Contribution of classic and alternative effector pathways in peanut-induced anaphylactic responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.     

IL-13 overexpression predisposes to anaphylaxis following antigen sensitization

Anaphylaxis represents an extreme form of allergic reaction. This acute-phase component of allergy and asthma is triggered by allergen-induced degranulation of mast cells following the cross-linking of cell surface-bound, allergen-specific IgE, resulting in the liberation of inflammatory mediators and the development of bronchoconstriction. We used IL-13 transgenic mice to investigate the role of this Th2 cell-derived cytokine in the onset of allergic disease. Strikingly, IL-13-transgenic mice were highly predisposed to fatal anaphylaxis following Ag sensitization. This response correlated with substantially elevated levels of circulating Ag-specific IgE, mast cell degranulation, and histamine release. Furthermore, allergen exposure also induced phenotypic changes typical of asthma, including pulmonary fibrosis, goblet cell hyperplasia, elevated Th2 cytokines, eosinophilia, and airways occluded by mucus and Charcot-Leyden crystals. Expression of IL-4 was not required for the induction of IgE-mediated responses. These data represent the first characterization of a functional role for IL-13-induced IgE in the generation of immediate hypersensitivity reactions and highlight the importance of IL-13 in the development of the symptoms of atopy. The systemic regulation of this response makes these mice an important resource for studying atopic responses.     

Theanine is a candidate amino acid for pharmacological stabilization of mast cells

The increasing occurrences of allergic disorders may be attributed to exposure to environmental factors that contribute to the pathogenesis of allergy. The health benefits of green tea have been widely reported but are largely unsubstantiated. Theanine is the major amino acid present in green tea. In this study, we investigated the role of theanine in both IgE- and non- IgE-induced allergic response. Theanine inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated passive cutaneous anaphylaxis was inhibited by the oral administration or pharmaceutical acupuncture of theanine. Histamine release from mast cells was decreased with the treatment of theanine. Theanine also repressed phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced TNF-α, IL-1β, IL-6, and IL-8 secretion by suppressing NF-κB activation. Furthermore, theanine suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. The current study demonstrates for the first time that theanine might possess mast cell-stabilizing capabilities.     

IL-9 promotes but is not necessary for systemic anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.     

IL-10 suppresses mast cell IgE receptor expression and signaling in vitro and in vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcepsilonRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcepsilonRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.     

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-α, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-α, IL-6, and IL-8 release from mast cells.     

Effect of myeloma IgE injections on passive and active cutaneous anaphylaxis in rats

The ability of injected rat IgE myeloma protein IR162 to inhibit passive and active cutaneous anaphylaxis in Lewis rats was investigated. IgE injected i.p. 24 hr before the sensitization with IgE anti-ovalbumin (OVA) completely inhibited both IgE- and IgG2a-induced passive cutaneous anaphylactic (PCA) reactions at a dose (2.5 mg/100 g body weight) that resulted in peak serum concentrations of 150 micrograms IgE IR162/ml. Peak IgE IR162 serum concentrations of 20 to 60 micrograms/ml inhibited the PCA reaction in approximately 50% of the rats. Intracutaneous injection of a mixture of myeloma IgE and anti-OVA IgE in a ratio of 100:1 or more also inhibited the PCA reaction. In contrast, the PCA reaction was not inhibited by seven daily doses of IgE beginning 24 hr after passive sensitization. Likewise, the cutaneous anaphylactic reaction elicited in rats 14 days after immunization with OVA and Bordetella pertussis was not prevented by daily injections of myeloma IgE despite a 1000- to 3000-fold excess of the myeloma IgE to anti-OVA IgE serum concentration. The data demonstrate that parenteral administration of myeloma IgE inhibits the PCA reaction only when given before passive sensitization and does not prevent cutaneous anaphylaxis in actively immunized rats. Because myeloma IgE failed to inhibit anaphylactic reactions in actively immunized rats, it is questionable whether administering human IgE-derived synthetic peptides or recombinant DNA-produced IgE fragments will be able to prevent allergic diseases by blocking the IgE Fc receptors on mast cells.     

Treatment with phosphodiester CpG-ODN ameliorates atopic dermatitis by enhancing TGF-β signaling

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG phosphorothioate (PS CpG-ODN) are known to decrease IgE synthesis in Th2 allergy responses. Nonetheless, the therapeutic role of PS CpG-ODN is limited due to cytotoxicity. Therefore, we developed a phosphodiester (PO) form of CpG-ODN (46O) with reduced toxicity but effective against allergies. In this study, we first compared the toxicity of 46O with CpG-ODNs containing a PS backbone (1826S). We also investigated the therapeutic efficacy and mechanism of 46O injected intravenously in a mouse model of ovalbumin (OVA)-induced atopic dermatitis (AD). To elucidate the mechanism of 46O underlying the inhibition of IgE production, IgE- and TGF-β-associated molecules were evaluated in CD40/IL-4- or LPS/IL-4-stimulated B cells. Our data showed that the treatment with 46O was associated with a lower hematological toxicity compared with 1826S. In addition, injection with 46O reduced erythema, epidermal thickness, and suppressed IgE and IL-4 synthesis in mice with OVA-induced AD. Additionally, 46O induced TGF-β production in LPS/IL-4-stimulated B cells via inhibition of Smad7, which suppressed IgE synthesis via interaction between Id2 and E2A. These findings suggest that enhanced TGF-β signaling is an effective treatment for IgE-mediated allergic conditions, and 46O may be safe and effective for treating allergic diseases such as AD and asthma.     

