IL-3 but not monomeric IgE regulates Fc?RI levels and cell survival in primary human basophils.
ABSTRACT: Binding of allergen-specific IgE to its primary receptor Fc?RI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and Fc?RI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate Fc?RI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in Fc?RI regulation and cell survival of primary human basophils. Fc?RI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for Fc?RI. Ultimately, we investigated the effect of IL-3 on basophil surface Fc?RI levels by protein and gene expression analysis. Surface levels of Fc?RI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of Fc?RI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates Fc?RI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface Fc?RI levels and to prevent prolonged cell survival in allergic inflammation.
Project description:Allergic disorders have now become a major worldwide public health issue, but the effective treatment options remain limited. We report a novel approach to block allergic reactivity by targeting the surface-bound IgE of the allergic effector cells via low-affinity anti-human IgE Abs with dissociation constants in the 10-6 to 10-8 M range. We demonstrated that these low-affinity anti-IgE mAbs bind to the cell surface-bound IgE without triggering anaphylactic degranulation even at high concentration, albeit they would weakly upregulate CD203c expression on basophils. This is in contrast to the high-affinity anti-IgE mAbs that trigger anaphylactic degranulation at low concentration. Instead, the low-affinity anti-IgE mAbs profoundly block human peanut- and cat-allergic IgE-mediated basophil CD63 induction indicative of anaphylactic degranulation; suppress peanut-, cat-, and dansyl-specific IgE-mediated passive cutaneous anaphylaxis; and attenuate dansyl IgE-mediated systemic anaphylaxis in human Fc?RI? transgenic mouse model. Mechanistic studies reveal that the ability of allergic reaction blockade by the low-affinity anti-IgE mAbs was correlated with their capacity to downregulate the surface IgE and Fc?RI level on human basophils and the human Fc?RI? transgenic mouse bone marrow-derived mast cells via driving internalization of the IgE/Fc?RI complex. Our studies demonstrate that targeting surface-bound IgE with low-affinity anti-IgE Abs is capable of suppressing allergic reactivity while displaying an excellent safety profile, indicating that use of low-affinity anti-IgE mAbs holds promise as a novel therapeutic approach for IgE-mediated allergic diseases.
Project description:Interleukin 4 (IL-4) is a critical cytokine implicated with TH2 immune reactions, which are linked to pathologic conditions of allergic diseases. In that context, the initiation of TH2 responses can critically depend on early basophil-derived IL-4 to activate T-cell responses, which then amplify IL-4 secretion. As a pleiotropic cytokine, IL-4 acts on a broad variety of hematopoietic and non-hematopoietic cells. However, the effect of IL-4 on basophils themselves, which are emerging as relevant players in allergic as well as autoimmune diseases, was only scarcely addressed so far. Here we used in vitro-differentiated mouse basophils to investigate the direct effects of IL-4 on cellular viability and surface expression of the high-affinity receptor for IgE, Fc?RI. We observed that IL-4 elicits pronounced pro-survival signaling in basophils, delaying spontaneous apoptosis in vitro to a degree comparable to the known pro-survival effects of IL-3. Our data indicate that IL-4-mediated survival depends on PI3K/AKT signaling and-in contrast to IL-3-seems to be largely independent of transcriptional changes but effectuated by post-translational mechanisms affecting BCL-2 family members among others. Additionally, we found that IL-4 signaling has a stabilizing effect on the surface expression levels of the critical basophil activation receptor Fc?RI. In summary, our findings indicate an important regulatory role of IL-4 on in vitro-differentiated mouse basophils enhancing their survival and stabilizing Fc?RI receptor expression through PI3K-dependent signaling. A better understanding of the regulation of basophil survival will help to define promising targets and consequently treatment strategies in basophil-driven diseases.
Project description:Basophils and mast cells have high affinity IgE receptors (Fc?RI) on their plasma membrane and play important roles in Fc?RI-associated allergic diseases, such as pollen allergy, food allergy, chronic spontaneous urticarial (CSU), and atopic dermatitis (AD). To date, several reports have revealed that high IgE antibody concentrations activate mast cells-which reside in tissue-in the absence of any antigens (allergens). However, IgE antibody-induced activation of basophils-which circulate in blood-has not been reported. Here, we investigated whether IgE antibodies may regulate functions of human peripheral basophils without antigens in vitro. We successfully removed IgE antibodies bound to Fc?RI on the surface of human peripheral basophils by treating with 0.1% lactic acid. We also demonstrated that high IgE antibody concentrations (>1 ?M) induced histamine release, polarization, and CD203c upregulation of IgE antibody-stripped basophils. Thus, high IgE antibody concentrations directly activate basophils, which express IgE-free Fc?RI on the cell surface. This mechanism may contribute to the pathogenesis of patients with AD and CSU who have higher serum IgE concentrations compared to healthy donors.
