Optimizing drug inhibition of IgE-mediated anaphylaxis in mice.

BACKGROUND: Administering allergens in increasing doses can temporarily suppress IgE-mediated allergy and anaphylaxis by desensitizing mast cells and basophils; however, allergen administration during desensitization therapy can itself induce allergic responses. Several small molecule drugs and nutraceuticals have been used clinically and experimentally to suppress these allergic responses. OBJECTIVES: This study sought to optimize drug inhibition of IgE-mediated anaphylaxis. METHODS: Several agents were tested individually and in combination for ability to suppress IgE-mediated anaphylaxis in conventional mice, FcεRIα-humanized mice, and reconstituted immunodeficient mice that have human mast cells and basophils. Hypothermia was the readout for anaphylaxis; therapeutic efficacy was measured by degree of inhibition of hypothermia. Serum mouse mast cell protease 1 level was used to measure extent of mast cell degranulation. RESULTS: Histamine receptor 1 (HR1) antagonists, ß-adrenergic agonists, and a spleen tyrosine kinase (Syk) inhibitor were best at individually inhibiting IgE-mediated anaphylaxis. A Bruton's tyrosine kinase (BTK) inhibitor, administered alone, only inhibited hypothermia when FcεRI signaling was suboptimal. Combinations of these agents could completely or nearly completely inhibit IgE-mediated hypothermia in these models. Both Syk and BTK inhibition decreased mast cell degranulation, but only Syk inhibition also blocked desensitization. Many other agents that are used clinically and experimentally had little or no beneficial effect. CONCLUSIONS: Combinations of an HR1 antagonist, a ß-adrenergic agonist, and a Syk or a BTK inhibitor protect best against IgE-mediated anaphylaxis, while an HR1 antagonist plus a ß-adrenergic agonist ± a BTK antagonist is optimal for inhibiting IgE-mediated anaphylaxis without suppressing desensitization.

Suppression of IgE-mediated anaphylaxis and food allergy with monovalent anti-FcεRIα mAbs.

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.

IgG1 protects against renal disease in a mouse model of cryoglobulinaemia.

Immunoglobulins protect against disease to a considerable extent by activating complement and stimulatory immunoglobulin crystallizable fragment receptors (Ig FcRs), and aggregating microbial pathogens. Yet IgG1, the predominant murine serum Ig isotype, cannot activate complement by the classical pathway, binds more avidly to an inhibitory than to stimulatory FcRs, and has limited ability to aggregate pathogens. In these regards, it resembles human IgG4 (ref. 4). We hypothesized that limited ability to activate effector mechanisms might protect against immune complex immunopathology. Here we show that IgG1-deficient (γ1(-)) mice, immunized with a potent antigen, develop lethal renal disease soon after they begin to produce antigen-specific antibody, whereas similarly immunized wild-type mice remain healthy. Surprisingly, renal disease in this model is complement and FcR independent and results from immune complex precipitation in glomerular capillaries, as in some cryoglobulinaemic humans. IgG3, which self-associates to form large immune complexes, accounts for more than 97% of the mouse Ig in this cryoglobulin; furthermore, glomerular disease develops when mice are injected with IgG3 anti-trinitrophenyl (TNP) monoclonal antibody followed by a TNP-labelled protein. Renal disease is prevented in both active and passive immunization models by antigen-specific IgG1; other isotypes are less potent at preventing disease. These observations demonstrate the adaptive significance of Ig isotypes that poorly activate effector mechanisms, reveal an immune-complex-dependent, complement- and FcR-independent nephrotoxic mechanism, and suggest that isotypes that poorly activate effector mechanisms may be useful for inhibiting immune complex immunopathology.

Rapid desensitization of humanized mice with anti-human FcεRIα monoclonal antibodies.

BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed ∼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.

Prevention of food allergy development and suppression of established food allergy by neutralization of thymic stromal lymphopoietin, IL-25, and IL-33.

