Basophil Expression Research Articles

In human basophils, IL-3 selectively induces RANKL expression that is modulated by IgER-dependent and IgER-independent stimuli.

Receptor activator of NF-κB ligand (RANKL) is expressed as either surface (hRANKL1, hRANKL2) or soluble (hRANKL3) form. RANKL is involved in multifaceted processes of immunoregulation and bone resorption such as they occur in rheumatoid arthritis (RA). Interestingly, activated basophils, which are effector cells in allergic inflammation, contribute to the progress of collagen-induced arthritis (CIA), a mouse model for RA. Here, we investigate under which conditions human basophils express RANKL. Among other stimuli, basophils were cultured with IL-3 alone. Alternatively, as a secondary stimulus, IgER-dependent or IgER-independent agents were added simultaneously either with IL-3 or after prolonged IL-3 culturing. Expression of RANKL protein and mRNA was analyzed by flow cytometry, ELISA, and real-time PCR. A coculture system was applied to investigate biological activity of basophil-derived RANKL. We show that in human basophils, IL-3 but no other stimulus induces de novo expression of soluble and surface RANKL, of which the latter enhances survival of MoDC. Upon simultaneous stimulation, IgER cross-linking reduces surface RANKL expression, while IgER-independent stimuli have no effect. This is in contrast to consecutive stimulation, as triggering with both IgER-dependent and IgER-independent stimuli enhances RANKL expression, particularly in its soluble form. Real-time PCR analysis shows that RANKL expression is mainly regulated at the mRNA level. This study identifies IL-3 as a potent inducer of RANKL expression in human basophils, suggesting them to interact with bone physiology and activation of immune cells. IgER-dependent and IgER-independent stimuli modulate the IL-3-mediated RANKL expression in a time- and stimulus-dependent fashion.

Efecto de la inmunoterapia en la activación de basófilos inducida por alergenos en pacientes con dermatitis atópica

Subcutaneous allergen-specific immunotherapy (IT) is a proved, highly effective treatment for respiratory IgE-mediated diseases. However, few studies have explored the immunological mechanisms of IT in patients with atopic dermatitis. To explore the immune response of atopic dermatitis patients receiving house dust mite (HDM) immunotherapy, according to humoral response and basophil activation. An open label study was done which assessed the severity of atopic dermatitis with SCORAD in 20 patients (10 with immunotherapy and 10 without it) every three months during two years. Serum samples were taken before the follow up, and at the first and second year of study to analyze CD63 basophil expression, total IgE levels and specific IgE and IgG4 to Der p and Der f. Ten patients with allergic rhinitis receiving IT and 5 non-allergic subjects were used as controls. CD63 expression after basophil stimulation with Der p was higher in atopic dermatitis patients than in rhinitis and non-allergic subjects. After the first and second year of treatment, CD63 expression was lower in atopic dermatitis active group than in the atopic dermatitis control group. We observed a correlation between SCORAD, IgG4 and CD63 expression. In patients with atopic dermatitis, basophil activation test could be a biomarker of clinical response and basophil modulation can result in a better clinical control.

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Relationship of IgE to basophil phenotypes in peanut-sensitized adults

Mast cell-derived IL-33 orchestrates antigen-driven tissue inflammation by promoting a unique inflammatory basophil response (HYP3P.352)

Abstract While IL-33 is commonly described as an epithelial derived cytokine, we previously showed that IgE activation of mast cells induces IL-33 expression. In vivo, IgE-primed mast cells serve as peripheral sensors for antigen (Ag) and drive an early edema followed by an antigen-driven tissue inflammation (ADTI). Histamine drives the edematous response but the mechanisms controlling local ADTI are unclear. Here, we sought to examine the roles of IL-33 and its receptor, ST2 in this process. Using a passive cutaneous anaphylaxis model, we show that mast cell-derived IL-33 is both sufficient and necessary for the ADTI response, which is dominated by CD45+Gr-1high neutrophils. ST2-expressing basophils are essential for ADTI and we show that IL-33 serves to activate them, rather than cause migration. Using microarray analysis to examine responses to either IgE/Ag or IL-33 activation, we show that IL-33 promotes an atypical basophil response characterized by neutrophilic chemokines. Interestingly, we find that histamine is necessary for both phases but, while H1R or H2R are required for edema, ADTI is dependent on basophil expression of H4R. In summary, our findings define a mechanism through which mast cells regulate local tissue responses to Ag. We propose a model whereby mast cell histamine release initially attracts basophils via H4R. Basophils are then activated by mast cell-derived IL-33, via ST2, to produce an atypical profile of cytokines and chemokines that recruit neutrophils.

