Cross-linking Of IgE Research Articles

Conditionally immortalized cell lines with mast cell potential

Abstract Prior to the establishment of definitive hematopoiesis, mast cells (MCs) derived from the yolk sac and fetal liver colonize diverse tissues, maintaining a presence throughout life. Following the establishment of definitive hematopoiesis, hematopoietic stem cells from the bone marrow (BM) also contribute to the MC pool. The diverse origins and tissue-resident nature of MCs complicate their study. Therefore, a system facilitating the expansion of MC progenitors and their controlled differentiation becomes crucial. This report outlines the development of conditionally immortalized MC progenitor cell lines based on two tissue sources, i.e. mouse fetal liver and adult bone marrow. To obtain cell lines, retroviral vectors encoding an estrogen-regulated Hox gene were introduced into progenitor cells derived from both tissues. These transduced cells can be expanded in their non-differentiated state for extended periods and upon estrogen removal, these cells differentiate into MCs in the presence of IL-3 and SCF. Resulting mast cells are functionally comparable to BM-derived MCs, initiating degranulation upon IgE cross-linking. Likewise, these MCs express MC-specific proteases such as chymase 1 and MCPT4. In conclusion, our findings highlight the efficacy of estrogen-regulated Hox genes in targeting MC progenitors in both fetal liver and adult BM. The established cell lines serve as reliable tools for studying MC biology, ontogeny and, possibly, disease conditions, such as allergies.

Generation of a virus-like particles based vaccine against IgE.

Anti-IgE immunotherapy with monoclonal antibodies represents a breakthrough in treatment of severe allergic diseases. However, drawbacks such as short half-life and high price are not negligible. Our objective is to develop an anti-IgE vaccine based on virus-like particles (VLPs) which can induce long-lasting neutralizing IgG anti-IgE antibodies reducing allergic responses without causing intrinsic mast cell activation due to IgE cross-linking. The vaccines were made by chemically coupling three synthetic mouse IgE-Fc fragments to plant-derived immunologically optimized CuMVTT VLPs. The immunogenicity of the vaccines was tested by immunizing naive or allergic mice either with the coupled vaccines or the VLP control followed by systemic or local allergen challenge. Mice immunized with the vaccines exhibited high titers of anti-IgE antibodies in the sera and high levels of anti-IgE secreting plasma cells in lymphoid organs. Moreover, free IgE in serum were reduced by the induced anti-IgE antibodies; therefore, less IgE was bound to FcεRI on the surface of basophils. In line with these reduced IgE levels on effector cells after vaccination, immunized mice were protected from challenge with allergens. Importantly, despite presence of anti-IgE antibodies, no signs of acute or chronic allergic response were seen in immunized allergic mice. The generated vaccines can effectively induce anti-IgE antibodies that did not cause allergic responses in sensitized mice but were able to decrease the level of free and cell bound IgE and protected sensitized animals from allergic responses upon allergen challenge.

An optimized method for IgE-mediated degranulation of human lung mast cells.

Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

Degranulation of human mast cells: modulation by P2 receptors' agonists.

Since the late 1970s, there has been an alarming increase in the incidence of asthma and its morbidity and mortality. Acute obstruction and inflammation of allergic asthmatic airways are frequently caused by inhalation of exogenous substances such as allergens cross-linking IgE receptors expressed on the surface of the human lung mast cells (HLMC). The degree of constriction of human airways produced by identical amounts of inhaled allergens may vary from day to day and even hour to hour. Endogenous factors in the human mast cell (HMC)'s microenvironment during allergen exposure may markedly modulate the degranulation response. An increase in allergic responsiveness may significantly enhance bronchoconstriction and breathlessness. This review focuses on the role that the ubiquitous endogenous purine nucleotide, extracellular adenosine 5'-triphosphate (ATP), which is a component of the damage-associated molecular patterns, plays in mast cells' physiology. ATP activates P2 purinergic cell-surface receptors (P2R) to trigger signaling cascades resulting in heightened inflammatory responses. ATP is the most potent enhancer of IgE-mediated HLMC degranulation described to date. Current knowledge of ATP as it relates to targeted receptor(s) on HMC along with most recent studies exploring HMC post-receptor activation pathways are discussed. In addition, the reviewed studies may explain why brief, minimal exposures to allergens (e.g., dust, cat, mouse, and grass) can unpredictably lead to intense clinical reactions. Furthermore, potential therapeutic approaches targeting ATP-related enhancement of allergic reactions are presented.