BACKGROUND: Food allergy (FA) is an increasing problem that has no approved treatment. The pro-TH2 cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) are associated with FA, and mAbs to these cytokines are reported to suppress murine FA development. OBJECTIVE: We sought to determine whether anti-pro-TH2 cytokine mAbs can block both FA maintenance and induction. METHODS: IgE-mediated FA was induced in BALB/c mice by oral gavage with medium-chain triglycerides (MCTs) plus egg white (EW) and was characterized by increased numbers of lamina propria TH2 cells, mast cells, and eosinophils, shock (hypothermia), mast cell degranulation (increased serum mouse mast cell protease 1), increased serum IgG1 anti-EW and IgE levels, and increased IL-4 and IL-13 secretion after MCT/EW challenge. Mice were injected with anti-IL-25, IL-33 receptor, and/or TSLP mAbs before initial oral gavage with MCT/EW to suppress FA development; treatment with the same mAbs was initiated after FA development to suppress established FA. RESULTS: Injection of an mAb to IL-25, IL-33 receptor, or TSLP strongly inhibited FA development. No single mAb to a pro-TH2 cytokine could suppress established FA, and optimal FA suppression required treatment with a cocktail of all 3 anti-pro-TH2 mAbs. Treatment with the 3-mAb cocktail during initial MCT/EW immunization induced EW tolerance. CONCLUSION: All of the pro-TH2 cytokines are required to induce our model of FA, whereas any pro-TH2 cytokine can maintain established FA. Pro-TH2 cytokines prevent oral tolerance. Combined treatment with antagonists to all 3 pro-TH2 cytokines or with an inhibitor of pro-TH2 cytokine production might be able to suppress established human FA.

Anti-FcγRIIB mAb suppresses murine IgG-dependent anaphylaxis by Fc domain targeting of FcγRIII.

BACKGROUND: The inhibitory receptor FcγRIIB is expressed on human and murine bone marrow-derived cells and limits inflammation by suppressing signaling through stimulatory receptors. OBJECTIVE: We sought to evaluate the effects of K9.361, a mouse IgG2a alloantibody to mouse FcγRIIB, on murine anaphylaxis. METHODS: Wild-type and FcγR-deficient mice were used to study anaphylaxis, which was induced by injection of 2.4G2 (rat IgG2b mAb that binds both FcγRIIB and the stimulatory receptor FcγRIII), by actively immunizing IgE-deficient mice and then challenging with the immunizing antigen, and by passive immunization with IgG or IgE anti-2,4,6-trinitrophenyl mAb, followed by injection of 2,4,6-trinitrophenyl-ovalbumin. Pretreatment with K9.361 was assessed for its ability to influence anaphylaxis. RESULTS: Unexpectedly, K9.361 injection induced mild anaphylaxis, which was both FcγRIIB and FcγRIII dependent and greatly enhanced by ß-adrenergic blockade. K9.361 injection also decreased expression of stimulatory Fcγ receptors, especially FcγRIII, and strongly suppressed IgG-mediated anaphylaxis without strongly affecting IgE-mediated anaphylaxis. The F(ab')2 fragment of K9.361 did not induce anaphylaxis, even after ß-adrenergic blockade, and did not deplete FcγRIII or suppress IgG-mediated anaphylaxis but prevented intact K9.361-induced anaphylaxis without diminishing intact K9.36 suppression of IgG-mediated anaphylaxis. CONCLUSION: Cross-linking FcγRIIB to stimulatory FcγRs through the Fc domains of an anti-FcγRIIB mAb induces and then suppresses IgG-mediated anaphylaxis without affecting IgE-mediated anaphylaxis. Because IgG- and IgE-mediated anaphylaxis can be mediated by the same cell types, this suggests that desensitization acts at the receptor rather than cellular level. Sequential treatment with the F(ab')2 fragment of anti-FcγRIIB mAb followed by intact anti-FcγRIIB safely prevents IgG-mediated anaphylaxis.

Human IgE-independent systemic anaphylaxis.

Anaphylaxis is a rapidly developing, life-threatening, generalized or systemic allergic reaction that is classically elicited by antigen crosslinking of antigen-specific IgE bound to the high-affinity IgE receptor FcεRI on mast cells and basophils. This initiates signals that induce cellular degranulation with release and secretion of vasoactive mediators, enzymes, and cytokines. However, IgE-independent mechanisms of anaphylaxis have been clearly demonstrated in experimental animals. These include IgG-dependent anaphylaxis, which involves the triggering of mediator release by IgG/antigen complex crosslinking of FcγRs on macrophages, basophils, and neutrophils; anaphylaxis mediated by binding of the complement-derived peptides C3a and C5a to their receptors on mast cells, basophils, and other myeloid cells; and direct activation of mast cells by drugs that interact with receptors on these cells. Here we review the mechanisms involved in these IgE-independent forms of anaphylaxis and the clinical evidence for their human relevance. We conclude that this evidence supports the existence of all 3 IgE-independent mechanisms as important causes of human disease, although practical and ethical considerations preclude their demonstration to the degree of certainty possible with animal models. Furthermore, we cite evidence that different clinical situations can suggest different mechanisms as having a primal role in anaphylaxis and that IgE-dependent and distinct IgE-independent mechanisms can act together to increase anaphylaxis severity. As specific agents become available that can interfere with mechanisms involved in the different types of anaphylaxis, recognition of specific types of anaphylaxis is likely to become important for optimal prophylaxis and therapy.

