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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.

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., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Staining cells or tissues with basic dyes was the mainstay of mast cell and basophil detection methods for more than a century following the first identification of these cell types using such methods. These techniques have now been largely supplanted by immunohistochemical procedures with monoclonal antibodies directed against unique constituents of these cell types. Immunohistochemistry with antibodies specific for the granule protease tryptase provides a more sensitive and discriminating means for detecting mast cells than using the classical histochemical procedures, and using antibodies specific for products of basophils (2D7 antigen and basogranulin) has allowed detection of basophils that infiltrate into tissues. The application of immunohistochemistry to detect more than one marker in the same cell has underpinned concepts of mast cell heterogeneity based on differential expression of chymase and other proteases. The double labeling procedures employed have also provided a means for investigating the expression of cytokines and a range of other products. Protocols are here set out that have been used for immunohistochemical detection of mast cells and basophils and their subpopulations in human tissues. Consideration is given to pitfalls to avoid and to a range of alternative approaches.

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-X L 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-X L 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-X L . Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-X L , 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-X L 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.

Recent studies have identified nonredundant roles for basophils in immune responses including allergy and protective immunity. It is well known that activated basophils release granule contents such as histamine and proteases as do mast cells. However, the functional significance of basophil-derived proteases remains poorly understood in contrast to those released from mast cells. For this study we generated a line of knockout (KO) mice deficient for mouse mast cell protease-11 (mMCP-11) that is preferentially expressed by basophils rather than mast cells. In spite of normal development of basophils, the mMCP-11-deficient mice showed amelioration of immunoglobulin E-mediated chronic allergic inflammation (IgE-CAI), with reduction of cutaneous swelling, microvascular permeability, and leukocyte infiltration in the skin lesion, when KO mice were compared with wild-type mice. Repeated administration of recombinant mMCP-11 in the skin induced infiltration of leukocytes, including basophils, in a tryptase activity-dependent manner. The transwell migration assay in vitro suggested that mMCP-11-mediated proteolytic products of serum protein promoted migration of basophils, eosinophils, and macrophages via 1 or more G protein-coupled receptors. Thus, basophil tryptase mMCP-11 is a crucial effector molecule for the induction of IgE-CAI. This is the first demonstration that the basophil-derived protease plays a significant role in vivo., (© 2016 by The American Society of Hematology.)

In human basophils, Syk expression is 10-fold lower than most other types of leukocytes. There are indirect studies that suggest that Syk protein is highly unstable (a calculated half-life less than 15 min) in human peripheral blood basophils. Therefore, in these studies, Syk stability was directly examined. Purified basophils were metabolically labeled and a pulse-chase experimental design showed Syk protein to be stable in the time frame of 12 h (95% likelihood that half-life is more than 12 h). However, its synthetic rate was very slow (∼10-fold slower) compared with CD34-derived basophils, which have been shown to express levels of Syk consistent with other mature circulating leukocytes. Syk mRNA expression was found to be 5-30-fold lower than other cell types (CD34-derived basophils, peripheral blood eosinophils, and plasmacytoid dendritic cells). Syk protein and mRNA levels, across cell types, were relatively concordant. Syk mRNA in basophils showed a half-life of 3.5 h, which was greater than that of interleukin-4 or Fc epsilon receptor I-α mRNA (∼2 h), but somewhat shorter than Fc epsilon receptor I-β mRNA (8 h). A comparison of miR expression between CD34-derived and peripheral blood basophils demonstrated only 1 significant increase, in miR-150 (77-fold). Transfection in human embryonic kidney cells of a stabilized form of miR-150 showed that it modified expression of c-Myb mRNA but not of Syk mRNA or protein. These results suggest that low Syk expression in basophils results, not from protein instability and perhaps not from mRNA stability. Instead, the results point to the transcriptional nature of an important point of regulation., (© Society for Leukocyte Biology.)

