Activation Of Human Basophils Research Articles

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.

Sputum basophils from allergic asthmatic patients do not express IL‐7Rα that is essential for TSLP signalling

AbstractBasophils are crucial in regulating allergic reactions via immediate secretion of multiple mediators upon IgE‐induced degranulation. IL‐3 regulates the development and activation of human basophils while epithelial cytokine thymic stromal lymphopoietin (TSLP) is another key regulator for murine basophils. Despite association of increased TSLP with exaggerated basophil responses in oesophageal biopsies, the effects of TSLP in regulating human basophil degranulation and activation are under debate. In this study, we aimed to examine whether human basophils responded to TSLP by co‐expression of TSLP receptors, TSLPR and IL‐7Rα (CD127), upon in vitro activation and in sputum of allergic asthmatic patients. Flow cytometric analysis of fresh basophils from healthy controls revealed no detectable TSLPR and CD127. Further flow cytometric analysis of basophils from healthy controls in vitro stimulated by multiple established basophil modulators for 24 hours showed induction of TSLPR but not CD127 by IL‐3 (10 ng/ml), anti‐IgE (10 μg/ml) and C5a (50 ng/ml). One‐hour stimulation of basophils from allergic asthmatic patients and healthy controls by TSLP (50 ng/ml) with or without IL‐3 (10 ng/ml) and anti‐IgE (10 μg/ml) had no effect on induction of CD63+ degranulated basophils. Similarly, TSLP (50 ng/ml) with or without IL‐3 (10 ng/ml) and anti‐IgE (10 μg/ml) did not induce IL‐4 and IL‐13 production by basophils from healthy controls. Further ex vivo analysis revealed that sputum basophils from allergic asthmatic patients did not express CD127. We conclude that human basophils from healthy controls and allergic asthmatic patients do not respond to TSLP as lacking CD127.

On the role of allergen-specific IgG subclasses for blocking human basophil activation

Successful treatment of IgE mediated allergies by allergen-specific immunotherapy (AIT) usually correlates with the induction of allergen-specific IgG4. However, it is not clear whether IgG4 prevents the allergic reaction more efficiently than other IgG subclasses. Here we aimed to compare allergen-specific monoclonal IgG1 and IgG4 antibodies in their capacity to inhibit type I allergic reactions by engaging FcγRIIb. We found that IgG1, which is the dominant subclass induced by viruses, binds with a similar affinity to the FcγRIIb as IgG4 and is comparable at blocking human basophil activation from allergic patients; both by neutralizing the allergen as well as engaging the inhibitory receptor FcγRIIb. Hence, the IgG subclass plays a limited role for the protective efficacy of AIT even if IgG4 is considered the best correlate of protection, most likely simply because it is the dominant subclass induced by classical AITs.

IgG Autoantibodies Against IgE from Atopic Dermatitis Can Induce the Release of Cytokines and Proinflammatory Mediators from Basophils and Mast Cells.

IgE-mediated release of proinflammatory mediators and cytokines from basophils and mast cells is a central event in allergic disorders. Several groups of investigators have demonstrated the presence of autoantibodies against IgE and/or FcεRI in patients with chronic spontaneous urticaria. By contrast, the prevalence and functional activity of anti-IgE autoantibodies in atopic dermatitis (AD) are largely unknown. We evaluated the ability of IgG anti-IgE from patients with AD to induce the in vitro IgE-dependent activation of human basophils and skin and lung mast cells. Different preparations of IgG anti-IgE purified from patients with AD and rabbit IgG anti-IgE were compared for their triggering effects on the in vitro release of histamine and type 2 cytokines (IL-4, IL-13) from basophils and of histamine and lipid mediators (prostaglandin D2 and cysteinyl leukotriene C4) from human skin and lung mast cells. One preparation of human IgG anti-IgE out of six patients with AD induced histamine release from basophils, skin and lung mast cells. This preparation of human IgG anti-IgE induced the secretion of cytokines and eicosanoids from basophils and mast cells, respectively. Human monoclonal IgE was a competitive antagonist of both human and rabbit IgG anti-IgE. Human anti-IgE was more potent than rabbit anti-IgE for IL-4 and IL-13 production by basophils and histamine, prostaglandin D2 and leukotriene C4 release from mast cells. Functional anti-IgE autoantibodies rarely occur in patients with AD. When present, they induce the release of proinflammatory mediators and cytokines from basophils and mast cells, thereby possibly contributing to sustained IgE-dependent inflammation in at least a subset of patients with this disorder.

Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions - a replication study

Background: Inhibition of human basophil activation by highly diluted histamine was reported to be a reliable experimental model to examine biological effects of high dilutions. However, independent replications did not always yield concordant results.
 Aims: We aimed at performing an independent replication of a former study [1] using rigorously controlled experimental conditions to minimise confounding factors.
 Materials and Methods: In 20 independent experiments, human basophils were treated with highly diluted histamine (15cH, 16cH, corresponding to 10-30-10-32 M) prior to activation by fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Controls were treated with analogously diluted water (15cH, 16cH). The dilutions were prepared freshly for each experiment in deionised water by successive steps of centesimal dilution and agitation (10 s vortex at high speed). Highly diluted samples were blinded and randomised. All samples were set in triplicates. Activated basophils were determined by flow cytometry using anti-CD203c. 20 independent systematic negative control (SNC) experiments were carried out to investigate possible systematic errors. 
 Results: No difference in basophil activation was observed between the highly diluted histamine samples and the highly diluted water controls. There was no evidence for a blood donor specificity of the results. The SNC experiments demonstrated the stability of the test system. Experimental variability within and between experiments was slightly reduced for the highly diluted histamine samples.
 Discussion: This study was designed as an independent reproduction of a former study [1]. Though we strictly adopted the experimental procedure described in [1], our results do not confirm the large inhibitory effects observed for histamine 15cH and 16cH. This lack of reproducibility might be due to minor differences in the experimental design, such as blinding and randomising of the samples, which we chose to perform in order to reduce the possibility of artifacts but was omitted in the former study.
 Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research.
 Keywords: Human basophils; histamine; high dilutions; flow cytometry
 Reference:
 [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.

Epithelial Cell-Associated Galectin-3 Activates Human Dendritic Cell Subtypes for Pro-Inflammatory Cytokines.

There is mounting evidence that galectin-3 is a prognostic and diagnostic biomarker associated with diverse diseases and conditions, including cancer, cardiovascular disease, autoimmunity, wound healing, allergic disease, and chronic inflammation in general. Yet, whether and exactly how galectin-3 may participate in the pathogenesis of these diseases remains poorly understood. Recently, we have linked the expression of galectin-3 on the A549 epithelial cell line –an adenocarcinoma, to the activation of human basophils for the release of histamine and secretion of IL-4 and IL-13. These responses proved dependent on cell-to-cell contact, basophil expression of IgE, were inhibited by n-acetyllactosamine, and were ablated when basophils were co-cultured with A549 clones lacking galectin-3 expression. While recombinant galectin-3 failed to activate basophils when in solution, microspheres expressing this lectin did so by mimicking the responses seen when using A549 cells. Given the IgE dependency of the basophil responses, and the fact that galectin-3 is long known to bind this immunoglobulin, we hypothesize that a similar mode of activation extends to other IgE-bearing cells. To investigate this possibility, we tested epithelial cell-associated galectin-3 for its capacity to activate human dendritic cells, including the plasmacytoid and myeloid subtypes as well as monocytes, all of which bind IgE. Indeed, results indicate that epithelial cell-associated galectin-3 activated these cells for robust production of TNF-α and IL-6 and up-regulated the expression of activation markers found on dendritic cells. Moreover, many of the same parameters previously observed for basophils applied to the findings herein, including evidence that matrix-bound galectin-3 (whether on epithelial cells or microspheres) facilitates this mode of activation. In contrast, IgE expression was dispensable for these galectin-3-dependent cytokine responses, implying that this lectin activates dendritic cells (and monocytes) by binding to a glycoprotein other than this immunoglobulin. Overall, these findings further demonstrate how galectin-3 mediates immune cell activation, providing novel insight into how this lectin may promote chronic inflammation underlying the pathogenesis of many diseases.

Interleukin 3-induced GITR promotes the activation of human basophils

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Bruton's tyrosine kinase inhibition effectively protects against human IgE-mediated anaphylaxis.

No known therapies can prevent anaphylaxis. Bruton's tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcεRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human CD34+ cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting FcεRI-mediated signaling.

