IL-3 in the development and function of basophils.

The ß common chain (ßc) cytokine family includes granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5, all of which use ßc as key signaling receptor subunit. GM-CSF, IL-3 and IL-5 have specific roles as hematopoietic growth factors. IL-3 binds with high affinity to the IL-3 receptor α (IL-3Rα/CD123) and then associates with the ßc subunit. IL-3 is mainly synthesized by different subsets of T cells, but is also produced by several other immune [basophils, dendritic cells (DCs), mast cells, etc.] and non-immune cells (microglia and astrocytes). The IL-3Rα is also expressed by immune (basophils, eosinophils, mast cells, DCs, monocytes, and megacaryocytes) and non-immune cells (endothelial cells and neuronal cells). IL-3 is the most important growth and activating factor for human and mouse basophils, primary effector cells of allergic disorders. IL-3-activated basophils and mast cells are also involved in different chronic inflammatory disorders, infections, and several types of cancer. IL-3 induces the release of cytokines (i.e., IL-4, IL-13, CXCL8) from human basophils and preincubation of basophils with IL-3 potentiates the release of proinflammatory mediators and cytokines from IgE- and C5a-activated basophils. IL-3 synergistically potentiates IL-33-induced mediator release from human basophils. IL-3 plays a pathogenic role in several hematologic cancers and may contribute to autoimmune and cardiac disorders. Several IL-3Rα/CD123 targeting molecules have shown some efficacy in the treatment of hematologic malignancies.

Response of peripheral blood basophils in subjects with chronic spontaneous urticaria during treatment with omalizumab.

BACKGROUND: Treatment of patients with asthma or food allergy with omalizumab results in several consistent changes in circulating basophils. The multiple basophil phenotypes observed in patients with chronic spontaneous urticaria (CSU) present some unique attributes that may not respond in a similar fashion to patients with asthma or food allergy. As part of a clinical study on the therapeutic outcomes of omalizumab treatment in CSU, the basophil compartment was examined for changes in characteristics predicted by prior studies. OBJECTIVE: This study sought to examine the changes in basophil function and its relationship to auto-antibodies in serum during treatment with omalizumab. METHODS: At multiple time points before and during omalizumab treatment of patients with CSU, basophil surface IgE and FcεRI expression, cellular spleen tyrosine kinase (SYK) expression, IgE-mediated histamine release (HR), and the presence of auto-antibodies in serum were determined. RESULTS: Three basophil phenotypes were enumerated in the clinical study and used to group results in this basophil study: subjects with (1) basopenia, (2) normal basophil numbers with normal IgE-mediated HR, and (3) normal basophil numbers with poor HR. Basopenia was highly associated with the presence of auto-antibodies to unoccupied FcεRI and basophil numbers did not change during treatment. Likewise, subjects who are basopenic showed no changes in SYK expression or HR during treatment. In basophils of subjects who are nonbasopenic, increases in SYK expression and HR showed the expected inverse relationship to starting SYK and HR levels. Treatment with omalizumab resulted in similar kinetics for decreases in surface FcεRI and IgE in all 3 groups. CONCLUSIONS: A unifying interpretation of the results revolves around the presence of auto-antibodies to FcεRI in CSU. If present, basopenia and an absence of changes in basophils during omalizumab treatment are observed. If auto-antibodies are absent, the changes in the basophil compartment are consistent with prior studies of asthma and food allergy. These group differences also are related to efficacy of the treatment for clinical outcomes, as found in the parent clinical study.

The efficacy of omalizumab treatment in chronic spontaneous urticaria is associated with basophil phenotypes.

