RESUMO
Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).
Assuntos
Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Urticária/genética , Vibração/efeitos adversos , Biópsia , Degranulação Celular/genética , Feminino , Histamina/sangue , Humanos , Líbano , Masculino , Mastócitos/fisiologia , Pessoa de Meia-Idade , Linhagem , Receptores Acoplados a Proteínas G/metabolismo , Pele/patologia , Urticária/sangue , Urticária/etiologiaRESUMO
Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the ß-subunit of the high-affinity IgE receptor (FcεRIß) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIß expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIß is a potential approach for mast cell-specific treatment of allergic diseases.
Assuntos
Degranulação Celular/efeitos dos fármacos , Dermatite Alérgica de Contato/terapia , Mastócitos/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Splicing de RNA/efeitos dos fármacos , Receptores de IgE/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Anafilaxia Cutânea Passiva/genética , Receptores de IgE/genéticaRESUMO
BACKGROUND: Clonal mast cell disorders are known to occur in a subset of patients with systemic reactions to Hymenoptera stings. This observation has prompted the question of whether clonal mast cell disorders also occur in patients with idiopathic anaphylaxis (IA). OBJECTIVE: We sought to determine the prevalence of clonal mast cell disorders among patients with IA, criteria to identify those patients who require a bone marrow biopsy, and whether the pathogenesis of IA involves a hyperresponsive mast cell compartment. METHODS: We prospectively enrolled patients with IA (≥3 episodes/y) who then underwent a medical evaluation that included a serum tryptase determination, allele-specific quantitative PCR (ASqPCR) for the KIT D816V mutation, and a bone marrow examination. Mast cells were cultured from peripheral blood CD34+ cells and examined for releasability after FcεRI aggregation. RESULTS: Clonal mast cell disease was diagnosed in 14% of patients referred with IA. ASqPCR for the KIT D816V mutation was a useful adjunct in helping identify those with systemic mastocytosis but not monoclonal mast cell activation syndrome. A modified overall clonal prediction model was developed by using clinical findings, a serum tryptase determination, and ASqPCR. There was no evidence of a hyperresponsive mast cell phenotype in patients with IA. CONCLUSION: Patients with clonal mast cell disease can present as having IA. Distinct clinical and laboratory features can be used to select those patients more likely to have an underlying clonal mast cell disorder (monoclonal mast cell activation syndrome or systemic mastocytosis) and thus candidates for a bone marrow biopsy.
Assuntos
Anafilaxia/genética , Anafilaxia/imunologia , Mastócitos/imunologia , Mastocitose Sistêmica/genética , Mastocitose Sistêmica/imunologia , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas c-kit , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Anafilaxia/patologia , Feminino , Humanos , Masculino , Mastócitos/patologia , Mastocitose Sistêmica/patologia , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologiaRESUMO
BACKGROUND: IL-6, levels of which are reported to be increased in association with mastocytosis, asthma, and urticaria, is used in conjunction with stem cell factor to generate CD34(+) cell-derived primary human mast cell (HuMC) cultures. Despite these associations, the effects on and mechanisms by which prolonged exposure to IL-6 alters HuMC numbers and function are not well understood. OBJECTIVES: We sought to study the effect of IL-6 on HuMC function, the mechanisms by which IL-6 exerts its effects, and the relationship of these findings to mastocytosis. METHODS: HuMCs were cultured in stem cell factor with or without IL-6. Responses to FcεRI aggregation and expression of proteases and receptors, including the soluble IL-6 receptor (sIL-6R), were then quantitated. Epigenetic changes in suppressor of cytokine signaling 3 (SOCS3) were determined by using methylation-specific PCR. Serum samples from healthy control subjects and patients with mastocytosis were assayed for IL-6, tryptase, and sIL-6R. RESULTS: IL-6 enhanced mast cell (MC) proliferation, maturation, and reactivity after FcεRI aggregation. IL-6 reduced expression of SOCS3, which correlated with methylation of the SOCS3 promoter and increased expression and activation of signal transducer and activator of transcription 3. IL-6 also suppressed constitutive production of sIL-6R, and serum levels of sIL-6R were similarly reduced in patients with mastocytosis. CONCLUSION: IL-6 increases MC proliferation and formation of a more reactive phenotype enabled by suppressing proteolytic cleavage of sIL-6R from IL-6R and downregulation of the SOCS3 autoinhibitory pathway. We suggest IL-6 blockade might ameliorate MC-related symptoms and pathology in patients with MC-related diseases associated with increased IL-6 levels, including mastocytosis.