MHC class II expression in human basophils: induction and lack of functional significance.

The antigen-presenting abilities of basophils and their role in initiating a Th2 phenotype is a topic of current controversy. We aimed to determine whether human basophils can be induced to express MHC Class II and act as antigen presenting cells for T cell stimulation. Isolated human basophils were exposed to a panel of cytokines and TLR-ligands and assessed for MHC Class II expression. MHC Class II was expressed in up to 17% of isolated basophils following incubation with a combination of IL-3, IFN-γ and GM-CSF for 72 hours. Costimulatory molecules (CD80 and CD86) were expressed at very low levels after stimulation. Gene expression analysis of MHC Class II-positive basophils confirmed up-regulation of HLA-DR, HLA-DM, CD74 and Cathepsin S. However, MHC Class II expressing basophils were incapable of inducing antigen-specific T cell activation or proliferation. This is the first report of significant cytokine-induced MHC Class II up-regulation, at both RNA and protein level, in isolated human basophils. By testing stimulation with relevant T cell epitope peptide as well as whole antigen, the failure of MHC Class II expressing basophils to induce T cell response was shown not to be solely due to inefficient antigen uptake and/or processing.

Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas. Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining. Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples. Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies. Disclosures: Vakkalanka: Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

GATA-1 regulates the generation and function of basophils

Developmental processes of hematopoietic cells are orchestrated by transcriptional networks. GATA-1, the founding member of the GATA family of transcription factors, has been demonstrated to play crucial roles in the differentiation of erythroid cells, magakaryocytes, eosinophils, and mast cells. However, the role of GATA-1 in basophils remains elusive. Here we show that basophils abundantly express Gata1 mRNAs, and that siRNA-mediated knockdown of Gata1 resulted in impaired production of IL-4 by basophils in response to the stimulation with IgE plus antigens. ΔdblGATA mice that carry the mutated Gata1 promoter and are widely used for functional analysis of eosinophils owing to their selective loss of eosinophils showed a decreased number of basophils with reduced expression of Gata1 mRNAs. The number of basophil progenitors in bone marrow was reduced in these mice, and the generation of basophils from their bone marrow cells in culture with IL-3 or thymic stromal lymphopoietin was impaired. ΔdblGATA basophils responded poorly ex vivo to stimulation with IgE plus antigens compared with wild-type basophils as assessed by degranulation and production of IL-4 and IL-6. Moreover, ΔdblGATA mice showed impaired responses in basophil-mediated protective immunity against intestinal helminth infection. Thus, ΔdblGATA mice showed numerical and functional aberrancy in basophils in addition to the known deficiency of eosinophils. Our findings demonstrate that GATA-1 plays a key role in the generation and function of basophils and underscore the need for careful distinction of the cell lineage responsible for each phenotype observed in ΔdblGATA mice.

Decrease in antigen‐specific CD63 basophil expression is associated with the development of tolerance to egg by SOTI in children

In the last decade, there have been an increasing number of studies on achieving tolerance to foods by specific oral tolerance induction (SOTI). Still, the underlying mechanism of SOTI is unknown. Our aim was to describe changes in CD63 expression on basophils following in vitro Ag-specific stimulation by basophil activation test (BAT), after SOTI with egg in a pediatric population. Ten children with persistent allergy to egg were included. Skin prick tests (SPTs) and open food challenges (OFCs) were performed before SOTI. Specific IgE determination and BAT with egg white (EW), ovomucoid (OM), and ovalbumin (OVA) were performed before and after 1 month of the buildup phase of SOTI. Total tolerance to egg was achieved in 9 cases and partial in one. After SOTI, there was a significant decrease in mean specific IgE levels (p < 0.05). CD63 expression also decreased (p < 0.05) in all patients. Decrease in Ag-specific basophil responsiveness is associated with the development of clinical tolerance by SOTI.

