The soluble isoform of human FcɛRI is an endogenous inhibitor of IgE-mediated mast cell responses.

BACKGROUND: The soluble isoform of FcɛRI, the high-affinity IgE receptor (sFcεRI), is a protein of the IgE network with poorly defined functions. OBJECTIVE: To define cellular sources and signals that result in the production of human sFcεRI and study its in vivo functions. METHODS: FcεRI-transfected human cell lines (MelJuso), human monocyte-derived dendritic cells (moDCs), and murine bone marrow-derived mast cells (MC) were stimulated by FcεRI cross-linking and release of sFcεRI was analyzed (ELISA, Western Blot). Lysosomal-associated membrane protein 1 degranulation assays and human basophil activation tests (BATs) were used to study IgE-dependent activation. Recombinant sFcεRI (rsFcεRI) was used to assess its role in murine models of anaphylaxis with WT (wild-type) and IgE-/- (IgE-deficient) mice. RESULTS: Antigen-specific cross-linking of IgE-loaded FcɛRI on MelJuso cells that express the trimeric or tetrameric receptor isoform induced the production of sFcεRI. Using MCs and moDCs, we confirmed that IgE/FcɛRI activation induces sFcɛRI release. We demonstrated that generation of sFcɛRI requires Src phosphorylation and endo/lysosomal acidification. In experimental mouse models, sFcɛRI diminishes the severity of IgE-mediated anaphylaxis. BATs confirmed that, comparable to the anti-IgE monoclonal antibody omalizumab, sFcɛRI is an inhibitor of the human innate IgE effector axis, implying that sFcɛRI and omalizumab potentially inhibit each other in vivo. CONCLUSION: sFcɛRI is produced after antigen-specific IgE/FcɛRI-mediated activation signals and functions as an endogenous inhibitor of IgE loading to FcɛRI and IgE-mediated activation. Our results imply, therefore, that sFcɛRI contributes to a negative regulatory feedback loop that aims at preventing overshooting responses after IgE-mediated immune activation.

Importance of basophils in eosinophilic asthma: the murine counterpart.

Several experimental studies in mice showed that basophils participate in the initiation of Th2 adaptive immune response, in addition to the effector phase. However, the role of basophils in allergic airway inflammation is less clear. The aim of this experiment was to assess the importance of basophils in recruiting inflammatory cells and, in particular, eosinophils in a murine model of asthma induced by Aspergillus fumigatus allergens. Additionally, bronchial reactivity was evaluated. Basophil depletion resulted in a reduction of inflammatory cells in the airways and eosinophil recruitment was significantly impaired. Also bronchial reactivity seemed to be impaired in basophil-depleted mice, but the result was not statistically significant. According to these preliminary data, basophils seem to influence the local eosinophilic response of allergic asthma.

IgE promotes type 2 innate lymphoid cells in murine food allergy.

BACKGROUND: Mast cells serve an important sentinel function at mucosal barriers and have been implicated as key early inducers of type 2 immune responses in food allergy. The generation of Th2 and IgE following food allergen ingestion is inhibited in the absence of mast cells. Group 2 innate lymphoid cells are also thought to play an important early role in nascent allergic responses. OBJECTIVE: To test whether IgE-mediated mast cell activation promotes intestinal ILC2 responses following ingestion of food allergens and whether ILC2 amplify food allergy. METHODS: Two different mouse models of food allergy, one using intraperitoneally ovalbumin (OVA)-primed BALB/c animals and the other using enterally peanut-sensitized inherently atopic IL4raF709 mice, were applied to test the contributions of IgE antibodies and mast cells to ILC2 responses. The effect of ILC2 on mast cell activation and on anaphylaxis was tested. RESULTS: ILC2 responses were significantly impaired in both models of food allergy in Igh7-/- mice harbouring a targeted deletion of the gene encoding IgE. A similar reduction in food allergen-induced ILC2 was observed in mast cell-deficient Il4raF709 KitW-sh mice, and this was partially corrected by reconstituting these animals using cultured bone marrow mast cells. Mast cells activated ILC2 for IL-13 production in an IL-4Rα-dependent manner. Activated ILC2 amplified systemic anaphylaxis by increasing target tissue sensitivity to mast cell mediators. CONCLUSIONS AND CLINICAL RELEVANCE: These findings support an important role for IgE-activated mast cells in driving intestinal ILC2 expansion in food allergy and reveal that ILC2, in turn, can enhance responsiveness to the mediators of anaphylaxis produced by mast cells. Strategies designed to inhibit IgE signalling or mast cell activation are likely to inhibit both type 2 immunity and immediate hypersensitivity in food allergy.

