Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

Identifying regulatory pathways of spleen tyrosine kinase expression in human basophils.

BACKGROUND: Expression levels of spleen tyrosine kinase (SYK), a critical signaling tyrosine kinase in basophils, are uniquely low relative to all other circulating leukocytes, and levels are highly variable in the population. OBJECTIVE: We sought to determine whether transcriptional regulation of SYK through unique silencing of the SYK gene determines its basophil-specific expression patterns. METHODS: Culture-derived basophils (CD34B cells) were derived from cultures of CD34+ progenitor cells by using 2 methods (G1 or G3). Peripheral blood basophils (PBBs; relative SYK protein level = 1), B cells (SYK = 8), CD34B-G1 cells (SYK = 11), and CD34B-G3 cells (SYK = 5) were examined by using assay for transposase-accessible chromatin sequencing (ATAC-seq) methods. In addition, the transcriptomes of 6 cell types, PBBs, peripheral blood eosinophils (SYK = 11), plasmacytoid dendritic cells (SYK = 30), CD34+ progenitors (SYK = 11), CD34B-G1 cells, and CD34B-G3 cells, were analyzed for patterns that matched patterns of SYK expression in these cells, with a focus on transcription factors. RESULTS: ATAC-seq showed that PBBs have multiple open regions in the SYK gene, suggesting a nonsilenced state with 1 region unique to PBBs (low SYK expression), 1 region unique to both PBBs (low SYK expression) and both G1 and G3 CD34B cells (high and moderate SYK expression, respectively), and 5 regions unique to B cells (high SYK expression). SYK expression across the 6 cell types explored showed a unique pattern that was matched to expression patterns of 3 transcription factors: Kruppel-like factor 5 (KLF5), zinc-finger protein 608 (ZNF608), and musculoaponeurotic fibrosarcoma protein (c-MAF). CONCLUSIONS: Two new potential regulatory pathways for SYK expression were identified. One appears independent of transcriptional regulation, and one appears to be dependent on transcriptional control in the SYK gene.

BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells.

Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.

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.

α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

Basophils act as initiator cells for the development of IgE-mediated chronic allergic inflammation (IgE-CAI). However, detailed mechanisms of initial recruitment of basophils into the skin have yet to be clarified. Selectins mediate leukocyte capture and rolling on the vascular endothelium for extravasation. Counter-receptor activity of selectins is regulated by α(1, 3) fucosyltransferases (FTs) IV and VII. To clarify the contribution of selectin ligands regulated by FTs for initial basophil recruitment, IgE-CAI was induced in mice deficient in FT-IV and/or FT-VII genes. Although FT-IV(-/-) and FT-VII(-/-) mice exhibited comparable skin responses to wild-type mice, the FT-IV(-/-)/FT-VII(-/-) mice showed significantly impaired inflammation. Although the transfer of basophils to FcRγ(-/-) mice induced IgE-CAI, this induction was completely absent when basophils from FT-IV(-/-)/FT-VII(-/-) mice were transferred. L-selectin, but not P- and E-selectin, blocking Abs inhibited skin inflammation in vivo. P-selectin glycoprotein-1 (PSGL-1) antibody also ameliorated skin inflammation, and basophils were bound to L-selectin in a PSGL-1-dependent manner, which was regulated by FT-IV/VII. Functional PSGL-1 generated by basophil FT-IV/VII and its subsequent binding to L-selectin could be one of the essential steps required for initial basophil recruitment and the development of IgE-CAI in mice.

KU812 basophils express urocortin, CRH-R, MC1R and steroidogenic enzymes and secrete progesterone.

Little is known about neuroendocrine regulation of human basophils by components of the hypothalamic-pituitary-adrenal (HPA) axis. Using the basophil cell line KU812 as an in vitro model, we show that these cells express urocortin 1-3, specific isoforms of the corticotropin-releasing hormone (CRH) receptor (CRH-R)1 and CRH-R2 but not CRH itself. The precursor for melanocortins and ß-endorphin, proopiomelanocortin, was not detectable, while the melanocortin-1 receptor was present at RNA and protein level in KU812 cells. KU812 basophils furthermore expressed key enzymes involved in steroidogenesis, that is, CYP11A1, CYP17 and CYP21A2. The relevance of steroidogenic enzyme expression in KU812 cells was confirmed by showing the presence of progesterone and 17OH-progesterone in conditioned media of these cells. Our data demonstrate the expression of some but not all components of the HPA axis in human basophils. These cells are not only target cells for multiple hormones of the HPA axis but may also generate neuroendocrine mediators autonomously.

Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment.

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 µg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 µg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

Isolation of 5-(hydroxymethyl)furfural from Lycium chinense and its inhibitory effect on the chemical mediator release by basophilic cells.

