Natsuko Kageyama-Yahara

IgE-induced degranulation of mucosal mast cells is negatively regulated via nicotinic acetylcholine receptors.

The autonomic nervous system is known to mediate mast cell activation. We investigated expression of nicotinic acetylcholine receptors (nAChRs) in mucosal-type mast cells and their contribution to the regulation of mast cell activation. Expression of mRNA of nAChR alpha4, alpha7, and beta2 subunits were detected in specially differentiated mucosal-type murine bone marrow-derived mast cells (mBMMCs). Pretreatment with non-specific nAChRs agonists, acetylcholine, nicotine and epibatidine and a specific alpha7 subunit agonist GTS-21 significantly inhibited antigen-induced degranulation of mBMMCs in a dose-dependent manner and GTS-21-induced inhibition was significantly blocked by alpha7 subunit antagonist, alpha-bungarotoxin. Furthermore, confocal microscopy also demonstrated surface binding of alpha-bungarotoxin on mBMMCs. Our findings indicate that mucosal mast cell activation may be negatively regulated mainly through nAChR alpha7 subunit, suggesting that nAChRs are involved in neuronal-mucosal mast cell interactions.

Oral Tolerance Induced by Transfer of Food Antigens via Breast Milk of Allergic Mothers Prevents Offspring from Developing Allergic Symptoms in a Mouse Food Allergy Model

We examined whether maternal exposure to food antigens during lactation and maternal allergic status would affect the development of food allergy in offspring. OVA-sensitized or OVA-nonsensitized BALB/c female mice were exposed or unexposed to OVA during lactation. After weaning, their offspring were systemically sensitized twice with OVA and repeatedly given OVA by oral intubation. While 97.1% of the mice breastfed by OVA-nonsensitized and OVA-unexposed mothers developed allergic diarrhea, 59.7% of the mice breastfed by OVA-exposed nonallergic mothers during lactation and 24.6% of the mice breastfed by OVA-exposed allergic mothers during lactation developed food allergy. Furthermore, OVA was detected in breast-milk from OVA-exposed nonallergic mothers during lactation (4.6 ± 0.5 μg/mL). In addition, OVA-specific IgG1 titers were markedly increased in breast milk from allergic mothers (OVA-sensitized and OVA-unexposed mother: 11.0 ± 0.5, OVA-sensitized and OVA-exposed mother: 12.3 ± 0.3). Our results suggest that oral tolerance induced by breast milk-mediated transfer of dietary antigens along with their specific immunoglobulins to offspring leads to antigen-specific protection from food allergy.

Therapeutic Effect of Kakkonto in a Mouse Model of Food Allergy with Gastrointestinal Symptoms

Background: The number of patients with food allergy has increased dramatically over the last several decades. However, there is no effective drug for food allergies. In the present study, we evaluated the effects of kakkonto, a traditional Japanese herbal medicine, in a mouse model of food allergy with gastrointestinal symptoms. Methods: BALB/c mice were systemically sensitized twice with ovalbumin (OVA) and then were repeatedly given OVA by oral intubation (OVA mice). Kakkonto was administered orally before the OVA challenges. Results: The OVA mice developed allergic diarrhea (91.8 ± 3.8% after 6 OVA challenges), and myeloperoxidase (MPO) activity was dramatically elevated in the colons of the OVA mice. Kakkonto significantly suppressed the occurrence of allergic diarrhea and MPO activity in the OVA mice. Furthermore, the number of mucosal mast cells was greatly increased in the proximal colons of the OVA mice, and this was also suppressed by kakkonto. Interestingly, mRNA expression of helper T cell type 1 (Th1) cytokines (IFN-γ) and Th2 cytokines (IL-4, IL-5 and IL-10) were significantly upregulated in the proximal colons of the OVA mice, an effect which was also reduced by kakkonto. Transcriptome analysis detected increased mRNA expression of suppressor of cytokine signaling-3 in the proximal colons of OVA mice, which was decreased by kakkonto administration. Conclusion: Kakkonto has immunosuppressive effects and interferes with the infiltration of mucosal mast cells in the colons of mice with induced food allergy, leading to improvement of allergic symptoms. Kakkonto has potential as a therapeutic drug for treatment of allergic symptoms induced by the disruption of intestinal mucosal immunity.

Pentagalloylglucose down‐regulates mast cell surface FcεRI expression in vitro and in vivo

Mast cell activation by immunoglobulin E (IgE)‐mediated stimuli is a central event in the pathogenesis of allergic disorders. The present report shows that treatment with pentagalloylglucose (PGG) resulted in a down‐regulation of FcεRI surface expression on mucosal‐type murine bone marrow‐derived mast cells (mBMMCs), which correlated with a reduction in IgE‐mediated activation of mBMMCs. Furthermore, PGG prevented development of allergic diarrhea in a food‐allergy mouse model and suppressed the up‐regulated FcεRI surface expression on mast cells derived from the food‐allergy mouse colon. These findings on PGG suggest its therapeutic potential for allergic diseases through suppressing the FcεRI surface expression.

High glucose-induced apoptosis in human coronary artery endothelial cells involves up-regulation of death receptors

BackgroundHigh glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs).MethodsHCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice.ResultsExposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels.ConclusionsIn association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis.