Distinct Roles for Neutrophils and Dendritic Cells in Inflammation and Autoimmunity in motheaten Mice

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.

New paraneoplastic syndrome in chronic basophilic leukemia

Chronic basophilic leukemia (CBL) is an extremely rare disorder. A female patient presented with recurrent attacks of chills, fever and abdominal pain was found to have simultaneous cyclic oscillation in leukocyte counts and C-reactive protein values. She was initially diagnosed with familial Mediterranean fever and treated with colchicine. Diagnosis of CBL was established by morphologic studies of peripheral blood and bone marrow. Her febrile attacks recurred with marked elevation in serum interleukin-6 (IL-6) level when basophil counts climbed to peak levels during cyclic oscillation. Molecular studies by real-time PCR showed IL-6 gene expression in neoplastic basophils separated by magnetic-activated cell sorting infiltrating the bone marrow, suggesting that IL-6 is released by neoplastic basophils of an underlying CBL, resulting in a new paraneoplastic syndrome that mimics autoinflammatory disorders.

TGR-1202 Suppresses AML and ALL Cells Via Selective Inhibition of PI3Kδ Kinase.

AbstractAbstract 2610 Background:Acute leukemia, characterized by the presence clonal hematopoietic cells in peripheral blood and bone marrow, comprises approximately 40% of newly diagnosed leukemias. First line treatment for acute leukemias with multi-agent cytotoxic chemotherapy is usually associated with significant toxicity. Advances in therapy have been slow, and nearly all effective therapies lead to prolonged marrow suppression and toxicities associated with subsequent cytopenias. Herein, we describe the biological and pharmacokinetic properties of TGR-1202, a novel small molecule PI3Kδ inhibitor with scope to be developed as a safe and effective therapy for acute myeloid (AML) and lymphoblastic (ALL) leukemia. Material & Methods:Activity of TGR-1202 against individual isoforms of the PI3K enzyme was determined via enzyme, cellular, and whole blood based assays. Potency of the compound was confirmed via leukemic cell viability and Annexin V/PI staining besides testing for inhibition of pAkt, a downstream kinase regulating cell survival and growth. These assays were conducted with cell lines (CCRF-CEM, HL-60, and MOLT-4) and patient derived cells. Anti-tumor efficacy of the compound was studied in vivo with the subcutaneous MOLT-4 xenograft model. Lastly, ADME and pharmacokinetic properties of the molecule were determined. Results:TGR-1202 demonstrated significant potency against PI3Kδ (22.2 nM) with several fold selectivity over the α (>10000), β (>50), and γ (>48) isoforms. Additionally, the compound inhibited B-cell proliferation (24.3 nM) and FcεR1 induced CD63 expression in human whole blood basophils (68.2 nM) indicating specificity towards the delta isoform. Viability testing demonstrated that the compound caused a dose-dependent inhibition in growth of immortalized as well as patient-derived AML and ALL cells. Reduction in viability was accompanied by a reduction in pAKT (>50% @ 0.3–1 μM) along with a significant induction in apoptosis in both cell lines (CCRF-CEM, HL-60, and MOLT-4) and patient samples. In tumor xenografts, oral administration of 150 mg/kg RP5264 salt over a 25-day period resulted in significant inhibition (>50%) of MOLT-4 tumor growth in mice. Pharmacokinetic studies across species indicated good oral absorption (>40% bioavailability for mice, rat, and dog) with favorable plasma concentrations (3–10 μM @ 20 mg/kg for mice, rat, and dog) relevant for efficacy. In addition, early toxicological evaluation of the molecule indicated a MTD > 500 mg/kg over a 14-day treatment period in Balb/c mice. Conclusions:TGR-1202, primarily, through its activity at the δ isoform of PI3K, has activity in both myeloid and lymphoid acute leukemia cell lines and primary patient tumors. Further evaluation of this molecule in the treatment of AML and ALL is justified, and current testing of TGR-1202 in various leukemia cell lines and within a variety of primary leukemias is ongoing. Disclosures:Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Niecestro:TG Therapeutics, Inc.: Consultancy, Equity Ownership. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership.