The effect of hot water extracts of LYCIUM CHINENSE fruits (LCF) on the ß-hexosaminidase (ß-hexo) release by IgE sensitized BSA stimulated rat basophilic leukemia (RBL-2H3) cells was investigated. The ethylacetate (EtOAc) layer of the extract has shown an inhibitory effect on ß-hexo release from RBL-2H3 cells at the antigen antibody binding stage. The water (H2O) fraction (EFW) of the chloroform (CHCl3) extract from the EtOAc layer also inhibited ß-hexo release at the same stage in a dose-dependent manner. With column chromatography preparation, proton and carbon nuclear magnetic resonance (¹H and ¹³C NMR) spectra, electron ionization mass spectrometer (EI-MS) spectra, and high-performance liquid chromatography (HPLC) analysis, the active component was determined to be 5-(hydroxymethyl)furfural (5-HMF). Thus, the 5-HMF showed an inhibitory effect on ß-hexo release at the antigen-antibody binding stage and the antibody-receptor binding stage. Furthermore, 5-HMF suppressed [Ca²+] I influx in the IgE-sensitized BSA-stimulated RBL-2H3 cells. Our results show that 5-HMF may be useful for the treatment or prevention of type I allergic diseases.

Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors.

Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.

Comparison of the anti-allergic activity of Syk inhibitors with optimized Syk siRNAs in FcepsilonRI-activated RBL-2H3 basophilic cells.

Spleen tyrosine kinase (Syk) binds ITAM-bearing receptors in a wide variety of cell types. One such example is the activation of mast cells, basophils and eosinophils via the stimulation of the FcepsilonRI receptor by IgE/allergen complexes. The possible role of Syk in inflammatory signaling cascades has led to the development of pharmacological agents designed to block the Syk catalytic domain as potential novel therapeutics. Whilst the enzymatic activity of Syk lends towards the design of small-molecule inhibitors, other attention has focused on the possibility of targeting Syk expression using anti-sense oligonucleotides as an alternate means of anti-inflammatory therapy. In this study, we compared the ability of multiple optimized Syk siRNA sequences and small-molecule Syk inhibitors to block FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion in the basophilic cell line RBL-2H3. We also characterized the specificity of each siRNA sequence with regards to off-target induction of the interferon-inducible gene IFIT1. We identified a single siRNA sequence, which displayed a favorable profile of efficient Syk knockdown, blockage of FcepsilonRI-mediated signal transduction, degranulation and TNFalpha secretion and a lack of IFIT1 induction. The effect of this siRNA was comparable to that of the Syk kinase domain inhibitors BAY61-3606 and R406. The identification of an active and specific Syk siRNA could be a basis for the development of therapeutic Syk siRNAs against inflammatory diseases.

Inhibitory effects of flavonoids isolated from Fragaria ananassa Duch on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3.

We isolated the 4 kinds of flavonoids from strawberry 'Nohime' and examined the effect of these flavonoids on the degranulation in RBL-2H3 cells. The flavonoids were found to suppress the degranulation from Ag-stimulated RBL-2H3 cells to different extents. To disclose the inhibitory mechanism of degranulation by flavonoids, we examined their effects on the intracellular free Ca(2+) concentration ([Ca(2+)]i) and the intracellular signaling pathway such as Lyn, Syk, and PLCgammas. The intracellular free Ca(2+) concentration ([Ca(2+)]i) was elevated by Fc epsilonRI activation, but these flavonoid treatments reduced the elevation of [Ca(2+)]i by suppressing Ca(2+) influx. Kaempferol strongly suppressed the activation of Syk and PLCgammas. It was thus suggested that suppression of Ag-stimulated degranulation by the flavonoids is mainly due to suppression of [Ca(2+)]i elevation and Syk activation. These results suggested that strawberry would be of some ameliorative benefit for the allergic symptoms.

Structural analysis of ARC-type inhibitor (ARC-1034) binding to protein kinase A catalytic subunit and rational design of bisubstrate analogue inhibitors of basophilic protein kinases.

The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy.

BACKGROUND: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. METHODS: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. RESULTS: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 µg/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. CONCLUSION: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation.

Basophil effector function and homeostasis during helminth infection.

Basophils are effector cells of the innate immune system that are associated with allergic inflammation and infections with helminth parasites. However, their development and in vivo functions are largely unknown. Here, we characterize basophil development, turnover, tissue localization, and effector function during infection with the helminth Nippostrongylus brasiliensis. Our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60 hours. N brasiliensis-induced basophilia is caused by increased de novo production of basophils in the bone marrow. Basophils were found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine, and in the lung parenchyma. Activated basophils promoted systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung, and contributed to efficient worm expulsion, demonstrating that basophils play a crucial role as effector cells in type 2 immune responses.

Induced loss of Syk in human basophils by non-IgE-dependent stimuli.

