RESUMO
Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.
Assuntos
Cromatina/genética , Suscetibilidade a Doenças , Estudo de Associação Genômica Ampla , Genômica , Mastócitos/imunologia , Mastócitos/metabolismo , Biomarcadores , Células Cultivadas , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Fibrinogênio/genética , Fibrinogênio/metabolismo , Perfilação da Expressão Gênica , Genômica/métodos , Histonas/metabolismo , Humanos , Hipersensibilidade/etiologia , Hipersensibilidade/metabolismo , Imunoglobulina E/imunologia , Inflamação/etiologia , Inflamação/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.
Assuntos
Dermatite/imunologia , Imunidade Inata/imunologia , Linfócitos/imunologia , Pele/imunologia , Animais , Comunicação Celular/imunologia , Células Cultivadas , Dermatite/genética , Dermatite/metabolismo , Derme/citologia , Derme/imunologia , Derme/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Imunidade Inata/genética , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-2/imunologia , Interleucina-2/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Pele/metabolismo , Gravação de VideoteipeRESUMO
BACKGROUND: Polyethylene glycol (PEG) is a nonprotein polymer that is present in its native (unbound) form as an excipient in a range of products. It is increasingly being utilized clinically in the form of PEGylated liposomal medications and vaccines. PEG is the cause of anaphylaxis in a small percentage of drug reactions; however, diagnosis of PEG allergy is complicated by the variable and poor diagnostic performance of current skin testing protocols. OBJECTIVE: We assessed the diagnostic performance of PEGylated lipid medications as an alternative to currently described tests that use medications containing PEG excipients. METHODS: Nine patients with a strong history of PEG allergy were evaluated by skin testing with a panel of PEG-containing medications and with a PEGylated lipid nanoparticle vaccine (BNT162b2). Reactivity of basophils to unbound and liposomal PEG was assessed ex vivo, and specificity of basophil responses to PEGylated liposomes was investigated with a competitive inhibition assay. More detailed information is provided in this article's Methods section in the Online Repository available at www.jacionline.org. RESULTS: Despite compelling histories of anaphylaxis to PEG-containing medications, only 2 (22%) of 9 patients were skin test positive for purified PEG or their index reaction-indicated PEG-containing compound. Conversely, all 9 patients were skin test positive or basophil activation test positive to PEGylated liposomal BNT162b2 vaccine. Concordantly, PEGylated liposomal drugs (BNT162b2 vaccine and PEGylated liposomal doxorubicin), but not purified PEG2000, consistently induced basophil activation ex vivo in patients with PEG allergy but not in nonallergic controls. Basophil reactivity to PEGylated nanoparticles competitively inhibited by preincubation of basophils with native PEG2000. CONCLUSION: Presentation of PEG on the surface of a lipid nanoparticle increases its in vivo and ex vivo allergenicity, and improves diagnosis of PEG allergy.
Assuntos
Basófilos , Hipersensibilidade a Drogas , Lipossomos , Polietilenoglicóis , Testes Cutâneos , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/efeitos adversos , Lipossomos/química , Feminino , Masculino , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/imunologia , Pessoa de Meia-Idade , Adulto , Basófilos/imunologia , Idoso , Anafilaxia/imunologia , Anafilaxia/diagnóstico , Anafilaxia/induzido quimicamente , Nanopartículas/químicaRESUMO
BACKGROUND: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common ß (ßc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. OBJECTIVE: To determine the pathogenesis of ßc receptor-mediated inflammation and remodeling in severe asthma and to investigate ßc antagonism as a therapeutic strategy for mixed granulocytic airway disease. METHODS: ßc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human ßc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of ßc was used to block ßc signaling. RESULTS: ßc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. CONCLUSIONS: ßc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-ßc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.
Assuntos
Asma , Receptores de Citocinas , Camundongos , Animais , Humanos , Receptores de Citocinas/metabolismo , Remodelação das Vias Aéreas , Pulmão , Citocinas/metabolismo , Camundongos Transgênicos , Inflamação , Alérgenos , Esteroides/uso terapêutico , Fibrose , PyroglyphidaeRESUMO
Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.
Assuntos
Mastócitos , RNA , Diferenciação CelularRESUMO
The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.
Assuntos
Medicina Clínica , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Citocinas/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Eosinófilos , BiologiaRESUMO
BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.
