RESUMEN
BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced by mast cells (MCs) on cross-linking of their high-affinity receptors for IgE by antigen that can amplify MC responses by binding to its S1P receptors. An acute MC-dependent allergic reaction can lead to systemic shock, but the early events of its development in lung tissues have not been investigated, and S1P functions in the onset of allergic processes remain to be examined. OBJECTIVE: We used a highly specific neutralizing anti-S1P antibody (mAb) and the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist JTE-013 to study the signaling contributions of S1P and S1PR2 to MC- and IgE-dependent airway allergic responses in mice within minutes after antigen challenge. METHODS: Allergic reaction was triggered by a single intraperitoneal dose of antigen in sensitized mice pretreated intraperitoneally with anti-S1P, isotype control mAb, JTE-013, or vehicle before antigen challenge. RESULTS: Kinetics experiments revealed early pulmonary infiltration of mostly T cells around blood vessels of sensitized mice 20 minutes after antigen exposure. Pretreatment with anti-S1P mAb inhibited in vitro MC activation, as well as in vivo development of airway infiltration and MC activation, reducing serum levels of histamine, cytokines, and the chemokines monocyte chemoattractant protein 1/CCL2, macrophage inflammatory protein 1α/CCL3, and RANTES/CCL5. S1PR2 antagonism or deficiency or MC deficiency recapitulated these results. Both in vitro and in vivo experiments demonstrated MC S1PR2 dependency for chemokine release and the necessity for signal transducer and activator of transcription 3 activation. CONCLUSION: Activation of S1PR2 by S1P and downstream signal transducer and activator of transcription 3 signaling in MCs regulate early T-cell recruitment to antigen-challenged lungs through chemokine production.
Asunto(s)
Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Lisofosfolípidos/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Linfocitos T/inmunología , Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Antígenos/inmunología , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Lisofosfolípidos/antagonistas & inhibidores , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Transgénicos , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Esfingosina/antagonistas & inhibidores , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
We previously demonstrated that TGF-ß1 suppresses IgE-mediated signaling in human and mouse mast cells in vitro, an effect that correlated with decreased expression of the high-affinity IgE receptor, FcεRI. The in vivo effects of TGF-ß1 and the means by which it suppresses mast cells have been less clear. This study shows that TGF-ß1 suppresses FcεRI and c-Kit expression in vivo. By examining changes in cytokine production concurrent with FcεRI expression, we found that TGF-ß1 suppresses TNF production independent of FcεRI levels. Rather, IgE-mediated signaling was altered. TGF-ß1 significantly reduced expression of Fyn and Stat5, proteins critical for cytokine induction. These changes may partly explain the effects of TGF-ß1, because Stat5B overexpression blocked TGF-mediated suppression of IgE-induced cytokine production. We also found that Stat5B is required for mast cell migration toward stem cell factor, and that TGF-ß1 reduced this migration. We found evidence that genetic background may alter TGF responses. TGF-ß1 greatly reduced mast cell numbers in Th1-prone C57BL/6, but not Th2-prone 129/Sv mice. Furthermore, TGF-ß1 did not suppress IgE-induced cytokine release and did increase c-Kit-mediated migration in 129/Sv mast cells. These data correlated with high basal Fyn and Stat5 expression in 129/Sv cells, which was not reduced by TGF-ß1 treatment. Finally, primary human mast cell populations also showed variable sensitivity to TGF-ß1-mediated changes in Stat5 and IgE-mediated IL-6 secretion. We propose that TGF-ß1 regulates mast cell homeostasis, and that this feedback suppression may be dependent on genetic context, predisposing some individuals to atopic disease.