The transcription factor NFIL3/E4BP4 regulates the developmental stage–specific acquisition of basophil function

Basophils are rare but important effector cells in many allergic disorders. Contrary to their early progenitors, the terminal developmental processes of basophils in which they gain their unique functional properties are unknown. We sought to identify a novel late-stage basophil precursor and a transcription factor regulating the terminal maturation of basophils. Using flow cytometry, transcriptome analysis, and functional assays, we investigated the identification and functionality of the basophil precursors as well as basophil development. We generated mice with basophil-specific deletion of nuclear factor IL-3 (NFIL3)/E4BP4 and analyzed the functional impairment of NFIL3/E4BP4-deficient basophils invitro and invivo using an oxazolone-induced murine model of allergic dermatitis. We report a new mitotic transitional basophil precursor population (referred to as transitional basophils) that expresses the FcεRIα chain at higher levels than mature basophils. Transitional basophils are less responsive to IgE-linked degranulation but produce more cytokines in response to IL-3, IL-33, or IgE cross-linking than mature basophils. In particular, we found that the expression of NFIL3/E4BP4 gradually rises as cells mature from the basophil progenitor stage. Basophil-specific deletion of NFIL3/E4BP4 reduces the expression of genes necessary for basophil function and impairs IgE receptor signaling, cytokine secretion, and degranulation in the context of murine atopic dermatitis. We discovered transitional basophils, a novel late-stage mitotic basophil precursor cell population that exists between basophil progenitors and postmitotic mature basophils. We demonstrated that NFIL3/E4BP4 augments the IgE-mediated functions of basophils, pointing to a potential therapeutic regulator for allergic diseases.

Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Multistep IgE Mast Cell Desensitization Is a Dose- and Time-Dependent Process Partially Regulated by SHIP-1

Multistep mast cell desensitization blocks the release of mediators following IgE crosslinking with increasing doses of Ag. Although its invivo application has led to the safe reintroduction of drugs and foods in IgE-sensitized patients at risk for anaphylaxis, the mechanisms of the inhibitory process have remained elusive. We sought to investigate the kinetics, membrane, and cytoskeletal changes and to identify molecular targets. IgE-sensitized wild-type murine (WT) and FcεRIα humanized (h) bone marrow mast cells were activated and desensitized with DNP, nitrophenyl, dust mites, and peanut Ags. The movements of membrane receptors, FcεRI/IgE/Ag, actin, and tubulin and the phosphorylation of Syk, Lyn, P38-MAPK, and SHIP-1 were assessed. Silencing SHIP-1 protein was used to dissect the SHIP-1 role. Multistep IgE desensitization of WT and transgenic human bone marrow mast cells blocked the release of β-hexosaminidase in an Ag-specific fashion and prevented actin and tubulin movements. Desensitization was regulated by the initial Ag dose, number of doses, and time between doses. FcεRI, IgE, Ags, and surface receptors were not internalized during desensitization. Phosphorylation of Syk, Lyn, p38 MAPK, and SHIP-1 increased in a dose-response manner during activation; in contrast, only SHIP-1 phosphorylation increased in early desensitization. SHIP-1 phosphatase function had no impact on desensitization, but silencing SHIP-1 increased β-hexoxaminidase release, preventing desensitization. Multistep IgE mast cell desensitization is a dose- and time-regulated process that blocks β-hexosaminidase, impacting membrane and cytoskeletal movements. Signal transduction is uncoupled, favoring early phosphorylation of SHIP-1. Silencing SHIP-1 impairs desensitization without implicating its phosphatase function.

The effect of soy processing on its allergenicity: Discrepancy between IgE binding and basophil stimulation tests