IgE epitope proximity determines immune complex shape and effector cell activation capacity.

BACKGROUND: IgE-allergen complexes induce mast cell and basophil activation and thus immediate allergic inflammation. They are also important for IgE-facilitated allergen presentation to T cells by antigen-presenting cells. OBJECTIVE: To investigate whether the proximity of IgE binding sites on an allergen affects immune complex shape and subsequent effector cell activation in vitro and in vivo. METHODS: We constructed artificial allergens by grafting IgE epitopes in different numbers and proximity onto a scaffold protein. The shape of immune complexes formed between artificial allergens and the corresponding IgE was studied by negative-stain electron microscopy. Allergenic activity was determined using basophil activation assays. Mice were primed with IgE, followed by injection of artificial allergens to evaluate their in vivo allergenic activity. Severity of systemic anaphylaxis was measured by changes in body temperature. RESULTS: We could demonstrate simultaneous binding of 4 IgE antibodies in close vicinity to each other. The proximity of IgE binding sites on allergens influenced the shape of the resulting immune complexes and the magnitude of effector cell activation and in vivo inflammation. CONCLUSIONS: Our results demonstrate that the proximity of IgE epitopes on an allergen affects its allergenic activity. We thus identified a novel mechanism by which IgE-allergen complexes regulate allergic inflammation. This mechanism should be important for allergy and other immune complex-mediated diseases.

Differential control of CD4(+) T-cell subsets by the PD-1/PD-L1 axis in a mouse model of allergic asthma.

Studies examining the role of PD-1 family members in allergic asthma have yielded conflicting results. Using a mouse model of allergic asthma, we demonstrate that blockade of PD-1/PD-L1 has distinct influences on different CD4(+) T-cell subsets. PD-1/PD-L1 blockade enhances airway hyperreactivity (AHR), not by altering the magnitude of the underlying Th2-type immune response, but by allowing the development of a concomitant Th17-type immune response. Supporting differential CD4(+) T-cell responsiveness to PD-1-mediated inhibition, naïve PD-1(-/-) mice displayed elevated Th1 and Th17 levels, but diminished Th2 cytokine levels, and ligation of PD-1 in WT cells limited cytokine production by in vitro polarized Th1 and Th17 cells, but slightly enhanced cytokine production by in vitro polarized Th2 cells. Furthermore, PD-1 ligation enhanced Th2 cytokine production by naïve T cells cultured under nonpolarizing conditions. These data demonstrate that different CD4(+) T-cell subsets respond differentially to PD-1 ligation and may explain some of the variable results observed in control of allergic asthma by the PD-1 family members. As the PD-1/PD-L1 axis limits asthma severity by constraining Th17 cell activity, this suggests that severe allergic asthma may be associated with a defective PD-1/PD-L1 regulatory axis in some individuals.

Identification of markers that distinguish IgE- from IgG-mediated anaphylaxis.

IgG-mediated anaphylaxis occurs in mice and may contribute to human reactions to infused drugs. To distinguish IgE- from putative IgG-mediated human anaphylaxis, we developed blood markers for murine anaphylaxis and evaluated their human relevance. Both IgG- and IgE-mediated anaphylaxis were characterized by decreased basophil and monocyte percentages and an increased neutrophil percentage in mouse blood. IgE- but not IgG-mediated murine anaphylaxis was accompanied by large increases in IL-4 secretion, plasma soluble IL-4 receptor-α (IL-4Rα) concentration, and T-cell membrane IL-4Rα expression. T-cell IL-4Rα expression also increased when mice that express human Fcε receptor Iα were sensitized with IgG-depleted serum from a peanut-allergic individual and challenged with peanut extract. Increased T-cell IL-4Rα expression is likely to also be a marker for human IgE-mediated anaphylaxis, because IgE-activated human basophils secrete IL-4, and IL-4 increases human T-cell IL-4Rα expression in vitro. Murine IgG- but not IgE-mediated anaphylaxis was characterized by decreased neutrophil Fcγ receptor III (FcγRIII) expression that was observed even when the antigen dose was insufficient to induce shock. Human neutrophils cultured with IgG immune complexes also lost FcγRIII. These observations suggest that decreased blood neutrophil FcγRIII expression without increased IL-4Rα expression can be used to determine whether and when IgG-mediated anaphylaxis occurs in man.