Project description:Antigen-mediated cross-linking of Immunoglobulin E (IgE) bound to mast cells/basophils via Fc?RI, the high affinity IgE Fc-receptor, is a well-known trigger of allergy. In humans, but not mice, dendritic cells (DCs) also express Fc?RI that is constitutively occupied with IgE. In contrast to mast cells/basophils, the consequences of IgE/Fc?RI signals for DC function remain poorly understood. We show that humanized mice that express Fc?RI on DCs carry IgE like non-allergic humans and do not develop spontaneous allergies. Antigen-specific IgE/Fc?RI cross-linking fails to induce maturation or production of inflammatory mediators in human DCs and Fc?RI-humanized DCs. Furthermore, conferring expression of Fc?RI to DCs decreases the severity of food allergy and asthma in disease-relevant models suggesting anti-inflammatory IgE/Fc?RI signals. Consistent with the improved clinical parameters in vivo, antigen-specific IgE/Fc?RI cross-linking on papain or lipopolysaccharide-stimulated DCs inhibits the production of pro-inflammatory cytokines and chemokines. Migration assays confirm that the IgE-dependent decrease in cytokine production results in diminished recruitment of mast cell progenitors; providing a mechanistic explanation for the reduced mast cell-dependent allergic phenotype observed in Fc?RI-humanized mice. Our study demonstrates a novel immune regulatory function of IgE and proposes that DC-intrinsic IgE signals serve as a feedback mechanism to restrain allergic tissue inflammation.
Project description:Mast cells and basophils are key effector cells of IgE-mediated anaphylactic reactions. The Chinese herbal formula, food allergy herbal formula 2 (FAHF-2), protects against peanut anaphylaxis in mice. However, the mechanisms underlying this effect are not fully elucidated.To investigate whether FAHF-2 inhibits mast cell/basophil numbers and IgE-mediated activation.Mice with peanut allergy (PNA mice) were treated with FAHF-2 intragastrically for 7 weeks and challenged intragastrically with peanut 1 day and 4 weeks posttreatment. Peripheral blood basophil numbers and peritoneal mast cell numbers and Fc?RI expression were determined. Direct effects of FAHF-2 on the murine mast cell line MC/9, and effects of 4 fractions and 3 compounds isolated from FAHF-2 on rat basophilic leukemia cells (RBL-2H3) and human skin mast cells degranulation and on the IgE-mediated spleen tyrosine kinase signaling pathway, were determined.Although all sham-treated PNA mice developed anaphylaxis, FAHF-2-treated PNA mice were protected against anaphylaxis after peanut challenge at 1 day and 4 weeks posttherapy. Reduction of peripheral blood basophils began after 1 week of treatment and continued for at least 4 weeks posttherapy. The number and Fc?RI expression of peritoneal mast cells were also significantly decreased 4 weeks posttherapy. FAHF-2-treated MC/9 cells showed significantly reduced IgE-induced Fc?RI expression, Fc?RI ? mRNA subunit expression, proliferation, and histamine release on challenge. Fraction 2 from FAHF-2 inhibited RBL-2H3 cell and human mast cell degranulation. Three compounds from fraction 2-berberine, palmatine, and jatrorrhizine-inhibited RBL-2H3 cell degranulation via suppressing spleen tyrosine kinase phosphorylation.Food allergy herbal formula 2 reduction of basophils and mast cell numbers as well as suppression of IgE-mediated mast cell activation may contribute to FAHF-2's persistent protection against peanut anaphylaxis.
Project description:The remarkably stable interaction of IgE with its high-affinity receptor Fc?RI on basophils and mast cells is critical for the induction of allergic hypersensitivity reactions. Because of the exceptionally slow dissociation rate of IgE-Fc?RI complexes, such allergic effector cells permanently display allergen-specific IgE on their surface and immediately respond to allergen challenge by releasing inflammatory mediators. We have recently described a novel macromolecular inhibitor that actively promotes the dissociation of IgE from Fc?RI through a molecular mechanism termed facilitated dissociation.Here we assessed the therapeutic potential of this non-immunoglobulin-based IgE inhibitor E2_79, a designed ankyrin repeat protein (DARPin), as well as a novel engineered biparatopic DARPin bi53_79, and directly compared them with the established anti-IgE antibody omalizumab.IgE-Fc?RI complex dissociation was analyzed in vitro by using recombinant proteins in ELISA and surface plasmon resonance, ex vivo by using human primary basophils with flow cytometry, and in vivo by using human Fc?RI ?-chain transgenic mice in a functional passive cutaneous anaphylaxis test.We show that E2_79-mediated removal of IgE from primary human basophils fully abrogates IgE-dependent cell activation and release of proinflammatory mediators ex vivo. Furthermore, we report that omalizumab also accelerates the dissociation of IgE from Fc?RI, although much less efficiently than E2_79. Using the biparatopic IgE targeting approach, we further improved the disruptive potency of E2_79 by approximately 100-fold and show that disruptive IgE inhibitors efficiently prevent passive cutaneous anaphylaxis in mice expressing the human Fc?RI ?-chain.Our findings highlight the potential of such novel IgE inhibitors as important diagnostic and therapeutic tools for management of allergic diseases.