The traditional herbal medicine saireito exerts its inhibitory effect on murine oxazolone-induced colitis via the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

Background: Ulcerative colitis is an intractable inflammatory colonic disease, and its etiology remains unclear. Saireito, a traditional herbal medicine, is widely used for treating ulcerative colitis in Japan. We analyzed the immunological characteristics of an oxazolone (OXZ)-induced colitis (OC) model and examined the effects of sareito on this model. Methods: OXZ was injected into the colon of BALB/c mice. Saireito was orally administered once a day for 3 consecutive days. Colitis was assessed by scoring the symptoms and macroscopic findings. The transcription patterns in the middle colon and spleen were analyzed with global transcriptome analysis and real-time polymerase chain reaction (PCR). Results: The above-mentioned scores were increased in the OC mice. The transcription levels of Th2 cytokines were significantly upregulated in the spleen and middle colon of the OC mice, whereas those of the Th1 cytokine interferon (IFN)-γ decreased in the spleen and increased in the middle colon. Saireito significantly ameliorated OC. In the middle colon of the saireito-treated mice, enhanced expression of Th2 cytokine mRNAs was markedly downregulated, while that of IFN-γ mRNA was further upregulated. In contrast, in the spleen, saireito had no effect on the transcription of either type of cytokine. After global transcriptome analysis, real-time PCR analysis revealed that saireito greatly downregulated the enhanced expression of the suppressor of cytokine signaling (SOCS)-3 mRNA in the middle colon of OC mice. Conclusions: Saireito exhibits inhibitory effects on OC by the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

Shikonin, a constituent of Lithospermum erythrorhizon exhibits anti-allergic effects by suppressing orphan nuclear receptor Nr4a family gene expression as a new prototype of calcineurin inhibitors in mast cells.

Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor.

Suppression of phospholipase Cγ1 phosphorylation by cinnamaldehyde inhibits antigen-induced extracellular calcium influx and degranulation in mucosal mast cells

Antigen-IgE-mediated mucosal mast-cell activation is critical in the development of food allergies. Cinnamaldehyde, a major constituent of Cinnamomi cortex, dose-dependently inhibited the antigen-IgE-induced degranulation of mucosal-type bone-marrow derived mast cells (mBMMCs) and RBL-2H3 cells. Cinnamaldehyde also suppressed the elevation of the intracellular Ca(2+) level that is induced by the extracellular Ca(2+) influx in antigen-IgE-stimulated mBMMCs. Furthermore, tyrosine phosphorylation of phospholipase C (PLC) γ1, which is a crucial activation switch for the intracellular Ca(2+) mobilization in mast cells, was attenuated by cinnamaldehyde. Together, our results demonstrated that cinnamaldehyde suppressed the intracellular Ca(2+) mobilization and the degranulation of mucosal mast cells by inhibiting the activity of the IgE receptor-PLCγ-Ca(2+) influx pathway. These findings suggest that cinnamaldehyde may have therapeutic potential in mucosal mast cell-related allergic diseases, such as food allergies.

Induction of Regulatory T Cells as a Novel Mechanism Underlying the Therapeutic Action of Kakkonto, a Traditional Japanese Herbal Medicine, in a Murine Food Allergy Model.

Background: The number of patients with food allergy (FA) has dramatically increased. Although satisfactory drug therapies for FA are not available, we have found that kakkonto, a traditional Japanese herbal medicine, suppressed the occurrence of allergic symptoms in an FA mouse model. Thus, we investigated whether kakkonto could regulate the activation and differentiation of T cells in the colon. Methods: BALB/c mice were systemically sensitized and then orally challenged with ovalbumin. FA mice were orally treated with kakkonto. Lamina propria (LP) cells from their colons were isolated and analyzed. Results: Kakkonto significantly reduced the proportion of CD69+ cells and the elevated helper T cell type 2-specific transcription factor GATA-3 mRNA expression in the LP CD4+ T cells, showing that kakkonto has a suppressive effect on the activation and Th2 differentiation of LP effector CD4+ T cells of the FA mouse colon. Furthermore, kakkonto significantly increased the proportion of Foxp3+CD4+ regulatory T cells in the LP CD4+ T cells of the FA mouse colon. Similarly, the number of Foxp3-positive cells was dramatically increased in the colonic mucosa of kakkonto-administered FA mice. However, the pharmacological effect and Foxp3+CD4+ regulatory T cell-inducing ability of kakkonto were not attenuated by the administration of an anti-CD25 monoclonal antibody in the FA model. Conclusions: The induction of Foxp3+CD4+CD25- regulatory T cells in the colon as a novel mechanism underlying the therapeutic action of kakkonto could be utilized for the development of a novel anti-FA drug.

Sphingosine kinase-1-dependent and -independent inhibitory effects of zanthoxyli fructus to attenuate the activation of mucosal mast cells and ameliorate food allergies in mice.

Food allergy (FA) is relatively a common disease in infants, but effective drug therapies are not yet available. Notably, mucosal mast cells, but not connective-tissue mast cells, play important roles in food allergic reactions via the release of inflammatory mediators. Therefore, we screened medicinal herb extracts for in vitro and in vivo antiallergic activity through inhibiting mucosal mast cell activation. As a result, both antigen-induced and calcium ionophore-induced degranulation was significantly inhibited by Zanthoxyli Fructus water extract (ZF) in mucosal-type murine bone marrow-derived mast cells (mBMMCs). ZF suppressed the antigen-induced [Ca2+]i elevation and the antigen-enhanced mRNA expression of TNF-α, IL-4, and IL-13. The transcriptome and real-time PCR analyses revealed that ZF greatly decreased the antigen-enhanced expression level of sphingosine kinase 1 (Sphk1), which plays a key role in the FcεRI-mediated immune responses in mast cells. Furthermore, ZF inhibited allergic symptoms in an ovalbumin-caused murine FA model and decreased the number of infiltrating mucosal mast cells and the enhanced mRNA expression levels of IL-4 and Sphk1 in the FA mice colons. These results indicate that ZF suppresses mucosal mast cell activities mainly through Sphk1-dependent mechanism, and ZF is utilized for the development of a novel, potent anti-FA agent.