TGR-1202: A Novel, Targeted PI3Kδ Inhibitor in Multiple Myeloma

AbstractAbstract 5018 Background:The bone marrow microenvironment contributes to the pathogenesis of Multiple Myeloma (MM) by promoting the oncogenic process including drug resistance. High expression levels of the PI3Kδ isoform in patient MM cells implicate this target as a novel and attractive intervention strategy aimed at attenuating the progression of the disease. Herein, we describe the biological and pharmacokinetic properties of TGR-1202, a novel and small molecule PI3Kδ inhibitor with scope to be developed as a clinical candidate for patients with relapsed and/or refractory MM. Material & Methods:Activity of TGR-1202 on individual PI3K isoforms was determined in enzyme, cell, and whole blood based assays. Potency of the compound was confirmed viaMM cell viability and apoptosis assays besides testing for inhibition of pAkt, a downstream kinase regulating cell survival and growth. Additionally, the compound was tested for potency in viability, apoptosis, and Akt phosphorylation assays using immortalized and MM patient derived primary cells. ADME and pharmacokinetic properties of the molecule were also determined. Results:TGR-1202 demonstrated significant potency against PI3Kδ (22. 2 nM) with several fold selectivity over the α (>10000), β (>50), and γ (>48) isoforms. Additionally, the compound inhibited B-cell proliferation (24. 3 nM) and FcεR1 induced CD63 expression in human whole blood basophils (68. 2 nM) indicating specificity towards the delta isoform. The addition of 5 μM TGR-1202 to dexamethasone (0. 55 μM) caused a dramatic shift in GI50 of MM-1S cells compared to that of either TGR-1202 (9. 7 μM) or dexamethasone (18. 3 μM) alone. Further viability testing demonstrated that the compound caused a dose-dependent inhibition (>50% @ 1–3 μM) in growth of immortalized (U266B1 and MM-1R) as well as patient-derived MM cells. Reduction in viability was accompanied by a reduction in pAKT (>80% @ 3 μM) along with induction of apoptosis in both cell lines and patient samples. Pharmacokinetic studies across species indicated good oral absorption (>40% bioavailability for mice, rat, and dog) with favorable plasma concentrations (3–10 μM @ 20 mg/kg for mice, rat, and dog) relevant for efficacy. Conclusions:Data demonstrate the therapeutic potential of TGR-1202 in MM viainhibition of the PI3Kδ pathway. Data from immortalized as well as primary patient-derived cells justify further evaluation of this molecule in MM. Combination therapy of TGR-1202 and dexamethasone in a mouse MM-1S xenograft model is currently ongoing. Disclosures:Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics: Employment. Sportelli:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.

GATA2 Is a Critical Transactivator for the Human IL1RL1/ST2 Promoter in Mast Cells/Basophils: OPPOSING ROLES FOR GATA2 and GATA1 IN HUMAN IL1RL1/ST2 GENE EXPRESSION

