RESUMO
IgE signaling through its high-affinity receptor FcεRI is central to the pathogenesis of numerous allergic disorders. Oral inhibitors of Bruton's tyrosine kinase (BTKis), which are currently Food and Drug Administration-approved for treating B cell malignancies, broadly inhibit the FcεRI pathway in human mast cells and basophils, and therefore may be effective allergen-independent therapies for a variety of allergic diseases. The application of these drugs to the allergy space was previously limited by the low kinase selectivity and subsequent toxicities of early-generation compounds. Fortunately, next-generation, highly selective BTKis in clinical development appear to have more favorable risk-benefit profiles, and their likelihood of being Food and Drug Administration-approved for an allergy indication is increasing. Recent clinical trials have indicated the remarkable and rapid efficacy of the second-generation BTKi acalabrutinib in preventing clinical reactivity to peanut ingestion in adults with peanut allergy. In addition, next-generation BTKis including remibrutinib effectively reduce disease activity in patients with antihistamine-refractory chronic spontaneous urticaria. Finally, several BTKis are currently under investigation in early clinical trials for atopic dermatitis and asthma. In this review, we summarize recent data supporting the use of these drugs as novel therapies in food allergy, anaphylaxis, urticaria, and other allergic disorders. We also discuss safety data derived from trials using both short-term and chronic dosing of BTKis.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Inibidores de Proteínas Quinases , Humanos , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/imunologia , Animais , Receptores de IgE/imunologia , Receptores de IgE/antagonistas & inibidores , Benzamidas/uso terapêutico , PirazinasRESUMO
Excessive reactions to allergens can induce systemic, life-threatening physiological dysfunction (anaphylaxis) in humans. The surface of mast cells expresses high-affinity IgE receptors that play a vital role during anaphylaxis. Alpha-linolenic acid (ALA) is an essential non-toxic fatty acid in humans. Since it has been reported having potential to regulate pro-inflammatory reactions, we postulated that ALA could inhibit anaphylaxis by down-regulating Lyn kinase phosphorylation. We found that local and systematic inflammation induced by albumin from chicken egg white (OVA) were attenuated by ALA in vivo. Furthermore, ALA inhibited IgE-mediated Ca2+ mobilization, degranulation, and cytokine release in Laboratory of Allergic Disease 2 (LAD2) cells. The western blot results showed that ALA down-regulate the FcεRI/Lyn/Syk signaling pathway by suppressing Lyn kinase activity. Therefore, ALA could serve as a therapeutic drug candidate for preventing IgE-mediated anaphylaxis.
Assuntos
Anafilaxia/induzido quimicamente , Alérgenos/metabolismo , Animais , Degranulação Celular , Humanos , Imunoglobulina E/metabolismo , Mastócitos/imunologia , Anafilaxia Cutânea Passiva , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de IgE/antagonistas & inibidores , Transdução de Sinais , Quinase Syk/metabolismo , Ácido alfa-Linolênico/efeitos adversos , Ácido alfa-Linolênico/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Autoantibodies (AAbs) against immunoglobulin E (IgE) antibodies (Abs) and their high-affinity receptor alpha subunits (FcεRIα) are key factors in the elicitation of type IIb autoimmune chronic spontaneous urticaria (type IIb aiCSU). In this study, we aimed to develop a new method to detect functional anti-FcεRIα and anti-IgE AAbs, which can crosslink the plural FcεRÐα molecules and IgE Abs on the surface of mast cells and basophils, in sera from aiCSU patients using the amplified luminescence proximity homogeneous assay (Alpha). METHODS: Sera were obtained from 14 aiCSU patients, as diagnosed by recurrent chronic spontaneous urticaria episodes and positive results for the autologous serum skin test and/or histamine release test (HRT). The AAbs to FcεRIα and IgE Abs were determined in sera from aiCSU patients using enzyme-linked immunosorbent assay (ELISA) and Alpha by cross-linking (AlphaCL) of IgE Abs and/or FcεRÐα. RESULTS: Serum anti-FcεRIα and anti-IgE AAb levels were not significantly different between aiCSU patients and healthy subjects in ELISA. Anti-FcεRIα AAbs were detected in 10 of 14 aiCSU patients who displayed positive (5/5) and negative (5/9) results in the HRT for anti-FcεRIα AAbs by AlphaCL, whereas no signals were observed in healthy subjects. Additionally, anti-IgE AAbs were detected in two of four aiCSU patients who displayed positive results in the HRT for anti-IgE AAbs. CONCLUSIONS: A new assay method using AlphaCL can detect anti-FcεRIα and anti-IgE AAbs with FcεRIα- and IgE-crosslinking abilities in sera from aiCSU patients. This simple and practical assay method may be available as a diagnostic tool for urticaria patients.