Developing a nonhuman primate basophil activation assay by flow cytometry

Abstract The Human Basophil Activation test (BAT) has been validated for many disease conditions, used for the diagnosis of allergy to drugs and foods, and for monitoring the role of basophils in immunotherapy. While several BAT kits are commercially available and widely used for humans, the BAT assay in nonhuman primate (NHP) is scarcely present in the literature. Given that NHPs are used as a nonclinical model in drug development and safety assessment when other animal species cannot be used, we developed a BAT using peripheral blood of cynomolgus macaques (Cyno). For human, the mAb panels 1) CCR3+SSClow,2) CD203c+SSClow, 3) CRTH2+CD3−, and 4) CD66−CD3−CD123+HLA-DR− have been used to identify total basophils. Following stimulation with allergens or a positive control (eg. anti-human FcɛRIα or IgE), the activated basophils can be gated as CD63+ and/or CD203+. For the Cyno BAT development, first, the anti-human mAbs noted above were tested for their cross-reactivity with Cyno. Second, the anti-human FcɛRIα and IgE mAbs were tested for their ability to crosslink and activate the Cyno basophils. Third, the basophil stimulation buffer (BSB) was formulated with calcium and heparin with or without IL-3 for blood collected in EDTA tubes. The FcɛRIα but not the IgE mAb, consistently activated basophils and upregulated CD63 expression. IL-3 added to BSB resulted in high background CD63 expression on unstimulated basophils and therefore was not used in the finalized method. Our data indicates that the only panel with all mAbs cross-reactive to Cyno was 4) CD66−CD3−CD123+HLA−. This panel identified the Cyno basophils that were SSCMed not SSCLow. In summary, a BAT has been successfully developed for use in exploratory NHP studies.

Selective suppression of oral allergen-induced anaphylaxis by Allergin-1 on basophils in mice.

Mast cells (MCs) play a critical role in oral allergen-induced anaphylaxis. However, the contribution of basophils to the anaphylaxis remains unclear. The inhibitory immunoreceptor Allergin-1 is highly expressed on MCs and basophils and inhibits FcεRI-mediated signaling in MCs. Here, we show that Allergin-1-deficient (Milr1-/-) mice developed more severe hypothermia, a higher mortality rate and a greater incidence of diarrhea than did wild-type (WT) mice in an oral ovalbumin (OVA)-induced food allergy model. MC-deficient Mas-TRECK mice, which had been reconstituted with either WT or Milr1-/- bone marrow-derived cultured MCs, did not develop hypothermia in this food allergy model. On the other hand, depletion of basophils by injection of anti-CD200R3 antibody rescued Milr1-/- mice from lethal hypothermia but not from diarrhea. In vitro analyses demonstrated that Allergin-1 inhibits IgE-dependent activation of both human and mouse basophils. Thus, Allergin-1 on basophils selectively suppresses oral allergen-induced anaphylaxis.

Interleukin 2 regulates the activation of human basophils

Basophils are important effector cells in allergic disorders and anti-parasitic immune response. A number of activators including interleukin 3 (IL-3) and IgE have been identified in the regulation of human basophils expressing mediators such as histamine and leukotriene C4 (LTC4) and cytokines, including IL-4 and IL-13. Human basophils express high levels of IL-2 receptors. However, the function of the IL-2 pathway in basophils remains unknown. Here, we identified that IL-2 induced the activation of human basophils in vitro to express a variety of inflammatory cytokines and chemokines including IL-5, IL-13, GM-CSF and CCL-17. This effect by IL-2 is confirmed by an upstream regulator analysis using Ingenuity pathway analysis. Of note, one of the top regulated cytokines, IL-5, was for the first time identified to be induced by IL-2 in human basophils rather than IL-3 or anti-IgE. Immunofluorescence analysis of skin specimens from bullous pemphigoid and eczema revealed that infiltrating basophils in skin lesions widely expressed IL-5 and GM-CSF. Together, our findings reveal IL-2 as a novel regulator of human basophils. This adds a new layer to support the importance of basophils in allergic disorders.