Assuntos
Interleucina-6/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Quimases/metabolismo , Metilação de DNA , Humanos , Interleucina-6/farmacologia , Mastócitos/efeitos dos fármacos , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. OBJECTIVES: We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. METHODS: Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. RESULTS: Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. CONCLUSION: Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.
Assuntos
Anafilaxia/genética , Anafilaxia/fisiopatologia , Estradiol/metabolismo , Pulmão/enzimologia , Óxido Nítrico Sintase Tipo III/imunologia , Óxido Nítrico/biossíntese , Anafilaxia/enzimologia , Anafilaxia/imunologia , Animais , Temperatura Corporal , Permeabilidade Capilar , Modelos Animais de Doenças , Estradiol/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Feminino , Expressão Gênica , Histamina/imunologia , Histamina/farmacologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/fisiopatologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/genética , Ovariectomia , Agregados Proteicos , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/química , Receptores de Estrogênio/imunologia , Receptores de IgE/antagonistas & inibidores , Receptores de IgE/química , Receptores de IgE/imunologia , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/química , Receptores de IgG/imunologia , ômega-N-Metilarginina/farmacologiaRESUMO
IL-33 is elevated in afflicted tissues of patients with mast cell (MC)-dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ(1) and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.
Assuntos
Terapia de Imunossupressão , Interleucinas/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Fenótipo , Actinas/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/farmacologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosfolipase C gama/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologiaAssuntos
Deficiência de GATA2 , Hipersensibilidade Imediata , Imunoglobulina E/imunologia , Feminino , Deficiência de GATA2/genética , Deficiência de GATA2/imunologia , Deficiência de GATA2/patologia , Humanos , Hipersensibilidade Imediata/genética , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/patologia , MasculinoRESUMO
Upon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene-I (RIG-I) pathway activates the latent transcription factor IRF-3, causing its nuclear translocation and the induction of many antiviral genes, including those encoding interferons. Here, we report a novel and distinct activity of IRF-3, in virus-infected cells, that induces apoptosis. Using genetically defective mouse and human cell lines, we demonstrated that, although both pathways required the presence of RIG-I, IPS1, TRAF3 and TBK1, only the apoptotic pathway required the presence of TRAF2 and TRAF6 in addition. More importantly, transcriptionally inactive IRF-3 mutants, such as the one missing its DNA-binding domain, could efficiently mediate apoptosis. Apoptosis was triggered by the direct interaction of IRF-3, through a newly identified BH3 domain, with the pro-apoptotic protein Bax, their co-translocation to the mitochondria and the resulting activation of the mitochondrial apoptotic pathway. Thus, IRF-3 is a dual-action cytoplasmic protein that, upon activation, translocates to the nucleus or to the mitochondrion and triggers two complementary antiviral responses of the infected cell.
Assuntos
Apoptose , Regulação Viral da Expressão Gênica , Fator Regulador 3 de Interferon/metabolismo , Proteína X Associada a bcl-2/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Citoplasma/metabolismo , Humanos , Camundongos , Transporte Proteico , RNA de Cadeia Dupla/metabolismo , Tretinoína/metabolismoRESUMO
Mast cells, activated by Ag via FcεRI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of FcεRI-mediated degranulation and cytokine production. The hyporesponsive phenotype was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Fator de Células-Tronco/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Degranulação Celular/imunologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Homeostase/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Imunofenotipagem , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3RESUMO
BACKGROUND: Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. OBJECTIVE: Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. METHODS: We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. RESULTS: Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. CONCLUSION: This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.
Assuntos
Degranulação Celular/genética , Hipersensibilidade Alimentar/epidemiologia , Síndrome de Job/epidemiologia , Mastócitos/imunologia , Fator de Transcrição STAT3/fisiologia , Adolescente , Adulto , Idoso , Animais , Células Cultivadas , Criança , Pré-Escolar , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Feminino , Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoglobulina E/metabolismo , Incidência , Lactente , Síndrome de Job/genética , Síndrome de Job/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transgenes/genética , Adulto JovemAssuntos
Adenosina Desaminase/genética , Imunoglobulina E/metabolismo , Imunodeficiência Combinada Severa/imunologia , Células Th2/imunologia , Adolescente , Adulto , Transplante de Medula Óssea , Células Cultivadas , Criança , Pré-Escolar , Estudos de Coortes , Estudos Transversais , Citocinas/metabolismo , Terapia de Reposição de Enzimas , Feminino , Terapia Genética , Humanos , Imunoglobulina E/genética , Memória Imunológica , Lactente , Masculino , Mutação/genética , Fenótipo , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Adulto JovemRESUMO
Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.
