Basophils exhibit antibacterial activity through extracellular trap formation.

Basophils are primarily associated with immunomodulatory functions in allergic diseases and parasitic infections. Recently, it has been demonstrated that both activated human and mouse basophils can form extracellular DNA traps (BETs) containing mitochondrial DNA and granule proteins. In this report, we provide evidence that, in spite of an apparent lack of phagocytic activity, basophils can kill bacteria through BET formation.

Biomarkers of the involvement of mast cells, basophils and eosinophils in asthma and allergic diseases.

Biomarkers of disease activity have come into wide use in the study of mechanisms of human disease and in clinical medicine to both diagnose and predict disease course; as well as to monitor response to therapeutic intervention. Here we review biomarkers of the involvement of mast cells, basophils, and eosinophils in human allergic inflammation. Included are surface markers of cell activation as well as specific products of these inflammatory cells that implicate specific cell types in the inflammatory process and are of possible value in clinical research as well as within decisions made in the practice of allergy-immunology.

Role of interleukins in the regulation of basophil development and secretion

It is well appreciated that differentiation, growth, and function of basophils are regulated by a network of cytokines, and that these cells express a unique composition of surface receptors including interleukin-binding sites. In the current article, most recent discoveries around cytokine regulation of basophils are discussed and compared with previous data.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein

Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release.

Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

Etiology and pathogenesis of adverse drug reactions

In clinical routine, adverse drug reactions (ADR) are common, and they should be included in the differential diagnosis in all patients undergoing drug treatment. Only part of those ADR are immune-mediated hypersensitivity reactions and thus true drug allergies. Far more common are non-immune-mediated ADR, e.g. due to the pharmacological properties of the drug or to the individual predisposition of the patient (enzymopathies, cytokine dysbalance, mast cell hyperreactivity). In true drug allergiesT cell- and immunoglobulin E (lgE)-mediated reactions dominate the clinical presentation. T cell-mediated ADR usually have a delayed appearance and include skin eruptions in most cases. Nevertheless, it should not be forgotten that they may involve systemic T cell activation and thus take a severe, sometimes lethal turn. Clinical danger signs are involvement of mucosal surfaces, blistering within the exanthematous skin areas and systemic symptoms, e.g. fever or malaise. Drug presentation via antigen-presenting cells to T cells can either involve the classical pathway of haptenization of endogenous proteins or be directly mediated via noncovalent binding to immune receptors (MHC molecules or T cell receptors), the so-called p-i concept. Flare-up reactions during the acute phase of T cell-mediated ADR should not be mistaken for true drug allergies, as they only occur in the setting of a highly activated T cell pool. IgE-mediated ADR are less frequent and involve mast cells and/or basophils as peripheral effector cells. Recent data suggest that certain patients with drug allergy have a preexistent sensitization although they have never been exposed to the culprit drug, probably due to cross-reactivity. Thus, allergic drug reactions on first encounter are possible. In general, the extent of cross-reactivity is higher in IgE-compared to T cell-mediated ADR. Based on a specific ethnic background and only for severe T cell-mediated ADR to certain drugs, a strong HLA association has been established recently.

Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

The basophil activation test in immediate-type drug allergy

Diagnosis of drug allergy involves first the recognition of sometimes unusual symptoms as drug allergy and, second, the identification of the eliciting drug. This is an often difficult task, as the clinical picture and underlying pathomechanisms are heterogeneous. In clinical routine, physicians frequently have to rely upon a suggestive history and eventual provocation tests, both having their specific limitations. For this reason both in vivo (skin tests) and in vitro tests are investigated intensively as tools to identify the disease-eliciting drug. One of the tests evaluated in drug allergy is the basophil activation test (BAT). Basophils with their high-affinity IgE receptors are easily accessible and therefore can be used as indicator cells for IgE-mediated reactions. Upon allergen challenge and cross-linking of membrane-bound IgE antibodies (via Fc-epsilon-RI) basophils up-regulate certain activation markers on their surface such as CD63 and CD203c, as well as intracellular markers (eg, phosphorylated p38MAPK). In BAT, these alterations can be detected rapidly on a single-cell basis by multicolor flow cytometry using specific monoclonal antibodies. Combining this technique with in vitro passive sensitization of donor basophils with patients' serum, one can prove the IgE dependence of a drug reaction. This article summarizes the authors' current experience with the BAT in the diagnostic management of immediate-type drug allergy mediated by drug-specific IgE antibodies.