Presently, many studies assess allergenicity via IgE immunoblotting and IgE binding tests; however, IgE detection does not always signal the manifestation of a clinical allergy. However, the capacity of food allergens to trigger basophils makes it possible to use the in vitro functional basophil activation test (BAT) to assess allergenicity. The effect of the Maillard reaction (MR) on the allergenic potential of processed soy proteins was evaluated by two IgE binding tests (Competitive ELISA and Inhibition ImmunoCAP), a Western Blot and a functional BAT; with the aim to analyze whether the sIgE binding results correspond to the functional assay results. The results between the IgE binding tests and the functional assay were in-line for 2 of the 6 studied patients. For one patient there was no correlation between any of the results. For the raw soy protein extract heated with glucose for 10 and 30 min (SH SPE + Glu and LH SPE + Glu, respectively), the results were in-line for only 3 out of the 6 patients. Thus, the present study shows a discrepancy between IgE binding tests and basophil stimulation when assessing the effect of soy processing on its allergenicity. Since IgE-binding capacity does not always correlate to IgE cross-linking capacity, the conclusions of the allergenic potential based on the IgE binding tests alone should be drawn with care and further studies on this matter would benefit from the inclusion of a functional assay such as the BAT.

Remibrutinib inhibits hives effector cells stimulated by serum from chronic urticaria patients independently of FcεR1 expression level and omalizumab clinical response.

Despite advances in the treatment of chronic urticaria, in a significant percentage of the patients symptoms are not fully controlled with conventional approaches. New strategies under development include blocking intracellular mediators of mast cell and basophil activation. We aim to investigate the effects of the Bruton's tyrosine kinase (BTK) inhibitor remibrutinib on human blood basophils and CD34+ -derived mast cells activation induced by serum obtained from chronic urticaria patients. Twenty-two patients with chronic spontaneous urticaria (mean age 52years, 27% women) and 22 patients with chronic inducible urticaria (46years, 27% women) were included in the study together with a sex-matched control group. Patients were classified as responders or non-responders to anti-IgE therapy on the basis of their clinical data, FcεR1a expression on blood basophils and total IgE levels. Changes on CD63 expression-as an activation marker-, were used to evaluate in vitro the response of basophils and mast cells to serum exposure and the inhibitory effects of remibrutinib. Remibrutinib inhibits degranulation induced by IgE cross-linking in mast cells and basophils and also the activation triggered by factors present in the sera of spontaneous and inducible chronic urticaria patients. Patient's serum induces a greater degranulation of effector cells than controls. Activation of mast cells and basophils by patient sera and remibrutinib effects were not related to omalizumab responsiveness. Remibrutinib inhibits activation of human basophils and mast cells induced in vitro by exposure to the serum of chronic urticaria patients independently of their response to omalizumab.

Antigenic Nanostructures based on Amoxicilloyl-bidendrons for Effector Cell Activation to Study Allergic Reactions to Amoxicillin

Amoxicillin (AX) is the most common betalactam that causes allergic reactions. To be immunogenic, AX requires protein haptenation to form multivalent conjugates of increased size, suitable for crosslinking of specific IgE (sIgE) bound on basophils or mast cells (MCs). Using well-defined nanostructures allowed the identification of the shortest distance between antigenic determinants for cell activation in a realistic model using AX as a hapten. In this study, we designed nanostructures that varied both the number of antigenic determinants and the distance between them in order to assess the role of valence in promoting effector cell activation in the context of allergy to AX.

An advanced in vitro human mucosal immune model to predict food sensitizing allergenicity risk: A proof of concept using ovalbumin as model allergen.

The global demand of sustainable food sources leads to introduction of novel foods on the market, which may pose a risk of inducing allergic sensitization. Currently there are no validated in vitro assays mimicking the human mucosal immune system to study sensitizing allergenicity risk of novel food proteins. The aim of this study was to introduce a series of sequential human epithelial and immune cell cocultures mimicking key immune events after exposure to the common food allergen ovalbumin from intestinal epithelial cell (IEC) activation up to mast cell degranulation. This in vitro human mucosal food sensitizing allergenicity model combines crosstalk between IEC and monocyte-derived dendritic cells (moDC), followed by coculture of the primed moDCs with allogenic naïve CD4+ T cells. During subsequent coculture of primed CD4+ T cells with naïve B cells, IgE isotype-switching was monitored and supernatants were added to primary human mast cells to investigate degranulation upon IgE crosslinking. Mediator secretion and surface marker expression of immune cells were determined. Ovalbumin activates IEC and underlying moDCs, both resulting in downstream IgE isotype-switching. However, only direct exposure of moDCs to ovalbumin drives Th2 polarization and a humoral B cell response allowing for IgE mediated mast cell degranulation, IL13 and IL4 release in this sequential DC-T cell-B cell-mast cell model, indicating also an immunomodulatory role for IEC. This in vitro coculture model combines multiple key events involved in allergic sensitization from epithelial cell to mast cell, which can be applied to study the allergic mechanism and sensitizing capacity of proteins.

