RESUMO
Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.
Assuntos
Interleucina-4 , Fosfatidilinositol 3-Quinases , Humanos , Camundongos , Animais , Interleucina-4/metabolismo , Imunoglobulina E , Antígenos CD40/genética , Antígenos CD40/metabolismo , Imunoglobulina G , Técnicas de Cultura de CélulasRESUMO
Rapid and robust recall or 'memory' responses are an essential feature of adaptive immunity. They constitute a defense against reinfection by pathogens, yet arguably do more harm than good in allergic disease. Immunoglobulin (Ig)E antibodies mediate the allergic reaction characterized by immediate hypersensitivity, a manifestation of IgE memory. The origin of IgE memory remains obscure, mainly due to the low proportion of IgE-expressing B cells in the total B cell population. The recent development of ultrasensitive methods for tracking these cells in vivo has overcome this obstacle, and their use has revealed unexpected pathways to IgE memory in the mouse. Here, we review these findings and consider their bearing on our understanding of IgE memory and allergic disease in man.
Assuntos
Linfócitos B/imunologia , Imunoglobulina E/imunologia , Memória Imunológica , Animais , Afinidade de Anticorpos/imunologia , Linfócitos B/metabolismo , Modelos Animais de Doenças , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Switching de Imunoglobulina , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Camundongos , Hipermutação Somática de ImunoglobulinaRESUMO
BACKGROUND: Naturally occurring IgE-specific IgG autoantibodies have been identified in patients with asthma and other diseases, but their spectrum of functions is poorly understood. OBJECTIVE: Address the hypothesis that: (i) IgG anti-IgE autoantibodies are detectable in the serum of all subjects but elevated in asthmatic patients regardless of atopic status as compared with controls; (ii) some activate IgE-sensitized basophils; and (iii) some inhibit allergen-induced basophil activation. METHODS: IgE-specific IgG autoantibodies were detected and quantified in sera using ELISA. Sera were examined for their ability to activate IgE-sensitized human blood basophils in the presence and absence of allergen using a basophil activation test, and to inhibit allergen binding to specific IgE on a rat basophilic cell line stably expressing human FcεRI. RESULTS: IgG autoantibodies binding to both free and FcεRI-bound IgE were detected in patients with atopic and non-atopic asthma, as well as controls. While some were able to activate IgE-sensitised basophils, others inhibited allergen-induced basophil activation, at least partly by inhibiting binding of IgE to specific allergen. CONCLUSION: Naturally occurring IgG anti-IgE autoantibodies may inhibit, as well as induce, basophil activation. They act in a manner distinct from therapeutic IgG anti-IgE antibodies such as omalizumab. They may at least partly explain why atopic subjects who make allergen-specific IgE never develop clinical symptoms, and why omalizumab therapy is of variable clinical benefit in severe atopic asthma.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Asma/imunologia , Autoanticorpos/imunologia , Basófilos/imunologia , Imunoglobulina G/imunologia , Alérgenos/imunologia , Animais , Anticorpos Anti-Idiotípicos/sangue , Antígenos de Plantas/imunologia , Asma/sangue , Autoanticorpos/sangue , Proteínas de Ligação ao Cálcio/imunologia , Linhagem Celular , Humanos , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Phleum/imunologia , Ratos , Receptores de IgE/imunologiaRESUMO
Allergic asthma and allergic rhinitis/conjunctivitis are characterized by a T(H)2-dominated immune response associated with increased serum IgE levels in response to inhaled allergens. Because IgE is a key player in the induction and maintenance of allergic inflammation, it represents a prime target for therapeutic intervention. However, our understanding of IgE biology remains fragmentary. This article puts together our current knowledge on IgE in allergic airway diseases with a special focus on the identity of IgE-secreting cells ("who"), their location ("where"), and the circumstances in which they are induced ("when"). We further consider the therapeutic implications of the insights gained.