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

It is not clear whether pseudoallergic reactions are caused by similar mechanisms as type I allergic reactions. 3‑Caffeoylquinic acid (chlorogenic acid) is an active ingredient in traditional Chinese medicines used for antibacterial, anti-inflammatory and cholagogic purposes. It is assumed to be the reason for the high allergic reaction rates associated with certain traditional Chinese medicine injection solutions. The aim of the present study was to investigate the possible mechanisms through which chlorogenic acid triggers pseudoallergic reactions. The fluidity of the cell membrane was investigated using fluorescence recovery after photobleaching. Western blot analysis was used to measure the phosphorylation levels of the Spleen tyrosine kinase (Syk) protein and Fluo‑3/AM fluorescent probes were used to investigate the influx of calcium ions. In addition, fluorescence microscopy and phalloidin were used to determine F‑actin depolymerization levels. The secretion rate of β‑hexosaminidase by RBL‑2H3 cells clearly increased following treatment with chlorogenic acid and the levels of cytoskeletal disintegration were also markedly increased. Furthermore, we detected an increase in the intracellular calcium ion concentration along with distinct changes in Syk protein phosphorylation and cellular F‑actin. These changes indicated that chlorogenic acid affected the restructuring of the cytoskeleton and played a role in cell degranulation. In conclusion, chlorogenic acid may lead to the aggregation of lipid rafts on the cell membrane surface by altering RBL‑2H3 cell membrane fluidity, thus triggering Syk‑related signal transduction and inducing a truncated type I like allergic reaction.

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.

A photoluminescence probe ARC-1185, possessing both high affinity towards basophilic protein kinases (PKs) and microsecond-scale luminescence lifetime when associated with a kinase, was used for the mapping of ARC-1185-PK complexes in living cells with time-gated luminescence microscopy.

Mouse mast cell protease 11 (mMCP-11) is the most recently identified member of the mouse mast cell tryptase family. This tryptase is preferentially produced by basophils in contrast to other members that are expressed by mast cells but not basophils. Although blood-circulating basophils have long been considered as minor and redundant relatives of tissue-resident mast cells, recent studies illustrated that basophils and mast cells play distinct roles in vivo. To explore the in vivo role of basophil-derived mMCP-11, here we prepared recombinant mMCP-11 and its protease-dead mutant. Subcutaneous injection of the wild-type mMCP-11 but not the mutant induced edematous skin swelling with increased microvascular permeability in a dose-dependent manner. No apparent infiltration of proinflammatory cells including neutrophils and eosinophils was detected in the skin lesions. The cutaneous swelling was abolished by the pretreatment of mice with indomethacin, a cyclooxygenase inhibitor, suggesting the major contribution of prostaglandins to the microvascular leakage. Of note, the cutaneous swelling was elicited even in mast cell-deficient mice, indicating that mast cells are dispensable for the mMCP-11-induced cutaneous swelling. Thus, basophil-derived mMCP-11 can induce microvascular leakage via prostaglandins in a mast cell-independent manner, and may contribute to the development of basophil-mediated inflammatory responses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

PR3, also called myeloblastin, is a neutrophil serine protease that promotes myeloid cell proliferation by cleaving the cyclin-dependent kinase inhibitor p21(cip1/waf1). In addition, it is the target of ANCA in GPA, a necrotizing vasculitis. Anti-PR3 ANCA binding to membrane-expressed PR3 triggers neutrophil activation, potentiating vascular inflammation. This study performed in RBL cells identifies the structural motifs of PR3 membrane anchorage and examines its impact on PR3 proinflammatory and proliferative functions. With the use of MD simulations and mutagenesis, we demonstrate that the mutations of four hydrophobic (F180, F181, L228, F229) or four basic (R193, R194, K195, R227) amino acids abrogated PR3 membrane anchorage. The hydrophobic patch-deficient PR3 mutant (PR34H4A) was still able to cleave the synthetic substrate Boc-Ala-Pro-Val in cell lysates. However, in contrast to WT PR3, PR34H4A was not expressed at the plasma membrane after degranulation and failed to cleave extracellular fibronectin, was not externalized after apoptosis and did not impair macrophage phagocytosis of apoptotic cells, did not promote myeloid cell proliferation and failed to cleave p21/waf1. PR3 membrane insertion appears to be pivotal for its proinflammatory activities, such as extracellular proteolysis and impairment of apoptotic cell clearance, but also for myeloid cell proliferation. Targeting membrane-associated PR3 might constitute a novel, anti-inflammatory therapeutic strategy in inflammatory disease especially in vasculitis, but this approach has to be validated in mature neutrophils.