SHP-1 Regulation of Mast Cell Function in Allergic Inflammation and Anaphylaxis

Allergic inflammation and severe allergic reactions (anaphylaxis) are important in allergen induced diseases. Bacterial products such as lipopolysaccharide (LPS) are ubiquitous and can facilitate allergen induced Th2 immune responses. Phosphatase SHP-1 is critical in regulating immunological homeostasis and in allergen induced Th2 immune responses in the lung. However, the mechanisms underlying the initiation of allergic inflammation and allergen induced anaphylaxis are still not completely elucidated and it is unclear whether SHP-1 plays any role in LPS-induced airway inflammation and in allergen-induced anaphylaxis. In this study we tested the hypothesis that phosphatase SHP-1 plays an important role in allergic inflammation and anaphylaxis and determined whether its effects are through regulation of mast cell functions. SHP-1 deficient (mev/+ and mev/mev) and mast cell deficient (Kit^W-sh) mice were examined in their responses to LPS airway stimulation and to ovalbumin (OVA) allergen induced systemic anaphylaxis. Compared to wild type mice, mev/+ mice had significantly enhanced LPS induced airway inflammation and OVA induced anaphylactic responses, including hypothermia and clinical symptoms. These changes were mast cell dependent as Kit^W-sh mice had reduced responses whereas adoptive transfer of mast cells restored the responses. However, T and B cells were not involved and macrophages did not play a significant role in LPS induced airway inflammation. Interestingly, basophil differentiation from SHP-1 deficient bone marrow cells was significantly reduced. These findings provided evidence that through regulation of mast cell functions SHP-1 plays a critical role as a negative regulator in allergic inflammation and in allergen induced anaphylaxis. In addition, SHP-1 seems to be required for normal basophil development.     

Another anti-allergic mechanism: antibody IgE deglycosylation induced by a substance extracted from human urine

Enzymically-deglycosylated antibody IgE lost its allergic activity in mouse systemic anaphylaxis, though the IgE kept its antibody activity. IgE antibody obtained from mice treated with a substance extracted from human urine was deglycosylated. This IgE also lost the allergic activity on the systemic anaphylaxis but kept its antibody activity. These findings strongly suggest that glycosylation of IgE has a close relation to the binding of the Fc receptor and that humans have another antiallergic mechanism: in vivo IgE antibody deglycosylation induced by the substance.     

Inhibitory effects of superoxide dismutase 3 on IgE production in B cells

Immunoglobulin E (IgE) functions as a first-line defense against parasitic infections. However, aberrant production of IgE is known to be associated with various life-threatening allergic diseases. Superoxide dismutase 3 (SOD3) has been found to suppress IgE in various allergic diseases such as allergic conjunctivitis, ovalbumin-induced allergic asthma, and dust mite-induced atopic dermatitis-like skin inflammation. However, the role of SOD3 in the regulation of IgE production in B cells remains elusive. In this study, we investigated the effect of SOD3 on LPS/IL-4 and anti-CD40/IL-4-mediated secretion of IgE in murine B cells. Our data showed that SOD3 can suppress both LPS/IL-4 and antiCD40/IL-7-induced IgE secretion in B cells isolated from both wild-type (SOD3^+/+) and SOD3 knock-out (SOD3^?/?) mice. Interestingly, B cells isolated from SOD3^?/? mice showed higher secretion of IgE, whereas, the use of DETCA, a known inhibitor of SOD3 activity, reversed the inhibitory effect of SOD3 on IgE production. Similarly, SOD3 was found to reduce the proliferation, IgE isotype switch, ROS level, and CCL17 and CCL22 productions in B cells. Furthermore, SOD3 was found to suppress both LPS/IL-4 and anti-CD40/IL-4-mediated activation of downstream signaling such as JAK1/JAK3, STAT6, NF-κB, p38, and JNK in B cells. Taken together, our data showed that SOD3 can be used as an alternative therapy to restrict IgE-mediated allergic diseases.     

An enteric helminth infection protects against an allergic response to dietary antigen

Although helminths induce a polarized Th2 response they have been shown, in clinical studies, to confer protection against allergies. To elucidate the basis for this paradox, we have examined the influence of an enteric helminth infection on a model of food allergy. Upon Ag challenge, mice fed peanut (PN) extract plus the mucosal adjuvant cholera toxin (CT) produced PN-specific IgE that correlated with systemic anaphylactic symptoms and elevated plasma histamine. PN-specific IgE was not induced in helminth-infected mice fed PN without CT. Moreover, when PN plus CT was fed to helminth-infected mice, both PN-specific IgE and anaphylactic symptoms were greatly diminished. The down-regulation of PN-specific IgE was associated with a marked reduction in the secretion of IL-13 by PN-specific T cells. When helminth-infected PN plus CT-sensitized mice were treated with neutralizing Abs to IL-10, the PN-specific IgE response and anaphylactic symptoms were similar to, or greater than, those seen in mice that receive PN and CT alone. Taken together, these results suggest that helminth-dependent protection against allergic disease involves immunoregulatory mechanisms that block production of allergen-specific IgE.     