Assuntos
Imunoglobulina E/imunologia , Plasmócitos/imunologia , Hipersensibilidade Respiratória/imunologia , Animais , Diferenciação Celular , Movimento Celular , Humanos , Terapia de Alvo Molecular , Hipersensibilidade Respiratória/terapiaRESUMO
The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Classe I de Fosfatidilinositol 3-Quinases , Complemento C5a/farmacologia , Fibroblastos/enzimologia , Teste de Complementação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Ligantes , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/enzimologia , Camundongos , Camundongos Mutantes , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transdução de SinaisRESUMO
Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Longevidade/genética , Animais , Biomarcadores/análise , Feminino , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Transdução de Sinais/genéticaRESUMO
IgE is secreted by plasma cells (PCs) and is central to allergic disease. Using an ex vivo tonsil B cell culture system, which mimics the Th2 responses in vivo, we have recently characterized the development pathway of human IgE-expressing PCs. In this system, as in mice, we reported the predisposition of IgE-expressing B cells to differentiate into PCs. To gain a comprehensive understanding of the molecular events involved in the differentiation of human IgE+ B cells into PCs we have used the Illumina HumanHT-12 v4 Expression BeadChip array to analyse the gene expression profile of ex vivo generated human IgE+ B cells at various stages of their differentiation into PCs. We also compared the transcription profiles of IgE+ and IgG1+ cells to discover isotype-specific patterns. Comparisons of IgE+ and IgG1+ cell transcriptional profiles revealed molecular signatures specific for IgE+ cells, which diverge from their IgG1+ cell counterparts upon differentiation into PCs. At the germinal center (GC) stage of development, unlike in some mouse studies of IgE biology, we observed similar rates of apoptosis and no significant differences in the expression of apoptosis-associated genes between the IgE+ and IgG1+ B cells. We identified a gene interaction network associated with early growth response 1 (EGR1) that, together with the up-regulated IRF4, may account for the predisposition of IgE+ B cells to differentiate into PCs. However, despite their swifter rates of PC differentiation, the transcription profile of IgE+ PCs is more closely related to IgE+ and IgG1+ plasmablasts (PBs) than to IgG1+ PCs, suggesting that the terminal differentiation of IgE+ cells is impeded. We also show that IgE+ PCs have increased levels of apoptosis suggesting that the IgE+ PCs generated in our in vitro tonsil B cell cultures, as in mice, are short-lived. We identified gene regulatory networks as well as cell cycle and apoptosis signatures that may explain the diverging PC differentiation programme of these cells. Overall, our study provides a detailed analysis of the transcriptional pathways underlying the differentiation of human IgE-expressing B cells and points to molecular signatures that regulate IgE+ PC differentiation and function.
Assuntos
Diferenciação Celular/genética , Imunoglobulina E/metabolismo , Plasmócitos/metabolismo , Transcriptoma/genética , Apoptose/genética , Células Cultivadas , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Centro Germinativo/imunologia , Humanos , Hipersensibilidade/metabolismo , Imunoglobulina G/metabolismo , Fatores Reguladores de Interferon/genética , Tonsila Palatina/patologia , FenótipoRESUMO
Class-switch recombination (CSR) is an essential B cell process that alters the isotype of antibody produced by the B cell, tailoring the immune response to the nature of the invading pathogen. CSR requires the activity of the mutagenic enzyme AID (encoded by AICDA) to generate chromosomal lesions within the immunoglobulin genes that initiate the class switching recombination event. These AID-mediated mutations also participate in somatic-hypermutation of the immunoglobulin variable region, driving affinity maturation. As such, AID poses a significant oncogenic threat if it functions outside of the immunoglobulin locus. We found that expression of the microRNA, miR-29b, was repressed in B cells isolated from tonsil tissue, relative to circulating naïve B cells. Further investigation revealed that miR-29b was able to directly initiate the degradation of AID mRNA. Enforced overexpression of miR-29b in human B cells precipitated a reduction in overall AID protein and a corresponding diminution in CSR to IgE. Given miR-29b's ability to potently target AID, a mutagenic molecule that can initiate chromosomal translocations and "off-target" mutations, we propose that miR-29b acts to silence premature AID expression in naïve B cells, thus reducing the likelihood of inappropriate and potentially dangerous deamination activity.
