05). The results of electrophysilogical tests of primary cultured DRG indicated that compared with blank control growp, the action potential threshold of neuron in NGF 100 ng/mL group significantly decreased ((-18.0±2.1) mV vs (-29.0±2.5) mV, t=12.26, P<0.05) and discharge frequency increased ((5.0±1.4)/800 ms vs (12.0±3.2)/800 ms, t=-8.40, P<0.05). Meanwhile, neuron voltage-gated K+ current density remarkably decreased, most were sustained delayed rectifier K+ current (IK) decreasing ((279.0±48.0) pA/pF vs (203.0±39.0) pA/pF, t=6.18, P<0.05). Conclusion Colonic stimulation in neonatal rats could cause intestinal master cells infiltration and degranulation, which induced changes of neuron electrophysilogical characters and resulted in visceral hypersensitivity after growing up. Key words: Irritable bowel syndrome; Hyperesthesia; Ganglion, spinal; Mast cells; Nerve growth factor

Cutting Edge: the BTLA-HVEM regulatory pathway interferes with protective immunity to intestinal Helminth infection.

Helminths exploit intrinsic regulatory pathways of the mammalian immune system to dampen the immune response directed against them. In this article, we show that infection with the parasitic nematode Strongyloides ratti induced upregulation of the coinhibitory receptor B and T lymphocyte attenuator (BTLA) predominantly on CD4(+) T cells but also on a small fraction of innate leukocytes. Deficiency of either BTLA or its ligand herpes virus entry mediator (HVEM) resulted in reduced numbers of parasitic adults in the small intestine and reduced larval output throughout infection. Reduced parasite burden in BTLA- and HVEM-deficient mice was accompanied by accelerated degranulation of mucosal mast cells and increased Ag-specific production of the mast cell-activating cytokine IL-9. Our combined results support a model whereby BTLA on CD4(+) T cells and additional innate leukocytes is triggered by HVEM and delivers negative signals into BTLA(+) cells, thereby interfering with the protective immune response to this intestinal parasite.

CGRP, a neurotransmitter of enteric sensory neurons, contributes to the development of food allergy due to the augmentation of microtubule reorganization in mucosal mast cells.

Neuro-immune interaction in the gut is substantially involved in the maintenance of intestinal immune homeostasis and the pathology of intestinal immune diseases. We have previously demonstrated that mucosal mast cells and nerve fibers containing CGRP, a neurotransmitter of intrinsic enteric sensory neurons, are markedly increased and exist in close proximity to each other in the colon of food allergy (FA) mice. In the present study, a CGRP-receptor antagonist BIBN4096BS significantly alleviated allergic symptoms in the murine FA model. In addition, the elevated numbers of mucosal mast cells in the proximal colon of FA mice were significantly decreased in that of BIBN4096BS-treated FA mice. Thus, we investigated the effects of CGRP on calcium-independent process in degranulation of mucosal mast cells since CGRP increases intracellular cAMP levels, but not Ca(2+) concentration. CGRP did not alter a calcium ionophore A23187-increased cytosolic Ca(2+) concentration in mucosal-type bone marrow-derived mast cells (mBMMCs), but did augment microtubule reorganization in resting and A23187-activated mBMMCs. Furthermore, CGRP alone failed to cause the degranulation of mBMMCs, but CGRP significantly enhanced the degranulation of mBMMCs induced by A23187. Together, these data indicate that CGRP- enhanced microtubule reorganization augments IgE-independent/non-antigenic stimuli-induced mucosal mast cell degranulation, thereby contributing to the development of FA.

Vagal Afferents Mediate Antinociception of Estrogen in a Rat Model of Visceral Pain: The Involvement of Intestinal Mucosal Mast Cells and 5-Hydroxytryptamine 3 Signaling

Estrogen reportedly facilitates visceral nociception at the spinal or supraspinal level. The present study was aimed to investigate whether estrogen modulates visceral pain through the vagal pathway. Ovariectomized rats received estradiol, which was administered subcutaneously (to act through both the vagal and spinal pathways) or intraduodenally (to preferentially act through the vagal pathway). Luminally applied estradiol induced a rapid and significant decrease in the visceromotor response to colorectal distension, with increased c-Fos expression in nodose ganglion neurons. Systemically injected estradiol increased visceromotor response and c-Fos expression in both nodose and dorsal root ganglion (T6-12) neurons. The antinociceptive effect of estrogen was abolished by surgical vagotomy or chemical denervation of vagal afferents. Both luminally and systemically administered estradiol elicited selective 5-hydroxytryptamine secretion from the duodenum. Granisetron, a 5-hydroxytryptamine 3 receptor antagonist, reversed the antinociceptive effect of estrogen. Intestinal mucosal mast cell stabilizers prevented estradiol-induced antinociception and 5-hydroxytryptamine secretion. Ultrastructural analysis revealed that estradiol caused piecemeal degranulation of intestinal mucosal mast cells. The actions of estradiol were inhibited by an estrogen receptor β antagonist and mimicked by an estrogen receptor β agonist. These results suggest that estrogen can trigger vagus-mediated antinociception, which is masked by its spinally mediated pronociception. PerspectiveThis study is the first to show a vagus-mediated estrogenic antinociception, in which the nongenomic estrogen receptor β–mediated, intestinal mucosal mast cell–derived 5-hydroxytryptamine/5-hydroxytryptamine 3 receptor pathway is involved. This work may provide new insights into the sex hormone modulation of visceral sensitivity related to irritable bowel syndrome and indicate potential therapeutic targets to manage this disease.