Assuntos
Autoanticorpos/imunologia , Urticária Crônica/sangue , Receptores de IgE/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Liberação de Histamina , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/sangue , Pele/química , Testes CutâneosRESUMO
Bruton's tyrosine kinase (BTK) is crucial for FcεRI-mediated mast cell activation and essential for autoantibody production by B cells in chronic spontaneous urticaria (CSU). Fenebrutinib, an orally administered, potent, highly selective, reversible BTK inhibitor, may be effective in CSU. This double-blind, placebo-controlled, phase 2 trial (EudraCT ID 2016-004624-35 ) randomized 93 adults with antihistamine-refractory CSU to 50 mg daily, 150 mg daily and 200 mg twice daily of fenebrutinib or placebo for 8 weeks. The primary end point was change from baseline in urticaria activity score over 7 d (UAS7) at week 8. Secondary end points were the change from baseline in UAS7 at week 4 and the proportion of patients well-controlled (UAS7 ≤ 6) at week 8. Fenebrutinib efficacy in patients with type IIb autoimmunity and effects on IgG-anti-FcεRI were exploratory end points. Safety was also evaluated. The primary end point was met, with dose-dependent improvements in UAS7 at week 8 occurring at 200 mg twice daily and 150 mg daily, but not at 50 mg daily of fenebrutinib versus placebo. Asymptomatic, reversible grade 2 and 3 liver transaminase elevations occurred in the fenebrutinib 150 mg daily and 200 mg twice daily groups (2 patients each). Fenebrutinib diminished disease activity in patients with antihistamine-refractory CSU, including more patients with refractory type IIb autoimmunity. These results support the potential use of BTK inhibition in antihistamine-refractory CSU.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Urticária Crônica/tratamento farmacológico , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Liberação de Histamina/efeitos dos fármacos , Piperazinas/uso terapêutico , Piridonas/uso terapêutico , Adolescente , Adulto , Angioedema/tratamento farmacológico , Autoimunidade/imunologia , Método Duplo-Cego , Resistência a Medicamentos/fisiologia , Feminino , Antagonistas dos Receptores Histamínicos H1/efeitos adversos , Humanos , Imunoglobulina E/imunologia , Masculino , Mastócitos/metabolismo , Pessoa de Meia-Idade , Piperazinas/efeitos adversos , Placebos/administração & dosagem , Piridonas/efeitos adversos , Receptores de IgE/antagonistas & inibidores , Transaminases/análise , Adulto JovemRESUMO
Antibody-mediated autoimmune diseases (AAID) involve several isotypes of autoreactive antibodies. In a growing number of AAID, autoreactive IgE are present with a significant prevalence and are often associated with the presence of IgG anti-IgE and/or anti-FcεRIα (high affinity IgE receptor α chain). FcεRI-bearing cells, such as basophils or mast cells, are key players in some of these AAID. Recent advances in the pathophysiology of these diseases led to the passed or current development of anti-IgE strategies that showed very potent effects in some of them. The present review centralizes the information on the relevance of autoreactive IgE and FcεRI-bearing cells in the pathophysiology of different AAID and the ones where the anti-IgE therapeutic strategy shows or may show some benefits for the patients.
Assuntos
Autoimunidade , Imunoglobulina E/imunologia , Especificidade de Órgãos/imunologia , Receptores de IgE/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Autoanticorpos/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Basófilos/imunologia , Basófilos/metabolismo , Biomarcadores , Degranulação Celular/genética , Degranulação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Gerenciamento Clínico , Suscetibilidade a Doenças/imunologia , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Terapia de Alvo Molecular , Especificidade de Órgãos/genética , Ligação Proteica , Receptores de IgE/antagonistas & inibidoresRESUMO
NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.