Asunto(s)
Inmunoglobulina E/inmunología , Mastocitos/metabolismo , Receptores de IgE/inmunología , Factor de Transcripción STAT5/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Movimiento Celular/inmunología , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunoglobulina E/metabolismo , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Receptores de IgE/biosíntesis , Receptores de IgE/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Factores de Necrosis Tumoral/biosíntesisRESUMEN
Anaphylaxis is a rapid, life-threatening hypersensitivity reaction. Until recently, it was mainly attributed to histamine released by mast cells activated by allergen crosslinking (XL) of FcεRI-bound allergen-specific IgE. However, recent reports established that anaphylaxis could also be triggered by basophil, macrophage, and neutrophil secretion of platelet-activating factor subsequent to FcγR stimulation by IgG/Ag complexes. We have investigated the contribution of Fyn and Lyn tyrosine kinases to FcγRIIb and FcγRIII signaling in the context of IgG-mediated passive systemic anaphylaxis (PSA). We found that mast cell IgG XL induced Fyn, Lyn, Akt, Erk, p38, and JNK phosphorylation. Additionally, IgG XL of mast cells, basophils, and macrophages resulted in Fyn- and Lyn-regulated mediator release in vitro. FcγR-mediated activation was enhanced in Lyn-deficient (knockout [KO]) cells, but decreased in Fyn KO cells, compared with wild-type cells. More importantly, Lyn KO mice displayed significantly exacerbated PSA features whereas no change was observed for Fyn KO mice, compared with wild-type littermates. Intriguingly, we establish that mast cells account for most serum histamine in IgG-induced PSA. Taken together, our findings establish pivotal roles for Fyn and Lyn in the regulation of PSA and highlight their unsuspected functions in IgG-mediated pathologies.
Asunto(s)
Anafilaxia/inmunología , Inmunoglobulina G/inmunología , Mastocitos/inmunología , Proteínas Proto-Oncogénicas c-fyn/inmunología , Familia-src Quinasas/inmunología , Alérgenos/genética , Alérgenos/inmunología , Anafilaxia/genética , Anafilaxia/patología , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Inmunoglobulina G/genética , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/inmunología , Mastocitos/patología , Ratones , Ratones Noqueados , Fosforilación/genética , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-fyn/genética , Receptores de IgG/genética , Receptores de IgG/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Familia-src Quinasas/genéticaRESUMEN
BACKGROUND: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined. OBJECTIVE: We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. METHODS: Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. RESULTS: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. CONCLUSION: S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
Asunto(s)
Amino Alcoholes/farmacología , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Mastocitos/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Asma/inducido químicamente , Asma/enzimología , Asma/metabolismo , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Quimiocina CCL2/metabolismo , Femenino , Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Humanos , Hiperplasia/tratamiento farmacológico , Hiperplasia/metabolismo , Inmunoglobulina E/metabolismo , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucinas/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Lisofosfolípidos/metabolismo , Mastocitos/metabolismo , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Ovalbúmina/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcεRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcεRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Bruton's tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcεRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.
Asunto(s)
Citocinas/biosíntesis , Mastocitos/metabolismo , Receptores de IgE/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Animales , Células Cultivadas , Colesterol/genética , Colesterol/metabolismo , Citocinas/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Mastocitos/citología , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores de IgE/genética , Factor de Transcripción STAT5/genética , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-beta1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-beta1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-beta1 had balancing effects on mast cell survival, migration, and FcepsilonRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-beta1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-beta1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.
Asunto(s)
Homeostasis/inmunología , Interleucina-4/fisiología , Mastocitos/inmunología , Mastocitos/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Receptores de Interleucina-4/antagonistas & inhibidores , Receptores de Interleucina-4/biosíntesis , Receptores de Interleucina-4/fisiología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/biosíntesisRESUMEN
Cellular and humoral constituents of the immune system differ significantly between children and adults, yet very little is known about the impact of early-life pathogen exposure on this immunologic transition. We examined CD4+ and CD8+ T cell subsets defined by CCR7 and CD45RA expression in two longitudinal pediatric cohorts experiencing divergent levels of pathogen burden. Using multiparameter flow cytometry, along with serological, cytokine, and transcriptomic data, we show that cumulative pathogen burden promotes the development of atypical CD8dim T cells with an innate-like profile (Granzyme Bhi, IFNγlow, TNFαlow, PLFZhi, ID2hi, IKZF2hi) in contrast to age-matched children residing in a low pathogen-exposure area who display a more conventional CD8bright profile (IFNγ+, TNFα+, CCL4+). Furthermore, these unconventional T cells had stunted proliferation, distinct transcriptional programs, and impaired T cell receptor signaling and were enriched in hallmark TNFα, NF-κB, and IL-6 gene signaling pathways, reminiscent of NK cells and type-1 innate lymphoid cells. Our findings suggest that these unconventional CD8dim T cells arise in a very particular immunological context and may provide a deeper understanding of the heterogeneity in human immune responses.
