The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human IgE-FcepsilonRI interaction.
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Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcepsilonRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcepsilonRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcepsilonRI interaction: Cepsilon3 represents the third constant domain of the IgE heavy chain, and alpha2 is the membrane-proximal Ig-like module from FcepsilonRIalpha. Native overlay experiments showed binding of human FcepsilonRIalpha to recombinant Cepsilon3 and of natural or recombinant human IgE to recombinant alpha2. Moreover, recombinant Cepsilon3 inhibited binding of natural IgE antibodies to alpha2, and preincubation of human IgE with alpha2 inhibited anti-IgE-triggered histamine release from human basophils. Isolated Cepsilon3 and alpha2 can now be used for the molecular and structural analysis of the IgE-FcepsilonRI interaction, as well as for diagnostic and therapeutic applications. (+info)
Establishment of antigen-specific IgE transgenic mice to study pathological and immunobiological roles of IgE in vivo.
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We have established transgenic mice that carry the genes coding for heavy and light chains of TNP-specific IgE. They produced high titers of TNP-specific IgE (20-40 microg/ml in serum) and their mast cells were heavily loaded with IgE. The level of FcepsilonRI expression on their mast cells was 6-8 times higher than that in non-transgenic littermates. The expression of low-affinity IgE receptor FcepsilonRII (CD23) on splenic B cells was also 6-8 times higher in the transgenic mice. Consistent with this, substantial amounts of IgE were detected on B cells in the transgenic mice. When challenged with i.v. administration of the corresponding antigen, the transgenic mice exhibited systemic anaphylactic symptoms such as a drastic drop of body temperature and extravasation of administered dye. Biphasic (immediate and delayed) ear swelling response was also elicited in a TNP-specific manner by epicutaneous antigen challenge without any prior sensitization. Thus, IgE produced in the transgenic mice was found to be biologically active to induce both local and systemic allergic reactions in vivo upon the challenge of the corresponding antigen. Taken together, the antigen-specific IgE transgenic mice established for the first time in this study appear to provide an attractive model system to study the pathological roles of IgE in acute and chronic phases of allergic inflammation as well as their immunobiological roles in vivo. They may also be useful to develop novel therapeutic strategies for atopic disorders. (+info)
Differences in nasal cellular infiltrates between allergic children and age-matched controls.
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Little is known about the cellular infiltrates in the nasal mucosa of children. This study was set up to compare the nasal cellular infiltrates in biopsy specimens from allergic children and controls. Atopic children were distinguished from controls on the basis of symptoms of allergic rhinitis and/or asthma, total serum immunoglobulin (Ig)E, family history and specific serum IgE to food and aeroallergens. Fifteen allergic patients (median age 4.3 yrs) and 15 age-matched nonallergic control subjects were evaluated. The number of cells positive for CD1a, CD4, CD8, CD19, CD68, chymase, tryptase, IgE and major basic protein was determined in the mucosa of the inferior turbinate. A significantly higher number of IgE-positive cells and mast cells was found in the epithelia of the allergic group. In the lamina propria, higher numbers of IgE-positive cells and eosinophils were found. Langerhans' cells positive for IgE were only seen in allergic children with specific serum IgE against aeroallergens. These children also had a higher number of IgE-positive mast cells compared to controls and atopic children without specific serum IgE. These results show that the nasal cellular infiltrates of allergic children differ from nonallergic control subjects. Prior to the detection of specific serum immunoglobulin E, cellular changes can be found in the nasal mucosa of atopic children. (+info)
Shifts in lung lymphocyte profiles correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model.
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T lymphocytes have a central regulatory role in the pathogenesis of asthma. We delineated the participation of lymphocytes in the acute allergic and chronic tolerant stages of a murine model of asthma by characterizing the various subsets of lymphocytes in bronchoalveolar lavage and lung tissue associated with these responses. Acute (10-day) aerosol challenge of immunized C57BL/6J mice with ovalbumin resulted in airway eosinophilia, histological evidence of peribronchial and perivascular airway inflammation, clusters of B cells and TCRgammadelta cells in lung tissue, increased serum IgE levels, and airway hyperresponsiveness to methacholine. In mice subjected to chronic (6-week) aerosol challenge with ovalbumin, airway inflammation and serum IgE levels were significantly attenuated and airway hyperresponsiveness was absent. The marked increases in lung B and T cell populations seen in the acute stage were also significantly reduced in the chronic stage of this model. Thus, acute ovalbumin challenge resulted in airway sensitization characteristic of asthma, whereas chronic ovalbumin challenge elicited a suppressed or tolerant state. The transition from antigenic sensitization to tolerance was accompanied by shifts in lymphocyte profiles in the lung and bronchoalveolar lavage fluid. (+info)
Short ragweed allergen induces eosinophilic lung disease in HLA-DQ transgenic mice.
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The human leukocyte antigen (HLA) restriction of the IgE response to different allergens in humans has been a subject of numerous published studies. However, the role and contribution of specific HLA class II molecules in the pathogenesis of allergic airway inflammation are unknown and difficult to assess. HLA-DQ6 and HLA-DQ8 transgenic mice lacking endogenous mouse class II gene expression were actively immunized and later challenged intranasally with short ragweed (SRW) allergenic extract. The HLA-DQ transgenic mice developed pulmonary eosinophilia and lung tissue damage. We also found an increase in total protein (TP) level and IL-5 production in bronchoalveolar lavage (BAL) fluid and an increase in SRW-specific Th2-type immunoglobulins (IgG1, IgG2b) and total serum IgE levels. Under similar treatment, DQ-negative full-sib control mice were normal. The allergic response could be significantly inhibited or abrogated in HLA-DQ mice by systemic treatment with anti-DQ mAb. The in vivo responses of HLA-DQ6 and HLA-DQ8 mice showed differences in terms of levels of eosinophilia, BAL protein, IL-5 concentration, and lung hyperreactivity to inhaled methacholine. These findings demonstrate the crucial role for specific HLA-DQ molecules in SRW-specific CD4(+) T-cell activation and resulting recruitment of eosinophils into the airways. (+info)
SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells.
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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. (+info)
Immunoglobulin E-independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions.
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Intradermal administration of short overlapping peptides derived from chain 1 of the cat allergen Fel d 1 (FC1P) that did not cross-link IgE, elicited isolated late asthmatic reactions with no visible early or late cutaneous response in 9/40 cat-allergic asthmatics. Four of the nine were human histocompatibility leukocyte antigen DR13-positive, as compared with only 1/31 nonreactors. The other five reactors expressed either DR1 or DR4. To confirm major histocompatibility complex restriction, fibroblast cell lines transfected with HLA-DR molecules were used to present FC1Ps to cat allergen-specific T cell lines derived from subjects before peptide injection. FC1P3 (peptide 28-44 of Fel d 1 chain 1) was recognized in the context of DR13 alleles (DRB1*1301, 1302) and induced specific T cell proliferation and IL-5 production. T cells from a DR1(+) responder proliferated and produced IL-5 in the presence of FC1P3 and DR1 (DRB1*0101) fibroblast cell lines, whereas T cells from a DR4(+) subject recognized FC1P2 (peptide 22-37) when presented by DRB1*0405. We conclude that short, allergen-derived peptides can directly initiate a major histocompatibility complex-restricted, T cell-dependent late asthmatic reaction, without the requirement for an early IgE/mast cell-dependent response, in sensitized asthmatic subjects. (+info)
Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum.
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The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders. (+info)