BACKGROUND: The mechanisms underlying disease pathogenesis in chronic spontaneous urticaria (CSU) and improvement with omalizumab are incompletely understood. OBJECTIVES: This study sought to examine whether the rate of clinical remission is concordant with baseline basophil features or the rate of change of IgE-dependent functions of basophils and/or plasmacytoid dendritic cells during omalizumab therapy. METHODS: Adults (n = 18) with refractory CSU were treated with omalizumab 300 mg monthly for 90 days. Subjects recorded daily urticaria activity scores, and clinical assessments with blood sampling occurred at baseline and on days 1, 3, 6, 10, 20, 30, 60, and 90 following omalizumab. At baseline, subjects were categorized by basophil functional phenotypes, determined by in vitro histamine release (HR) responses to anti-IgE antibody, as CSU-responder (CSU-R) or CSU-non-responder (CSU-NR), as well as basopenic (B) or nonbasopenic (NB). RESULTS: CSU-R/NB subjects demonstrated the most rapid and complete symptom improvement. By day 6, CSU-R/NB and CSU-NR/NB had increased anti-IgE-mediated basophil HR relative to baseline, and these shifts did not correlate with symptom improvement. In contrast, CSU-NR/B basophil HR did not change during therapy. The kinetics of the decrease in surface IgE/FcεRI was similar in all 3 phenotypic groups and independent of the timing of the clinical response. Likewise, plasmacytoid dendritic cells' surface IgE/FcεRI decline and TLR9-induced IFN-α responses did not reflect clinical change. CONCLUSIONS: Changes in basophil IgE-based HR, surface IgE, or FcεRI bear no relationship to the kinetics in the change in clinical symptoms. Baseline basophil count and basophil functional phenotype, as determined by HR, may be predictive of responsiveness to omalizumab.

Activation of Human Basophils by A549 Lung Epithelial Cells Reveals a Novel IgE-Dependent Response Independent of Allergen.

Evidence for epithelial cell (EC)-derived cytokines (e.g., thymic stromal lymphopoietin [TSLP]) activating human basophils remains controversial. We therefore hypothesize that ECs can directly activate basophils via cell-to-cell interaction. Basophils in medium alone or with IL-3 ± anti-IgE were coincubated with TSLP, IL-33, or IL-25. Analogous experiments cocultured basophils (1-72 h) directly with EC lines. Supernatants were tested for mediators and cytokines. Abs targeting receptors were tested for neutralizing effects. Lactic acid (pH 3.9) treatment combined with passive sensitization tested the role of IgE. Overall, IL-33 augmented IL-13 secretion from basophils cotreated with IL-3, with minimal effects on histamine and IL-4. Conversely, basophils (but not mast cells) released histamine and marked levels of IL-4/IL-13 (10-fold) when cocultured with A549 EC and IL-3, without exogenous allergen or IgE cross-linking stimuli. The inability to detect IL-33 or TSLP, or to neutralize their activity, suggested a unique mode of basophil activation by A549 EC. Half-maximal rates for histamine (4 h) and IL-4 (5 h) secretion were slower than observed with standard IgE-dependent activation. Ig stripping combined with passive sensitization ± omalizumab showed a dependency for basophil-bound IgE, substantiated by a requirement for cell-to-cell contact, aggregation, and FcεRI-dependent signaling. A yet unidentified IgE-binding lectin associated with A549 EC is implicated after discovering that LacNAc suppressed basophil activation in cocultures. These findings point to a lectin-dependent activation of basophil requiring IgE but independent of allergen or secreted cytokine. Pending further investigation, we predict this unique mode of activation is linked to inflammatory conditions whereby IgE-dependent activation of basophils occurs despite the absence of any known allergen.

Suppression of the immunologic response to peanut during immunotherapy is often transient.

BACKGROUND: Studies suggest that oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) for food allergy hold promise; however, the immunologic mechanisms underlying these therapies are not well understood. OBJECTIVE: We sought to generate insights into the mechanisms and duration of suppression of immune responses to peanut during immunotherapy. METHODS: Blood was obtained from subjects at baseline and at multiple time points during a placebo-controlled trial of peanut OIT and SLIT. Immunologic outcomes included measurement of spontaneous and stimulated basophil activity by using automated fluorometry (histamine) and flow cytometry (activation markers and IL-4), measurement of allergen-induced cytokine expression in dendritic cell (DC)-T-cell cocultures by using multiplexing technology, and measurement of MHC II and costimulatory molecule expression on DCs by using flow cytometry. RESULTS: Spontaneous and allergen-induced basophil reactivity (histamine release, CD63 expression, and IL-4 production) were suppressed during dose escalation and after 6 months of maintenance dosing. Peanut- and dust mite-induced expression of TH2 cytokines was reduced in DC-T-cell cocultures during immunotherapy. This was associated with decreased levels of CD40, HLA-DR, and CD86 expression on DCs and increased expression of CD80. These effects were most striking in myeloid DC-T-cell cocultures from subjects receiving OIT. Many markers of immunologic suppression reversed after withdrawal from immunotherapy and in some cases during ongoing maintenance therapy. CONCLUSION: OIT and SLIT for peanut allergy induce rapid suppression of basophil effector functions, DC activation, and TH2 cytokine responses during the initial phases of immunotherapy in an antigen-nonspecific manner. Although there was some interindividual variation, in many patients suppression appeared to be temporary.

