Flow-Cytometric Analysis of Human Basophil Degranulation

Quantification of human basophils and their degranulation were performed using a flow-cytometric system. It was shown that the number of basophils among total leukocytes, before and after the effect of degranulating agents, can be obtained rapidly and reproducibly, following alcian blue, staining. Dose-response curves of degranulation by anti-IgE and anti-IgG₄ were studied, and it was shown that these antibodies can induce basophil degranulation in a Ca⁺⁺ dependent manner. It was also shown that degranulation is completed within 10 min.
Values obtained by flow-cytometry and microscopic evaluation, as well as comparison made between percent basophil degranulation and percent histamine release, agreed well with each other in our system. Flow-cytometric analysis of human basophil degranulation provides a new reliable method to assess in vitro the morphological basis of immediate hyper-sensitivity.     

Diverse innate stimuli activate basophils through pathways involving Syk and IκB kinases

Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE cross-linking but also by endogenous “innate” factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFβ, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases.

Basophils are rare circulating granulocytes activated by immunoglobulin E (IgE)-mediated cross-linking of the high affinity receptor for IgE (FcεRI), which induces their degranulation and synthesis of the type 2 cytokines, interleukin 4 (IL-4) and IL-13 in autoimmune, allergic diseases and helminthiases (1). Basophils are also sensitive to innate signals, including the cytokine IL-3, the alarmins IL-18, IL-33, and Thymic stromal Lymphopoietin (TSLP), the prostaglandin D2 (PGD2), ATP, and various chemokines or growth factors (1–4). Some of these stimuli might regulate basophil immunoregulatory functions during homeostasis (5). Secretion of IL-4 and histamine release by human basophils is sensitive to FK506 (a calcineurin inhibitor), while secretion of IL-13 is not affected, indicating that distinct signaling pathways regulate the expression of these type 2 cytokines (6–8). By contrast, activation of basophils by IgE or IL-18/33 involves distinct receptors and upstream signaling pathways but results in expression of both IL-4 and IL-13 (5, 7, 9–11). Despite common evolutionary ancestry, it is currently thought that IL-4 and IL-13 show divergent functions in immunity and physiology (12, 13). Basophil IL-4 secretion has been implicated in T helper type 2 (Th2) differentiation and M2 skewing. Basophils are an important source of IL-13 in the lungs, where they control the phenotype of alveolar macrophages during development (1, 5).The Il4/13 locus is subject to a differential regulation between distinct cell types but has been mostly studied in T cells. The locus contains elements regulated by cAMP, c-fos/AP-1, NFAT, NF-κB, GATA3, STAT5, and STAT6 that have been associated with the regulation of Il4/13 expression (6, 14–19). Basophils produce IL-4 and IL-13 after the cross-linking of their surface-bound IgE by antigen or by exposure to IL-3. IgE- or IL-3-mediated basophil activation are both controlled by the tyrosine kinase Syk (6, 20, 21). IgE-induced cytokine expression is also promoted by IκB kinase (IKK) through NF-κB-dependent or -independent signals (22, 23).To date, studies of murine basophils have focused on cultured bone marrow-derived basophils (BMBa) or primary bone marrow (BM) basophils, with circulating mature basophils too few in number for useful studies (6). We used B8 × 4C13R IL-4/IL-13 triple reporter mice (24) to follow and compare the ex vivo responses of primary mature basophils to 96 common stimuli and found that basophils were highly sensitive to type 2 and epithelial-derived cytokines and growth factors. Single-cell analysis revealed that basophils displayed distinct phenotypes upon stimulation with IL-3, expressing IL-4, and/or IL-13, Ly6C, or the degranulation marker CD63 (25). During helminth infection, basophils were found to be hyper- and hyporeactive to distinct stimuli (“Hp-basophils”). Purified Hp-basophils were also sensitive to homeostatic growth factors and antiviral response elements. Basophil reactivity was always sensitive to Syk and IKKs specific inhibitors. In conclusion, we found basophils to be sensitive to a complex array of distinct innate stimuli that worked through core common signaling pathways.     

Mast cells derived from human induced pluripotent stem cells are useful for allergen tests

Background

Several methods have been developed to detect allergen-specific IgE in sera. The passive IgE sensitization assay using human IgE receptor-expressing rat cell line RBL-2H3 is a powerful tool to detect biologically active allergen-specific IgE in serum samples. However, one disadvantage is that RBL-2H3 cells are vulnerable to high concentrations of human sera. Only a few human cultured cell lines are easily applicable to the passive IgE sensitization assay. However, the use of human induced pluripotent stem cells (iPSCs) to generate human mast cells (MCs) has not yet been reported.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.     