Lipid-rich enteral nutrition regulates mucosal mast cell activation via the vagal anti-inflammatory reflex

Nutritional stimulation of the cholecystokinin-1 receptor (CCK-1R) and nicotinic acetylcholine receptor (nAChR)-mediated vagal reflex was shown to reduce inflammation and preserve intestinal integrity. Mast cells are important early effectors of the innate immune response; therefore modulation of mucosal mast cells is a potential therapeutic target to control the acute inflammatory response in the intestine. The present study investigates intestinal mast cell responsiveness upon nutritional activation of the vagal anti-inflammatory reflex during acute inflammation. Mucosal mast cell degranulation was induced in C57/Bl6 mice by administration of Salmonella enterica LPS. Lipid-rich enteral feeding prior to LPS significantly decreased circulatory levels of mouse mast cell protease at 30 min post-LPS compared with isocaloric low-lipid nutrition or fasting. CCK-1R blockage reversed the inhibitory effects of lipid-rich feeding, whereas stimulation of the peripheral CCK-1R mimicked nutritional mast cell inhibition. The effects of lipid-rich nutrition were negated by nAChR blockers chlorisondamine and α-bungarotoxin and vagal intestinal denervation. Accordingly, release of β-hexosaminidase by MC/9 mast cells following LPS or IgE-ovalbumin complexes was dose dependently inhibited by acetylcholine and nicotine. Application of GSK1345038A, a specific agonist of the nAChR α7, in bone marrow-derived mast cells from nAChR β2-/- and wild types indicated that cholinergic inhibition of mast cells is mediated by the nAChR α7 and is independent of the nAChR β2. Together, the present study reveals mucosal mast cells as a previously unknown target of the nutritional anti-inflammatory vagal reflex.

TRPM2 contributes to antigen-stimulated Ca2+ influx in mucosal mast cells

Food allergy (FA) is a common allergic disease without any currently available effective drug therapies. Mucosal mast cells (MMCs) play a particularly important role in FA, and the increase in their cytosolic Ca(2+) concentration ([Ca(2+)]cyt) is considered to be a principal component of the degranulation process. However, the mechanisms governing Ca(2+) influx remain poorly understood in MMCs. Recent reports have highlighted the functions of the transient receptor potential melastatin 2 (TRPM2) channel in immunocytes, including its role in monocyte chemokine production and macrophage phagocytic activity. Although TRPM2 gene expression has been demonstrated in mast cells, the significance of such expression remains virtually unknown. In this study, we found that antigen-stimulated degranulation was significantly reduced in mucosal-type bone marrow-derived mast cells (mBMMCs) prepared from TRPM2-knockout (TRPM2-KO) mice (TRPM2-KO mBMMCs) and was suppressed following the administration of three TRPM2 inhibitors with different chemical structures, including econazole, flufenamic acid (FFA), and 2-aminoethoxydiphenyl borate. Furthermore, the antigen-stimulated increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs and was also suppressed by the TRPM2 inhibitors econazole and FFA. In addition, thapsigargin-induced increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs. These results suggest that TRPM2 may participate in antigen-induced extracellular Ca(2+) influx and subsequent degranulation. In addition, TRPM2 inhibitors were shown to improve food allergic reactions in a mouse model. Together, these results suggest that TRPM2 inhibitors suppress MMC degranulation via regulation of the increase in [Ca(2+)]cyt. Thus, TRPM2 may play a key role in degranulation by modulating intracellular Ca(2+) in MMCs.

In vitro modelling of rat mucosal mast cell function in Trichinella spiralis infection

Intestinal infection with the parasitic nematode, Trichinella spiralis, provides a robust context for the study of mucosal mast cell function. In rats, mucosal mast cells are exposed to parasites during the earliest stage of infection, affording an opportunity for mast cells to contribute to an innate response to infection. During secondary infection, degranulation of rat mucosal mast cells coincides with expulsion of challenge larvae from the intestine. The goal of this study was to evaluate the rat bone marrow-derived mast cells (BMMC) and the rat basophilic leukaemia cell line (RBL-2H3) as models for mucosal mast cells, using parasite glycoproteins and antibody reagents that have been tested extensively in rats in vivo. We found that BMMC displayed a more robust mucosal phenotype. Although T. spiralis glycoproteins bound to mast cell surfaces in the absence of antibodies, they did not stimulate degranulation, nor did they inhibit degranulation triggered by immune complexes. Parasite glycoproteins complexed with specific monoclonal IgGs provoked release of rat mast cell protease II (RMCPII) and β-hexosaminidase from both cell types in a manner that replicated results observed previously in passively immunized rats. Our results document that RBL-2H3 cells and BMMC model rat mucosal mast cells in the contexts of innate and adaptive responses to T. spiralis.