Assuntos
Linfócitos B/enzimologia , Citidina Desaminase/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Ativação Enzimática , Técnicas de Silenciamento de Genes , Genoma Humano , Células HEK293 , Humanos , Switching de Imunoglobulina , Imunoglobulina E/metabolismo , MicroRNAs/genética , Tonsila Palatina/citologia , Recombinação Genética/genéticaRESUMO
Immunoglobulin E (IgE) antibodies play a central role in the allergic response: interaction with FcεRI on mast cells and basophils leads to immediate hypersensitivity reactions upon allergen challenge, while interaction with CD23/FcεRII, expressed on a variety of cells, regulates IgE synthesis among other activities. The receptor-binding IgE-Fc region has recently been found to display remarkable flexibility, from acutely bent to extended conformations, with allosteric communication between the distant FcεRI and CD23 binding sites. We report the structure of an anti-IgE antibody Fab (8D6) bound to IgE-Fc through a mixed protein-carbohydrate epitope, revealing further flexibility and a novel extended conformation with potential relevance to that of membrane-bound IgE in the B cell receptor for antigen. Unlike the earlier, clinically approved anti-IgE antibody omalizumab, 8D6 inhibits binding to FcεRI but not CD23; the structure reveals how this discrimination is achieved through both orthosteric and allosteric mechanisms, supporting therapeutic strategies that retain the benefits of CD23 binding.
Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Anti-Idiotípicos/metabolismo , Imunoglobulina E/química , Imunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Linfócitos B/imunologia , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Mastócitos/imunologia , Ligação Proteica , Conformação ProteicaAssuntos
Alérgenos , Arachis/imunologia , Linfócitos B , Humanos , Imunoglobulina E , Proteínas de Plantas , TranscriptomaRESUMO
Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the ß isoform of phosphoinositide 3-kinase (PI3Kß) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kß alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kß and PI3Kδ, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3Kß by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase-coupled receptor (FcγR) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the ß isoform of PI3K. PI3Kß-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kß and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kß as a potential therapeutic target in inflammatory disease.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Antígenos CD2/genética , Antígenos CD2/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Rearranjo Gênico do Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região de Junção de Imunoglobulinas/genética , Região de Junção de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neutrófilos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Espécies Reativas de Oxigênio/metabolismo , Receptores de IgG/metabolismo , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
B cell development is controlled by a series of checkpoints that ensure that the immunoglobulin (Ig)-encoding genes produce a functional B cell receptor (BCR) and antibodies. As part of this process, recombination-activating gene (Rag) proteins regulate the in-frame assembly of the Ig-encoding genes. The BCR consists of Ig proteins in complex with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Igalpha and Igbeta chains. Whereas the activation of the tyrosine kinases Src and Syk is essential for BCR signaling, the pathways that act downstream of these kinases are incompletely defined. Previous work has revealed a key role for the p110delta isoform of phosphatidylinositol 3-kinase (PI3K) in agonist-induced BCR signaling; however, early B cell development and mature B cell survival, which depend on agonist-independent or "tonic" BCR signaling, are not substantially affected by a deficiency in p110delta. Here, we show that p110alpha, but not p110beta, compensated in the absence of p110delta to promote early B cell development in the bone marrow and B cell survival in the spleen. In the absence of both p110alpha and p110delta activities, pre-BCR signaling failed to suppress the production of Rag proteins and to promote developmental progression of B cell progenitors. Unlike p110delta, however, p110alpha did not contribute to agonist-induced BCR signaling. These studies indicate that either p110alpha or p110delta can mediate tonic signaling from the BCR, but only p110delta can contribute to antigen-dependent activation of B cells.
Assuntos
Linfócitos B/citologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Análise de Variância , Animais , Linfócitos B/imunologia , Western Blotting , Classe I de Fosfatidilinositol 3-Quinases , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Organismos Livres de Patógenos EspecíficosRESUMO
Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.