Antigen-induced Anaphylaxis Research Articles

Elucidation of Mast Cell Activation Mechanism Mediated by Purinergic Signaling

Mast cells (MCs) are immune cells that are distributed in all tissues throughout the body, and their cytoplasm is rich in granules containing histamine and tryptase. When MCs recognize antigens through IgE bound to FcεRI, they release these mediators by degranulation. Because degranulation induces various type I allergic reactions, such as anaphylactic shock and hay fever, elucidation of the control mechanism of degranulation is important to the development of a therapeutic strategy for allergic diseases. It is known that the antigen-induced degranulation response is fine-tuned by various humoral factors via the activation of G protein-coupled receptors. We found that extracellular ATP enhanced antigen-dependent and -independent MC degranulation via activation of ionotropic P2X4 receptors. P2X4 receptor activation itself had no effect on MC degranulation, but significantly enhanced antigen-triggered degranulation. Stimulation of the P2X4 receptor potentiated the FcεRI-mediated tyrosine kinase signaling cascade. In addition to antigen-induced responses, P2X4 receptor signaling also affected antigen-independent MC responses. Thus, co-stimulation of ATP and Gi-coupled receptor agonists, such as prostaglandin E2 (PGE2) and adenosine, resulted in synergistic degranulation. The significance of P2X4 receptor signaling in allergic and inflammatory responses in vivo was confirmed by impaired responses of antigen-induced passive anaphylaxis and PGE2-induced increases in vascular permeability in P2rx4 knockout mice compared to that of wild-type mice. These results suggest that the P2X4 receptor is a potential therapeutic target for both antigen-dependent and -independent allergic reactions.

Dysregulation of intestinal epithelial CFTR-dependent Cl- ion transport and paracellular barrier function drives gastrointestinal symptoms of food-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of systemic and intestinal symptoms. Murine-based and clinical studies have revealed a role for histamine and H1R and H2R-pathway in the systemic response; however, the molecular processes that regulate the gastrointestinal (GI) response are not as well defined. In the present study, by utilizing an IgE-mast cell (MC)-dependent experimental model of oral antigen-induced anaphylaxis, we define the intestinal epithelial response during a food-induced anaphylactic reaction. We show that oral allergen-challenge stimulates a rapid dysregulation of intestinal epithelial transcellular and paracellular transport that was associated with the development of secretory diarrhea. Allergen-challenge induced (1) a rapid intestinal epithelial Cftr-dependent Cl- secretory response and (2) paracellular macromolecular leak that was associated with modification in epithelial intercellular junction proteins claudin-1, 2, 3 and 5, E-cadherin and desmosomal cadherins. OVA-induced Cftr-dependent Cl- secretion and junctional protein degradation was rapid occurring and was sustained for 72 h following allergen-challenge. Blockade of both the proteolytic activity and Cl- secretory response was required to alleviate intestinal symptoms of food-induced anaphylaxis. Collectively, these data suggest that the GI symptom of food-induced anaphylactic reaction, secretory diarrhea, is a consequence of CFTR-dependent Cl- secretion and proteolytic activity.

Deletion of ΔdblGata motif leads to increased predisposition and severity of IgE-mediated food-induced anaphylaxis response.