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.

The study of receptor-mediated signaling in human basophils is often limited by the availability of selective pharmacological agents. The early signaling reaction mediated by FcεRI aggregation is thought to require the activity of Bruton's tyrosine kinase (btk), an enzyme that has been identified as important in B cells signaling because mutations lead to X-linked agammaglobulinemia. This study uses the btk selective irreversible inhibitor, PCI-32765, to explore the role of btk in a variety of functions associated with the activation of human basophils. Nine endpoints of basophil activation were examined: induced cell surface expression of CD63, CD203c, CD11b; induced secretion of histamine, LTC4, IL-4 and IL-13; the cytosolic calcium response; and the induced loss of syk kinase. Four stimuli were examined; anti-IgE antibody, formyl-met-leu-phe (FMLP), C5a and IL-3. For stimulation with anti-IgE, PCI-32765 inhibited CD63, histamine, LTC4 and IL-4 secretion with an IC50 of 3-6 nM (with 100% inhibition at 50 nM) and it inhibited CD203c and CD11b and the cytosolic calcium response with and IC50 of 30-40 nM. Fifty percent occupancy of btk with PCI-32765 occurred at ~10nM. Consistent with btk functioning downstream or in parallel to syk activation, PCI-32765 did not inhibit the loss of syk induced by anti-IgE in overnight cultures. Finally, PCI-32765 did not significantly inhibit basophil activation by FMLP or C5a and did not inhibit IL-13 release induced by IL-3. These results suggest that btk is specifically required for IgE-mediated activation of human basophils., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The effect of hot water extracts of LYCIUM CHINENSE fruits (LCF) on the β-hexosaminidase (β-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 β-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 β-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 β-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., (© Georg Thieme Verlag KG Stuttgart · New York.)

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

Tryptases and chymases are the major proteins stored and secreted by mast cells, and they have various biological functions. However, the nature of proteases produced by basophils has been poorly characterized, particularly in mice. mMCP-11 is the most recently discovered mast cell tryptase in mice and was originally identified as Prss34, which is transcribed in some mast cell-like cell lines and at the early stage in the culture of BMMC with IL-3. Curiously, Prss34 is preferentially expressed in the BM and spleen among normal tissues in contrast to other mast cell tryptases. Therefore, it remains elusive what types of cells express mMCP-11 in vivo. Here, we show that mMCP-11 is highly expressed by primary basophils and to a much lesser extent, by some mast cells. Prss34 transcripts were detected abundantly in primary and cultured basophils and very weakly in peritoneal mast cells or cultured BMMC. Conversely, transcripts for mMCP-6 and mMCP-7 tryptases were preferentially expressed by cultured and peritoneal mast cells but not basophils. We established a mMCP-11-specific mAb and showed that mMCP-11 proteins are indeed expressed by primary basophils and those infiltrating the affected tissues during allergic inflammation and parasitic infections. Some primary mast cells also expressed mMCP-11 proteins, albeit at a much lower level. Thus, basophils rather than mast cells are the major source of mMCP-11. This is the first study to demonstrate that mouse basophils produce a trypsin-like protease.

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.