Basophils: emerging roles in the pathogenesis of allergic disease.

After approximately 130 years since their discovery as rare granulocytes that circulate in blood, basophils are just now gaining respect as significant contributors in the pathogenesis underlying allergic inflammation and disease. While long known for secreting preformed and newly synthesized mediators and for selectively infiltrating tissue during immunoglobulin E (IgE)-mediated inflammation, their role has largely been viewed as redundant to that of tissue mast cells in functioning as effector cells. This line of thought has persisted even though it has been known in humans for approximately 20 years that basophils additionally produce relatively large quantities of cytokines, e.g. interleukin-4 (IL-4)/IL-13, that are central for the manifestations of allergic disease. Studies using novel IL-4 reporter mice have significantly added to the in vivo importance of basophils as IL-4 producing cells, with recent findings indicating that these cells also function as antigen-presenting cells essential in initiating T-helper 2 responses. If confirmed and translated to humans, these provocative findings will give new meaning to the role basophils have in allergic disease, and in immunology overall.

Modulation of dendritic cell innate and adaptive immune functions by oral and sublingual immunotherapy.

Sublingual (SLIT) and oral immunotherapy (OIT) are promising treatments for food allergy, but underlying mechanisms are poorly understood. Dendritic cells (DCs) induce and maintain Th2-type allergen-specific T cells, and also regulate innate immunity through their expression of Toll-like receptors (TLRs). We examined how SLIT and OIT influenced DC innate and adaptive immune responses in children with IgE-mediated cow's milk (CM) allergy. SLIT, but not OIT, decreased TLR-induced IL-6 secretion by myeloid DCs (mDCs). SLIT and OIT altered mDC IL-10 secretion, a potent inhibitor of FcεRI-dependent pro-inflammatory responses. OIT uniquely augmented IFN-α and decreased IL-6 secretion by plasmacytoid DCs (pDCs), which was associated with reduced TLR-induced IL-13 release in pDC-T cell co-cultures. Both SLIT and OIT decreased Th2 cytokine secretion to CM in pDC-T, but not mDC-T, co-cultures. Therefore, SLIT and OIT exert unique effects on DC-driven innate and adaptive immune responses, which may inhibit allergic inflammation and promote tolerance.

Scratching the surface of allergic transfusion reactions.

Allergic transfusion reactions (ATRs) are a spectrum of hypersensitivity reactions that are the most common adverse reaction to platelets and plasma, occurring in up to 2% of transfusions. Despite the ubiquity of these reactions, little is known about their mechanism. In a small subset of severe reactions, specific antibody has been implicated as causal, although this mechanism does not explain all ATRs. Evidence suggests that donor, product, and recipient factors are involved, and it is possible that many ATRs are multifactorial. Further understanding of the mechanisms of ATRs is necessary so that rationally designed and cost-effective prevention measures can be developed.

The safety and efficacy of sublingual and oral immunotherapy for milk allergy.