BackgroundPrevious studies have revealed an important role for the transcription factor GATA-1 in mast cell maturation and degranulation. However, there have been conflicting reports with respect to the requirement of GATA-1 function in mast cell dependent inflammatory processes. Herein, we examine the requirement of GATA-1 signaling in mast cell effector function and IgE-mast cell-dependent anaphylaxis.ObjectiveTo study the requirement of GATA-1 dependent signaling in the development and severity of IgE-mast cell-dependent anaphylaxis in mice.MethodsWild type (Balb/c) and mutant ΔdblGata (Balb/c) mice were employed to study the role of GATA-1 signaling in in vitro IgE-mediated activation of bone marrow derived mast cells (BMMCs). Murine models of passive IgE-mediated and oral antigen-induced IgE-mediated anaphylaxis were employed in mice. Frequency of steady state mast cells in various tissues (duodenum, ear, and tongue), peritoneal cavity, and clinical symptoms (diarrhea, shock, and mast cell activation) and intestinal Type 2 immune cell analysis including CD4+ Th2 cells, type 2 innate lymphoid cells (ILC2), and IL-9 secreting mucosal mast cells (MMC9) were assessedResultsIn vitro analysis revealed that ΔdblGata BMMCs exhibit a reduced maturation rate, decreased expression of FcεRIα, and degranulation capacity when compared to their wildtype (WT) counterparts. These in vitro differences did not impact tissue resident mast cell numbers, total IgE, and susceptibility to or severity of IgE-mediated passive anaphylaxis. Surprisingly, ΔdblGata mice were more susceptible to IgE-mast cell-mediated oral antigen induced anaphylaxis. The increased allergic response was associated with increased Type 2 immunity (antigen-specific IgE, and CD4+ TH2 cells), MMC9 cells and small intestine (SI) mast cell load.ConclusionDiminished GATA-1 activity results in reduced in vitro mast cell FcεRIα expression, proliferation, and degranulation activity. However, in vivo, diminished GATA-1 activity results in normal homeostatic tissue mast cell levels and increased antigen-induced CD4+ Th2 and iMMC9 cell levels and heightened IgE-mast cell mediated reactions.

Allergic Airway-Induced Hypersensitivity Is Attenuated by Bergapten in Murine Models of Inflammation

Bergapten (5-methoxypsoralen, 5-MOP) is a plant-derived furocoumarin with demonstrated anti-inflammatory action. The present study investigated its effects on allergic inflammation in two related pathways of mast cell degranulation. Compound 48/80 and lipopolysaccharide (LPS) were used to activate the IgE-independent pathway while bovine serum albumin (BSA) was used as allergen for the IgE-dependent pathway. The modulatory effect of bergapten on mast cell degranulation, neutrophil extravasation, protein concentration, lung histopathology, and oxidative stress was assessed. Bergapten at 10, 30, and 100 μg/ml for 15 min stabilized mast cells in rat mesenteric tissue from disruption in vitro and when administered in vivo at 3, 10, and 30 mg kg−1 for 1 h protected mice from fatal anaphylaxis induced by compound 48/80. Similarly, treatment of LPS-challenged mice with bergapten (3, 10, and 30 mg kg−1) for 24 h significantly decreased neutrophil infiltration into bronchoalveolar lavage fluid, mean protein concentration, and inflammatory cell infiltration of pulmonary tissues when compared to the saline-treated LPS-challenged control. In addition, lung histology of the bergapten-treated LPS-challenged mice showed significantly less oedema, congestion, and alveolar septa thickening when compared to the saline-treated LPS-challenged disease control. LPS-induced oxidative stress was significantly reduced through increased tissue activities of catalase and superoxide dismutase and reduced malondialdehyde levels on treatment with bergapten. In the triple antigen-induced active anaphylaxis, daily administration of bergapten at 3, 10, and 30 mg kg−1 for 10 days, respectively, protected previously sensitized and challenged mice against anaphylactic shock. Overall, our study demonstrates the ability of bergapten to attenuate allergic airway-induced hypersensitivity in murine models of inflammation, suggesting its possible therapeutic benefit in this condition.

Fructo-Oligosaccharide Alleviates Soybean-Induced Anaphylaxis in Piglets by Modulating Gut Microbes.

Soybean-induced anaphylaxis poses a severe threat to the health of humans and animals. Some commensal bacteria, such as Lactobacillus and Bifidobacteria, can prevent and treat allergic diseases. Prebiotic oligosaccharides, a food/diet additive, can enhance health and performance via modulating gut microbes and immune responses. The purpose of this study was to examine whether fructo-oligosaccharides (FOS) could alleviate soybean-induced anaphylaxis by modulating gut microbes. Piglets (21 days of age) were sensitized with a diet containing 5% soybean and 30% peeled soybean meal. The treatment with 0.6% FOS started 1 day prior to sensitization and continued everyday thereafter. Blood was collected for measurements of immune indices. The DNA samples isolated from fresh intestinal contents of the middle jejunum (M-jejunum), posterior jejunum (P-jejunum), ileum, and cecum were used for gene sequencing based on 16S rRNA. Our results showed that there was an increase of glycinin-specific IgG, β-conglycinin-specific IgG, total serum IgG and IgE, and occurrence of diarrhea in piglets sensitized with soybean antigen. There was a decrease in interleukin 4 (IL-4) and IL-10 and an increase of interferon-γ (IFN-γ) in piglets with FOS treatment, compared with the piglets without FOS treatment. Improvement of intestinal microbes was indicated mostly by the increase of Lactobacillus and Bifidobacteria in M-jejunum and the decrease of Proteobacteria in P-jejunum and ileum. The correlation analysis indicated that FOS treatment decreased those closely related to the key species of gut microbes. These results suggest that FOS can alleviate soybean antigen-induced anaphylaxis, which is associated with increased Lactobacillus and Bifidobacteria in M-jejunum and declined Proteobacteria in P-jejunum and ileum of piglets.

