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.

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.

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.

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.

Decreases in human dendritic cell-dependent T(H)2-like responses after acute in vivo IgE neutralization.

BACKGROUND: Dendritic cells (DCs) and other professional antigen-presenting cells express a variant of the high-affinity IgE receptor known as alphagamma(2), which, on the basis of in vitro findings, has long been implicated to function in facilitating allergen uptake and presentation to T(H) cells. OBJECTIVES: To use omalizumab as an in vivo tool to neutralize IgE binding to circulating dendritic cells and to assess whether this results in altered DC-dependent T-cell responsiveness to allergen ex vivo. METHODS: Subjects with cat allergy were enrolled in a 3.5-month, double blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing for allergic asthma. Blood plasmacytoid and myeloid DCs were assessed at baseline and posttreatment for expression of surface IgE, FcepsilonRIalpha, and induction of CD4(+)T-cell proliferation and cytokine responses to cat allergen. RESULTS: IgE expression on plasmacytoid and myeloid DCs from omalizumab-treated subjects (n = 12) decreased by > or =95% posttreatment (P = .0005), whereas FcepsilonRIalpha expression decreased by 66% and 48%, respectively (P = .0005). Cat allergen-induced proliferation in DC/T-cell cocultures observed at baseline was suppressed approximately 20% to 40% postomalizumab treatment (P = .001). Multiplexing for cytokines in plasmacytoid DC/T-cell cocultures also showed decreases in IL-5, IL-13, and IL-10 (P < .05), whereas IL-2 and IFN-gamma were unaltered or slightly increased. These changes were not evident in placebo-control subjects (n = 4). CONCLUSION: IgE likely facilitates allergen presentation by dendritic cells in vivo and is also important in regulating DC-dependent T-cell cytokines during effector phases of allergic disease.

Interferons modulate Fc epsilon RI-dependent production of autoregulatory IL-10 by circulating human monocytoid dendritic cells.

BACKGROUND: Immature human blood monocytoid dendritic cells (mDCs) express high-affinity receptors for IgE (Fc epsilon RI), yet their exact function and regulation remain poorly understood. OBJECTIVE: We sought to characterize Fc epsilon RI-dependent cytokine responses and their regulation in circulating human blood mDCs. METHODS: Fc epsilon RI-dependent cytokine responses of circulating mDCs were studied by using anti-Fc epsilon RI alpha stimulation. Plasmacytoid dendritic cell (pDC) cross-regulation through Toll-like receptor 9 on these responses was investigated by examining the effects of exogenous IFN-alpha pretreatment and by coculturing pDCs and mDCs stimulated with CpG. Culture supernatants were analyzed by means of ELISA to determine cytokine levels. Cell markers were determined by means of flow cytometry. RESULTS: mDCs express marked levels of Fc epsilon RI (net mean fluorescence intensity, 196 +/- 49; n = 4). After Fc epsilon RI-dependent activation in mDCs, TNF-alpha (2189 +/- 864 pg/10(6) mDCs, n = 3) levels were upregulated within 4 hours, whereas IL-10 (112 +/- 47 pg/10(6) mDCs, n = 3) levels were detectable only after 24 hours of incubation. After adding IL-10-neutralizing antibody, TNF-alpha Fc epsilon RI-dependent responses were significantly augmented (3903 +/- 197 pg/10(6) mDCs, P < .01, n = 3). Conversely, recombinant IL-10 dose-dependently inhibited Fc epsilon RI-mediated TNF-alpha responses up to 86% +/- 3% (n = 3, P < .001). Pretreatment of mDCs with IFN-alpha (100 U/mL) enhanced Fc epsilon RI-dependent secretion of IL-10 by 3.2-fold (183 +/- 11 pg/10(6) mDCs, n = 4) compared with that seen in untreated cells (57 +/- 33 pg/10(6) mDCs, P < .001, n = 4). In pDC/mDC cocultures pretreated with CpG, Fc epsilon RI-dependent IL-10 secretion by mDCs was similarly augmented by 3-fold. CONCLUSIONS: Autocrine secretion of IL-10, a critical autoregulator of Fc epsilon RI-dependent proinflammatory responses in mDCs, is cross-regulated by IFN-alpha, a major product of Toll-like receptor 9 responses in pDCs that normally promotes T(H)1 immunity.