T helper 1 (Th1)-Th2 cell balance is key to host defense and its dysregulation has pathophysiological consequences. Basophils are important in Th2 cell differentiation. However, the factors controlling the onset and extent of basophil-mediated Th2 cell differentiation are unknown. Here, we demonstrate that Lyn kinase dampened basophil expression of the transcription factor GATA-3 and the initiation and extent of Th2 cell differentiation. Lyn-deficient mice had a marked basophilia, a constitutive Th2 cell skewing that was exacerbated upon in vivo challenge of basophils, produced antibodies to a normally inert antigen, and failed to appropriately respond to a Th1 cell-inducing pathogen. The Th2 cell skewing was dependent on basophils, immunoglobulin E, and interleukin-4, but was independent of mast cells. Our findings demonstrate that basophil-expressed Lyn kinase exerts regulatory control on Th2 cell differentiation and function.

We describe a hitherto unreported laboratory observation, namely the selective preservation of basophils, in a case of severe dapsone-induced agranulocytosis. Dapsone is known to be metabolised by peroxidases to nitroso derivatives in non-hemopoietic cells where these can in turn act as haptens. Our observation that basophils are selectively spared in this syndrome supports the hypothesis that leucocyte peroxidases function in a similar way to facilitate the pathogenesis of this form of agranulocytosis in susceptible individuals.

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.

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

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.

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., (Copyright © 2008 Clinical Cytometry Society.)

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.

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.

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.

Background: Previous studies have suggested that expression levels of spleen tyrosine kinase (syk) or phosphatidylinositol 5' phosphatase (SHIP) may explain certain extreme human basophil phenotypes., Objective: This study is designed to explore whether variability in syk and SHIP expression levels in the general population, alone or in concert, can account for the variability in basophil function., Methods: A survey of maximum responsiveness to IgE-mediated stimulation, sensitivity, and expression levels of 6 early signaling elements was performed on 36 subjects' basophils., Results: Of the 6 signaling elements, only syk and SHIP showed a correlation with maximum histamine release or cellular sensitivity. In a multiple regression, syk and SHIP together could account for 67% of population variance, although most of the variance was explained by syk expression. The pattern of expression variance syk>>SHIP1>SHIP2 approximately lyn approximately p85 approximately cbl suggested a process that primarily modulated syk levels. IL-3 is known to modulate syk levels, but we found that a 3-day incubation with IL-3 resulted in increased expression of other signaling elements to a greater degree: cbl>SHIP1>SHIP2 approximately lyn approximately p85 > or = syk, opposite the pattern in the population survey. In contrast, 18-hour stimulation with anti-IgE antibody led to marked downregulation of syk expression, modest downregulation of Fc epsilon RI expression, weak downregulation of lyn expression, and no effect on 23 other signaling elements., Conclusion: Unlike studies in mice, we conclude that expression of syk is a good preconditioning predictor of basophil function in the general population., Clinical Implications: The finding that expression of syk levels may strongly influence functional responses of basophils suggests a mechanism underlying the severity of atopic diseases.

Signal Transducer and Activator of Transcription (STAT) proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation after cytokine stimulation. One mechanism by which STAT signaling is regulated is by dephosphorylation through the action of protein tyrosine phosphatases (PTP). We have identified PTP-Basophil like (PTP-BL) as a STAT PTP. PTP-BL dephosphorylates STAT proteins in vitro and in vivo, resulting in attenuation of STAT-mediated gene activation. In CD4(+) T cells, PTP-BL deficiency leads to increased and prolonged activation of STAT4 and STAT6, and consequently enhanced T helper 1 (Th1) and Th2 cell differentiation. Taken together, our findings demonstrate that PTP-BL is a physiologically important negative regulator of the STAT signaling pathway.

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.