Helminth infection protects mice from anaphylaxis via IL-10-producing B cells

Modulation of the immune system by infection with helminth parasites, including schistosomes, is proposed to reduce the levels of allergic responses in infected individuals. In this study we investigated whether experimental infection with Schistosoma mansoni could alter the susceptibility of mice to an extreme allergic response, anaphylaxis. We formally demonstrate that S. mansoni infection protects mice from an experimental model of systemic fatal anaphylaxis. The worm stage of infection is shown to mediate this protective effect. In vivo depletion studies demonstrated an imperative role for B cells and IL-10 in worm-mediated protection. Furthermore, worm infection of mice increases the frequency of IL-10-producing B cells compared with that in uninfected mice. However, transfer of B cells from worm-infected mice or in vitro worm-modulated B cells to sensitized recipients exacerbated anaphylaxis, which was attributed to the presence of elevated levels of IL-4-producing B cells. Worm-modulated, IL-10-producing B cells from IL-4-deficient, but not IL-5-, IL-9- or IL-13-deficient, mice conferred complete resistance to anaphylaxis when transferred to naive mice. Therefore, we have dissected a novel immunomodulatory mechanism induced by S. mansoni worms that is dependent on an IL-10-producing B cell population that can protect against allergic hypersensitivity. These data support a role for helminth immune modulation in the hygiene hypothesis and further illustrate the delicate balance between parasite induction of protective regulatory (IL-10) responses and detrimental (IL-4) allergic responses.     

IgG-mediated systemic anaphylaxis to protein antigen can be induced even under conditions of limited amounts of antibody and antigen

Systemic anaphylaxis is an acute, severe, and potentially fatal allergic reaction. Two classes of antibodies, IgE and IgG, contribute to the development of anaphylaxis in mice, through different mechanisms with distinct usage of effector cells and chemical mediators. Larger quantities of antibody and antigen are reportedly required to induce IgG-mediated anaphylaxis than IgE-mediated one, suggesting that the former may not happen as frequently as the latter in real life. To readdress this issue, we established in the present study a novel mouse model of passive IgG-mediated systemic anaphylaxis to a native protein antigen, ovalbumin (OVA), rather than artificially haptenated protein antigens used in previous studies. Passive sensitization of mice with a cocktail of but not individual IgG1 mAbs specific to distinct OVA epitopes elicited systemic anaphylaxis in response to OVA challenge. Importantly, much smaller doses of antibody and antigen than previously reported were sufficient for the induction of IgG-mediated systemic anaphylaxis. Moreover, a relatively small dose of antigen could induce severe anaphylaxis through both IgE- and IgG-mediated mechanisms when mice had been passively sensitized with antigen-specific IgE and IgG. These results strongly suggest that IgG-mediated systemic anaphylaxis is not rare among antibody-mediated systemic anaphylaxis, in contrast to previous thought, and significantly contributes to active systemic anaphylaxis in real life, at least in mice.     

Role of mast cells and basophils in IgE responses and in allergic airway hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.     

IFN-gamma-mediated inhibition of human IgE synthesis by IL-21 is associated with a polymorphism in the IL-21R gene

IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.     

Chondroitin sulfate intake inhibits the IgE-mediated allergic response by down-regulating Th2 responses in mice

Chondroitin sulfate (CS) was administered orally to BALB/c mice immunized intraperitoneally with ovalbumin (OVA) and/or dinitrophenylated OVA. The titers of antigen-specific IgE and IgG1 in mouse sera were determined. The antigen-specific IgE production by mice fed ad libitum with CS was significantly inhibited. We also examined the effect of feeding CS on immediate-type hypersensitivity. One hour after antigen stimulation, the ears of mice fed with CS swelled less than those of the control mice. Furthermore, the rise in serum histamine in the mice fed with CS under active systemic anaphylaxis was significantly lower than that in the controls. We next examined the pattern of cytokine production by splenocytes from mice followed by re-stimulation with OVA in vitro. The splenocytes from the mice fed with CS produced less interleukin (IL)-5, IL-10, and IL-13 than those from the control group. In contrast, the production of interferon-gamma and IL-2 by the splenocytes of mice fed with CS was not significantly different from those in the control mice. In addition, the production of transforming growth factor-beta from the splenocytes of mice fed with CS was significantly higher than that of the control mice. Furthermore, we showed that the percentages of CD4(+) cells, CD8(+) cells, and CD4(+)CD25(+) cells in the splenocytes of mice fed with CS are significantly higher than those of the control. These findings suggest that oral intake of CS inhibits the specific IgE production and antigen-induced anaphylactic response by up-regulating regulatory T-cell differentiation, followed by down-regulating the Th2 response.