Epithelial Cell-Associated Galectin-3 Activates Human Dendritic Cell Subtypes for Pro-Inflammatory Cytokines.

There is mounting evidence that galectin-3 is a prognostic and diagnostic biomarker associated with diverse diseases and conditions, including cancer, cardiovascular disease, autoimmunity, wound healing, allergic disease, and chronic inflammation in general. Yet, whether and exactly how galectin-3 may participate in the pathogenesis of these diseases remains poorly understood. Recently, we have linked the expression of galectin-3 on the A549 epithelial cell line -an adenocarcinoma, to the activation of human basophils for the release of histamine and secretion of IL-4 and IL-13. These responses proved dependent on cell-to-cell contact, basophil expression of IgE, were inhibited by n-acetyllactosamine, and were ablated when basophils were co-cultured with A549 clones lacking galectin-3 expression. While recombinant galectin-3 failed to activate basophils when in solution, microspheres expressing this lectin did so by mimicking the responses seen when using A549 cells. Given the IgE dependency of the basophil responses, and the fact that galectin-3 is long known to bind this immunoglobulin, we hypothesize that a similar mode of activation extends to other IgE-bearing cells. To investigate this possibility, we tested epithelial cell-associated galectin-3 for its capacity to activate human dendritic cells, including the plasmacytoid and myeloid subtypes as well as monocytes, all of which bind IgE. Indeed, results indicate that epithelial cell-associated galectin-3 activated these cells for robust production of TNF-α and IL-6 and up-regulated the expression of activation markers found on dendritic cells. Moreover, many of the same parameters previously observed for basophils applied to the findings herein, including evidence that matrix-bound galectin-3 (whether on epithelial cells or microspheres) facilitates this mode of activation. In contrast, IgE expression was dispensable for these galectin-3-dependent cytokine responses, implying that this lectin activates dendritic cells (and monocytes) by binding to a glycoprotein other than this immunoglobulin. Overall, these findings further demonstrate how galectin-3 mediates immune cell activation, providing novel insight into how this lectin may promote chronic inflammation underlying the pathogenesis of many diseases.

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models.

Previously, we demonstrated a negative correlation between histamine release to histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP-1 in human basophils. The present study was conducted to investigate whether suppressing SHIP-1 using small interfering (si)RNA technology would alter the releasability of culture-derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro-34 medium containing cytokines: mast cells with IL-6 and stem cell factor (100 ng/ml each) for 6-8 weeks and basophils with IL-3 (6.7 ng/ml) for 2-3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP-1 or a negative control siRNA. Changes in SHIP-1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP-induced histamine release. siRNA knockdown of SHIP-1 in mast cells ranged from 31% to 82%, mean 65 +/- 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP-1 knockdown in basophils ranged from 34% to 69%, mean 51.8 +/- 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP-1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.

Toll-like receptor 9 suppression in plasmacytoid dendritic cells after IgE-dependent activation is mediated by autocrine TNF-alpha.

BACKGROUND: Functional significance for the alphagamma(2) variant of the high-affinity IgE receptor (FcepsilonRI) reportedly expressed on human dendritic cell subtypes remains poorly understood. Studies show that immature plasmacytoid dendritic cells (pDCs) secrete large quantities of TNF-alpha and IL-6 when directly stimulated with anti-IgE antibody. This mode of activation, however, reduces Toll-like receptor 9 (TLR9) expression in pDCs and their ability to mount an IFN-alpha response when subsequently activated with oligodeoxynucleotide containing CpG. OBJECTIVE: To investigate the mechanisms underlying this IgE-dependent suppression of TLR9 and innate immune responsiveness in pDCs by focusing on autocrine cytokine responses. METHODS: pDCs were isolated from blood by using blood dendritic cell antigen 4 selection. Cytokine responses to anti-IgE antibody-dependent and/or CpG-dependent stimulation were measured by using ELISA. TLR9 expression was determined by using quantitative RT-PCR and Western blotting. RESULTS: The time required for downregulating TLR9 expression in pDCs after anti-IgE stimulation correlated with the induction and duration of TNF-alpha secreted by these cells. Pretreatment of pDCs with recombinant TNF-alpha (but not IL-6 or IL-10) markedly suppressed TLR9 expression. Functional response to CpG (ie, IFN-alpha induction) was also inhibited with TNF-alpha pretreatment (inhibitory concentration(50) = approximately 200 pg/mL). Finally, an antibody that neutralizes TNF-alpha activity completely restored TLR9 expression during anti-IgE stimulation and significantly improved IFN-alpha secretion on subsequent activation with CpG. CONCLUSION: Autocrine TNF-alpha secretion resulting from IgE/FcepsilonRI-dependent activation plays a critical role in suppressing TLR9-dependent responses in pDCs that normally promote T(H)1 activity.