C5a receptor 1-/- mice are protected from the development of IgE-mediated experimental food allergy.

Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Reprograming immunity to food allergens

A novel immune-modulatory therapy utilizing targeted delivery of cytokines to draining lymph nodes effectively reprograms Th2 allergic responses towards a Th1 and tolerogenic profile, resulting in protection from peanut antigen-induced anaphylaxis.

Platelet Activation is Increased in IgE-mediated Systemic Anaphylaxis but not in IgE-mediated Oral Antigen-induced Anaphylaxis in Mice

Platelet Activation is Increased in IgE-mediated Systemic Anaphylaxis but not in IgE-mediated Oral Antigen-induced Anaphylaxis in Mice

Role of Histamine-releasing Factor in Allergic Inflammatory Reactions.

Mast cells are effector cells in immunoglobulin E (IgE)-mediated immediate hypersensitivity and allergic diseases such as asthma and food allergy. Mast cells are activated by the aggregation of the IgE-bound high-affinity IgE receptor FcεRI with multivalent antigen. Activated mast cells secrete proinflammatory mediators such as histamine, serotonin, and proteases and produce cytokines and chemokines. However, it has been reported that mast cells are activated by crosslinking of FcεRI with monomeric IgE in the absence of antigen. We have recently demonstrated that histamine-releasing factor (HRF) is involved in IgE-mediated mast cell activation both in vitro and in vivo. HRF binds to a subset of IgE and IgG molecules [HRF-reactive antibodies (Abs)]. The Fab, but not Fc, portions of the IgE and IgG molecules are HRF-binding sites, and the N-terminal 19-residue (N19) and H3 portions of HRF are HRF-reactive Ab-binding sites. We observed that both N19 and H3 tagged with glutathione S transferase (GST) (GST-N19 and GST-H3) can inhibit the interaction between HRF and HRF-reactive Abs. Using acute- and late-phase passive cutaneous anaphylaxis mouse models, it was shown that HRF initiates mast cell activation through HRF-reactive, but not HRF-nonreactive, IgE in vivo. Antigen-induced passive cutaneous anaphylaxis was inhibited by pretreatment with GST-N19 and GST-H3. We demonstrated that pretreatment with GST-N19 before antigen challenge inhibited antigen-induced mast cell-dependent airway inflammation. In addition, GST-N19 partially inhibited Aspergillus fumigatus extract-induced IgE-dependent airway inflammation. However, GST-N19 did not inhibit T cell-dependent airway inflammation. These results suggest that mast cells are target cells for HRF to initiate IgE- and mast cell-dependent airway inflammation.

Experimental food-induced anaphylaxis is driven by C5aR1 actvivation on mast cells