The IL1RL1/ST2 gene encodes a receptor for IL-33. Signaling from IL1RL1/ST2 induced by IL-33 binding was recently identified as a modulator of the Th2 response. The target cells for IL-33 are restricted in some hematopoietic lineages, including mast cells, basophils, eosinophils, Th2 cells, natural killer cells, and dendritic cells. To clarify the molecular mechanisms of cell type-specific IL1RL1/ST2 expression in mast cells and basophils, transcriptional regulation of the human IL1RL1/ST2 promoter was investigated using the mast cell line LAD2 and the basophilic cell line KU812. Reporter assays suggested that two GATA motifs just upstream of the transcription start site in the ST2 promoter are critical for transcriptional activity. These two GATA motifs possess the capacity to bind GATA1 and GATA2 in EMSA. ChIP assay showed that GATA2, but not GATA1, bound to the ST2 promoter in LAD2 cells and that histone H3 at the ST2 promoter was acetylated in LAD2 cells, whereas binding of GATA1 and GATA2 to the ST2 promoter was detected in KU812 cells. Knockdown of GATA2 mRNA by siRNA reduced ST2 mRNA levels in KU812 and LAD2 cells and ST2 protein levels in LAD2 cells; in contrast, GATA1 siRNA transfection up-regulated ST2 mRNA levels in KU812 cells. The ST2 promoter was transactivated by GATA2 and repressed by GATA1 in coexpression analysis. When these siRNAs were introduced into human peripheral blood basophils, GATA2 siRNA reduced ST2 mRNA, whereas GATA1 siRNA up-regulated ST2 mRNA. These results indicate that GATA2 and GATA1 positively and negatively control human ST2 gene transcription, respectively.

Identification of Basophils as a Major Source of Hepatocyte Growth Factor in Chronic Myeloid Leukemia: A Novel Mechanism of BCR-ABL1-Independent Disease Progression

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.

Marked Differences in the Signaling Requirements for Expression of CD203c and CD11b versus CD63 Expression and Histamine Release in Human Basophils

Many techniques are being used to examine the status of circulating human basophils including the enhanced expression of a variety of cell surface proteins. There is accumulating evidence that there are at least two compartments containing these activation marker proteins but there are only some indications for the signaling requirements for each of the compartments. This study began with published reports by other investigators who potentially dissociated CD63 expression from anaphylactic degranulation with the p38 inhibitor, SB203580, a possible falsification of a previously proposed hypothesis regarding CD63 expression. To explore the signaling requirements for CD63, a variety of pharmacological agents were used to inhibit or enhance 4 endpoints of basophil activation. First, it was found that inhibition of both histamine release and CD63 expression with SB203580 was concordant. But it was also found that this agent had no effect on increased expression of CD203c and CD11b. Actin polymerization inhibitors caused marked enhancement of CD63 expression (concordant with their effects on degranulation) with no effect on expression of CD203c and CD11b. The third generation syk inhibitor, NVP-QAB205, showed a 5-fold lower potency for inhibiting expression of CD203c and CD11b than for CD63. Finally, while desensitization of CD11b and CD203c expression occurs, it is slower than desensitization of the CD63 response. Taken together, these various observations demonstrate a marked difference in the early signaling requirements for the CD11b/CD203c compartment and CD63 degranulation and provide support for the hypothesis that CD11b and CD203c reside in a similar compartment.

Roles for the High Affinity IgE Receptor, FcεRI, of Human Basophils in the Pathogenesis and Therapy of Allergic Asthma: Disease Promotion, Protection or Both?

The role of basophils, the rarest of blood granulocytes, in the pathophysiology of allergic asthma is still incompletely understood. Indirect evidence generated over many decades is consistent with a role for basophils in disease promotion. Recent improvements in procedures to purify and analyze very small numbers of human cells have generally supported this view, but have also revealed new complexities.This chapter focuses on our analyses of Fcε R1 function in basophils in the context of understanding and treating human allergic asthma. In long-term studies, we demonstrated that asthmatic subjects have higher circulating numbers of basophils than non-atopic non-asthmatic subjects and that their basophils show higher rates of both basal and anti-IgE or antigen-stimulated histamine release. These results hint at a direct role for basophils in promoting asthma. Supporting this interpretation, the non-releaser phenotype that we linked to the excessive proteolysis of Syk via the ubiquitin/proteasomal pathway is less common in basophils from asthmatic than non-asthmatic donors. The discovery of a basophil-specific pathway regulating Syk levels presents a clear opportunity for therapy. Another route to therapy was revealed by evidence that basophil FcεRI signaling can be downregulated by co-crosslinking the ITAM-containing IgE receptor, FcγRI, to the ITIM-containing IgG receptor, FcγRIIB. Based on this discovery, hybrid co-crosslinking fusion proteins are being engineered as potential therapies targeting basophils. A third distinguishing property of human basophils is their high dependence on IgE binding to stabilize membrane FcεRI. The circulating IgE scavenging mAb, Omalizumab, reduces FcεRI expression in basophils from asthmatics by over 95% and produces a substantial impairment of IL-4, IL-8 and IL-13 production in response to the crosslinking of residual cell surface IgE-FcεRI. A search for small molecule inhibitors that similarly impair high affinity IgE binding to basophils may yield reagents that mimic Omalizumab’s therapeutic benefits without the potential for immune side effects.Although studies on allergen and FcεRI-mediated basophil activation all point to a role in promoting disease, a case can also be made for an alternative or additional role of basophil FcεRI in protection against allergic asthma. Human basophils have high affinities for IgE, they upregulate receptor levels over a >100-fold range as circulating IgE levels increase and they have short half-lives in the circulation. Thus, when allergen is absent, basophil FcεRI could serve as scavengers of serum IgE and therefore protectors against mast cell IgE-mediated inflammatory responses. Further studies are clearly needed to determine if FcεR-expressing basophils play pathogenic or protective roles – or both – in human allergic asthma and other IgE-mediated inflammatory disorders.