Previous studies of secretion from basophils have demonstrated the phenomenon called nonspecific desensitization, the ability of one IgE-mediated stimulus to alter the cell's response to other non-cross-reacting IgE-mediated stimuli, and a process that would modify phosphatidylinositol 3,4,5-phosphate levels was speculated to be responsible for nonspecific desensitization. The current studies examined the changes and characteristics of SHIP1 phosphorylation as a measure of SHIP1 participation in the reaction. Based on the earlier studies, two predictions were made that were not observed. First, the kinetics of SHIP1 phosphorylation were similar to reaction kinetics of other early signals and returned to resting levels while nonspecific desensitization remained. Second, in contrast to an expected exaggerated SHIP phosphorylation, cells in a state of nonspecific desensitization showed reduced SHIP phosphorylation (compared with cells not previously exposed to a non-cross-reacting Ag). Discordant with expectations concerning partial recovery from nonspecific desensitization, treatment of cells with DNP-lysine to dissociate bound DNP-HSA, either enhanced or had no effect on SHIP phosphorylation following a second Ag. These experiments also showed a form of desensitization that persisted despite dissociation of the desensitizing Ag. Recent studies and the results of these studies suggest that loss of early signaling components like syk kinase may account for some of the effects of nonspecific desensitization and result in a form of immunological memory of prior stimulation. Taken together, the various characteristics of SHIP phosphorylation were not consistent with expectations for a signaling element involved in nonspecific desensitization, but instead one which itself undergoes nonspecific desensitization.

Background: Exposure of human basophils to allergens results in a rapid secretion of histamine, LTC(4), IL-4 and IL-13, which dominate both the symptomology of allergic diseases and support the underlying Th2/IgE predominance associated with these reactions. The IgE-dependent release of these mediators in basophils crucially involves PI 3-kinase and the subsequent activation of p38 MAPK and ERK1&2. Here, we investigated the role of the third major member of the mitogen activated kinase family, namely the c-Jun amino terminal kinase (JNK), which is rapidly activated following IgE receptor cross-linking in murine mast cells., Methods: Human basophils were highly purified by magnetic cell sorting. The activities of various intracellular signaling components, in basophils that had been stimulated under various conditions, were assessed by Western blotting. Mediator secretions were also determined using either spectrofluorometric analysis (histamine) or ELISA (LTC(4), IL-4 and IL-13)., Results: Our results show that while JNK is moderately expressed in human basophils, it is not consistently phosphorylated upon anti-IgE stimulation. Phosphorylation of the transcription factor c-Jun, a downstream target of JNK, was also undetected in contrast to p38 MAPK and ERK1&2, which were clearly activated following anti-IgE stimulation of the cells. Additionally, inhibitors of the JNK pathway failed to prevent basophil mediator release and had no effect on the phosphorylation of p38 MAPK or ERK1&2 at concentrations which were specific for JNK blockade., Conclusions: These data suggest major differences in utilizing various members of the mitogen-activated kinase family in the signal transduction cascade of IgE-receptor-bearing cells., (Copyright (c) 2005 S. Karger AG, Basel)

Background: To date, there is no well-established test available that can be used to measure functional properties of modified allergens (allergoids). Due to the cross-linking process, the IgE-binding capacity of the allergens, normally necessary for their characterization, is lost. The aim of this study was to test whether the rat basophilic leukaemia (RBL) cell assay (beta-hexosaminidase release by rat basophils upon allergen stimulation) can be adopted to characterize allergoids and to evaluate the assay for testing allergoids and native allergens as well., Methods: Mice were immunized with native and modified Phleumpratense extracts in the presence of alum. Their sera were used to sensitize RBL-2H3 cells and measure basophil stimulation induced by different allergen extracts in the presence or absence of various additives., Results: Sera containing specific IgE against both extract formulations were obtained. Native as well as modified extracts induced dose-dependent beta-hexosaminidase release from RBL cells. Both extracts were used to evaluate the characteristics of the assay, which showed high precision. Storage conditions were chosen to enhance extract degradation, which could be read directly from the altered stimulatory capacity of the extracts. Additives turned out to have diverse effects on the assay, whereas phenol had no measurable effect, alum had an inhibitory effect and glycerol elevated basophil activation., Conclusions: For the first time, a reliable, precise in vitro assay is available that is able to directly measure the properties of modified allergen extracts after their production process. The test is well evaluated and its advantages and limitations are discussed in this report., (Copyright (c) 2005 S. Karger AG, Basel)