Basophils are rapidly mobilized following initial aeroallergen encounter in naïve mice and provide a priming source of IL-4 in adaptive immune responses.

Chronic aeroallergen inhalation elicits the expansion of IL-4-producing Th2 cells and the production of IgE antibodies. In sensitized subjects, who have established IgE and Th2 responses, re-exposure to allergen leads to rapid recruitment of basophils, which are thought to be important effectors of late phase allergic reactions. Several investigations of responses to parasites and injected antigens have identified an additional role for basophils as innate immune effectors during initial antigen encounter in immunologically naïve hosts. These cells constitutively express IL-4 and promote Th2 polarized adaptive responses to such antigens. Their early recruitment and modulation of cellular immune responses to natural inhaled allergens in the airways has been scarcely investigated. In this study, basophils were enumerated in lung tissue, blood and spleen from BALB/c mice in the first days after inhalation of an aqueous extract of the allergen, Aspergillus fumigatus (Af). Af inhalation induced rapid increases in basophil numbers in the lung, blood and spleen. This was Rag-1-, MyD88- and IL-3-independent. The basophils expressed abundant IL-4. Their depletion during Af sensitization resulted in an attenuated induction of both IL-4 producing Th lymphocytes and specific IgE and IgG1 responses to an inhaled protein antigen, ovalbumin, which was co-administered. Our results suggest that basophils are rapidly recruited to the airways of naïve mice following initial fungal allergen exposure, produce IL-4 and influence the development of the adaptive immune response.

Direct effects of IL-4 on mast cells drive their intestinal expansion and increase susceptibility to anaphylaxis in a murine model of food allergy.

Interleukin (IL)-4 has critical roles in allergic disorders, including food hypersensitivity. The direct effects of the cytokine on the survival and function of mast cells, the key effectors of food anaphylaxis, have not been established. In this study, we demonstrate that IL-4 induces a marked intestinal mastocytosis in mice. This phenotype is reproduced in animals expressing Il4rαF709, an activating variant of the IL-4 receptor α-chain (IL-4Rα). Il4rαF709 mice exhibit enhanced anaphylactic reactions but unaltered physiological responses to vasoactive mediators. IL-4 induces Bcl-2 and Bcl-X(L) and enhances survival and stimulates proliferation in cultured bone marrow-derived mast cells (BMMC). These effects are STAT6 (signal transducer and activator of transcription factor 6)-dependent and are amplified in Il4rαF709 BMMC. In competitive bone marrow chimeras, Il4rαF709 mast cells display a substantial competitive advantage over wild-type mast cells, which, in turn, prevail over IL-4Rα⁻/⁻ mast cells in populating the intestine, establishing a cell-intrinsic effect of IL-4 in intestinal mast cell homeostasis. Our results demonstrate that IL-4-signaling is a key determinant of mast cell expansion in food allergy.

Immunoglobulin E antibodies enhance pulmonary inflammation induced by inhalation of a chemical hapten.

BACKGROUND: Occupational exposure to chemicals is an important cause of asthma. Recent studies indicate that IgE antibodies enhance sensitization to chemicals in the skin. OBJECTIVE: We investigated whether IgE might similarly promote the development of airway inflammation following inhalation of a contact sensitizer. METHODS: A model of chemical-induced asthma is described in which introduction of the low-molecular-weight compound, trinitrobenzene sulphonic acid (TNBS), via the respiratory tract was used for both sensitization and challenge. The role of IgE antibodies in the immune response to inhaled TNBS in this model was assessed by comparing the responses of wild-type (WT) and IgE-deficient (IgE(-/-)) mice on the BALB/c background. Reconstitution of circulating IgE levels by intravenous injection of IgE antibodies into IgE(-/-) mice before sensitization was performed to confirm the role of IgE in any differences observed between the responses of WT and IgE(-/-) mice. RESULTS: Intranasal challenge of TNBS-sensitized (but not sham-sensitized control mice) induced intense pulmonary inflammation. Macrophages, eosinophils and lymphocytes, including T, B, natural killer and natural killer T cells, were recruited to the airway and the animals displayed bronchial hyperresponsiveness (BHR) to methacholine. Serum levels of murine mast cell protease-1 (mMCP-1) were elevated suggesting mast cell activation. In contrast, the development of airway inflammation, recruitment of lymphocytes, induction of BHR and production of mMCP-1 were all significantly attenuated in IgE-deficient mice. Reconstitution of IgE(-/-) mice with IgE (of unrelated antigen specificity) before sensitization partially restored these features of asthma. CONCLUSION: Our data indicate that IgE antibodies non-specifically enhance the development of airway inflammation induced by exposure to chemical antigens.

