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.

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.

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.

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.

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.

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.

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.

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.

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.

A review of the structure and function of the T-cell receptor-T3 complex.

Of fundamental importance in understanding the events involved in T cell activation is the identification and characterization of the relevant cell surface molecules. Antigen-induced stimulation and subsequent activation of the T cell are initiated through interactions with the T cell antigen receptor. Several lines of evidence have demonstrated the intimate association between the T cell antigen receptor and T3, thus forming the so-called T3-T cell receptor complex. First, in immunoprecipitates with either anti-T3 monoclonal antibodies, or with anti-T cell receptor antibodies, five polypeptide chains have been detected. These are two disulfide bridged variable glycoproteins (alpha and beta chains) and three invariable structures the T3-gamma, delta, and epsilon chains with molecular weights of 25, 20, and 20 kdaltons, respectively. Second, mutants of a T leukemic cell line which were selected for the loss of the T3 complex from their surface by treatment with an anti-T3 antibody and complement concomitantly lost expression of the clonotypic heterodimer. Third, monoclonal antibodies directed at either the T cell receptor alpha and beta chains or at the T3 chains affect T cell functions in an identical fashion. Thus, we see that the complex formed between the T cell receptor and the T3 molecules is functionally as well as structurally central to the immune response. The structure, biosynthesis, and regulation of gene expression of the T3-T cell receptor complex will be discussed. An attempt will be made to relate the structural information to the function of the T3-T cell receptor complex.

Further biochemical studies of the human B-cell differentiation antigens B1 and B2.

Two human B lymphocyte-associated antigens, B1 and B2, were studied by immunoprecipitation using monoclonal antibodies. B1 was found to be a nonglycosylated protein of MW 35 kD, phosphorylated at serine and threonine. B2, a 140 kD MW antigen, was characterized as a glycoprotein without phosphorylated sites. Both proteins were found to contain portions which could be labeled with 125I-iodonaphthylazide. The B1 protein displayed hydrophobic properties whereas B2 had a more amphiphilic character.

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.

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.

Purification, preliminary characterization, and immunological comparison of hog lens leucine aminopeptidase (EC 3.4.11.1) with hog kidney and beef lens aminopeptidases.

Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508-513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53-84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.

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.

A T3-like protein complex associated with the antigen receptor on murine T cells.

Antigen recognition by human T lymphocytes and initiation of T-cell activation are mediated by a group of integral membrane proteins, the T-cell antigen receptor (TCR) and the T3 complex. The polypeptides which comprise T3 (a gamma-chain of relative molecular mass (Mr) 25,000 (25K), and delta and epsilon chains of 20K each) are physically associated with the TCR chains. Surface expression of the complex requires the presence of all the component T3 and TCR proteins. In contrast to the human system, murine T3 has not been identified using antibodies. Here we describe a murine T3-like protein complex. It appears to be more complicated than human T3, containing three monomeric glycoproteins (21-28K), two of which have N-linked carbohydrate side chains and a novel family of TCR-associated homo- and heterodimers. The 28K protein is identified as the murine T3 delta-chain. The 21K protein is phosphorylated on cell activation with concanavalin A (Con A).

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx.

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca2+ (Ca2+i) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a nonvariable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca2+i was dependent on the presence of extracellular Ca2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca2+i, indicating that cross-linking is not necessary to induce a Ca2+ response. Alterations in plasma membrane potential and La3+ blocked the Ca2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca2+ channel.

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes.

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.