Activation of mast cells and basophils is accompanied by the production of reactive oxygen and nitrogen species that regulate diverse signaling pathways leading to the release of inflammatory mediators and production of a variety of cytokines. Although the functional pathways of reactive oxygen and nitrogen species in vivo are not completely understood, some novel metabolic pathways can be envisioned based on recent findings that protein tyrosine phosphatases can be regulated by reversible oxidation. In this review, we describe major sources and targets of reactive oxide and nitrogen species in mast cells and basophils. Direct and indirect regulations of class I and II Cys-based protein tyrosine phosphatases (LMW-PTP, PTEN, PTP-PEST, SHP-2, PTP1B, PTPalpha, PTPepsilon, DEP-1, TC45, SHP-1, HePTP and LAR) are discussed. The combined data highlight the role of redox-regulated protein tyrosine phosphatases as targets in the development of new ways of therapeutic intervention in allergies and inflammatory diseases.

Background: Ongoing secretion from human basophils is a balance of activation and deactivation events. Recent studies have focused on downregulatory steps that appear to modify the presence of the activated state of various signaling molecules. We now examine downregulation regulated by mechanisms related to proteasome processing., Objective: To determine the long-term effects of FcepsilonRI aggregation on expression of syk kinase., Methods: Peripheral blood basophils were examined for changes in the expression of syk kinase after stimulation with optimal and suboptimal stimulation., Results: Stimulation results in a 20% loss of syk in 1 hour and an 80% loss of syk in longer incubations (>18 hours). Loss of syk in this time frame can occur at levels of stimulation that do not result in observable mediator release. Loss of syk occurs after stimulation with either anti-IgE antibody or antigen. Activation is shown to result in c-Cbl phosphorylation, and its association with syk and immunoblotting reveals the appearance of a ladder of syk species with molecular weights that are consistent with ubiquitylation of syk. Stimulation in the presence of a proteasome inhibitor such as lactacystin A results in the sustained presence of very high-molecular-weight ubiquitylated species, although it does not alter the presence of the syk ladder., Conclusions: Although the loss of syk is probably too slow to account for downregulation of ongoing secretion of histamine or leukotriene C4 release, it may lead to longer-term alterations in basophil function that explain characteristics of clinical procedures like rapid drug desensitization.

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.

The aim of the present study was to determine whether inhibition of cyclic nucleotide phosphodiesterase (PDE) modulates the stimulated generation of the cytokines, interleukin-4 (IL-4) and IL-13, from human basophils. This was addressed by evaluating the effects of both nonselective and selective inhibitors of PDEs on the generation of cytokines from basophils. The nonselective PDE inhibitors, isobutyl-methylxanthine (IBMX) and theophylline, attenuated the IgE-mediated generation of IL-4 and IL-13 and, also, the release of histamine from basophils. The effects of the isoform-selective inhibitors, 8-methoxymethyl-IBMX (PDE 1 inhibitor), siguazodan (PDE3 inhibitor), rolipram (PDE4 inhibitor), denbufylline (PDE4 inhibitor), Org 30029 (mixed PDE3 and 4 inhibitor) and zaprinast (PDE5 inhibitor), were studied. Of these selective compounds, only rolipram, denbufylline and Org 30029 inhibited the IgE-dependent generation of IL-4, IL-13 and histamine from basophils to a statistically significant (P<0.05) degree. The effects of isoform-selective inhibitors on basophils activated by IL-3 were evaluated. The IL-3-induced generation of IL-4, IL-13 and histamine was inhibited to a statistically significant (P<0.05) extent, only by compounds that act as inhibitors of PDE4. These data suggest that inhibition of PDE4 can regulate the generation of cytokines from human basophils.