Abstract Food-induced anaphylaxis is a serious allergic reaction mainly caused by antigen cross-linking of IgE-loaded Fce-receptors on mast cells (MCs). Such MC activation leads to the release of pro-inflammatory mediators which results in the manifestation of the disease. Here, we demonstrate that the complement fragment C5a plays an important and until now not recognized role in the development and severity of food allergy. We subjected C5aR1−/− and wild-type (wt) Balb/c mice to oral ovalbumin (OVA)-induced anaphylaxis. We observed that 78% of wt but only 9% of C5aR1−/− mice suffered from diarrhea and hypothermia after the 7th oral OVA challenge. Importantly, peritoneal MCs strongly expressed C5aR1 after the 7th oral OVA treatment. OVA-specific serum IgE and MCPT-1 levels were significantly decreased in C5aR1-deficient as compared with wt mice. To directly examine the impact of the C5a/C5aR1 axis on MC function, we generated bone marrow-derived MCs (BMMCs) and tested their activation and degranulation potency in response to FceR1 cross-linking. Degranulation of C5aR1−/− BMMCs was reduced by 50% and IL-6 production was decreased by 35% as compared with wt cells. Also, FceR1 cross-linking markedly upregulated C5aR1 MC expression. Further, in contrast to wt mice, C5aR1−/− mice were largely protected from histamine-induced temperature drop in vivo. In line with this finding, C5aR targeting protected human endothelial cells from histamine-induced barrier dysfunction and disruption of tight junction proteins in vitro. Our findings identify a critical role of the C5a/C5aR1 axis in IgE-mediated oral antigen-induced anaphylaxis. C5aR1 may serve as a novel therapeutic target to suppress the inflammatory response in food-induced anaphylaxis.

Nucleoside 5′-O-monophosphorothioates as modulators of the P2Y14 receptor and mast cell degranulation

Mast cells (MCs) are long-lived resident cells known for their substantial role in antigen-induced anaphylaxis and other immunoglobulin E-mediated allergic reactions as well as tumor promotion. MCs' activation results in the release of pro-inflammatory factors such as histamine, tryptase, tumor necrosis factor or carboxypeptidase A stored in secretory granules. IgE-dependent hypersensitivity has been thought to be the major pathway mediating degranulation of mast cells, but the P2Y14 nucleotide receptor activated by UDP-glucose (UDPG) may also enhance this process. In this study we identified thymidine 5'-O-monophosphorothioate (TMPS) as a molecule inhibiting UDPG-induced degranulation in a rat mast cell line (RBL-2H3). Additionally, TMPS diminished UDPG-evoked intracellular calcium mobilization in a stable HEK293T cell line overexpressing the P2Y14 receptor. Therefore, we demonstrate that the use of thymidine 5'-O-monophosphorothioate might be a novel anti-inflammatory approach based on preventingmast cell activation.

Calcium-binding protein, spermatid-specific 1 is expressed in human salivary glands and contains an anti-inflammatory motif.

Salivary glands are involved in the production and exocrine and endocrine secretion of biologically active proteins, polypeptides, and hormones involved in growth and differentiation, homeostasis, and digestion. We have previously studied the prohormone submandibular rat 1 (SMR1), product of the Vcsa1 gene, which is highly expressed in the testes and salivary glands of rats, and can be cleaved to produce polypeptides with analgesic, erectile function, and anti-inflammatory activities. Humans lack the Vcsa1 gene, but homologous sequences and functions for analgesia and erectile function exist in the human genes Prol1, SMR3a, and SMR3b located on the human chromosomal region close to where Vcsa1 lies in the rat. Here we show the human protein calcium-binding protein spermatid-specific 1 (CABS1) contains a similar sequence to the anti-inflammatory sequence in rat SMR1, thus CABS1 may be another human gene with homologous function to Vcsa1. Using Western blot and PCR, we discovered that the human protein CABS1, previously thought to only be expressed in the testes, is also expressed in the salivary glands and lung, in a tissue-specific manner. Peptides derived from CABS1 were tested in an in vivo mouse model of lipopolysaccharide (LPS)-induced neutrophilia and an ex vivo rat model of antigen-induced intestinal anaphylaxis and significantly reduced both neutrophil accumulation in bronchoalveolar lavage fluid and antigen-induced ileal contractions, respectively. Thus human CABS1 has a peptide motif homologous to the anti-inflammatory peptide sequence of rat SMR1. Whether this similarity of CABS1 extends to the neuroendocrine regulation of the anti-inflammatory activity seen for SMR1 remains to be determined.