Assuntos
Gliotoxina/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptor Notch2/antagonistas & inibidores , Receptor Notch3/agonistas , Adulto , Idoso , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzazepinas/administração & dosagem , Benzazepinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Feminino , Gliotoxina/administração & dosagem , Humanos , Lectinas Tipo C/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismo , Receptores de IgE/antagonistas & inibidores , Elementos Reguladores de Transcrição , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Asthma is characterized by airway hyperresponsiveness and allergic inflammation, detrimentally affecting the patients' quality of life. The development of new drugs for the treatment of asthma is warranted to alleviate these issues. Recent studies have demonstrated that sirtuin2 (SIRT2) aggravates asthmatic inflammation by up-regulation of T-helper type 2 responses and macrophage polarization. However, effects of SIRT2 on mast cell activation remain obscure. In this study, we investigated the effects of AGK2, an inhibitor for SIRT2, on mast cell-mediated allergic airway inflammation. Pre-treatment with AGK2 inhibited degranulation of mast cells by suppressing the FcεRI signaling pathway and intracellular calcium influx. The expression of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4, IL-5, IL-6, and IL-8, was inhibited via regulation of transcription factors such as NF-κB and NRF2. These effects of AGK2 were verified in passive cutaneous anaphylaxis and acute lung injury animal models. AGK2 attenuated Evans blue pigmentation by inhibiting mast cell activation and lung barrier dysfunction by inhibiting inflammatory responses in these animal models. In the ovalbumin (OVA)-induced allergic airway inflammation murine model, AGK2 alleviated allergic asthma symptoms such as lung histological changes (immune cell and mast cell infiltration, collagen deposition, and α-smooth muscle actin expression) and serum immunoglobulins (Ig) levels (IgE, OVA-specific IgE, IgG1, and IgG2a). Moreover, AGK2 reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-4, IL-5, and IL-6) and inflammatory mediators (myeloperoxidase, eosinophil peroxidase, and tumor growth factor-α) in the bronchoalveolar lavage fluid and lung tissues. In addition, the anti-fibrotic effects of AGK2 were verified using lung epithelial cells and TGF-ß/Smad reporter stable cells. In conclusion, our findings suggest that SIRT2 plays a role in mast cell-mediated airway inflammatory disease. Therefore, AGK2 is a good potential candidate for treating allergic asthma and lung inflammation.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Furanos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Pulmão/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Quinolinas/farmacologia , Receptores de IgE/antagonistas & inibidores , Sirtuína 2/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Degranulação Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Liberação de Histamina/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Masculino , Mastócitos/enzimologia , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de IgE/metabolismo , Transdução de Sinais , Sirtuína 2/metabolismoRESUMO
Background: As the number of allergic disease increases, studies to identify new treatments take on new urgency. Epigallocatechin gallate (EGCG), a major component of green tea, has been shown to possess a wide range of pharmacological properties, including anti-inflammation and anti-viral infection. In previous study, gallic acid (GA), a part of EGCG, has shown anti-allergic inflammatory effect. To improve on preliminary evidence that GA has allergy mitigating effect, we designed SG-SP1 based on GA, and aimed to assess the effects of SG-SP1 on mast cell-mediated allergic inflammation using various animal and in vitro models. Methods: For in vitro experiments, various types of IgE-stimulated mast cells (RBL-2H3: mast cell-like basophilic leukemia cells, and primary cultured peritoneal and bone marrow-derived mast cells) were used to determine the role of SG-SP1 (0.1-1 nM). Immunoglobulin (Ig) E-induced passive cutaneous anaphylaxis and ovalbumin-induced systemic anaphylaxis, standard animal models for immediate-type hypersensitivity were also used. Results: For in vitro, SG-SP1 reduced degranulation of mast cells by down-regulating intracellular calcium levels in a concentration-dependent manner. SG-SP1 decreased expression and secretion of inflammatory cytokines in activated mast cells. This suppressive effect was associated with inhibition of the phosphorylation of Lyn, Syk and Akt, and the nuclear translocation of nuclear factor-κB. Due to the strong inhibitory effect of SG-SP1 on Lyn, the known upstream signaling to FcεRI-dependent pathway, we confirmed the direct binding of SG-SP1 to FcεRI, a high affinity IgE receptor by surface plasmon resonance experiment. Oral administration of SG-SP1 hindered allergic symptoms of both anaphylaxis models evidenced by reduction of hypothermia, serum IgE, ear thickness, and tissue pigmentation. This inhibition was mediated by the reductions in serum histamine and interleukin-4. Conclusions: We determined that SG-SP1 directly interacts with FcεRI and propose SG-SP1 as a therapeutic candidate for mast cell-mediated allergic inflammatory disorders via inhibition of FcεRI signaling.
