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Background: Clinical management of allergic diseases has been hampered by the lack of safe and convenient tests to reliably identify culprit allergens and to closely follow changes in disease activity over time. Because allergy diagnosis is a complex and laborious multistep procedure, there is an urgent need for simpler but still functionally accurate ex vivo assays allowing objective diagnosis, substantiating treatment choices, and quantifying therapeutic responses., Objective: In this study, we sought to develop a novel functional cell-based assay that relies on passive sensitization of allergic effector cells with patient serum, circumventing current limitations in allergy diagnosis., Methods: We genetically engineered a conditional homeobox B8 (Hoxb8)-immortalized progenitor line from the bone marrow of mice that are transgenic for the human high-affinity IgE receptor (FcεRIα). These cells can be reproducibly differentiated into mature Hoxb8 mast cells within 5 days of culture in virtually unlimited numbers., Results: We demonstrate that the established Hoxb8 mast cell assay can be used to accurately measure total IgE levels, identify culprit allergens, longitudinally monitor allergen-specific immunotherapy, and potentially determine the time point of tolerance induction upon allergen-specific immunotherapy in patients with allergy. To facilitate the analysis of large testing volumes, we demonstrate a proof-of-concept for a high-throughput screening application based on fluorescent cell barcoding using the engineered Hoxb8 mast cells., Conclusions: Our results indicate that this novel mast cell assay could represent a valuable tool to support clinicians in the identification of IgE-mediated allergies and in the quantification of treatment efficacy as well as duration of therapeutic response., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cultured human mast cells are a useful tool for research into innate immune responses as well as allergic mechanisms. Mast cells cultured from peripheral blood can provide information on immune mechanisms of known, selected individuals. With the method presented here, eight million mast cells can be cultured from ca. one million stem cells purified from one unit (450 mL) of human peripheral blood. Culture with IgE and IL4 optimizes an allergic phenotype of the 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., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background: MicroRNAs (miRNAs) represent important post-transcriptional regulators with a dynamic expression profile during health and disease., Objectives: We explored the miRNA profile of human mast cells (MCs) during sen-sitization with IgE, during activation through IgE, and relat ed it to prostaglandin D2 synthesis and histamine release., Method: We investigated the expression pattern of 762 miRNAs during the IgE-mediated sensitization and activation of MCs cultured from CD133+ stem cells that were isolated from allergic asthmatic patients and nonatopic controls., Results: IgE-mediated sensitization increased the expression of miRNA-210 eight-fold. This increase was sustained during IgE-mediated MC activation. Furthermore, we confirmed the increase of the miRNA-132/212 cluster after MC activation. Predicted target genes of miRNA-210/132/212 were enriched in several pathways known to be involved in MC activation. Histamine release was significantly higher in MCs from allergic patients when compared to controls, and a number of miRNAs correlated with histamine release and prostaglandin D2 synthesis during MC activation., Conclusion: The miRNAs and analysis presented here can help to elucidate the role of miRNAs in mediator release during MC activation. We speculate that miRNA-210 could be important in MC sensitization that leads to allergic symptoms., (© 2019 S. Karger AG, Basel.)

Background: Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol., Methods: Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR., Results: The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01)., Conclusion: Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa., (© 2015 John Wiley & Sons Ltd.)

Mast cell activation releases the mediators associated with type I allergy. As such, the study of mast cell activation is critical for understanding the allergic reaction, and for developing methods to control it. Importantly, another ligand receptor pair (compound 48/80 and MRGPRX2) that activates mast cells in addition to allergen-IgE-FcεRI has been identified. As mast cells mature in tissue from hematopoietic stem cells, their physiology and pathophysiology is difficult to study. Mast cell lines and mast cells cultured from stem cells are often studied instead of tissue mast cells. There has been some progress in the description of the mechanism of the activation of mast cells, substances limiting mast cell activation and in the catalogue of proteases that mast cells express. Basophil granulocytes express FcεRI, bind IgE and respond to allergen crosslinking in a very similar fashion to mast cells. In the recent literature, basophils were mistakenly described as antigen-presenting cells; this has convincingly been disputed in a number of subsequent publications. Their function in physiology and pathophysiology is not known, but they are frequently used to document allergic sensitisation in the basophil activation test. Significant progress has been made in documenting the relevance of basophil activation as a second-line test in allergy diagnosis. Basophil reactivity and sensitivity may reflect symptom severity and allergen threshold, and are used to document and monitor allergy. The physiology and pathophysiology of allergic effector cells remain an important area of research., (© 2016 S. Karger AG, Basel.)