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

BACKGROUND: Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. OBJECTIVE: We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. METHODS: Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. RESULTS: Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. CONCLUSION: A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

BACKGROUND: Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. OBJECTIVE: We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. METHODS: Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. RESULTS: Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. CONCLUSION: IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology.

Proton Treatment Suppresses Exosome Production in Head and Neck Squamous Cell Carcinoma.

Proton therapy (PT) is emerging as an effective and less toxic alternative to conventional X-ray-based photon therapy (XRT) for patients with advanced head and neck squamous cell carcinomas (HNSCCs) owing to its clustered dose deposition dosimetric characteristics. For optimal efficacy, cancer therapies, including PT, must elicit a robust anti-tumor response by effector and cytotoxic immune cells in the tumor microenvironment (TME). While tumor-derived exosomes contribute to immune cell suppression in the TME, information on the effects of PT on exosomes and anti-tumor immune responses in HNSCC is not known. In this study, we generated primary HNSCC cells from tumors resected from HNSCC patients, irradiated them with 5 Gy PT or XRT, and isolated exosomes from cell culture supernatants. HNSCC cells exposed to PT produced 75% fewer exosomes than XRT- and non-irradiated HNSCC cells. This effect persisted in proton-irradiated cells for up to five days. Furthermore, we observed that exosomes from proton-irradiated cells were identical in morphology and immunosuppressive effects (suppression of IFN-γ release by peripheral blood mononuclear cells) to those of photon-irradiated cells. Our results suggest that PT limits the suppressive effect of exosomes on cancer immune surveillance by reducing the production of exosomes that can inhibit immune cell function.

Induction and suppression of allergic diarrhea and systemic anaphylaxis in a murine model of food allergy.

BACKGROUND: The clinical manifestations of food allergy include diarrhea and systemic anaphylaxis (shock), which can occur together or by themselves in different subjects. Although ingested food antigens need to be absorbed to induce shock, it is not known whether they need to be absorbed to induce diarrhea. OBJECTIVE: We sought to identify mechanisms that determine whether food allergy induces diarrhea versus shock and determine whether diarrhea requires absorption of ingested antigens. METHODS: These issues were studied in mice in active, passive, and hybrid immunization models. The active model was used to determine the allergic diarrhea susceptibility of J chain- and polymeric immunoglobulin receptor-deficient mice, which are unable to secrete IgA. The hybrid model was used to determine whether intravenously administered antigen-specific IgG antibody, which is not secreted into the gut, can protect against allergic diarrhea, as well as shock. RESULTS: Shock, but not diarrhea, was induced in naive mice by using intravenous IgE anti-trinitrophenyl (TNP) antibody, followed by oral TNP-BSA, whereas both were induced in mice presensitized with intraperitoneal ovalbumin/alum plus oral ovalbumin. More TNP-BSA was required to induce shock than diarrhea in presensitized mice, and intravenous IgG anti-TNP antibody, which is not secreted into the gut, protected these mice against both diarrhea and shock. Consistent with this, chicken ovalbumin-immunized J chain- and polymeric immunoglobulin receptor-deficient mice, which have high serum IgA levels but little intestinal IgA, resisted diarrhea induction. CONCLUSION: Intestinal immunity and oral antigen dose determine whether diarrhea, systemic anaphylaxis, or both are induced, and ingested antigen must be absorbed to induce either response.

Acoustic metamaterials-driven transdermal drug delivery for rapid and on-demand management of acute disease.

Transdermal drug delivery provides convenient and pain-free self-administration for personalized therapy. However, challenges remain in treating acute diseases mainly due to their inability to timely administrate therapeutics and precisely regulate pharmacokinetics within a short time window. Here we report the development of active acoustic metamaterials-driven transdermal drug delivery for rapid and on-demand acute disease management. Through the integration of active acoustic metamaterials, a compact therapeutic patch is integrated for penetration of skin stratum corneum and active percutaneous transport of therapeutics with precise control of dose and rate over time. Moreover, the patch device quantitatively regulates the dosage and release kinetics of therapeutics and achieves better delivery performance in vivo than through subcutaneous injection. As a proof-of-concept application, we show our method can reverse life-threatening acute allergic reactions in a female mouse model of anaphylaxis via a multi-burst delivery of epinephrine, showing better efficacy than a fixed dosage injection of epinephrine, which is the current gold standard 'self-injectable epinephrine' strategy. This innovative method may provide a promising means to manage acute disease for personalized medicine.