New Mechanistic Advances in FcεRI-Mast Cell-Mediated Allergic Signaling.

Mast cells originate from the CD34+/CD117+ hematopoietic progenitors in the bone marrow, migrate into circulation, and ultimately mature and reside in peripheral tissues. Microbiota/metabolites and certain immune cells (e.g., Treg cells) play a key role in maintaining immune tolerance. Cross-linking of allergen-specific IgE on mast cells activates the high-affinity membrane-bound receptor FcεRI, thereby initiating an intracellular signal cascade, leading to degranulation and release of pro-inflammatory mediators. The intracellular signal transduction is intricately regulated by various kinases, transcription factors, and cytokines. Importantly, multiple signal components in the FcεRI-mast cell–mediated allergic cascade can be targeted for therapeutic purposes. Pharmacological interventions that include therapeutic antibodies against IgE, FcεRI, and cytokines as well as inhibitors/activators of several key intracellular signaling molecues have been used to inhibit allergic reactions. Other factors that are not part of the signal pathway but can enhance an individual’s susceptibility to allergen stimulation are referred to as cofactors. Herein, we provide a mechanistic overview of the FcεRI-mast cell–mediated allergic signaling. This will broaden our scope and visions on specific preventive and therapeutic strategies for the clinical management of mast cell–associated hypersensitivity reactions.

Systematic comparisons of various markers for mast cell activation in RBL-2H3 cells.

Mast cell activation plays a key role in various allergic diseases and anaphylaxis. Several methods/techniques can be used for detection of mast cell activation. However, there was no previous systematic evaluation to compare the efficacy of each method/technique. The present study thus systematically compared various markers for mast cell activation induced by IgE cross-linking. The widely used RBL-2H3 mast cells were sensitized with anti-DNP (dinitrophenyl) IgE overnight and activated with DNP-BSA (bovine serum albumin) for up to 4h. The untreated cells and those with anti-DNP IgE sensitization but without DNP-BSA activation served as the controls. Intracellular calcium level gradually increased to ~2-fold at 1h, reached its peak (~5-fold) at 2h, and returned to the basal level at 3-h post-activation. The increases in cellular tryptase level (by Western blotting) (~0.3- to 0.4-fold) and average cell size (~2.5-fold) and decrease of nucleus/cytoplasm ratio (~0.4- to 0.5-fold) were marginal at all time-points. By contrast, β-hexosaminidase release and CD63 expression (by both flow cytometry and immunofluorescence detection/localization), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence detection/localization) stably and obviously increased (~10-fold as compared with the untreated control and sensitized-only cells or detectable only after activation). Based on these data, the stably obvious increases (by ≥ 10-fold) in β-hexosaminidase release, CD63 expression (by both flow cytometry and immunofluorescence staining), secreted tryptase level (by Western blotting), and tryptase expression (by immunofluorescence staining) are recommended as the markers of choice for the in vitro study of mast cell activation using RBL-2H3 cells.

Molecular and immunological characterization of cysteine protease from Phaseolus vulgaris and evolutionary cross-reactivity.