Cultured human mast cells are a useful tool for research into innate immune responses as well as allergic mechanisms. Mast cells cultured from peripheral blood can provide information on immune mechanisms of known, selected individuals. With the method presented here eight million mast cells can be cultured from ca. one million stem cells purified from one unit (450 mL) of human peripheral blood. Culture with IgE and IL4 optimizes an allergic phenotype of the mast cells.

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22,275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [-0.4795 log ng/ml ± 0.092 (SE)] and controls (-0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma., (© 2013 John Wiley & Sons Ltd.)

Objective: The contribution of affinity, clonality, and concentration of individual IgE species to effector cell response has recently been characterized in a model with recombinant human IgE on passively sensitized basophils. This study extends the dependence of effector cell degranulation on IgE concentration to mast cells cultured with IgE for 2 weeks., Methods: Human mast cells cultured for 7 weeks from peripheral blood stem cells were matured for 2 weeks with interleukin-4 (IL-4) and recombinant human IgE consisting of two clones specific for Dermatophagoides pteronyssinus 2 (Derp2) (7% + 7%) and unspecific IgE at 0.8, 8, 80, and 800 kU/L. The density of the IgE receptor, FcϵRI, and mast cell function were measured after challenging with recombinant Derp2 at 14 concentrations from 10 fg/mL to 100 pg/mL. CD63 expression, histamine release, and Prostaglandin D2 (PGD(2)) synthesis were measured, and maximal expression and mast cell sensitivity were calculated., Results: At 800 kU/L IgE, FcϵRI expression varied more than at 80, 8, and 0.8 kU/L IgE. There was a trend toward increased maximal expression of CD63, histamine release, and PGD(2) secretion with increasing IgE concentration. At 0.1 kU/L specific IgE, the LC50 increased up to fivefold, least so for PGD(2)., Conclusions: Human mast cells cultured with rhIgE of known composition are a sensitive model for studying factors governing effector cell degranulation that is close to the in vivo situation. This model can be used to study effects of IgE concentration, clonality, and affinity and may help predict the optimal immunologic treatment for a given patient.

Objective: We determined the density of FcεRI on mast cells cultured from cord (CBMC) and peripheral blood (PBMC) and studied the kinetics of the response through FcεRI., Methods: Mast cells were cultured from CD133+ progenitors from peripheral or cord blood. FcεRI was stabilized by culture with 2 μg/ml IgE. Cells were activated by addition of anti-FcεRI antibody (1 ng/ml-10 μg/ml). Maximal activation, sensitivity, and cooperativity were determined., Results: All cultures were homogeneous for tryptase and metachromasy. All cells expressing FcεRI could be activated by cross-linking FcεRI to upregulate CD63. PBMC bind 203,000 molecules of IgE/cell. Stabilization of FcεRI with IgE doubled the number of CD63+ cells (p = 0.0001) and increased the sensitivity (from 0.083 to 0.013 μg/ml anti-FcεRI) and the slope factor (from 10.8 to 68) of PBMC but not of CBMC. Anti-IgE reversed these effects (p = 0.0002) but did not reduce activation levels below that of cell lines not stabilized with IgE., Conclusion: Baseline expression of FcεRI is independent of anti-IgE. The fraction of PBMC that binds high levels of IgE can be activated through FcεRI., (Copyright © 2011 S. Karger AG, Basel.)

Functional, mature human mast cells have been generated by in vitro differentiation of CD133(+)/CD34(+) progenitor cells isolated from e.g. cord blood, peripheral blood, bone marrow or fetal liver. However, the protocols published so far require long term cultivation, i.e. up to 15 weeks for mast cell differentiation, which makes such approaches not only laborious but also costly. Here, we have developed a protocol for generating functional human mast cells from peripheral blood already within 7 weeks. Human CD133(+) progenitors were isolated from buffy coat preparations of peripheral blood and cultured in the presence of stem cell factor (SCF) and IL-6 for 7 weeks. IL-3 was added to the culture medium during the first 3 weeks, and fetal calf serum (FCS) added during the last week. In vitro differentiated CD133(+) cells exhibited multiple characteristics of mature mast cells. Thus, cells contained tryptase and expressed functional levels of FcepsilonRI. Anti-IgE stimulation induced significant release of histamine and PGD(2) and also of chemokines including MCP-1, IL-8, MIP-1alpha, and MIP-1beta. The fact that our in vitro differentiated mast cells are derived from a generally available source of progenitor cells makes this novel protocol widely applicable to any patient group, irrespective of age. Moreover, this progenitor source is more readily available than e.g. bone marrow or cord blood-derived progenitors. Consequently, our protocol has great potential in studies on mast cell biology and mast cell pathology, and e.g. on evaluation of drug effects.