Inhibiting phosphatase SHIP-1 enhances suboptimal IgE-mediated activation of human blood basophils but inhibits IgE-mediated activation of cultured human mast cells

IgE-mediated activation of basophil granulocytes and mast cells follows a bell-shaped dose-response curve. The decreased activation at supraoptimal allergen stimulation is thought to be associated with SH2-containing inositol-5′-phosphatase 1 (SHIP-1). SHIP-1 phosphorylation is inversely related to IgE-mediated releasability of basophils. This study sought to clarify the regulatory role of SHIP-1 in degranulation of basophil granulocytes and mast cells by selective inhibition of the phosphatase function of SHIP-1with 3-α-aminocholestane (3-α-AC).Six grass pollen allergic patients, six non-responder patients and six cultured human primary mast cell lines were included. The effect of 3-α-AC (1–60 μM, 30 min, 37 °C) was analyzed at individual suboptimal, optimal and supra-optimal allergen concentrations. The activity, upregulation of CD63, measured at different conditions was compared to evaluate the maximal effect of selective SHIP-1 inhibition. Basophils of five non-responder patients were treated with 3-α-AC (10 μM, 30 min, 37 °C).At high concentrations (>60 μM) of 3-α-AC, cells appeared to enter apoptosis. The median reactivity increased from 27.1% to 44.9% CD63+ basophils at 10 μM of 3-α-AC and suboptimal allergen stimulation (p = 0.0153). There was no effect on blood basophils of 3-α-AC at optimal or supra-optimal allergen concentrations. In contrast, treatment with more than 6 μM 3-α-AC significantly inhibited mast cell reactivity. 10 μM 3-α-AC reduced median reactivity from 32.85% to 16.5% CD63+ mast cells (p = 0.0465). Treatment with 3-α-AC did not increase response of basophils of non-responder patients.Modulating blood basophils with 3-α-AC enhanced reactivity only at suboptimal allergen concentration, and basophils from non-responders did not regain responsiveness to IgE stimulation. 3-α-AC inhibited the IgE response of mast cells in a dose dependent manner.

Galectin-3 is essential for IgE-dependent activation of human basophils by A549 lung epithelial cells

Using novel co-culture model systems, galectin-3 is shown to facilitate basophil secretion of IL-4/IL-13, with implications that this unique mode of activation may be linked to a variety of diseases ranging from asthma to cancer.

High concentration of IgE antibodies induce activation of human peripheral basophils

High concentration of IgE antibodies induce activation of human peripheral basophils

Activation of Human Peripheral Basophils in Response to High IgE Antibody Concentrations without Antigens.

Basophils and mast cells have high affinity IgE receptors (FcεRI) on their plasma membrane and play important roles in FcεRI-associated allergic diseases, such as pollen allergy, food allergy, chronic spontaneous urticarial (CSU), and atopic dermatitis (AD). To date, several reports have revealed that high IgE antibody concentrations activate mast cells—which reside in tissue—in the absence of any antigens (allergens). However, IgE antibody-induced activation of basophils—which circulate in blood—has not been reported. Here, we investigated whether IgE antibodies may regulate functions of human peripheral basophils without antigens in vitro. We successfully removed IgE antibodies bound to FcεRI on the surface of human peripheral basophils by treating with 0.1% lactic acid. We also demonstrated that high IgE antibody concentrations (>1 μM) induced histamine release, polarization, and CD203c upregulation of IgE antibody-stripped basophils. Thus, high IgE antibody concentrations directly activate basophils, which express IgE-free FcεRI on the cell surface. This mechanism may contribute to the pathogenesis of patients with AD and CSU who have higher serum IgE concentrations compared to healthy donors.

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

The soluble isoform of FcɛRI, the high-affinity IgE receptor (sFcεRI), is a protein of the IgE network with poorly defined functions. To define cellular sources and signals that result in the production of human sFcεRI and study its invivo functions. 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. 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 invivo. 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.

Characterization of a hybrid protein designed with segments of allergens from Blomia tropicalis and Dermatophagoides pteronyssinus

BackgroundSensitization to allergens of the house dust mites Dermatophagoides pteronyssinnus and Blomia tropicalis is an important risk factor for asthma and allergic diseases. Allergen-specific immunotherapy is currently based on natural allergen extracts, however, in the last years recombinant allergens with different modifications have shown promising immunological properties that may be advantageously applied for developing novel allergy vaccines. MethodsA hybrid molecule (MAVAC-BD-2) containing epitopes of B. tropicalis (Blo t 5, Blo t 8 and Blo t 10) and D. pteronyssinus (Der p 1, Der p 2, Der p 7 and Der p 8) allergens was constructed, expressed in Escherichia coli and purified by affinity chromatography. Its folding was analyzed by circular dichroism. Antibody reactivities were evaluated by ELISA and non-denaturing dot blot assays using a battery of sera from mite allergic patients and non-allergic subjects. ELISA inhibition and dot blot assays with monoclonal antibodies were used to detect B-cell epitopes. Human basophil activation and induction of IgG-blocking antibodies in mice immunized with the hybrid protein were also evaluated. ResultsMAVAC-BD-2, expressed as a 22.8 kDa protein, showed a lower frequency and strength of IgE reactivity compared to Blo t 5, Der p 1, Der p 2 and the extracts of B. tropicalis and D. pteronyssinus. MAVAC-BD-2 inhibited 26% of IgE reactivity to Der p 2 and Blo t 5, reacted with anti-Der p 1 and anti-Der p 2 monoclonal antibodies and did not induce relevant basophil activation. MAVAC-BD-2 immunized mice produced specific antibodies that reacted against mite extracts and the purified allergens, as well as IgG antibodies that blocked the human IgE reactivity to mite extracts. ConclusionMAVAC-BD-2 has hypoallergenic characteristics and in mice induces IgG antibodies that block the human IgE reactivity to mite extracts.