A commonly consumed legume in India, the kidney bean (Phaseolus vulgaris) is associated with allergy. We report molecular and immunological characterization of cysteine protease allergen and its cross-reactivity. In silico allergenicity assessment and phylogenetic analysis of kidney bean cysteine protease showed significant sequence homology (upto 67%) with allergens from kiwi, papaya, soybean, ragweed pollen and mites. Physicochemical properties and motif-analysis depicted cysteine protease as probable allergen. Multiple sequence alignment and phylogenetic analysis indicated structural conservation between kidney bean and homologous cysteine protease sequences. The gene was cloned, expressed and affinity purified. Cysteine protease was resolved at 42 kDa and exhibited high IgE binding (up to 89%) with hypersensitive sera. Cysteine protease showed functional property on cross-linking IgE receptors and upregulated expression of CD203c on activated basophils. In inhibition studies, 8.4ng of cysteine protease was required for 50% self-inhibition, whereas significant inhibition was also observed with kidney bean (52 ng), black gram (155 ng), chick pea (437 ng), mesquite pollen (36 ng), house dust mite (64.85 ng), Alternaria alternata (78.8ng) and Curvularia lunata (73.6ng) extracts. ConSurf analysis indicated conserved active site and catalytic residues in mature domain among proteases from legumes, fruits, pollens, mites and fungus. In summary, P. vulgaris cysteine protease was molecularly characterized having functional activity. This study demonstrated, cross-reactivity between food and aeroallergens based on evolutionary conservancy that showed its clinical importance as cross-reactive allergen. PRACTICAL APPLICATIONS: Adaptation of sustainable lifestyle has led to a surge in consumption of plant-based foods especially legumes. Their high nutritional content lowers the risk of developing cardiovascular diseases, diabetes, obesity, and stroke. Kidney beans, a commonly consumed legume in Indian subcontinent, have a potential to be used as nutraceutical and functional food. Despite its alimentary nature, it elicits allergic reactions. Being a major sensitizer, trivial information regarding its allergic components has led to an urgent need for exploring its allergen repertoire. Our study reported biochemical and immunological characterization of its major cysteine protease allergen. Cysteine proteases are major cross-reactive allergens from insects, fruits and fungal sources. Identification and molecular characterization of such immunodominant allergens by RDT offers the prospect of using recombinant proteins for accurate diagnosis and therapeutic purposes. This study suggests that a potential major cross-reactive allergen may aid in developing allergy management interventions for a wide range of allergenic sources.

The Allergen-Specific IgE Concentration Is Important for Optimal Histamine Release From Passively Sensitized Basophils.

BackgroundThe basophil histamine release (HR) assay can be used for allergy diagnosis in addition to the conventional measurement of allergen-specific IgE (sIgE). Passive sensitization of basophils increases the versatility and allows testing the biological relevance of allergen-induced IgE cross-linking in any serum unbiased by the cellular component. However, not all the patient sera perform equally well and we hypothesized that the absolute level and fraction of sIgE affect the performance. Choosing birch pollen allergy as a model, we investigated the concentration of sIgE needed for successful passive sensitization using soluble- or matrix-fixed Bet v 1.MethodsTwenty-eight sera with Bet v 1 sIgE [7 sera within each allergy class (1: 0.1–0.70 kUA/L, 2: 0.71–3.50 kUA/L, 3: 3.51–17.50 kUA/L, and 4+: >17.50 kUA/L)] and a negative control serum pool were used to passively sensitize donor basophils, obtained from buffy coat blood (n = 3). The cells were incubated (30 min) with a soluble allergen (rBet v 1 from 0.2 to 50 ng/ml), matrix-fixed allergen (ImmunoCAP™ containing recombinant Bet v 1), or phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (maximal HR) and released histamine was quantified fluorometrically.ResultsThe lowest level of Bet v 1 sIgE generating a detectable HR (HR > 10% of maximal release) in all the 3 runs was found to be 1.25 kUA/L (corresponding to allergy class 2, 0.71–3.50 kUA/L). Furthermore, sera from allergy classes 3 and 4+ ascertained a significant reproducible HR: 42/42 vs. 5/21 in allergy class 1 and 15/21 in allergy class 2. Using ImmunoCAP™s containing Bet v 1 as a matrix-fixed allergen system, similar results were obtained where the lowest sIgE concentration mediating an HR was 1.68 kUA/L and 7/7 for both allergy classes 3 and 4+.ConclusionThe results demonstrate that the IgE titer is strikingly robust in predicting the ability to sensitize basophils and produce a measurable HR.

An Evaluation of the Mechanisms of Galacto-Oligosaccharide (GOS)-Induced IgE Cross-Linking on Basophils in GOS Allergy.

The prebiotics, galacto-oligosaccharides (GOS), are small carbohydrate molecules with 1–7 galactose units linked to glucose and have been shown to trigger IgE-mediated anaphylaxis in some cases following ingestion. It is still an unresolved question of how GOS cross-links IgE on basophils. In this study, we examined whether human galectins, a class of lectins that bind specifically to β-galactoside carbohydrates, are involved in GOS-induced basophil activation. Basophil activation test to GOS and control allergen, Blomia tropicalis (Blo t) extract were performed in the presence or absence of four sugar-based galectin inhibitors (lactose, thiodigalactoside [TDG], TD139, and GB1107) and one peptide-based inhibitor, G3-C12. Results showed that TD139, GB1107, and G3-C12 did not display a specific inhibitory effect on GOS-induced basophil activation as compared to control allergen. An inhibitory effect of lactose and TDG on GOS-induced basophil activation was observed and varied between subjects with up to 100% inhibition at low doses of GOS. The results of competitive ELISA suggest that the inhibitory effects of high dose lactose and TDG on the basophil activation is likely due to the cross-reactivity of GOS-specific IgE to lactose and TDG. Basophil activation is performed using purified basophils suggested that cell surface receptors on other blood cells were not required to induce basophil activation. In conclusion, our results suggest that GOS, a low molecular weight sugar, is able to cross-link IgE independently.