Assuntos
Anafilaxia/tratamento farmacológico , Anafilaxia/metabolismo , Anti-Inflamatórios/administração & dosagem , Ácido Gálico/análogos & derivados , Ácido Gálico/administração & dosagem , Mastócitos/metabolismo , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Receptores de IgE/antagonistas & inibidores , Anafilaxia/induzido quimicamente , Animais , Anti-Inflamatórios/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Degranulação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ácido Gálico/metabolismo , Imunoglobulina E/efeitos adversos , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Ovalbumina/efeitos adversos , Ratos , Ratos Sprague-Dawley , Receptores de IgE/metabolismoRESUMO
BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed â¼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.
Assuntos
Anafilaxia/imunologia , Anticorpos Monoclonais/farmacologia , Dessensibilização Imunológica/métodos , Receptores de IgE/antagonistas & inibidores , Anafilaxia/prevenção & controle , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Mast cells express high-affinity IgE receptor (FcεRI) on their surface, cross-linking of which leads to the immediate release of proinflammatory mediators such as histamine but also late-phase cytokine secretion, which are central to the pathogenesis of allergic diseases. Despite the growing evidences that mammalian target of rapamycin (mTOR) plays important roles in the immune system, it is still unclear how mTOR signaling regulates mast cell function. In this study, we investigated the effects of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as an mTOR agonist on FcεRI-mediated allergic responses of mast cells. Our data showed that administration of 3BDO decreased ß-hexosaminidase, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI cross-linking, which was associated with an increase in mTOR complex 1 (mTORC1) signaling but a decrease in activation of Erk1/2, Jnk, and mTORC2-Akt. In addition, we found that a specific Akt agonist, SC79, is able to fully restore the decrease of ß-hexosaminidase release in 3BDO-treated BMMCs but has no effect on IL-6 release in these cells, suggesting that 3BDO negatively regulates FcεRI-mediated degranulation and cytokine release through differential mechanisms in mast cells. The present data demonstrate that proper activation of mTORC1 is crucial for mast cell effector function, suggesting the applicability of the mTORC1 activator as a useful therapeutic agent in mast cell-related diseases.
Assuntos
4-Butirolactona/análogos & derivados , Degranulação Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores de IgE/antagonistas & inibidores , 4-Butirolactona/farmacologia , Animais , Mastócitos/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.
Assuntos
Degranulação Celular , Imunoglobulina E/metabolismo , Leucemia Basofílica Aguda/imunologia , Antígenos Comuns de Leucócito/metabolismo , Mastócitos/imunologia , Receptores de IgE/metabolismo , Animais , Sistemas CRISPR-Cas , Reagentes de Ligações Cruzadas/química , Integrinas/metabolismo , Leucemia Basofílica Aguda/metabolismo , Leucemia Basofílica Aguda/patologia , Antígenos Comuns de Leucócito/genética , Mastócitos/metabolismo , Ratos , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/genética , Células Tumorais CultivadasRESUMO
We found out 2',3'-dihydroxypuberulin from South American medicinal plant, V. thapsus L., as a candidate of an anti-allergic lead which inhibits the expression of high-affinity receptor of IgE (FcεRI) on the surface of mast cells. Furthermore, the analysis of structure-activity relationship by using synthesized 2',3'-dihydroxypuberulin analogs revealed that both hydroxy groups in the side chain and both of methyl moieties on phenolic hydroxy groups were crucial for potent activity, but absolute configuration of C-3' position wasn't. The active principle, 2',3'-dihydroxypuberulin, was disclosed to down-regulate the mRNA level of ß-chain of FcεRI, different from previous reported active natural product reducing γ-chain level.