During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors. To investigate the significance of different progenitors, we have compared MC generated from CD133(+) progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD(2). The generated metachromatic (<99%) MC were 75% tryptase(+), regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcepsilonRI was comparable. The density of c-kit/CD117 receptors on CBMC was higher that of PBMC (p<0.001). The density of CD203c and FcepsilonRI was higher on PBMC (p<0.001). PBMC contained more histamine (p<0.001), expressed more FcepsilonRI (p<0.001) and released more histamine (p<0.001) and PGD(2) (p<0.001) upon ligation of FcepsilonRI, than CBMC. Culture with IL-4 increased expression of tryptase, FcepsilonRI, CD117 and CD203c, secretion of histamine and PGD(2) of PBMC, and histamine secretion of CBMC. Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used.

Background: Mast cells have long been recognized as the principal cell type that initiates the inflammatory response characteristic of acute allergic type 1 reactions. Our goal has been to further characterize maturation of progenitors to mast cells., Methods: Mast cells were cultured from human cord blood derived CD133(+) progenitors. Mast cell function was tested using histamine release. During differentiation mast cells surface marker expression was monitored by flow cytometry., Results: CD133(+) progenitors expressed the early haematopoietic and myeloid lineage markers CD34, CD117, CD13 and CD33. Mature mast cells expressed CD117, CD13 and CD33, and expression of the high affinity immunoglobulin E receptor FcepsilonRI increased during culture. Cytokine receptors interleukin (IL)-5R, IL-3R, granulocyte-macrophage-colony stimulating factor (GM-CSF)R and IL-18R were expressed at high levels during maturation. Chemokine receptors CXCR4 and CXCR2 were highly expressed on both newly purified CD133(+) cells and mature cells., Conclusion: Human mast cells can be cultured from a CD34(+)/CD117(+)/CD13(+)/CD33(+) progenitor cell population in cord blood that is tryptase and chymase negative. Developing and mature mast cells express a wide range of chemokine and cytokine receptors. We found high levels of expression of CD123, IL-5R and GM-CSF receptors, also found on eosinophils and basophils, and high levels of expression of the receptor for the inflammatory cytokine IL-18.

Background: Atopy is closely associated with the cellular T helper type-2 (Th2) phenotype, that is dominated by the pleiotrophic cytokine IL-4. The cellular source of IL-4 has yet to be determined, although basophils have been proposed. Eosinophils and mast cells are likely contenders investigated here, and the eosinophil-like leukaemia line AML14.3D10 is compared to eosinophils as an in vitro culturable model for eosinophils. Lectins can cross-link-specific surface glycoproteins and are found in the ingested (processed foods) and inhaled (airborne pollen grains) human environment. Therefore it is of interest to determine whether lectins can elicit the release of IL-4 from Th2-associated granulocytes other than basophils., Method: This study investigated the ability of eosinophils, AML14.3D10 and mast cells to secrete preformed IL-4 in response to stimulation with lectins, and explored molecular mechanisms underlying the interaction., Results: Purified eosinophils and basophils, and cultured mast cells and AML14.3D10 cells were incubated with 1 micro m lectin. Agglutination was scored by microscopy. IL-4 secretion was measured by enzyme-linked immunosorbent assay. Biotinylated lectins were used to determine binding to cells by flow cytometry and in lectin blots of sodium dodecyl sulphate (SDS) gels., Discussion: Purified human eosinophils, AML14.3D10 cells and cultured mast cells secrete IL-4 with a pattern similar to that found in basophils when stimulated with a panel of reactive and unreactive lectins. The lectin SNA induces IL-4 secretion from mast cells and basophils, but not from eosinophils or AML14.3D10. Eosinophils appear to secrete only pre-formed IL-4, whereas mast cells may synthesize IL-4 on ligation with the lectin LCA. Lectins that agglutinate the granulocytes investigated do not necessarily induce secretion of IL-4. Lectins that elicit secretion of IL-4 bind more to eosinophils than unreactive lectins as determined by flow cytometry and lectin blotting of SDS gels., Conclusion: As granulocytes with functions related to that of basophils, eosinophils, AML14.3D10 and cultured mast cells respond to stimulation with lectins similarly to basophils. This emphasizes the possibility that eosinophils and mast cells may be linked in their cellular heritage as the cellular partners, and lectins as ligands, may contribute to the maintenance of a Th2-favoured microenvironment that is thought to underlie the allergic march.