Assuntos
Neoplasias Hematológicas/metabolismo , Mastócitos/fisiologia , Mastocitose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Apoptose , Carcinogênese , Proliferação de Células , Sobrevivência Celular , Reparo do DNA , Neoplasias Hematológicas/genética , Humanos , Hidrazinas/farmacologia , Mastocitose/genética , Camundongos , Camundongos Knockout , Mutação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The culture of mast cells from human tissues such a cord blood, peripheral blood or bone marrow aspirates has advanced our understanding of human mast cells (huMC) degranulation, mediator production and response to pharmacologic agents. However, existing methods for huMC culture tend to be laborious and expensive. Combining technical approaches from several of these protocols, we designed a simplified and more cost effective approach to the culture of mast cells from human cell populations including peripheral blood and cryopreserved cells from lymphocytapheresis. On average, we reduced by 30-50 fold the amount of culture media compared to our previously reported method, while the total MC number generated by this method (2.46±0.63×106 vs. 2.4±0.28×106, respectively, from 1.0×108 lymphocytapheresis or peripheral blood mononuclear blood cells [PBMCs]) was similar to our previous method (2.36±0.70×106), resulting in significant budgetary savings. In addition, we compared the yield of huMCs with or without IL-3 added to early cultures in the presence of stem cell factor (SCF) and interlukin-6 (IL-6) and found that the total MC number generated, while higher with IL-3 in the culture, did not reach statistical significance, suggesting that IL-3, often recommended in the culture of huMCs, is not absolutely required. We then performed a functional analysis by flow cytometry using standard methods and which maximized the data we could obtain from cultured cells. We believe these approaches will allow more laboratories to culture and examine huMC behavior going forward.
Assuntos
Antígenos CD34/metabolismo , Separação Celular/métodos , Leucaférese , Mastócitos/metabolismo , Células-Tronco/metabolismo , Antígenos CD34/imunologia , Biomarcadores/metabolismo , Orçamentos , Degranulação Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Separação Celular/economia , Forma Celular , Células Cultivadas , Redução de Custos , Análise Custo-Benefício , Criopreservação , Meios de Cultura/metabolismo , Citometria de Fluxo , Humanos , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Leucaférese/economia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores de IgE/metabolismo , Fator de Células-Tronco/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Fatores de Tempo , Fluxo de TrabalhoRESUMO
Neoplastic accumulation of mast cells in systemic mastocytosis (SM) associates with activating mutations in the receptor tyrosine kinase KIT. Constitutive activation of tyrosine kinase oncogenes has been linked to imbalances in oxidant/antioxidant mechanisms in other myeloproliferative disorders. However, the impact of KIT mutations on the redox status in SM and the potential therapeutic implications are not well understood. Here, we examined the regulation of reactive oxygen species (ROS) and of the antioxidant protein DJ-1 (PARK-7), which increases with cancer progression and acts to lessen oxidative damage to malignant cells, in relationship with SM severity. ROS levels were increased in both indolent (ISM) and aggressive variants of the disease (ASM). However, while DJ-1 levels were reduced in ISM with lower mast cell burden, they rose in ISM with higher mast cell burden and were significantly elevated in patients with ASM. Studies on mast cell lines revealed that activating KIT mutations induced constant ROS production and consequent DJ-1 oxidation and degradation that could explain the reduced levels of DJ-1 in the ISM population, while IL-6, a cytokine that increases with disease severity, caused a counteracting transcriptional induction of DJ-1 which would protect malignant mast cells from oxidative damage. A mouse model of mastocytosis recapitulated the biphasic changes in DJ-1 and the escalating IL-6, ROS and DJ-1 levels as mast cells accumulate, findings which were reversed with anti-IL-6 receptor blocking antibody. Our findings provide evidence of increased ROS and a biphasic regulation of the antioxidant DJ-1 in variants of SM and implicate IL-6 in DJ-1 induction and expansion of mast cells with KIT mutations. We propose consideration of IL-6 blockade as a potential adjunctive therapy in the treatment of patients with advanced mastocytosis, as it would reduce DJ-1 levels making mutation-positive mast cells vulnerable to oxidative damage.