Assuntos
Antialérgicos/química , Cumarínicos/química , Mastócitos/efeitos dos fármacos , Receptores de IgE/antagonistas & inibidores , Verbascum/química , Antialérgicos/isolamento & purificação , Cumarínicos/isolamento & purificação , Regulação para Baixo , Humanos , Estrutura Molecular , Receptores de IgE/genética , Relação Estrutura-AtividadeRESUMO
Activation of high affinity receptor for IgE (FcεRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca2+-dependent process known as anaphylactic degranulation. Degranulation inhibition has been proposed as a strategy to control allergies and chronic inflammation conditions. Cannabinoids are important inhibitors of inflammatory reactions but their effects on IgE/Ag-mediated MCs responses are not well described. In this study, we analyzed the effect of the endocannabinoid anandamide (AEA), the selective CB2 receptor agonist HU308, and the GPR55 receptor agonist lysophosphatidylinositol (LPI) on FcεRI-induced activation in murine bone marrow-derived mast cells (BMMCs). Our results show that AEA, HU380 and LPI inhibited FcεRI-induced degranulation in a concentration-dependent manner. This effect was mediated by CB2 and GPR55 receptor activation through a mechanism insensitive to pertussis toxin. Degranulation inhibition was prevented by CB2 and GPR55 antagonism, but not by CB1 receptor blockage. AEA also inhibited calcium-dependent cytokine mRNA synthesis induced by FcεRI crosslinking, without affecting early phosphorylation events. In addition, AEA, HU308 and LPI inhibited intracellular Ca2+ rise in response to IgE/Ag. CB2 and GPR55 receptor antagonism could not prevent the inhibition produced by AEA and HU308, but partially blocked the one caused by LPI. These results indicate that AEA inhibits IgE/Ag-induced degranulation through a mechanism that includes the participation of CB2 and GPR55 receptors acting in close crosstalk, and show that CB2-GPR55 heteromers are important negative regulators of FcεRI-induced responses in MCs.
Assuntos
Ácidos Araquidônicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Citocinas/biossíntese , Endocanabinoides/farmacologia , Mastócitos/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB2 de Canabinoide/fisiologia , Receptores de Canabinoides/fisiologia , Receptores de IgE/antagonistas & inibidores , Animais , Camundongos , Camundongos Endogâmicos C57BL , Toxina Pertussis/farmacologia , Receptor CB2 de Canabinoide/química , Receptores de Canabinoides/química , Receptores de IgE/fisiologiaRESUMO
Mast cells (MCs) are important effectors in allergic reactions since they produce a number of pre-formed and de novo synthesized pro-inflammatory compounds in response to the high affinity IgE receptor (FcεRI) crosslinking. IgE/Antigen-dependent degranulation and cytokine synthesis in MCs have been recognized as relevant pharmacological targets for the control of deleterious inflammatory reactions. Despite the relevance of allergic diseases worldwide, efficient pharmacological control of mast cell degranulation has been elusive. In this work, the xanthone jacareubin was isolated from the heartwood of the tropical tree Callophyllum brasilense, and its tridimensional structure was determined for the first time by X-ray diffraction. Also, its effects on the main activation parameters of bone marrow-derived mast cells (BMMCs) were evaluated. Jacareubin inhibited IgE/Ag-induced degranulation in a dose-response manner with an IC50â¯=â¯46â¯nM. It also blocked extracellular calcium influx triggered by IgE/Ag complexes and by the SERCA ATPase inhibitor thapsigargin (Thap). Inhibition of calcium entry correlated with a blockage on the reactive oxygen species (ROS) accumulation. Antioxidant capacity of jacareubin was higher than the showed by α-tocopherol and caffeic acid, but similar to trolox. Jacareubin shown inhibitory actions on xanthine oxidase, but not on NADPH oxidase (NOX) activities. In vivo, jacareubin inhibited passive anaphylactic reactions and TPA-induced edema in mice. Our data demonstrate that jacareubin is a potent natural compound able to inhibit anaphylactic degranualtion in mast cells by blunting FcεRI-induced calcium flux needed for secretion of granule content, and suggest that xanthones could be efficient anti-oxidant, antiallergic, and antiinflammatory molecules.
Assuntos
Anafilaxia/metabolismo , Cálcio/metabolismo , Mastócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgE/antagonistas & inibidores , Xantonas/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Difração de Raios X , Xantonas/isolamento & purificaçãoRESUMO
Chronic spontaneous urticaria (CSU) is a condition presenting as the spontaneous occurrence of itchy weals with or without angio-oedema for > 6 weeks. A patient with severe chronic spontaneous urticaria who developed resistance to omalizumab is described. The patient's D-dimer plasma levels strictly paralleled the disease activity despite the administration of anti-IgE therapy.