Regulation of human basophil activation. IV. Dissociation between cationic dye binding and histamine release: role of Ca2+ ions*

In previous studies we observed that in vitro histamine release from human basophils could be dissociated from the loss of affinity of basophil granules for a cationic dye, toluidine blue. In the present study we further explored the intracellular signals leading to the decrease in toluidine blue positive basophil (TB+) numbers, with or without histamine release. Since Ca2+ mobilization is a crucial event in secretion and particularly in histamine release, we studied the role of Ca2+ in histamine release as compared to TB+ decrease. In the presence of external Ca2+ (2 mM): i) Ca2+ channel antagonists verapamil and nifedipine up to 10 microM were without effect on IgE-mediated histamine release and TB+ decrease; ii) loading of the leucocytes with Quin2 or preincubation with TMP-8, an internal Ca2+ antagonist, significantly inhibited the release of histamine and the decrease of TB+ basophils. In the absence of added external Ca2+:i) histamine release was abolished whereas the decrease of TB+ was not modified, even in the presence of EGTA;ii) the decrease of TB+ could be inhibited by prolonged EGTA preincubation, by Quin2 loading and incubation with TMB-8. We conclude that histamine release requires both external Ca2+ influx and mobilization of internal Ca2+. In contrast, no influx of external Ca2+ is required for TB+ decrease in which, however, internal Ca2+ mobilization appears to play an important role.     

Tryptase genetics and anaphylaxis

Tryptases secreted by tissue mast cells and basophils can enter the bloodstream. In human subjects tryptases are encoded by several genes and alleles, including alpha, beta, gamma, and delta. Common variations include complete absence of alpha genes. Until recently, alpha tryptase was considered to be the major tryptase secreted at baseline and in mastocytosis. However, lack of alpha tryptase genes has little effect on circulating tryptase levels, which are now thought mainly to consist of inactive pro-beta tryptase secreted constitutively rather than stored in granules with mature tryptases. Pro-beta tryptase levels thus might reflect total body mast cell content. In contrast, mature beta tryptase can increase transiently in severe systemic anaphylaxis and confirm the diagnosis. However, it might fail to increase in food anaphylaxis or might increase nonspecifically in samples acquired after death. Thus pro- and mature beta tryptase measurements are useful but associated with false-negative and false-positive results, which need to be considered in drawing clinical conclusions in cases of suspected anaphylaxis.     

CD63 expression on basophils as a tool for the diagnosis of pollen-associated food allergy: sensitivity and specificity

BACKGROUND: Basophil activation is associated with the expression of CD63. Because allergens can induce basophil activation by cross-linking specific IgE, increased CD63 expression has been proposed as a novel in vitro test for immediate type allergy. OBJECTIVE: We compared the CD63-based basophil activation test (BAT) in the diagnosis of allergy to carrot, celery and hazelnut with skin prick tests (SPT) and measurement of allergen-specific IgE. METHODS: Twenty-nine patients with a history of an oral allergy syndrome induced by carrot, celery or hazelnut (n = 20 for each allergen) and 20 controls were studied. SPT were performed with standardized and native carrot, celery and hazelnut extracts. Allergen-specific IgE was determined by the CAP FEIA method and basophil activation was determined by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. RESULTS: SPT with native carrot, celery and hazelnut showed sensitivities of 100%, 100% and 90%, and specificities of 80%, 80% and 90%. SPT with commercial extracts of the same allergens gave sensitivities of 85%, 80% and 85%, and specificities of 80%, 80% and 90%. Sensitivity of allergen-specific IgE and the BAT for carrot, celery and hazelnut was 80% vs. 85%, 70% vs. 85%, and 80% vs. 90%, with corresponding specificities of 80% vs. 85%, 80% vs. 80%, and 95% vs. 90%. The cut-off for a positive BAT was 10% CD63+ basophils. Moreover, there was a positive correlation between IgE reactivity and the number of CD63+ basophils for all food allergens (carrot: r = 0.69, celery: r = 0.67, hazelnut: r = 0.66). CONCLUSIONS: Quantification of basophil activation by CD63 expression is a valuable new in vitro method for diagnosis of immediate type food sensitization. Although double-blind placebo-controlled food challenges remain the gold standard, the CD63-based BAT may supplement routine diagnostic tests such as SPT or allergen-specific IgE in the future.     

Basophil activation in idiopathic mixed cryoglobulinemia

We studied basophil activation in patients affected by idiopathic mixed cryoglobulinemia in order to investigate the role of the basophil cell in the pathogenesis of the vasculitis typical of this disease. We selected 13 patients and we performed thein vitro basophil degranulation test with cryoprecipitate; in 5 cases the test was positive when it was performed in whole blood, while it was negative when it was performed with cells washed and resuspended in Tyrode's buffer. In 6 subjects it was not possible to perform the test because of the very low number of circulating basophil cells. Finally, in 2 subjects thein vitro basophil degranulation test with cryoprecipitate was negative; this result may be related to the very low levels of complement factors in the sera of these subjects. These findings sugges that the pathogenetic mechanism of vasculitis in idiopathic mixed cryoglobulinemia could be started by basophil activation.