IgE regulation and roles in asthma pathogenesis.

Asthma and the predisposition to produce IgE are inherited as linked traits in families. In patients IgE levels correlate with asthma severity and bronchial hyperresponsiveness. The concept that IgE plays a critical role in asthma pathogenesis has driven the development of IgE blockers, which are currently being introduced into clinical use. This review focuses on the mechanisms whereby IgE participates both in immediate hypersensitivity responses in the airways and in the induction of chronic allergic bronchial inflammation. The molecular genetic events that give rise to IgE production by B cells and the cellular and cytokine factors that support IgE production in the bronchial mucosal microenvironment are discussed. It is clear that much remains to be learned regarding the roles of IgE in asthma and the genetic and environmental influences that lead to its production. Over the next few years, the emerging experience with anti-IgE in patients will provide a more complete understanding of the mechanisms whereby IgE contributes to disease, as well as the therapeutic potential of its inhibition.

An obligate role for T-cell receptor alphabeta+ T cells but not T-cell receptor gammadelta+ T cells, B cells, or CD40/CD40L interactions in a mouse model of atopic dermatitis.

BACKGROUND: We recently described a murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). The skin lesions in these mice were characterized by a dermal infiltrate consisting of eosinophils and T cells and by increased expression of the TH2 cytokines IL-4 and IL-5. Epicutaneous sensitization induces a rise in the levels of serum total IgE and OVA-specific antibodies, further indicating that it elicits a predominantly TH2 response. OBJECTIVE: This study was undertaken to assess the roles of T cells, B cells, and CD40L-CD40 interactions in AD. METHODS: Mice with targeted gene deletions were sensitized with OVA. Histologic and immunohistochemical examinations, as well as measurements of IL-4 mRNA, were performed on OVA-sensitized skin. Total and antigen-specific serum IgE levels were determined. RESULTS: RAG2(-/-) mice, which lack both T and B cells, did not exhibit cellular infiltration, induction of dermal IL-4 mRNA, or elevation of serum IgE after OVA sensitization; all of these features were present in B-cell-deficient IgH(-/-) mice. T-cell receptor alpha(-/-) mice did not display cellular infiltration, IL-4 mRNA expression, or increased IgE levels after OVA sensitization, but these responses were elicited in T-cell receptor delta(-/-) mice after sensitization. Absence of CD40 had no effect on these responses. CONCLUSION: These results suggest that alphabeta T cells, but not gammadelta T cells, B cells, or CD40L-CD40 interactions, are critical for skin inflammation and the TH2 response in AD.

Regulation of the IgE isotype switch: new insights on cytokine signals and the functions of epsilon germline transcripts.

In allergic responses, B cells are driven to undergo an immunoglobulin isotype switch, shifting from IgM to IgE synthesis. This process involves the rearrangement of germline DNA in the immunoglobulin heavy-chain locus and is stimulated by cytokines (IL-4 and IL-13) and CD40 activation. It is now evident that cytokine-induced 'germline' epsilon-RNA transcripts associate with DNA in the genomic switch region (S epsilon) to form DNA-RNA hybrid structures, which target nucleases in for deletional switch recombination. Alterations in cytokine production and signaling affect the efficiency of this process and are associated with inherited predisposition to allergy.

CD40L, but not CD40, is required for allergen-induced bronchial hyperresponsiveness in mice.