Assuntos
Antioxidantes/metabolismo , Mastocitose/metabolismo , Proteína Desglicase DJ-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transferência Adotiva , Adulto , Animais , Linhagem Celular , Espaço Extracelular/metabolismo , Homeostase , Humanos , Mastócitos/metabolismo , Mastocitoma/patologia , Mastocitose/sangue , Camundongos , Pessoa de Meia-Idade , Mutação/genética , Proteína Desglicase DJ-1/sangue , Proteína Desglicase DJ-1/genética , Proteólise , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Espécies Reativas de Oxigênio/sangue , Receptores de Interleucina-6/metabolismo , Transcrição GênicaRESUMO
Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in HPS1 which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the HPS1 mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcÉRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcÉRI and intracytoplasmic proteases and exhibited less mediator release following FcÉRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients' serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with HPS1 rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by HPS1 transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.
Assuntos
Síndrome de Hermanski-Pudlak/diagnóstico , Síndrome de Hermanski-Pudlak/metabolismo , Mastócitos/metabolismo , Mastócitos/ultraestrutura , Adulto , Biomarcadores , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Quimiotaxia , Análise por Conglomerados , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Síndrome de Hermanski-Pudlak/genética , Humanos , Mesilato de Imatinib/farmacologia , Imunofenotipagem , Pulmão/metabolismo , Pulmão/patologia , Masculino , Mastócitos/efeitos dos fármacos , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Fenótipo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Adulto JovemRESUMO
Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1â»/â» and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1â»/â» mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1â»/â» mice. Furthermore, contrary to expectations, Trpc1â»/â» BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1â»/â» BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1â»/â» mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.
Assuntos
Anafilaxia/imunologia , Mastócitos/imunologia , Canais de Cátion TRPC/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Alérgenos/imunologia , Animais , Sinalização do Cálcio/genética , Degranulação Celular/genética , Células Cultivadas , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
BACKGROUND: While a number of the consequences of mast cell degranulation within tissues have been documented including tissue-specific changes such as bronchospasm and the subsequent cellular infiltrate, there is little known about the immediate effects of mast cell degranulation on the associated vasculature, critical to understanding the evolution of mast cell dependent inflammation. OBJECTIVE: To characterize the microcirculatory events that follow mast cell degranulation. METHODOLOGY/PRINCIPAL FINDINGS: Perturbations in dermal blood flow, temperature and skin color were analyzed using laser-speckle contrast imaging, infrared and polarized-light colorimetry following cold-hand immersion (CHI) challenge in patients with cold-induced urticaria compared to the response in healthy controls. Evidence for mast cell degranulation was established by documentation of serum histamine levels and the localized release of tryptase in post-challenge urticarial biopsies. Laser-speckle contrast imaging quantified the attenuated response to cold challenge in patients on cetirizine. We found that the histamine-associated vascular response accompanying mast cell degranulation is rapid and extensive. At the tissue level, it is characterized by a uniform pattern of increased blood flow, thermal warming, vasodilation, and recruitment of collateral circulation. These vascular responses are modified by the administration of an antihistamine. CONCLUSIONS/SIGNIFICANCE: Monitoring the hemodynamic responses within tissues that are associated with mast cell degranulation provides additional insight into the evolution of the acute inflammatory response and offers a unique approach to assess the effectiveness of treatment intervention.
Assuntos
Degranulação Celular/fisiologia , Temperatura Baixa , Mastócitos/metabolismo , Mastócitos/patologia , Urticária/fisiopatologia , Adolescente , Adulto , Idoso , Degranulação Celular/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fluxo Sanguíneo Regional/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Triptases/sangue , Adulto JovemRESUMO
Mast cells are considered the primary initiators of allergic diseases as a consequence of the release of multiple inflammatory mediators on activation. Although predominately activated through antigen-mediated aggregation of IgE-occupied-FcÉRI, they can also be induced to release mediators by other receptors and environmental stimuli. Based on studies conducted in the RBL 2H3 rodent mast cell line, the transient receptor potential melastatin 8 (TRPM8) cation channel has been implicated in the activation of mast cells in response to cold and, by inference, the development of urticaria. Here we investigated the expression and role of TRPM8 receptor, in both human and mouse non-transformed cells, with the aim of exploring the potential link between TRPM8 and the pathology of cold urticaria in humans. Although expressed in mouse mast cells, we found no evidence of TRPM8 expression in human mast cells or functional mutations in TRPM8 in cold urticaria patients. Furthermore, neither mouse nor human primary cultured mast cells degranulated in response to cold challenge or TRPM8 agonists and mast cell reactivity was unaffected in Trpm8(-/-) mice. From these data, we conclude that TRPM8 is unlikely to directly regulate mast cell activation in cold urticaria. Thus, alternative mechanisms likely exist for the pathogenesis of this disease.