Ingested allergens must be absorbed systemically to induce systemic anaphylaxis.

BACKGROUND: IgE-mediated food allergy is a common cause of enteric disease and is responsible for approximately 100 systemic anaphylaxis deaths in the United States each year. IgG antibodies can protect against IgE-mediated systemic anaphylaxis induced by injected antigens by neutralizing antigens before they can bind to mast cell-associated IgE. OBJECTIVE: We have investigated whether IgA and IgG antibodies can similarly protect against systemic, IgE-mediated anaphylaxis induced by ingested antigens and, if so, whether IgA and IgG antibodies protect by neutralizing antigens before or after their systemic absorption. METHODS: Murine passive and active anaphylaxis models were used to study the abilities of serum versus gut lumenal IgA antibodies and serum IgG antibodies to inhibit systemic anaphylaxis induced by ingested allergens in normal mice, mice deficient in the ability to secrete IgA into the intestines, and mice in which intestinal IL-9 overexpression has induced intestinal mastocytosis and increased intestinal permeability. RESULTS: IgE-mediated systemic anaphylaxis and mast cell degranulation induced by antigen ingestion are suppressed by both serum antigen-specific IgA and IgG, but not by IgA within the gut lumen. CONCLUSION: Systemic rather than enteric antibodies protect against systemic anaphylaxis induced by ingested antigen. This implies that ingested antigens must be absorbed systemically to induce anaphylaxis and suggests that immunization protocols that increase serum levels of antigen-specific, non-IgE antibodies should protect against severe food allergy.

Endogenously produced IL-4 nonredundantly stimulates CD8+ T cell proliferation.

T cell proliferation and survival are regulated by the cytokine receptor common gamma-chain-associated cytokines IL-2, IL-7, and IL-15, while IL-4, another gamma-chain-associated cytokine, is thought to primarily affect T cell quality rather than quantity. In contrast, our experiments reveal that endogenously produced IL-4 is a direct, nonredundant, and potent stimulator of CD8(+) T cell proliferation in Ag- and pathogen-induced CD8(+) T cell responses. These stimulatory effects of IL-4 are observed in both BALB/c and C57BL/6 mice and activate both naive and memory/activated phenotype CD8(+) T cells, although the former are stimulated less than are the latter. IL-4 effects are IL-7- and IL-15-independent, but MHC class I-dependent stimulation appears to be required for the mitogenic effect of IL-4 on naive phenotype CD8(+) T cells. Thus, endogenously produced IL-4 is an important regulator of quantitative as well as qualitative aspects of T cell immunity.

Nonredundant roles for B cell-derived IL-10 in immune counter-regulation.

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8(+) T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.

Basophils initiate IL-4 production during a memory T-dependent response.

Experiments were performed to characterize and identify the cellular sources of the secondary interleukin (IL)-4 response to a T cell-dependent antigen. Mice were primed by immunization with goat anti-mouse immunoglobulin (Ig)D antibody (GaMD), which stimulates naive CD4+ T cells to secrete IL-4 in 3-4 d. When challenged with goat serum 14 d after immunization, GaMD-primed mice generated an IL-4 response that exceeded the primary response by approximately 100-fold, started in <2 h, and lasted for 4 d. Studies with 4get mice, in which cells with an accessible Il4 gene express a green fluorescent protein (GFP), revealed CD4+ memory T cells, natural killer T cells, basophils, mast cells, and eosinophils as possible rapid producers of IL-4. GFP(+)CD4+ T cells and basophils expanded more in the spleen than the other cell types during the primary response to GaMD. Quantitation of in vivo IL-4 production by the in vivo cytokine capture assay after individual cell types were selectively stimulated or deleted demonstrated that basophils and memory CD4+ T cells account for most of the secondary IL-4 response, with basophils initiating that response through IgE/FcepsilonRI-mediated signaling but secreting IL-4 for <4 h and memory T cells secreting IL-4 within 4 h and continuing to secrete this cytokine for 4 d.