BACKGROUND: Oral immunotherapy (OIT) and sublingual immunotherapy (SLIT) are potential therapies for food allergy, but the optimal method of administration, mechanism of action, and duration of response remain unknown. OBJECTIVE: We sought to explore the safety and efficacy of OIT and SLIT for the treatment of cow's milk (CM) allergy. METHODS: We randomized children with CM allergy to SLIT alone or SLIT followed by OIT. After screening double-blind, placebo-controlled food challenges and initial SLIT escalation, subjects either continued SLIT escalation to 7 mg daily or began OIT to either 1000 mg (the OITB group) or 2000 mg (the OITA group) of milk protein. They were challenged with 8 g of milk protein after 12 and 60 weeks of maintenance. If they passed the 60-week challenge, therapy was withdrawn, with challenges repeated 1 and 6 weeks later. Mechanistic correlates included end point titration skin prick testing and measurement of CM-specific IgE and IgG(4) levels, basophil histamine release, constitutive CD63 expression, CD203c expression, and intracellular spleen tyrosine kinase levels. RESULTS: Thirty subjects with CM allergy aged 6 to 17 years were enrolled. After therapy, 1 of 10 subjects in the SLIT group, 6 of 10 subjects in the SLIT/OITB group, and 8 of 10 subjects in the OITA group passed the 8-g challenge (P = .002, SLIT vs OIT). After avoidance, 6 of 15 subjects (3 of 6 subjects in the OITB group and 3 of 8 subjects in the OITA group) regained reactivity, 2 after only 1 week. Although the overall reaction rate was similar, systemic reactions were more common during OIT than during SLIT. By the end of therapy, titrated CM skin prick test results and CD63 and CD203c expression decreased and CM-specific IgG(4) levels increased in all groups, whereas CM-specific IgE and spontaneous histamine release values decreased in only the OIT group. CONCLUSION: OIT was more efficacious for desensitization to CM than SLIT alone but was accompanied by more systemic side effects. Clinical desensitization was lost in some cases within 1 week off therapy.

Basophils beyond allergic and parasitic diseases.

Basophils bind IgE via FcεRI-αßγ2, which they uniquely share only with mast cells. In doing so, they can rapidly release mediators that are hallmark of allergic disease. This fundamental similarity, along with some morphological features shared by the two cell types, has long brought into question the biological significance that basophils mediate beyond that of mast cells. Unlike mast cells, which mature and reside in tissues, basophils are released into circulation from the bone marrow (constituting 1% of leukocytes), only to infiltrate tissues under specific inflammatory conditions. Evidence is emerging that basophils mediate non-redundant roles in allergic disease and, unsuspectingly, are implicated in a variety of other pathologies [e.g., myocardial infarction, autoimmunity, chronic obstructive pulmonary disease, fibrosis, cancer, etc.]. Recent findings strengthen the notion that these cells mediate protection from parasitic infections, whereas related studies implicate basophils promoting wound healing. Central to these functions is the substantial evidence that human and mouse basophils are increasingly implicated as important sources of IL-4 and IL-13. Nonetheless, much remains unclear regarding the role of basophils in pathology vs. homeostasis. In this review, we discuss the dichotomous (protective and/or harmful) roles of basophils in a wide spectrum of non-allergic disorders.

The XIST lncRNA is a sex-specific reservoir of TLR7 ligands in SLE.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9 times more women than men. Activation of Toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long noncoding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFN-α production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from women with SLE compared with controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN inducible, suggesting that XIST is a driver, rather than a consequence, of IFN in SLE. Overall, our work elucidated a role for XIST RNA as a female sex-specific danger signal underlying the sex bias in SLE.

Allergic agonists in apheresis platelet products are associated with allergic transfusion reactions.

BACKGROUND: The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet (PLT) products associated with ATRs and controls. STUDY DESIGN AND METHODS: Using bead-based and standard enzyme-linked immunosorbent assays, we tested supernatants from 20 consecutive apheresis PLT transfusions associated with ATRs and 30 control products for concentrations of mediators in three categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth and/or priming factors of basophils and mast cells. RESULTS: Median concentrations of the direct allergic agonists C5a, brain-derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6, 41.8, and 13.9% higher, respectively, in the supernatant of apheresis PLT products that were most strongly associated with ATRs (p < 0.05 for each mediator). Other direct agonists (macrophage inflammatory protein-1α, monocyte chemotactic protein-1, eotaxin-1, interleukin-8) were similar between groups. Concentrations of acute inflammatory mediators and basophil growth and/or priming factors were also similar between groups (p > 0.2 for all associations). CONCLUSION: The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis PLT products. Acute inflammatory proteins and basophil and/or mast cell growth and priming factors do not appear to be associated with apheresis PLT products that cause ATRs.

SHP-1 deficient mast cells are hyperresponsive to stimulation and critical in initiating allergic inflammation in the lung.

Phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1)-deficient mice display an allergic asthma phenotype that is largely IL-13 and STAT6 dependent. The cell types responsible for the Th2 phenotype have not been identified. We hypothesized that SHP-1 deficiency leads to mast cell dysregulation and increased production and release of mediators and Th2 cytokines, leading to the allergic asthma phenotype. We examined SHP-1 regulation of mast cell differentiation, survival, and functional responses to stimulation using bone marrow-derived mast cells from viable motheaten (mev) mice. We assessed pulmonary phenotypical changes in mev mice on the mast cell-deficient Kit(W-Sh) genetic background. The results showed that SHP-1 deficiency led to increased differentiation and survival, but reduced proliferation, of mast cells. SHP-1-deficient mast cells produced and released increased amounts of mediators and Th2 cytokines IL-4 and -13 spontaneously and in response to H(2)O(2), LPS, and Fc epsilonI cross-linking, involving c-Kit-dependent and -independent processes. The Fc epsilonRI signaling led to binding of SHP-1 to linker for activation of T cells 2 and enhanced linker for activation of T cells 2 phosphorylation in mev bone marrow-derived mast cells. Furthermore, the number of mast cells in the lung tissue of mev mice was increased and mast cell production and release of Th2 cytokines were distinctly increased upon Fc epsilonRI stimulation. When backcrossed to the Kit(W-Sh) background, mev mice had markedly reduced pulmonary inflammation and Th2 cytokine production. These findings demonstrate that SHP-1 is a critical regulator of mast cell development and function and that SHP-1-deficient mast cells are able to produce increased Th2 cytokines and initiate allergic inflammatory responses in the lung.

The S1 Subunit of the SARS-CoV-2 Spike Protein Activates Human Monocytes to Produce Cytokines Linked to COVID-19: Relevance to Galectin-3.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly evolved into a pandemic -the likes of which has not been experienced in 100 years. While novel vaccines show great efficacy, and therapeutics continue to be developed, the persistence of disease, with the concomitant threat of emergent variants, continues to impose massive health and socioeconomic issues worldwide. Studies show that in susceptible individuals, SARS-CoV-2 infection can rapidly progress toward lung injury and acute respiratory distress syndrome (ARDS), with evidence for an underlying dysregulated innate immune response or cytokine release syndrome (CRS). The mechanisms responsible for this CRS remain poorly understood, yet hyper-inflammatory features were also evident with predecessor viruses within the ß-coronaviridae family, namely SARS-CoV-1 and the Middle East Respiratory Syndrome (MERS)-CoV. It is further known that the spike protein (S) of SARS-CoV-2 (as first reported for other ß-coronaviruses) possesses a so-called galectin-fold within the N-terminal domain of the S1 subunit (S1-NTD). This fold (or pocket) shows structural homology nearly identical to that of human galectin-3 (Gal-3). In this respect, we have recently shown that Gal-3, when associated with epithelial cells or anchored to a solid phase matrix, facilitates the activation of innate immune cells, including basophils, DC, and monocytes. A synthesis of these findings prompted us to test whether segments of the SARS-CoV-2 spike protein might also activate innate immune cells in a manner similar to that observed in our Gal-3 studies. Indeed, by immobilizing S components onto microtiter wells, we show that only the S1 subunit (with the NTD) activates human monocytes to produce a near identical pattern of cytokines as those reported in COVID-19-related CRS. In contrast, both the S1-CTD/RBD, which binds ACE2, and the S2 subunit (stalk), failed to mediate the same effect. Overall, these findings provide evidence that the SARS-CoV-2 spike protein can activate monocytes for cytokines central to COVID-19, thus providing insight into the innate immune mechanisms underlying the CRS and the potential for therapeutic interventions.

Basophils from allergy to cancer.

Human basophils, first identified over 140 years ago, account for just 0.5-1% of circulating leukocytes. While this scarcity long hampered basophil studies, innovations during the past 30 years, beginning with their isolation and more recently in the development of mouse models, have markedly advanced our understanding of these cells. Although dissimilarities between human and mouse basophils persist, the overall findings highlight the growing importance of these cells in health and disease. Indeed, studies continue to support basophils as key participants in IgE-mediated reactions, where they infiltrate inflammatory lesions, release pro-inflammatory mediators (histamine, leukotriene C4: LTC4) and regulatory cytokines (IL-4, IL-13) central to the pathogenesis of allergic diseases. Studies now report basophils infiltrating various human cancers where they play diverse roles, either promoting or hampering tumorigenesis. Likewise, this activity bears remarkable similarity to the mounting evidence that basophils facilitate wound healing. In fact, both activities appear linked to the capacity of basophils to secrete IL-4/IL-13, with these cytokines polarizing macrophages toward the M2 phenotype. Basophils also secrete several angiogenic factors (vascular endothelial growth factor: VEGF-A, amphiregulin) consistent with these activities. In this review, we feature these newfound properties with the goal of unraveling the increasing importance of basophils in these diverse pathobiological processes.