Anti-eosinophil activity and clinical efficacy of the CRTH2 antagonist OC000459 in eosinophilic esophagitis

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic, Th2-type inflammatory disease. Chemoattractant receptor-homologous molecule on Th2 cells (CRTH2) is a prostaglandin D(2) (PGD(2)) receptor, expressed by Th2 cells and other inflammatory cells, including eosinophils and basophils, that mediates chemotaxis and activation. OC000459 is a selective CRTH2 antagonist and would be expected to suppress eosinophilic tissue inflammation. The purpose of this study was to evaluate the efficacy and safety of an OC000459 monotherapy in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE. METHODS: In this randomized, double-blind, placebo-controlled trial, 26 adult patients (m/f = 22/4; mean age 41 years, range 22-69 years) with active EoE, dependent or resistant to corticosteroids, were treated either with 100 mg OC000459 (n = 14) or placebo (n = 12) twice daily. Pre- and post-treatment disease activity was assessed clinically, endoscopically, histologically, and via biomarkers. The primary end point was the reduction in esophageal eosinophil infiltration. RESULTS: After an 8-week OC000459 treatment, the esophageal eosinophil load decreased significantly, from 114.83 to 73.26 eosinophils per high-power field [(eos/hpf), P = 0.0256], whereas no reduction was observed with placebo (102.80-99.47 eos/hpf, P = 0.870). With OC000459, the physician's global assessment of disease activity improved from 7.13 to 5.18 (P = 0.035). OC000459 likewise reduced extracellular deposits of eosinophil peroxidase and tenascin C, the effects not seen with placebo. No serious adverse events were observed. CONCLUSIONS: An 8-week treatment with the CRTH2-antagonist, OC000459, exerts modest, but significant, anti-eosinophil and beneficial clinical effects in adult patients with active, corticosteroid-dependent or corticosteroid-refractory EoE and is well tolerated.

IL-33 is a mediator rather than a trigger of the acute allergic response in humans

BACKGROUND IL-33 enhances FcεRI-induced mediator release in human basophils without inducing degranulation itself. In contrast, studies in mice suggested that in the presence of high IgE levels, IL-33 triggers degranulation and anaphylaxis of similar severity as specific allergen. Consistent with this view, sera of atopic patients contain elevated levels of IL-33 after anaphylaxis. In this study, we determined whether IL-33 is potentially anaphylactogenic in humans with high IgE levels by regulating exocytosis independent of FcεRI cross-linking. Furthermore, we investigated whether IL-33 is released upon allergen provocation in vivo. METHODS In subjects with high serum IgE levels, we measured IL-33-induced histamine/LTC4 in vitro, CD63 translocation ex vivo, and responsiveness of mast cells in vivo by skin prick test (SPT). In asthma patients, release of IL-33 and its correlation with early (tryptase)- and late-phase markers (IL-13 levels, eosinophil numbers) of the allergic response were assessed in bronchoalveolar lavage fluids (BALFs) after allergen challenge. RESULTS IL-33 itself does not trigger basophil degranulation in vitro and ex vivo, even in subjects with high serum IgE levels, and negative SPTs demonstrate that skin mast cells do not degranulate in response to IL-33. However, in response to allergen challenge, IL-33 is rapidly released into BALFs at levels that do not correlate with other immediate- and late-phase parameters. CONCLUSION IL-33 is unlikely an independent trigger of anaphylaxis even in subjects with high IgE levels. However, the rapid release of IL-33 upon allergen provocation in vivo supports its role as a mediator of immediate allergic responses.