RESUMEN
The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.
Asunto(s)
Inmunidad Humoral , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Animales , Anticuerpos/metabolismo , Recuento de Células , Técnicas de Inactivación de Genes , Centro Germinal/citología , Centro Germinal/inmunología , Interleucina-4/metabolismo , Ratones , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Proteínas Proto-Oncogénicas c-fyn/deficiencia , Proteínas Proto-Oncogénicas c-fyn/genética , Transducción de Señal/inmunología , Bazo/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Mast cells are central players in immune surveillance and activation, positioned at the host-environment interface. Understanding the signaling events controlling mast cell function, especially those that maintain host homeostasis, is an important and still less understood area of mast cell-mediated disease. With respect to allergic disease, it is well established that IgE and its high affinity receptor FcεRI are major mediators of mast cell activation. However, IgG-mediated signals can also modulate mast cell activities. Signals elicited by IgG binding to its cognate receptors (FcγR) are the basis for autoimmune disorders such as lupus and rheumatoid arthritis. Using knowledge of IgE-mediated mast cell signaling, recent work has begun to illuminate potential overlap between FcεRI and FcγR signal transduction. Herein we review the importance of Src family kinases in FcεRI and FcγR signaling, the role of the transcription factor STAT5, and impingement of the regulatory cytokines IL-4, IL-10, and TGFß1 upon this network.
RESUMEN
Systemic exacerbation of allergic responses, in which mast cells play a critical role, results in life-threatening anaphylactic shock. Sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors, is a new addition to the repertoire of bioactive lipids secreted by activated mast cells. Yet little is known of its role in human mast cell functions and in anaphylaxis. We show that S1P(2) receptors play a critical role in regulating human mast cell functions, including degranulation and cytokine and chemokine release. Immunoglobulin E-triggered anaphylactic responses, including elevation of circulating histamine and associated pulmonary edema in mice, were significantly attenuated by the S1P(2) antagonist JTE-013 and in S1P(2)-deficient mice, in contrast to anaphylaxis induced by administration of histamine or platelet-activating factor. Hence, S1P and S1P(2) on mast cells are determinants of systemic anaphylaxis and associated pulmonary edema and might be beneficial targets for anaphylaxis attenuation and prophylaxis.
Asunto(s)
Anafilaxia/fisiopatología , Mastocitos/fisiología , Edema Pulmonar/fisiopatología , Receptores de Lisoesfingolípidos/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos/fisiología , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Hipersensibilidad/fisiopatología , Inmunoglobulina E/inmunología , Inmunoglobulina E/farmacología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Roedores , Piel/metabolismo , Fenómenos Fisiológicos de la Piel , Receptores de Esfingosina-1-FosfatoRESUMEN
Mast cells are well known as principle effector cells of type I hypersensitivity responses. Beyond this role in allergic disease, these cells are now appreciated as playing an important role in many inflammatory conditions. This review summarizes the support for mast cell involvement in resisting bacterial infection, exacerbating autoimmunity and atherosclerosis, and promoting cancer progression. A commonality in these conditions is the ability of mast cells to elicit migration of many cell types, often through the production of inflammatory cytokines such as tumor necrosis factor. However, recent data also demonstrates that mast cells can suppress the immune response through interleukin-10 production. The data encourage those working in this field to expand their view of how mast cells contribute to immune homeostasis.