Novel IL-9-Secreting innate helper cells promote oral antigen-induced anaphylaxis (P6249)

Abstract IgE-mediated food allergy is manifested by an overactive T-helper 2 immune response to dietary antigens in the gastrointestinal (GI) tract. Interleukin (IL)-9 promote intestinal mastocytosis which allow the development of oral antigen-induced anaphylaxis. However, little is known about the primary cellular sources of IL-9 or the mechanisms that promote intestinal mastocytosis. Here, we report the identification of multifunctional IL-9-producing innate helper cells (IHC9) that secrete prodigious amounts of TH2 cytokines and mast cell protease-1 (MCPt-1) in response to IL-33 and antigen/IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induces significant accumulations of both intestinal IHC9 and CD4+TH2 cells, which correlate positively with symptoms and susceptibility to oral antigen-induced anaphylaxis. IHC9 development requires T cells and IL-4R, not IL-9R, signaling. The failure of irradiated mice reconstituted with STAT6-deficient bone marrow to generate IHC9 was accompanied by reduced serum MCPt-1, intestinal TH2 inflammation, mastocytosis, and goblet cell hyperplasia and loss of allergic diarrhea. Furthermore, inhibition of food allergy symptoms in mice ablated of IHC9 by anti-FcϵRIα antibody treatment could be reversed by adoptive transfer of IHC9. Thus, IHC9 represent a new type of innate helper cell lineage and function in promoting intestinal TH2 inflammation and mastocytosis that drive IgE-mediated anaphylaxis.

Monitoring neutrophils and platelets during casein-induced anaphylaxis in an experimental BALB/c mouse model

With respect to the cellular players, mast cells and basophils have been well studied in experimental murine systemic anaphylaxis models, but the role of neutrophils and platelets is not fully understood today. We tested the hypothesis that neutrophils and platelets might participate in an antigen-induced anaphylaxis model. BALB/c mice were sensitized intraperitoneally with alum-adsorbed casein. A period of 2 weeks later, mice were challenged with 100 μg casein intravenously and immediate hypersensitivity reactions were assessed by rectal temperature measurements and monitoring the physical activity. Subsequently, leucocytes were counted in the peripheral blood as well as quantified in situ in typical shock organs like lung, liver and spleen, heart and kidney. Mice sensitized with casein showed casein-specific IgG1, IgE, and IgG2a. When sensitized mice were specifically challenged with casein they developed immediate hypersensitivity reactions including drop of temperature and reduced activity. Furthermore, pronounced peripheral neutropenia and reduced platelet counts correlated with the severity of the hypersensitivity reactions. In the histological analyses of collected tissues we observed lung interstitial neutrophilia using Gr-1 staining. These events occurred specifically in mice sensitized and challenged with casein, in contrast to control groups. On the basis of our data we suggest that in addition to mast cells and basophils, neutrophils and platelets participate in the anaphylactic response in this BALB/c mouse model. Platelet and neutrophils expressing relevant immunoglobulin receptors may therefore have a synergistic effect with allergen specific IgE as well as IgG antibodies in food-induced anaphylaxis. We suggest that management of these cells could be of clinical importance to handle anaphylaxis.

Intestinal Mast Cell Levels Control Severity of Oral Antigen-Induced Anaphylaxis in Mice

Food-triggered anaphylaxis can encompass a variety of symptoms that affect multiple organ systems and can be life threatening. The molecular distinction between non-life-threatening and life-threatening modes of such anaphylaxis has not yet been delineated. In this study, we sought to identify the specific immune functions that regulate the severity of oral antigen-induced anaphylaxis. We thus developed an experimental mouse model in which repeated oral challenge of ovalbumin-primed mice induced an FcεRI- and IgE-dependent oral antigen-triggered anaphylaxis that involved multiple organ systems. Strikingly, the severity of the systemic symptoms of anaphylaxis (eg, hypothermia) positively correlated with the levels of intestinal mast cells (r = -0.53; P < 0.009). In addition, transgenic mice with both increased intestinal and normal systemic levels of mast cells showed increased severity of both intestinal and extra-intestinal symptoms of IgE-mediated passive as well as oral antigen- and IgE-triggered anaphylaxis. In conclusion, these observations indicate that the density of intestinal mast cells controls the severity of oral antigen-induced anaphylaxis. Thus, an awareness of intestinal mast cell levels in patients with food allergies may aid in determining their susceptibility to life-threatening anaphylaxis and may eventually aid in the treatment of food-triggered anaphylaxis.