Project description: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 Fc?RI-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.
Project description:The cell type(s) mediating the maternal influence on allergic disease in children remain unclear. We set out to define the relationship between maternal allergy and frequencies of cord blood (CB) basophils, and plasmacytoid dendritic cells (pDCs); to characterize surface-bound IgE and Fc?RI expressions on these cells; and to investigate the association between maternal and CB serum IgE levels with surface-bound IgE and Fc?RI expressions.One hundred and three mother/infant dyads were recruited prenatally, and maternal allergic history was recorded. Maternal blood was collected prior to delivery, and CB was collected after birth. Flow cytometry was used to identify CB basophils and pDCs and to determine surface-bound IgE and Fc?RI expressions.Frequencies of CB basophils and pDCs were low and not related to maternal history of allergy. Percentages of CB basophils with surface-bound IgE were significantly higher in infants of allergic mothers compared with infants of non-allergic mothers (median, 59.60% vs. 19.70%, p = 0.01). IgE on CB basophils correlated with CB IgE levels (r = 0.72, p < 0.0001), but not with maternal IgE levels (r = 0.26, p = 0.06). IgE on CB pDCs was low and not significantly associated with maternal or CB IgE levels. Similarly, Fc?RI expression by CB basophils and pDCs was not significantly associated with maternal or CB IgE levels.Frequencies of CB basophils and pDCs are not influenced by maternal allergy. CB basophils and pDCs have surface-bound IgE and express Fc?RI; however, only IgE on CB basophils appears influenced by maternal allergy.
Project description:IgE and IgE receptors (Fc?RI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (Fc?RIIIA and Fc?RIV) expressed by neutrophils, rather than on IgE and Fc?RI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express Fc?RIIIA or Fc?RIV, but Fc?RIIA. We therefore investigated the possible role of Fc?RIIA in allergy by generating novel Fc?RIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, Fc?RIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking Fc?RIIA in vivo abolished these reactions. We identified mast cells to be responsible for Fc?RIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for Fc?RIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after Fc?RIIA engagement. IgG and Fc?RIIA may therefore contribute to allergic and anaphylactic reactions in humans.
Project description:Clinical desensitization of patients to drugs involves progressive exposure to escalating doses of drug over a period of 24 h. In prior studies, this method was re-capitulated in vitro to also demonstrate loss of mast cell or basophil responsiveness. However, most signalling studies of human basophils have identified changes in signalling by using other methods of inducing cellular desensitization.This study examined two well-described endpoints of basophil desensitization, loss of syk or Fc?RI expression, under conditions of subthreshold desensitization.The loss of Fc?RI and syk was examined in human basophils.It was shown that both loss of syk and Fc?RI/IgE occurred during an escalating series of stimulation (anti-IgE Ab) and that expression loss occurred despite the presence of little histamine release. If basophils were first cultured for 3 days in 10 ng/mL IL-3, the concentration-dependence of histamine release shifted to 100-fold lower concentrations of stimulus. However, loss of syk did not show any change in its EC50 while loss of Fc?RI also shifted 100-fold. From the perspective of early signal element activation, the marked shift in the EC50 for histamine release was not accompanied by similar shifts in the EC50s for several signalling elements. The EC50s for phospho-Src, phospho-SHIP1, phospho-Syk, or phospho-Cbl did not change while the EC50s for phospho-Erk and the cytosolic calcium response did shift 100-fold.These studies show that under normal conditions, subthreshold desensitization leads to loss of two critical signalling molecules (Fc?RI and syk) but under at least one condition, treatment with IL-3, it is possible to markedly blunt the loss of syk, but not Fc?RI, while executing a proper subthreshold titration. These data also suggest that IL-3 modifies only the sensitivity of signalling elements that are downstream of syk activation.