Dendritic Nanostructures for Effector Cell Activation to Study Allergic Reactions to Amoxicillin

Amoxicillin (AX) is the penicillin currently regarded as the main culprit in eliciting allergic reactions. According to hapten hypothesis, AX immunogenicity can only be gained by forming covalent conjugates with proteins. Structural properties of conjugates could be nano-engineered to be appropriate for crosslinking of specific IgE (sIgE) bound to high-affinity receptor on basophils or mast cells (MCs).

IL-27 signaling negatively regulates FcɛRI-mediated mast cell activation and allergic response.

IL-27 is a member of the IL-12 family, exerting both anti- and pro-inflammatory activity in a cell-dependent and disease context-specific manner. Antigen-mediated cross-linking of IgE on mast cells triggers a signaling cascade that results in mast cell degranulation and proinflammatory cytokine production, which are key effectors in allergic reactions. Here, we show that the activation of mast cells is negatively regulated by IL-27 signaling. We found that mice lacking IL-27Rα (WSX-1) displayed increased sensitivity to IgE-mediated skin allergic response and chronic airway inflammation. The bone marrow-derived mast cells (BMMCs) of IL-27Rα-deficient mouse showed greater high-affinity receptor Fc epsilon RI (FcεRI)-mediated activation with significantly enhanced degranulation and cytokine production. Mechanistically, the dysregulated signaling in IL-27Rα-/- mast cells is associated with increased activation of Grb2-PLC-γ1-SLP-76, PI3K/Akt/IκBα signaling and decreased phosphorylation level of SH2 domain-containing protein phosphatase1 (SHP1). Furthermore, IL-27 treatment could inhibit mast cell activation directly, and retrovirus-based IL-27 expression in lung attenuated the airway inflammation in mice. Collectively, our findings reveal that IL-27 signaling negatively regulates mast cell activation and its mediated allergic response.

E-Cadherin restricts mast cell degranulation in mice.

Crosslinking of FcεRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FcεRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdhfl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdhfl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdhfl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation.

IgE cross-linking induces activation of human and mouse mast cell progenitors

The concept of innate and adaptive effector cells that are repleted by maturing inert progenitor cell populations is changing. Mast cells develop from rare mast cell progenitors populating peripheral tissues at homeostatic conditions, or as a result of induced recruitment during inflammatory conditions. Because FcεRI-expressing mast cell progenitors are the dominating mast cell type during acute allergic lung inflammation invivo, we hypothesized that they are activated by IgE cross-linking. Mouse peritoneal and human peripheral blood cells were sensitized and stimulated with antigen, or stimulated with anti-IgE, and the mast cell progenitor population analyzed for signs of activation by flow cytometry. Isolated peritoneal mast cell progenitors were studied before and after anti-IgE stimulation at single-cell level by time-lapse fluorescence microscopy. Lung mast cell progenitors were analyzed for their ability to produce IL-13 by intracellular flow cytometry in a mouse model of ovalbumin-induced allergic airway inflammation. Sensitized mouse peritoneal mast cell progenitors demonstrate increased levels of phosphorylation of tyrosines on intracellular proteins (total tyrosine phosphorylation), and spleen tyrosine kinase (Syk) phosphorylation after antigen exposure. Anti-IgE induced cell surface-associated lysomal-associated membrane protein-1 (LAMP-1) in naive mast cell progenitors, and prompted loss of fluorescence signal and altered morphology of isolated cells loaded with lysotracker. In human mast cell progenitors, anti-IgE increased total tyrosine phosphorylation, cell surface-associated LAMP-1, and CD63. Lung mast cell progenitors from mice with ovalbumin-induced allergic airway inflammation produce IL-13. Mast cell progenitors become activated by IgE cross-linking and may contribute to the pathology associated with acute allergic airway inflammation.