Assuntos
Anafilaxia , Receptores de IgE , Humanos , Camundongos , Animais , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Mastócitos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunoglobulina E , Inflamação/metabolismo , Degranulação CelularRESUMO
BACKGROUND: Natural killer T (NKT) cells express a T-cell receptor that recognizes endogenous and environmental glycolipid antigens. Several subsets of NKT cells have been identified, including IFN-γ-producing NKT1 cells, IL-4-producing NKT2 cells, and IL-17-producing NKT17 cells. However, little is known about the factors that regulate their differentiation and respective functions within the immune system. OBJECTIVE: We sought to determine whether the polycomb repressive complex 2 protein enhancer of zeste homolog 2 (Ezh2) restrains pathogenicity of NKT cells in the context of asthma-like lung disease. METHODS: Numbers of invariant natural killer T (iNKT) 1, iNKT2, and iNKT17 cells and tissue distribution, cytokine production, lymphoid tissue localization, and transcriptional profiles of iNKT cells from wild-type and Ezh2 knockout (KO) iNKT mice were determined. The contribution of NKT cells to development of spontaneous and house dust mite-induced airways pathology, including airways hyperreactivity (AHR) to methacholine, was also assessed in wild-type, Ezh2 KO, and Ezh2 KO mice lacking NKT cells. RESULTS: Ezh2 restrains development of pathogenic NKT cells, which induce spontaneous asthma-like disease in mice. Deletion of Ezh2 increased production of IL-4 and IL-13 and induced spontaneous AHR, lung inflammation, mucus production, and IgE. Increased IL-4 and IL-13 levels, AHR, lung inflammation, and IgE levels were all dependent on iNKT cells. In house dust mite-exposed animals Ezh2 KO resulted in enhanced AHR that was also dependent on iNKT cells. CONCLUSION: Ezh2 is a central regulator of iNKT pathogenicity and suppresses the ability of iNKT cells to induce asthma-like pathology.
Assuntos
Asma/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Pulmão/imunologia , Células T Matadoras Naturais/imunologia , Animais , Asma/genética , Asma/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-13/genética , Interleucina-13/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/patologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/imunologiaAssuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Xenoenxertos , Pólipos Nasais/complicações , Pólipos Nasais/tratamento farmacológico , Receptores de Citocinas/imunologia , Rinite/complicações , Rinite/tratamento farmacológico , Sinusite/complicações , Sinusite/tratamento farmacológico , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Pólipos Nasais/patologia , Células Th2/imunologia , Transcriptoma , Resultado do TratamentoRESUMO
BACKGROUND: Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D3 (25OHD3), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE: We sought to assess whether the vitamin D3 metabolites 25OHD3 and 1α,25(OH)2D3 can repress IgE-dependent mast cell activation through mast cell-25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell-VDR activity. METHODS: We measured the extent of vitamin D3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro, as well as the vitamin D3-induced curtailment of PCA responses in WBB6F1-Kit(W/W-v) or C57BL/6J-Kit(W-sh/W-sh) mice engrafted with mast cells that did or did not express VDR or CYP27B1. RESULTS: Here we show that mouse and human mast cells can convert 25OHD3 to 1α,25(OH)2D3 through CYP27B1 activity and that both of these vitamin D3 metabolites suppressed IgE-induced mast cell-derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro. Furthermore, epicutaneously applied vitamin D3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo; a response that required functional mast cell-VDRs and mast cell-CYP27B1. CONCLUSION: Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD3 to dampen IgE-mediated mast cell activation in vitro and in vivo.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Anafilaxia/metabolismo , Calcifediol/metabolismo , Mastócitos/metabolismo , Receptores de Calcitriol/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Anafilaxia/genética , Anafilaxia/patologia , Animais , Linhagem Celular , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Humanos , Imunoglobulina E , Mastócitos/patologia , Camundongos , Camundongos Knockout , Receptores de Calcitriol/genéticaRESUMO
Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on ß-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 µM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1-16 µM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 µM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.
RESUMO
Allergic contact dermatitis (ACD) is a prevalent and poorly controlled inflammatory disease caused by skin infiltration of T cells and granulocytes. The beta common (ßc) cytokines GM-CSF, IL-3, and IL-5 are powerful regulators of granulocyte function that signal through their common receptor subunit ßc, a property that has made ßc an attractive target to simultaneously inhibit these cytokines. However, the species specificity of ßc has precluded testing of inhibitors of human ßc in mouse models. To overcome this problem, we developed a human ßc receptor transgenic mouse strain with a hematopoietic cellâspecific expression of human ßc instead of mouse ßc. Human ßc receptor transgenic cells responded to mouse GM-CSF and IL-5 but not to IL-3 in vitro and developed tissue pathology and cellular inflammation comparable with those in wild-type mice in a model of ACD. Similarly, Il3-/- mice developed ACD pathology comparable with that of wild-type mice. Importantly, the blocking anti-human ßc antibody CSL311 strongly suppressed ear pinna thickening and histopathological changes typical of ACD and reduced accumulation of neutrophils, mast cells, and eosinophils in the skin. These results show that GM-CSF and IL-5 but not IL-3 are major mediators of ACD and define the human ßc receptor transgenic mouse as a unique platform to test the inhibitors of ßc in vivo.