Background: Alpha and beta-tryptase levels in serum are clinical tools for the evaluation of systemic anaphylaxis and systemic mastocytosis. Basophils and mast cells are known to produce these proteins., Objective: The current study examines the effect of the alpha,beta-tryptase genotype on basophil tryptase levels and the type of tryptase stored in these cells., Methods: Tryptase extracted from purified peripheral blood basophils from 20 subjects was examined by using ELISAs measuring mature and total tryptase and by using an enzymatic assay with tosyl-Gly-Pro-Lys-p-nitroanilide. Tryptase genotypes (4:0, 3:1, and 2:2 beta/alpha ratios) were assessed by using a hot-stop PCR technique with alpha,beta-tryptase-specific primers. Total alpha,beta-tryptase mRNA was measured by means of competitive RT-PCR, and ratios of alpha to beta-tryptase mRNA were measured by means of hot-stop RT-PCR., Results: Tryptase in all but one of the basophil preparations was mature and enzymatically active. Tryptase quantities in basophils were less than 1% of those in tissue mast cells. Tryptase genotypes (beta/alpha) among the 20 donors were 4:0 in 7, 3:1 in 7, and 2:2 in 6. Tryptase protein and mRNA levels per basophil were not affected by the tryptase genotype., Conclusion: Basophils from healthy subjects contain modest amounts of mature and enzymatically active tryptase unaffected by the tryptase genotype.

Basophils are effector cells of allergic reactions. These cells produce and store a number of vasoactive and immunomodulatory mediators. During an allergic reaction, basophils can release their mediator substances into the extracellular space and thus contribute to the clinical picture and symptoms in allergy. The phenotypic hallmark of basophils is expression of high-affinity IgE receptors (Fc epsilon RI) on their cell surface together with expression of the activation-linked molecule CD203c. This ectoenzyme is located both on the plasma membrane and in the cytoplasmic compartment of basophils. Cross-linking of the Fc epsilon RI by an allergen or anti-IgE antibody results in a rapid upregulation of intracellular CD203c molecules to the cell surface and is accompanied by mediator release. CD203c is therefore a promising target molecule for a flow cytometry-based test to analyze sensitized individuals and patients with type I allergy. In the present article, we review the current knowledge of CD203c with special regard to its tissue distribution and regulation in basophil activation. In addition, we discuss the application of CD203c in allergy diagnosis., (Copyright 2004 S. Karger AG, Basel)

Previous studies demonstrated that after stimulation of human basophils with a polyclonal anti-IgE Ab, early signaling elements showed sustained phosphorylation, whereas later elements were transient, suggesting that a region of down-regulation involved inhibition of phosphatidylinositol (PI) 3 kinase or its products. However, the current studies show that under some conditions, syk phosphorylation is transient. Generally, stimulation with a variety of Ags makes this early form of down-regulation more apparent. An exploration of the conditions needed to induce early down-regulation indicates that both the nature of aggregation and the cell surface density of IgE play roles. It was also found that the previously described late form of down-regulation (PI3 kinase product transience) can occur in cells displaying early down-regulation (transient syk phosphorylation), but this phenomenon is revealed by testing for subsequent down-regulation of the response to non-cross-reacting stimuli, altering their ability to induce phosphorylation of Akt or extracellular signal-regulated kinase. In contrast, phosphorylation of syk kinase, in response to a non-cross-reacting stimulus, was relatively unaffected by prior stimulation. The magnitude of cross-desensitization of the Akt or extracellular signal-regulated kinase response was a function of the strength of the first stimulus. Mediator release showed a similar cross-desensitization effect. Therefore, stimulation induces two forms of down-regulation, one operating before or at the level of syk phosphorylation, possibly characterizing the process formerly known as specific desensitization, and one that operates in the region of PI3 kinase, accounting for the process formerly known as nonspecific desensitization, which is dependent on the strength of stimulus.