Exposure to food allergens through inflamed skin promotes intestinal food allergy through the thymic stromal lymphopoietin-basophil axis.

BACKGROUND Exposure to food allergens through a disrupted skin barrier has been recognized as a potential factor in the increasing prevalence of food allergy. OBJECTIVE We sought to test the immunologic mechanisms by which epicutaneous sensitization to food allergens predisposes to intestinal food allergy. METHODS Mice were epicutaneously sensitized with ovalbumin or peanut on an atopic dermatitis-like skin lesion, followed by intragastric antigen challenge. Antigen-specific serum IgE levels and T(H)2 cytokine responses were measured by ELISA. Expression of type 2 cytokines and mast cell proteases in the intestine were measured by using real-time PCR. Accumulation of basophils in the skin and mast cells in the intestine was examined by using flow cytometry. In vivo basophil depletion was achieved by using diphtheria toxin treatment of Baso-DTR mice. For cell-transfer studies, the basophil population was expanded in vivo by means of hydrodynamic tail vein injection of thymic stromal lymphopoietin (TSLP) cDNA plasmid. RESULTS Sensitization to food allergens through an atopic dermatitis-like skin lesion is associated with an expansion of TSLP-elicited basophils in the skin that promote antigen-specific T(H)2 cytokine responses, increased antigen-specific serum IgE levels, and accumulation of mast cells in the intestine, promoting the development of intestinal food allergy. Critically, disruption of TSLP responses or depletion of basophils reduced the susceptibility to intestinal food allergy, whereas transfer of TSLP-elicited basophils into intact skin promoted disease. CONCLUSION Epicutaneous sensitization on a disrupted skin barrier is associated with accumulation of TSLP-elicited basophils, which are necessary and sufficient to promote antigen-induced intestinal food allergy.

Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

In vitro differentiation of mouse granulocytes. Programmed Cell Death: Methods and Protocols

Granulocytes are central players of the immune system and, once activated, a tightly controlled balance between effector functions and cell removal by apoptosis guarantees maximal host benefit with least possible collateral damage to healthy tissue. Granulocytes are end-differentiated cells that cannot be maintained in culture for prolonged times. Isolating primary granulocytes is inefficient and challenging when working with mice, and especially so for the lowly abundant eosinophil and basophils subtypes. Here we describe an in vitro protocol to massively expand mouse derived myeloid progenitors and to differentiate them ‘on demand’ and in large numbers into mature neutrophils or basophils.

The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease

The basophil activation test (BAT) has become a pervasive test for allergic response through the development of flow cytometry, discovery of activation markers such as CD63 and unique markers identifying basophil granulocytes. Basophil activation test measures basophil response to allergen cross-linking IgE on between 150 and 2000 basophil granulocytes in <0.1 ml fresh blood. Dichotomous activation is assessed as the fraction of reacting basophils. In addition to clinical history, skin prick test, and specific IgE determination, BAT can be a part of the diagnostic evaluation of patients with food-, insect venom-, and drug allergy and chronic urticaria. It may be helpful in determining the clinically relevant allergen. Basophil sensitivity may be used to monitor patients on allergen immunotherapy, anti-IgE treatment or in the natural resolution of allergy. Basophil activation test may use fewer resources and be more reproducible than challenge testing. As it is less stressful for the patient and avoids severe allergic reactions, BAT ought to precede challenge testing. An important next step is to standardize BAT and make it available in diagnostic laboratories. The nature of basophil activation as an ex vivo challenge makes it a multifaceted and promising tool for the allergist. In this EAACI task force position paper, we provide an overview of the practical and technical details as well as the clinical utility of BAT in diagnosis and management of allergic diseases.