Dietary Intervention With Specific Non-digestible Oligosaccharide Mixtures Is Effective During Induction Of Murine Cow'S Milk Allergy But Not Suitable As Treatment In The Same Allergic Disease Model

RationaleIn this study, the effects of dietary supplementation with specific non-digestible oligosaccharides (prebiotics) either as single components or in mixtures on the outcome of the murine allergic response when provided before and during oral sensitization with whey were investigated. In addition, the feasibility to treat allergic mice with these prebiotics was addressed.MethodsMice were fed diets containing 1% single prebiotics, combinations of scGOS (short chain galacto-oligosaccharides), lcFOS (long chain fructo- oligosaccharides) and/or pAOS (pectin-derived acidic oligosaccharides) or a control diet. The diets were provided two weeks prior to and during oral whey-sensitization (prevention) or for four weeks starting one week after the last sensitization (treatment). The acute allergic skin response (ITH) and antigen-induced anaphylaxis (AiA) were determined one hour after intradermal whey-challenge. Whey-IgE/G1/G2a and mouse mast cell protease-1 (mMCP-1) were determined in serum.ResultsIn the whey-sensitized preventive intervention groups, only combination of scGOS/lcFOS (9:1) and scGOS/lcFOS/pAOS (9:1:2) strongly reduced both ITH (p<0.001) and AiA compared to controls. scGOS/lcFOS/pAOS was most effective, whereas single components were not. mMCP-1 tended to be reduced in the whey-sensitized mice fed scGOS/lcFOS(/pAOS), although the whey-IgE/G1/G2a remained high. In the treatment groups, allergic disease was confirmed prior to the diet intervention by presence of whey-IgE, ITH and AiA. Four weeks of dietary intervention with scGOS/lcFOS(/pAOS) did not alter the allergenic response compared to controls.ConclusionsDietary supplementation with scGOS/lcFOS(/pAOS) provided as prevention reduced the allergic effector response. However, these diets were reluctant to treat allergy in the chosen intervention protocol in the murine model of IgE-mediated hypersensitivity. RationaleIn this study, the effects of dietary supplementation with specific non-digestible oligosaccharides (prebiotics) either as single components or in mixtures on the outcome of the murine allergic response when provided before and during oral sensitization with whey were investigated. In addition, the feasibility to treat allergic mice with these prebiotics was addressed. In this study, the effects of dietary supplementation with specific non-digestible oligosaccharides (prebiotics) either as single components or in mixtures on the outcome of the murine allergic response when provided before and during oral sensitization with whey were investigated. In addition, the feasibility to treat allergic mice with these prebiotics was addressed. MethodsMice were fed diets containing 1% single prebiotics, combinations of scGOS (short chain galacto-oligosaccharides), lcFOS (long chain fructo- oligosaccharides) and/or pAOS (pectin-derived acidic oligosaccharides) or a control diet. The diets were provided two weeks prior to and during oral whey-sensitization (prevention) or for four weeks starting one week after the last sensitization (treatment). The acute allergic skin response (ITH) and antigen-induced anaphylaxis (AiA) were determined one hour after intradermal whey-challenge. Whey-IgE/G1/G2a and mouse mast cell protease-1 (mMCP-1) were determined in serum. Mice were fed diets containing 1% single prebiotics, combinations of scGOS (short chain galacto-oligosaccharides), lcFOS (long chain fructo- oligosaccharides) and/or pAOS (pectin-derived acidic oligosaccharides) or a control diet. The diets were provided two weeks prior to and during oral whey-sensitization (prevention) or for four weeks starting one week after the last sensitization (treatment). The acute allergic skin response (ITH) and antigen-induced anaphylaxis (AiA) were determined one hour after intradermal whey-challenge. Whey-IgE/G1/G2a and mouse mast cell protease-1 (mMCP-1) were determined in serum. ResultsIn the whey-sensitized preventive intervention groups, only combination of scGOS/lcFOS (9:1) and scGOS/lcFOS/pAOS (9:1:2) strongly reduced both ITH (p<0.001) and AiA compared to controls. scGOS/lcFOS/pAOS was most effective, whereas single components were not. mMCP-1 tended to be reduced in the whey-sensitized mice fed scGOS/lcFOS(/pAOS), although the whey-IgE/G1/G2a remained high. In the treatment groups, allergic disease was confirmed prior to the diet intervention by presence of whey-IgE, ITH and AiA. Four weeks of dietary intervention with scGOS/lcFOS(/pAOS) did not alter the allergenic response compared to controls. In the whey-sensitized preventive intervention groups, only combination of scGOS/lcFOS (9:1) and scGOS/lcFOS/pAOS (9:1:2) strongly reduced both ITH (p<0.001) and AiA compared to controls. scGOS/lcFOS/pAOS was most effective, whereas single components were not. mMCP-1 tended to be reduced in the whey-sensitized mice fed scGOS/lcFOS(/pAOS), although the whey-IgE/G1/G2a remained high. In the treatment groups, allergic disease was confirmed prior to the diet intervention by presence of whey-IgE, ITH and AiA. Four weeks of dietary intervention with scGOS/lcFOS(/pAOS) did not alter the allergenic response compared to controls. ConclusionsDietary supplementation with scGOS/lcFOS(/pAOS) provided as prevention reduced the allergic effector response. However, these diets were reluctant to treat allergy in the chosen intervention protocol in the murine model of IgE-mediated hypersensitivity. Dietary supplementation with scGOS/lcFOS(/pAOS) provided as prevention reduced the allergic effector response. However, these diets were reluctant to treat allergy in the chosen intervention protocol in the murine model of IgE-mediated hypersensitivity.