In the general population, Syk expression in human basophils is highly variable and correlates well with the IgE-mediated responsiveness of these cells. Previous studies established that IgE-mediated stimulation results in loss of Syk expression. The current studies investigated whether stimulation through other receptors results in loss of Syk. Two classes of stimulation were examined, those that operate through the kinase Syk and those that operate through a GTP-binding protein. These studies demonstrated that aggregation of leukocyte Ig-like receptor LILRA-2 resulted in phosphorylation of Syk and c-Cbl, was inhibited by a third generation Syk inhibitor with an expected IC(50), and induced histamine release in strict proportion to release induced by anti-IgE Ab. Stimulation of LILRA-2 for 18 h resulted in modest loss of Syk that correlated with the more profound loss of Syk induced by anti-IgE Ab. Human recombinant histamine-releasing factor has also recently been shown to induce Syk phosphorylation and in the current studies has also been shown to induce loss of Syk in 18-h cultures. fMLP stimulation for 18 h was also found to induce modest loss of Syk. fMLP induced phosphorylation of c-Cbl that was sustained for at least 45 min. Phosphorylation of c-Cbl was inhibited by a Syk kinase inhibitor but with an IC(50) that was not consistent with Syk activity, suggesting another kinase was responsible for Cbl phosphorylation following fMLP. These studies demonstrate that it is possible to induce the loss of Syk expression in human basophils by a non-IgE-dependent mechanism and even by a mechanism that does directly involve Syk in the reaction complex.

Recombinant allergens promote expression of aminopeptidase-n (CD13) on basophils in allergic patients.

IgE-dependent activation of basophils is associated with upregulation of several surface molecules. We recently identified the surface enzyme aminopeptidase N (CD13) as a novel activation antigen on human basophils. In the present study, we asked whether CD13 can be employed as a novel marker of allergen-induced activation of basophils in allergic individuals. Patients allergic to major allergens from grass pollen (Phl p 1, Phl p 5), birch pollen (Bet v 1), or house dust mites (Der p 2), were examined. Blood basophils were exposed to various concentrations of recombinant allergens for 15 minutes, and examined for expression of CD13 by multicolor flow cytometry. The allergen-induced upregulation of CD13 was compared with allergen-dependent increases in expression of CD63 and CD203c. Exposure to recombinant allergens resulted in an increase in expression of CD13 on basophils in all sensitized individuals, whereas no increase in CD13 was seen in healthy controls. The effects of the recombinant allergens on CD13-expression were dose- and time-dependent, were not observed in the absence of extracellular calcium, and were counteracted by preincubation of basophils with the PI3-kinase-targeting drugs staurosporin and LY294002. There was a good correlation between allergen-induced upregulation of CD13, CD63, and CD203c on basophils. In aggregate, our data show that recombinant allergens promote expression of CD13 on basophils in sensitized individuals. The functional significance and diagnostic implications of this observation remain to be determined.

Non-apoptotic functions of granzymes.

Granzymes (granule enzymes) are proteases released from cytotoxic lymphocyte granules into target cells to protect mammals from virus infection and transformed cells. Once released into the cytoplasm of the target cell, granzymes activate specific pathways to induce cell death. Although the induction of target cell death has been considered the central function for these proteases, accumulating evidence suggests that granzymes also possess additional non-death-related functions. Thus, some granzymes can achieve direct antiviral activities through the cleavage of proteins encoded by viruses as well as host factors required for the viral life cycle. The presence of elevated concentrations of circulating granzymes in various inflammatory processes and granzyme-mediated cleavage of extracellular substrates suggest that these proteases may have extracellular effects relevant to virus and tumor rejection and the pathogenesis of chronic inflammatory diseases. Here, we discuss the current knowledge of the substrates and the proposed non-apoptotic functions of granzymes, with special interest in non-death-related functions of granzymes inside the target cell.

Initial Fc epsilon RI-mediated signal strength plays a key role in regulating basophil signaling and deactivation.

BACKGROUND: Mediator releases after high-affinity IgE receptor (Fc(epsilon)RI) cross-linking in basophils and mast cells crucially govern the symptoms of allergic disease and amplify underlying T(H)2-type responses. Interestingly, the dose-response curve for Fc(epsilon)RI activation is bell-shaped, with supraoptimal stimulation leading to reduced mediator release. OBJECTIVE: We sought to characterize the mechanisms responsible for this control of Fc(epsilon)RI-triggered basophil activation. METHODS: Human basophils were purified by means of Ficoll density centrifugation, elutriation, and negative selection with immunomagnetic beads. Various intracellular signal protein activities were assessed by means of Western blotting, and mediator releases were analyzed either spectrofluorometrically (histamine) or by means of ELISA (IL-4 and IL-13). RESULTS: Supraoptimal anti-IgE concentrations led to lower mediator release than optimal concentrations but simultaneously to considerably faster histamine release kinetics. In parallel, basophil signaling proteins (Syk, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinases 1 and 2) were more rapidly phosphorylated at higher anti-IgE concentrations but more transiently activated in the supraoptimal range. This endogenous regulation most likely involved src homology 2 domain-containing inositol 5' phosphatase (SHIP), which was highly phosphorylated after supraoptimal anti-IgE triggering compared with lower stimulus concentrations. Conversely, N-formyl-methionyl-leucyl-phenylalanine-stimulated basophils failed to phosphorylate SHIP in the supraoptimal concentration range and did not display a bell-shaped dose-response curve. CONCLUSION: The kinetics of IgE-mediated signaling and mediator release in primary human Fc(epsilon)RI(+) cells varies substantially according to the magnitude of stimulation, and SHIP most likely plays an important role in terminating these events. CLINICAL IMPLICATIONS: The speed of allergic symptom generation depends on the degree of IgE receptor triggering, which is downregulated by SHIP, a potential target for allergy therapy.