Activation of Human Basophils by A549 Lung Epithelial Cells Reveals a Novel IgE-Dependent Response Independent of Allergen.

Evidence for epithelial cell (EC)-derived cytokines (e.g., thymic stromal lymphopoietin [TSLP]) activating human basophils remains controversial. We therefore hypothesize that ECs can directly activate basophils via cell-to-cell interaction. Basophils in medium alone or with IL-3 ± anti-IgE were coincubated with TSLP, IL-33, or IL-25. Analogous experiments cocultured basophils (1-72 h) directly with EC lines. Supernatants were tested for mediators and cytokines. Abs targeting receptors were tested for neutralizing effects. Lactic acid (pH 3.9) treatment combined with passive sensitization tested the role of IgE. Overall, IL-33 augmented IL-13 secretion from basophils cotreated with IL-3, with minimal effects on histamine and IL-4. Conversely, basophils (but not mast cells) released histamine and marked levels of IL-4/IL-13 (10-fold) when cocultured with A549 EC and IL-3, without exogenous allergen or IgE cross-linking stimuli. The inability to detect IL-33 or TSLP, or to neutralize their activity, suggested a unique mode of basophil activation by A549 EC. Half-maximal rates for histamine (4 h) and IL-4 (5 h) secretion were slower than observed with standard IgE-dependent activation. Ig stripping combined with passive sensitization ± omalizumab showed a dependency for basophil-bound IgE, substantiated by a requirement for cell-to-cell contact, aggregation, and FcεRI-dependent signaling. A yet unidentified IgE-binding lectin associated with A549 EC is implicated after discovering that LacNAc suppressed basophil activation in cocultures. These findings point to a lectin-dependent activation of basophil requiring IgE but independent of allergen or secreted cytokine. Pending further investigation, we predict this unique mode of activation is linked to inflammatory conditions whereby IgE-dependent activation of basophils occurs despite the absence of any known allergen.

Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation.

IntroductionThe microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli.MethodsTo determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation.ResultsWe uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type.ConclusionsIndividual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy.

Human basophil chemotaxis and activation are regulated via the histamine H4 receptor.

IgE-mediated cross-linking of FcεRI results in the release of mediators stored in basophil granules, such as histamine and proteases, and in the de novo synthesis of sulfidoleukotrienes. In this study, we investigated the role of the histamine receptors, in particular that of the histamine H4 receptor (H4R), in modulating human basophil function. The mRNA expression of the histamine receptors was measured by real-time PCR. Migration of basophils was assessed using the modified Boyden chamber technique. The expression levels of CD63 and CD203c on the cell surface and the sulfidoleukotriene release were determined by flow cytometry and ELISA, respectively. We could show that highly purified basophils express the H1R, H2R, and H4R but not the H3R mRNA. Human basophils expressed higher H4R mRNA levels as compared to the expression levels of the H1R (P < 0.01). Histamine and the H4R agonist ST-1006 initiated active migration of basophils (P < 0.001). A significant reduction in FcεRI cross-linking-mediated surface expression of CD63 and CD203c was observed on basophils after pre-incubation with histamine or the specific H4R agonist ST-1006 (P < 0.01). The synthesis and release of sulfidoleukotrienes from basophils after activation with different stimuli, by FcεRI cross-linking or by stimulation with hymenoptera venom allergens, were significantly reduced by histamine or the H4R agonist ST-1006 (P < 0.05-0.001). These data imply that the H4R regulates IgE-dependent processes in human basophils and provides a novel function of the H4R preventing an overwhelming immune reaction by engagement of a negative feedback loop.