Assuntos
Antialérgicos/uso terapêutico , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Omalizumab/uso terapêutico , Urticária/sangue , Doença Crônica , Resistência a Medicamentos , Feminino , Humanos , Deficiência de IgA , Pessoa de Meia-Idade , Receptores de IgE/antagonistas & inibidores , Urticária/tratamento farmacológico , Urticária/imunologiaRESUMO
Natural antibodies are defined as antibodies detected in a healthy individual without active immunization. These antibodies are specific for exoantigens, as well as for autoantigens, mostly without any pathogenic role. Most of the studies conducted with natural (auto-) antibodies have been performed using affinity purified antibodies from individual sera or polyclonal Ig-preparations such as Intravenous Ig (IVIg). For in-depth analysis of such autoantibodies affinity-purified Ig-preparations from healthy individuals are of no use, as they are oligoclonal or polyclonal. Thus, there is a need of human monoclonal autoantibodies. Human monoclonal autoantibodies can be produced from B cells isolated from humans; however, this requires the screening of a large number of antibodies to identify one among them specific to an antigen. Using the phage display technology we generated such autoantibodies against the alpha subunit of the high-affinity IgE receptor (FcεRIα). Here we describe the step-by-step protocol for the generation of such libraries and the isolation of autoantibodies by affinity panning.
Assuntos
Anticorpos/imunologia , Anticorpos/isolamento & purificação , Autoanticorpos/imunologia , Autoanticorpos/isolamento & purificação , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/imunologia , Linfócitos B/imunologia , Separação Celular/métodos , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , Biblioteca Gênica , Voluntários Saudáveis , Humanos , Imunoglobulina G/imunologia , Imunoglobulinas Intravenosas/imunologia , Imunoglobulinas Intravenosas/uso terapêutico , Leucócitos Mononucleares/imunologiaRESUMO
SUMMARY: Monoclonal anti-IgE antibodies (omalizumab) are able to induce clinically significant benefits in patients with severe chronic spontaneous urticaria (CS). Those results led clinicians and investigators to reconsider a possible pathogenic role not previously supported for IgE and its receptors in this disease, and to investigate additional approaches for understanding its pathogenesis. IgE antibodies to unknown environmental allergens able to trigger chronic urticaria are not generally regarded as the etiologic factor for the disease. Other proposed mechanisms for the production of wheals and angioedema in CSU include IgG autoantibodies and CD4-positive T cells directed to the high-affinity IgE receptor, autoantibodies to IgE itself, IgE autoantibodies directed to thyroid and nuclear autoantigens, highly cytokinergic IgE, and histamine-releasing factors able to bind to IgE and cause mast cell activation. It is expected that a better knowledge on the mechanisms leading to CSU and the clarification of the immunological effects of anti-IgE will provide novel therapies for this frequent condition.
Assuntos
Antialérgicos/uso terapêutico , Autoimunidade/efeitos dos fármacos , Imunoglobulina E/imunologia , Omalizumab/uso terapêutico , Urticária/tratamento farmacológico , Animais , Antialérgicos/efeitos adversos , Autoanticorpos/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , Citocinas/antagonistas & inibidores , Citocinas/imunologia , Liberação de Histamina/efeitos dos fármacos , Humanos , Imunidade Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Omalizumab/efeitos adversos , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/imunologia , Urticária/diagnóstico , Urticária/imunologiaRESUMO
Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE.
Assuntos
Antiasmáticos/farmacologia , Imunoglobulina E/metabolismo , Modelos Moleculares , Omalizumab/farmacologia , Receptores de IgE/antagonistas & inibidores , Sítio Alostérico , Substituição de Aminoácidos , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Omalizumab/química , Omalizumab/genética , Omalizumab/metabolismo , Maleabilidade , Mutação Puntual , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Receptores de IgE/química , Receptores de IgE/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 µmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.
Assuntos
Anafilaxia/tratamento farmacológico , Anafilaxia/imunologia , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Receptores de IgE/imunologia , Ácido Vanílico/análogos & derivados , Anafilaxia/induzido quimicamente , Animais , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Dinitrofenóis/antagonistas & inibidores , Relação Dose-Resposta a Droga , Hipersensibilidade/imunologia , Imunoglobulina E/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Masculino , Mastócitos/imunologia , Camundongos , NF-kappa B/metabolismo , Ovalbumina/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Ratos , Receptores de IgE/antagonistas & inibidores , Albumina Sérica/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , p-Metoxi-N-metilfenetilamina/antagonistas & inibidoresRESUMO
Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab-Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.