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. We show that stimulation of polymorphonuclear leukocytes (PMNL), rat basophilic leukemia (RBL)-1, or transfected HeLa cells with arachidonic acid (AA) caused prominent 5-LO product formation that coincided with the activity of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase. 5-LO product formation in AA-stimulated PMNL and RBL-1 cells was independent of Ca2+. However, in HeLa cells expressing a 5-LO mutant lacking potential 5-LO phosphorylation sites, removal of Ca2+ caused a prominent loss of 5-LO activity. For Mono Mac 6 (MM6) cells, AA failed to activate ERKs, and AA-induced 5-LO product formation was only minute. Also, activation of ERKs by phorbol esters did not lead to prominent 5-LO product synthesis. Instead, 5-LO activation in MM6 cells required Ca2+ or alternative signaling pathways induced by hyperosmotic stress. In summary, mechanisms for activation of 5-LO differ considerably between cell types.

We present a detailed mathematical model of the phosphorylation and dephosphorylation events that occur upon ligand-induced receptor aggregation, for a transfectant expressing FcepsilonRI, Lyn, Syk and endogenous phosphatases that dephosphorylate exposed phosphotyrosines on FcepsilonRI and Syk. Through model simulations we show how changing the ligand concentration, and consequently the concentration of receptor aggregates, can change the nature of a cellular response as well as its amplitude. We illustrate the value of the model in analyzing experimental data by using it to show that the intrinsic rate of dephosphorylation of the FcepsilonRI gamma immunoreceptor tyrosine-based activation motif (ITAM) in rat basophilic leukemia (RBL) cells is much faster than the observed rate, provided that all of the cytosolic Syk is available to receptors.

Background: In human basophils, FcepsilonRI signal initiation, leading to histamine release, relies on activation of Syk protein tyrosine kinase. Basophils from approximately 10% of unselected donors do not degranulate in response to FcepsilonRI cross-linking. Their unresponsiveness has been linked to the absence of Syk protein despite apparently normal levels of Syk mRNA., Objective: The aim of this study was to explore pathways of Syk protein degradation as a possible posttranslational mechanism for downregulating Syk protein levels in human basophils and other leukocytes., Methods: Highly purified basophils, lymphocytes, and monocytes were incubated in the presence or absence of a panel of cell-permeable inhibitors of proteolytic degradation pathway(s). Subsequently, the protein level of Syk tyrosine kinase was determined by means of Western blotting. In vitro assays were conducted through use of immunoprecipitated basophil Syk and a rabbit reticulocyte lysate system., Results: Three inhibitors of proteasome-mediated degradation-PSI, lactacystin, and ALLN-substantially increased Syk levels in releaser basophils and restored Syk expression in nonreleaser basophils. Caspase inhibitors were less effective, and inhibitors of calpain-mediated proteolysis had no effect. Among other leukocytes tested, only naive CD4(+) T cells had more Syk after proteasome inhibitor treatment. In vitro ubiquitination assays demonstrated that Syk is readily ubiquitinated in vitro and also that Syk ubiquitination is associated with a substantial decrease in total levels of Syk protein., Conclusion: These data provide evidence for a ubiquitin/proteasome-dependent mechanism that contributes to Syk regulation in human basophils and might also be relevant to naive T cells. Understanding this regulatory pathway might lead to strategies for suppressing allergic inflammation while preserving essential Syk-mediated functions in other hematopoietic cells.

Cross-linking the high affinity IgE receptor on the rat basophil leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk, phospholipase C (PLC), protein kinase C (PKC) and intracellular calcium mobilization. In an effort to look at pathways downstream of calcium mobilization, ionomycin-mediated granule release was studied. The kinase inhibitors PP1 (src family), GF109203X (PKC), PD98059 (MEK1/2), and U0126 (MEK1/2) substantially inhibited ionomycin-mediated granule release, while the p38 kinase inhibitor SB203580 did not. Both p38 and erk were phosphorylated upon ionomycin treatment, but only extracellular regulated kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the MEK inhibitors, thus, correlating with the granule release data. Interestingly, while GF109203X alone had no affect on erk activation, combining it with U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is MEK independent and PKC dependent. Experiments in which ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of calcium and controlling mast cell degranulation.