Isolation of Human Basophils.

Isolating human basophils from blood has long been hampered by the fact that these granulocytes represent just 1% or less of the circulating leukocyte population. We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils for use in a variety of assays to assess the in vitro biology of these immune cells, both in IgE -dependent and -independent responses.

Toll-like receptor 2 ligands activate human basophils for both IgE-dependent and IgE-independent secretion.

BACKGROUND: Toll-like receptor (TLR) molecules play a critical role in directing the course of acquired immunity, including that associated with allergic disease, by recognizing specific microbial products that activate immune cells for effector functions. OBJECTIVE: We investigated whether human basophils express 2 such molecules (TLR2 and TLR4), and assessed whether putative ligands for these receptors activate nuclear factor kappaB (NFkappaB) and modulate mediator release and cytokine secretion either alone or in response to stimulation. METHODS: Toll-like receptor expression was assessed by using RT-PCR and flow cytometry. Immunoblotting detected nuclear NFkappaB. Automated fluorometry, RIA, and ELISA detected concurrent changes in histamine, leukotriene C 4 , and cytokine, respectively, after culture with specific ligands. RESULTS: mRNA and protein for TLR2 and TLR4 were detected in basophils. However, in assessing nuclear localization of NFkappaB as a measure of functional receptor responses, basophils selectively reacted only to peptidoglycan, a TLR2 ligand, and not to LPS, a TLR4 ligand. Likewise, basophils secreted both IL-4 and IL-13 in direct response to peptidoglycan but not to LPS. Although neither ligand induced histamine or leukotriene C 4 release, several TLR2-specific ligands augmented the secretion of these mediators (and cytokine) in response to IgE-dependent activation and of IL-13 in response to IgE-independent stimulation. Finally, a selective inhibitor of NFkappaB did not prevent these enhancing effects mediated by TLR2 ligands. CONCLUSION: These data suggest that innate immune responses mediated through TLR2 play a role in augmenting allergic reactions, in part by modulating basophil cytokine secretion and mediator release independently of NFkappaB activation.

TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression.

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.

IFN-alpha inhibits IL-3 priming of human basophil cytokine secretion but not leukotriene C4 and histamine release.

BACKGROUND: Innate immune responses play a critical role in determining the course of acquired immunity, including that associated with allergic disease. Type I interferons, which are generated early in these reactions, are important soluble factors that prime for TH1-like activity. OBJECTIVE: Because human basophils secrete IL-4 and IL-13 in response to both IgE-dependent and IgE-independent stimuli, we tested whether IFN-alpha, a major type I IFN, affects the production of these TH2 cytokines and/or mediator release from these cells. METHODS: Basophils isolated from blood were treated with IFN-alpha in the presence and absence of IL-3 priming before stimulating through the IgE receptor to release histamine, leukotriene C4, and IL-4. Effects of IFN-alpha on IL-3-mediated IL-13 secretion and basophil survival were also tested. IFN-alpha receptor expression was determined by RT-PCR. RESULTS: IFN-alpha specifically inhibited the effects IL-3 has on basophil cytokine secretion. Enhanced secretion of IL-4 resulting from IL-3 priming was significantly inhibited in cells concurrently cultured with IFN-alpha. This effect was specific for cytokine generation, because histamine and leukotriene C4 were unaffected. Furthermore, IFN-alpha blocked IL-13 secretion directly induced by IL-3. Although IFN-beta also possessed some inhibitory activity, IFN-gamma (a type II IFN) had no effect on basophil cytokine secretion. Basophils constitutively expressed mRNA for the type I IFN receptor, and IFN-alpha did not affect basophil viability with regard to inhibition of cytokine secretion. CONCLUSIONS: These results support the belief that early innate immune responses resulting in IFN-alpha production negatively regulate allergic responses by also inhibiting priming of basophil cytokine release.