PEST‐domain‐enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice

PEST-domain-enriched tyrosine phosphatase (PEP) is a protein tyrosine phosphatase exclusively expressed in hematopoietic cells. It is a potent negative regulator of T-cell receptor signalling that acts on receptor-coupled protein tyrosine kinases. PEST-domain-enriched tyrosine phosphatase is also expressed in mast cell and is positively regulated by glucocorticoids, but its function is unknown. In this communication, the function of PEP is analysed in mast cells. Signal transduction cascades following IgE receptor cross-linking were compared in bone marrow-derived mast cells (BMMC) from PEP(-/-) and PEP(+/+) mice. Furthermore, antigen-induced passive systemic anaphylaxis (PSA) was analysed in PEP(+/+) and PEP(-/-) mice. Bone marrow-derived mast cells from PEP(-/-) mice showed impaired PLCγ1 phosphorylation and Ca(2+) mobilization. Additionally, mice deficient in PEP showed impaired mast cell degranulation and were less susceptible to PSA. Treatment of wild-type BMMC or mice with an Au(I)-phosphine complex that selectively inhibits PEP activity produced defects in Ca(2+) signalling pathway and reduced anaphylaxis similar to that caused by the deletion of the PEP gene. Glucocorticoid that negatively regulates a wide range of mast cell action increased PEP expression and only partially inhibited anaphylaxis. However, glucocorticoid potently inhibited anaphylaxis when combined with the PEP inhibitor. PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

Tyrosine kinase inhibitor sunitinib relieves systemic and oral antigen‐induced anaphylaxes in mice

Systemic and oral antigen-induced anaphylaxes are mediated by immunoglobulin (Ig) E and mast cells, but there is no satisfactory treatment for the life-threatening allergic reaction. We investigated the potential of the multitargeted receptor tyrosine kinase inhibitor sunitinib to relieve anaphylactic reactions in food allergy and systemic anaphylaxis. Efficacy of oral sunitinib on oral and parenteral antigen-induced anaphylaxes in Balb/c mice was evaluated. IgE-dependent degranulation and growth of rat basophilic leukemia RBL2H3 and bone marrow-derived mast cells (BMMCs) in response to sunitinib were investigated. Daily administration of sunitinib throughout antigen challenges prevented oral antigen-induced anaphylaxis including diarrhea, anaphylactic symptoms, and hypothermia. The mouse mast cell protease (MMCP)-1 concentration in serum and mast cell number in intestinal tissue after challenge were also decreased by the treatment. Spleen cells from sunitinib-treated mice contained smaller numbers of antigen-specific IgG-producing cells and secreted lower amounts of both Th1 and Th2 cytokines than those of the control mice, whereas the levels of antigen-specific antibodies in serum were not decreased. The reactions and MMCP-1 release in oral antigen-induced anaphylaxis and passive systemic anaphylaxis were attenuated even by a single predose of sunitinib. Degranulation and growth of RBL2H3 cells and BMMCs were greatly reduced by sunitinib. These results suggested that sunitinib relieves systemic and oral antigen-induced anaphylaxes by the prevention of mast cell activation and hyperplasia in intestinal tissue directly and indirectly through an immunosuppressive effect. Sunitinib and its related kinase inhibitors might be potential drugs for the treatment of food allergy and systemic anaphylaxis.