Asthma is characterized by immunoglobulin (Ig) E production, infiltration of the respiratory mucosa by eosinophils (EOSs) and mononuclear cells, and bronchial hyperresponsiveness (BHR). Interaction of CD40 on B cells and antigen presenting cells, with its ligand (CD40L) expressed transiently on activated T cells, is known to augment both T cell-driven inflammation and humoral immune responses, especially IgE production. Considering both the prominent role of inflammation in asthma and the association of the disease with IgE, we hypothesized that CD40-CD40L interactions would be important in pathogenesis. To test this hypothesis, we subjected wild-type (WT) mice and animals lacking either CD40 or CD40L to repeated inhalation of Aspergillus fumigatus (Af ) antigen. Af-treated WT mice displayed elevated IgE levels, bronchoalveolar lavage and pulmonary tissue eosinophilic inflammation, and BHR. IgE production was markedly suppressed in both the CD40 -/- and CD40L -/- strains. However, pulmonary inflammation did not appear to be inhibited by either of these mutations. Paradoxically, development of BHR was prevented by the lack of CD40L but not by the absence of CD40. We conclude that CD40/CD40L interactions, although critical in the induction of IgE responses to inhaled allergen, are not required for the induction of EOS-predominant inflammation. CD40L, but not CD40, is necessary for the development of allergen-induced BHR.

SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells.

SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.

The expression of murine B cell CD23, in vivo, is regulated by its ligand, IgE.

CD23, the low-affinity IgE receptor, is believed to participate in immune responses by mediating antigen capture for presentation by B cells and by shedding fragments with immunomodulatory properties. The number of CD23 molecules on B cells is increased during allergic responses and infection with helminths. This can be attributed in part to regulation of CD23 expression by cytokines, including IL-4. In addition, there is evidence that CD23 can be induced on cultured B cells by its ligand, IgE. In the current study we use IgE-deficient (IgE-/-) mice to establish the effects of IgE on CD23 expression by B cells in vivo, in the absence of allergic or parasitic stimuli. The spleens of IgE-/- and wild-type mice contained similar proportions of CD23+ B lymphocytes. However, cells from IgE-/- mice were found to have nearly 3-fold less CD23 on their surface. The mutant B cells had a corresponding defect in their ability to bind IgE. CD23 could be normally induced on IgE-/- B cells after culture with IL-4 or CD40 ligand, indicating that these cells had no inherent defect in CD23 biosynthesis. CD23 expression and IgE-binding capacity were both restored when splenocytes from IgE-/- mice were cultured in the presence of IgE. IgE-induced up-regulation of CD23 could be elicited in vivo as well. In IgE-/- mice, i.v. infusion of IgE corrected CD23 expression to wild-type levels. Our results demonstrate that IgE directly participates in CD23 regulation in vivo. This positive feedback loop may constitute a mechanism for the amplification of ongoing allergic responses.

Molecular and biological characterization of the murine leukotriene B4 receptor expressed on eosinophils.

The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane-spanning G protein-coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351-amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y7) and a leukotriene B4 (LTB4) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [3H]LTB4 with a dissociation constant of 0.6 +/- 0.1 nM. Furthermore, LTB4 induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [35S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5-exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon gamma stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB4 receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.

A murine model of allergic rhinitis: studies on the role of IgE in pathogenesis and analysis of the eosinophil influx elicited by allergen and eotaxin.

BACKGROUND: Allergic rhinitis is a prevalent disease with significant morbidity. Studies of its pathophysiology in human subjects have been limited. Nasal biopsy specimens are difficult to obtain, and nasal secretions incompletely reflect the cellular and molecular events in the mucosa. IgE-mediated mast cell activation and the elaboration of factors promoting eosinophil development and chemotaxis are likely to participate in pathogenesis. OBJECTIVES: We sought to develop a murine model of allergic rhinitis, to use it to assess the role of IgE in pathogenesis, and to study the effects of IL-5 and eotaxin in the nasal mucosa. METHODS: A protein extract of Aspergillus fumigatus (Af) was instilled intranasally in mice. Histologic changes were examined in wild-type and IgE-deficient (IgE-/-) animals. The effect of eotaxin administration was assessed in wild-type and IL-5 transgenic mice. RESULTS: Af-treated mice developed a nasal mucosal eosinophil influx comparable to that described for humans. This histology was distinct from that observed in a murine model of Af-induced asthma. The pathology appeared over a time course similar to that reported for human subjects. There was no difference in the intensity of the mucosal inflammatory infiltrate of Af-treated IgE-/- mice compared with wild-type mice. Eotaxin was able to recruit eosinophils to the mucosa but only in IL-5 transgenic animals. CONCLUSION: We describe a murine model for allergic rhinitis with an eosinophilic infiltrate comparable to that found in human disease and have demonstrated that rhinitis can arise in the absence of IgE. We have shown that the eosinophil influx can be induced by eotaxin in the presence of IL-5.