Basophil CD203c Levels Are Increased at Baseline and Can Be Used to Monitor Omalizumab Treatment in Subjects with Nut Allergy

Rationale: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. Methods: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. Results: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. Conclusions: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

Regulation of Syk kinase and FcRβ expression in human basophils during treatment with omalizumab

In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FcepsilonRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRbeta/FcepsilonRIalpha is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment. We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgE-mediated histamine release and disproportionately alters the relative presence of FcRbeta and FcepsilonRIalpha. Syk, FcepsilonRIalpha, and FcRbeta expression was monitored during the treatment of subjects with omalizumab. Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRbeta/FcepsilonRIalpha ratio in patients treated with omalizumab. In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FcepsilonRI alpha and beta subunits in basophils is influenced by factors extrinsic to the cell.

CD203c expression on human basophils is associated with asthma exacerbation

CD203c is a basophil cell surface marker used to diagnose and monitor various allergic diseases, but its relationship to asthma is not clear. We determined whether CD203c expression levels are associated with stable and exacerbated asthma. We used flow cytometry to compare spontaneous expression levels of surface markers on basophils from patients with stable or exacerbated asthma and from healthy subjects. Longitudinal changes in these expression levels were measured after basophil stimulation by IgE-dependent or IgE-independent mechanisms and compared with patients' asthma status. Spontaneous expression levels of CD203c were significantly higher on basophils from patients with asthma exacerbation than patients with stable asthma or healthy subjects. In contrast, no differences in spontaneous expression levels of CD63 or CD69 were observed among the 3 groups. Anti-IgE-induced expression of CD203c significantly increased in basophils during asthma exacerbation (P = .005). Low concentrations of Dermatophagoides pteronyssinus or IL-3 induced higher expression levels of CD203c during asthma exacerbation than during clinical improvement; induction of CD203c expression by these antigens therefore correlates with asthma control. In the patients with clinical improvement, there was a correlation between spontaneous CD203c expression levels and the percent predicted values of FEV(1) (r = -0.761; P = .022). Asthma exacerbation was accompanied by increased expression of CD203c on basophils that decreased significantly during remission. Basophil expression levels of CD203c might therefore be used to monitor asthma in patients.

Involvement of hypoxia‐inducible factor‐1 HiF(1α) in IgE‐mediated primary human basophil responses

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE-mediated activation of primary human basophils results in protein accumulation of the alpha-subunit of hypoxia-inducible factor 1alpha (HIF-1alpha), which is differentially regulated compared with signals controlling histamine release. HIF-1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3-kinase, were critical for IgE-mediated accumulation of HIF-1alpha, although the latter crucially affected degranulation. Abrogating HIF-1alpha expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF-1alpha protein alters IgE-induced ATP depletion in basophils, thus also supporting the production of the pro-allergic cytokine IL-4.