The ethyl acetate extract of Cordyceps militaris inhibits IgE-mediated allergic responses in mast cells and passive cutaneous anaphylaxis reaction in mice

Cordyceps militaris has been used as a traditional herbal medicine for treating allergy in East Asia. We investigated the anti-allergic efficacy of Cordyceps militaris and its mechanism of action. β-Hexosaminidase release of mast cells, a key parameter of degranulation, was evaluated. Anti-allergic potential of Cordyceps militaris was studied using passive cutaneous anaphylaxis (PCA) in vivo. The anti-allergic mechanism of Cordyceps militaris was investigated by immunoblotting analysis, RT-PCR and other biological approaches in mast cells. GSCM EtOAc extract (GSCME) inhibited antigen-induced degranulation with a IC50 value (28.5 μg/ml) in RBL-2H3 cells and antigen-induced passive cutaneous anaphylaxis (PCA) response with a ED50 value (665 mg/kg) in vivo. The release of interleukin (IL-4) and tumor necrosis factor (TNF-α were decreased by GSCME in RBL-2H3 cells. In order to elucidate the anti-allergic mechanisms of GSCME in mast cells, we examined the activated levels of signaling molecules. GSCME inhibited the phosphorylation Syk, ERK, p38 and JNK expression. Identified genistein, daidzein, genistein 7-O-β-d-glucoside 4″-O-methylate, genistein 4'-O-β-d-glucoside 4″-O-methylate, glycitein 7-O-β-d-glucoside 4″-O-methylate, daidzein 7-O-β-d-glucoside 4″-O-methylate and adenosine in GSCME, inhibited antigen-induced degranulation in RBL-2H3 cells. Our study suggests that GSCME might be used as a therapeutic agent for allergic diseases.

Differential roles for the IL-9/IL-9 receptor α-chain pathway in systemic and oral antigen–induced anaphylaxis

The cytokine IL-9 has been implicated in allergic reactions, including food allergy, but its contribution to parenteral versus oral antigen-induced anaphylaxis remains unclear. We sought to delineate the contribution of the IL-9/IL-9 receptor alpha-chain (IL-9R) pathway to parenteral and oral antigen-induced anaphylaxis. Wild-type, IL-9-deficient (Il9(-/-)), and IL-9R-deficient (Il9R(-/-)) mice were subjected to passive and active parenteral and oral antigen (ovalbumin [OVA])-induced anaphylaxis. Severity of systemic anaphylaxis was gauged by decreased body temperature; intestinal anaphylaxis was assessed based on secretory diarrhea, intestinal mastocytosis, and serum murine mast cell protease 1 level. Specific immunoglobulin isotypes or immunoglobulin receptor-blocking antibodies were administered before challenge to define the role of the IgE and IgG pathways. Repeated oral antigen challenge of OVA-sensitized wild-type mice induced anaphylaxis with both systemic and intestinal involvement; both were IgE dependent and attenuated in Il9(-/-) and Il9R(-/-) mice. In contrast, parenteral OVA challenge of OVA-sensitized wild-type mice induced systemic anaphylaxis, which was independent of the IL-9/IL-9R pathway. Strikingly, the IL-9/IL-9R pathway had no role in either the IgG or IgE component of parenteral antigen-induced or anti-IgE and anti-FcgammaRII/III mAb-induced systemic anaphylaxis. Parenteral antigen-induced murine systemic anaphylaxis is mediated by both IgG- and IgE-dependent pathways, and both can occur independently of IL-9/IL-9R signaling. In contrast, oral antigen-induced intestinal and systemic anaphylaxis is strictly IgE mediated and requires IL-9/IL-9R signaling. These studies indicate differential involvement of the IL-9/IL-9R pathway in systemic and oral antigen-induced anaphylaxis.