IgE regulates mouse basophil Fc epsilon RI expression in vivo.

The binding of IgE to high-affinity IgE receptors (Fc epsilon RI) on the surface of mast cells and basophils primes these cells to secrete a panel of proinflammatory mediators upon subsequent exposure to specific Ag. We now find that the level of Fc epsilon RI expression on bone marrow basophils in mice infected with the nematode Strongyloides venezuelensis exhibits a strong positive correlation with the serum concentration of IgE, as was previously reported for human blood basophils. Moreover, the administration of IgE in vivo can significantly upregulate Fc epsilon RI expression on mouse basophils, and genetically IgE-deficient (IgE -/-) mice exhibit a dramatic (approximately 81%) reduction of basophil Fc epsilon RI expression compared with the corresponding normal (IgE +/+) mice. The finding that IgE can be a major regulator of mouse basophil Fc epsilon RI expression in vivo identifies a potentially important mechanism for enhancing the expression of effector cell function in IgE-dependent allergic reactions or immunologic responses to parasites.

Allergen-induced bronchial hyperreactivity and eosinophilic inflammation occur in the absence of IgE in a mouse model of asthma.

In patients with asthma, elevations of IgE correlate both with allergic inflammation of the airways and with bronchial hyperreactivity (BHR). Several investigations, using mouse models of this disease, have indicated a central role for IgE in the pathogenesis of the eosinophilic inflammation as well as in the obstructive airway physiology of BHR. Some diagnostic studies and therapeutic strategies for asthma are based on the putative role of IgE in asthma pathogenesis. Here, we use mice with a null mutation of the C epsilon locus to show that bronchial inflammation and BHR in response to allergen inhalation both can occur in the absence of IgE. We demonstrate that the eosinophilic bronchial inflammation elicited in an established mouse model of hypersensitivity to Aspergillus fumigatus (Af) is accompanied by the asthmatic physiology of BHR. Wild-type and IgE-deficient mice were sensitized intranasally with Af extract. Both groups of animals developed bronchoalveolar lavage eosinophilia and pulmonary parenchymal eosinophilia. This was accompanied by increased serum levels of total and Af-specific IgE in the wild-type animals only. This Af-sensitization protocol resulted in significant BHR in both wild-type mice and IgE-deficient mice. Interestingly, unsensitized IgE-deficient mice had increased bronchial responsiveness compared with unsensitized wild-type controls. We conclude that BHR and airways inflammation can be fully expressed via IgE-independent mechanisms. These may involve the activation of mast cells by factors other than IgE as well as a mucosal lymphocyte-mediated immune response to allergen.

IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions.

The binding of immunoglobulin E (IgE) to high affinity IgE receptors (Fc(epsilon)RI) expressed on the surface of mast cells primes these cells to secrete, upon subsequent exposure to specific antigen, a panel of proinflammatory mediators, which includes cytokines that can also have immunoregulatory activities. This IgE- and antigen-specific mast cell activation and mediator production is thought to be critical to the pathogenesis of allergic disorders, such as anaphylaxis and asthma, and also contributes to host defense against parasites. We now report that exposure to IgE results in a striking (up to 32-fold) upregulation of surface expression of Fc(epsilon)RI on mouse mast cells in vitro or in vivo. Moreover, baseline levels of Fc(epsilon)RI expression on peritoneal mast cells from genetically IgE-deficient (IgE -/-) mice are dramatically reduced (by approximately 83%) compared with those on cells from the corresponding normal mice. In vitro studies indicate that the IgE-dependent upregulation of mouse mast cell Fc(epsilon)RI expression has two components: an early cycloheximide-insensitive phase, followed by a later and more sustained component that is highly sensitive to inhibition by cycloheximide. In turn, IgE-dependent upregulation of Fc(epsilon)RI expression significantly enhances the ability of mouse mast cells to release serotonin, interleukin-6 (IL-6), and IL-4 in response to challenge with IgE and specific antigen. The demonstration that IgE-dependent enhancement of mast cell Fc(epsilon)RI expression permits mast cells to respond to antigen challenge with increased production of proinflammatory and immunoregulatory mediators provides new insights into both the pathogenesis of allergic diseases and the regulation of protective host responses to parasites.