Differential Effects of Alarmins on Human and Mouse Basophils.

Epithelial-derived alarmins (IL-33, TSLP, and IL-25) play an upstream role in the pathogenesis of asthma. Basophil-derived cytokines are a pivotal component of allergic inflammation. We evaluated the in vitro effects of IL-33, TSLP, and IL-25, alone and in combination with IL-3 on purified peripheral blood human basophils (hBaso) and bone marrow-derived mouse basophils (mBaso) in modulating the production of IL-4, IL-13, CXCL8 or the mouse CXCL8 equivalents CXCL1 and CXCL2. IL-3 and IL-33, but not TSLP and IL-25, concentration-dependently induced IL-4, IL-13, and CXCL8 release from hBaso. IL-3 synergistically potentiated the release of cytokines induced by IL-33 from hBaso. In mBaso, IL-3 and IL-33 rapidly induced IL-4 and IL-13 mRNA expression and protein release. IL-33, but not IL-3, induced CXCL2 and CXCL1 from mBaso. Differently from hBaso, TSLP induced IL-4, IL-13, CXCL1 and CXCL2 mRNA expression and protein release from mBaso. IL-25 had no effect on IL-4, IL-13, and CXCL1/CXCL2 mRNA expression and protein release even in the presence of IL-3. No synergism was observed between IL-3 and either IL-25 or TSLP. IL-3 inhibited both TSLP- and IL-33-induced CXCL1 and CXCL2 release from mBaso. Our results highlight some similarities and marked differences between the effects of IL-3 and alarmins on the release of cytokines from human and mouse basophils.

Biological screening of a unique drug library targeting MRGPRX2.

Background: Allergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2. Objective: Our aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs. Methods: We conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds. Results: The alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate). Conclusion: Our findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.

Basophils beyond effector cells of allergic inflammation.

Despite being first described in humans nearly 130 years ago, the basophil granulocyte has received little recognition other than being the least common leukocyte circulating in blood. Even after its identity as the source of histamine released by blood cells in response to reaginic IgE, its role in allergic disease has largely been viewed as redundant to that of the tissue mast cell. This line of thought, however, is changing with evidence that has emerged during the last 15 years. Not only have these rare cells been shown to constitute a significant source of cytokines (IL-4 and IL-13) vital to the pathogenesis of allergic disease, but by doing so, may very well modulate T-helper 2-type inflammation at the level of T-cell/dendritic cell interactions. This novel concept combined with the fact that basophils selectively infiltrate allergic lesion sites has sparked greater interest in this once overlooked immune cell, both in adaptive as well as in innate immunity.

Human basophils secrete IL-3: evidence of autocrine priming for phenotypic and functional responses in allergic disease.

Although IL-3 is commonly recognized for its growth factor-like activity, in vitro studies have long demonstrated a unique capacity for this cytokine to also augment the proinflammatory properties and phenotype of human basophils. In particular, basophils secrete mediators that are hallmarks in allergic disease, including vasoactive amines (e.g., histamine), lipid metabolites (e.g., leukotriene C(4)), and cytokines (e.g., IL-4/IL-13), which are all markedly enhanced with IL-3 pretreatment. This priming phenomenon is observed in response to both IgE-dependent and IgE-independent stimulation. Additionally, IL-3 directly activates basophils for IL-13 secretion and enhanced CD69 expression, two markers that are elevated in allergic subjects. Lymphocytes are commonly thought to be the source of the IL-3 that primes for these basophil responses. However, we demonstrate herein for the first time that basophils themselves rapidly produce IL-3 (within 4 h) in response to IgE-dependent activation. More importantly, our findings definitively show that basophils rapidly bind and utilize the IL-3 they produce, as evidenced by functional and phenotypic activity that is inhibited in the presence of neutralizing anti-IL-3 receptor (CD123) Abs. We predict that autocrine IL-3 activity resulting from low-level IgE/FcepsilonRI cross-linking by specific allergen represents an important mechanism behind the hyperreactive nature of basophils that has long been observed in allergic disease.