Assuntos
Dermatite de Contato , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Animais , Citocinas , Eosinófilos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
OBJECTIVES/HYPOTHESIS: The efficacy of short-term oral corticosteroids in chronic rhinosinusitis without nasal polyps (CRSsNP) is unknown. The aim of this controlled study was to assess the immediate and long-term outcomes from a short course of a commonly used oral corticosteroid, prednisolone, in well-defined CRSsNP patients. STUDY DESIGN: Prospective, observational controlled study. METHODS: A prospective-controlled study of CRSsNP patients treated with prednisolone at 0.5 mg/kg tapered over 10 days and non-prednisolone treated CRSsNP patients (controls) and follow-up at 2, 6, and 12 months. Baseline and follow-up SinoNasal Outcome Test (SNOT)-22, nasal endoscopy (Lund-Kennedy), and sinus CT scan scores (Lund-Mackay) were compared. RESULTS: At 2 months, there was a significant improvement in the SNOT-22, nasal endoscopy, and sinus CT scan scores in the prednisolone group (P < .0001) compared with controls (p = ns, Mann-Whitney U test). 52.5% of prednisolone-treated CRSsNP patients had improved symptoms and did not require sinus surgery at 12 months compared with 14.3% of controls (P < .001). Side-effects were reported in 8.9% of prednisolone-treated patients. Patients who benefited from prednisolone had a median symptom duration of 7.25 (99% confidence, upper limit of 11) months compared with 18 months in those requiring surgery. CONCLUSIONS: Short-term oral prednisolone significantly improved all three clinical measures of disease in CRSsNP patients and avoided surgical intervention in 52.5% patients in the first 12 months. Patients with symptoms for less than 11 months were most likely to benefit. The side-effects of oral steroids require careful consideration and further studies are needed to ascertain appropriate dosage and treatment duration. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E2618-E2626, 2021.
Assuntos
Prednisolona/administração & dosagem , Rinite/tratamento farmacológico , Sinusite/tratamento farmacológico , Esteroides/administração & dosagem , Administração Oral , Adolescente , Adulto , Idoso , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Invariant and semi-invariant T cells are emerging as important regulators of host environment interactions at barrier tissues such as the airway and skin. In contrast to conventional T cells, invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells express T cell receptors of very limited diversity. iNKT and MAIT cells recognise antigens presented by the MHC class 1-like monomorphic molecules CD1d and MR1, respectively. Both iNKT cells and MAIT cells have been identified in the skin and airways and can rapidly produce cytokines after activation. Numerous studies have implicated iNKT cells in the pathology of both skin and airway disease, but conflicting evidence in human disease means that more studies are necessary to resolve the exact roles of iNKT in inflammation. The functions of MAIT cells in skin and lung inflammation are even less well defined. We herein describe the current literature on iNKT and MAIT cells in allergic and non-allergic skin diseases (dermatitis and psoriasis) and airway diseases (asthma, chronic obstructive pulmonary disease, rhinitis, and chronic rhinosinusitis).
Assuntos
Suscetibilidade a Doenças , Inflamação/etiologia , Inflamação/metabolismo , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores , Dermatite/etiologia , Dermatite/metabolismo , Dermatite/patologia , Regulação da Expressão Gênica , Antígenos HLA/imunologia , Humanos , Inflamação/diagnóstico , Ativação Linfocitária/imunologia , Doenças Respiratórias/etiologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/patologia , Transdução de SinaisRESUMO
The mast cells have been suggested to be a cellular source of nitric oxide (NO) which level is increased in the pathogenesis of asthma. However, isoforms of the NO generating enzyme, nitric oxide synthase (NOS), in primary human mast cells have not been defined due to the lack of a suitable model. We hence examined directly the expression of NOS mRNA and proteins in primary human cultured mast cells (HCMC). Mature HCMC were cultured from CD34+ progenitors isolated from buffy coat preparations and were subjected to IgE sensitisation, IgE receptor mediated activation and cytokines induced stimulation. While expression of NOS mRNA was detected by conventional reverse transcription-polymerase chain reaction (RT-PCR) and quantitatively analyzed with real-time RT-PCR, expression of NOS proteins was detected by immunostaining. In non-stimulated HCMC incubated in medium alone, expressions of NOS were not detected. While overnight incubation of HCMC with IgE significantly increased the expression of NOS2 and NOS3, only NOS2 expression was up-regulated after overnight incubation with a mixture of TNF-alpha, IFN-gamma and IL-1beta. Cross-linking of IgE with anti-IgE further increased NOS2 expression with a concomitant decrease in NOS3 expression. NOS1 was not detected in all treatments. In conclusion, we have shown for the first time that NOS2 and NOS3 expressions are induced in primary human mast cells following appropriate stimulations. Comparisons between the differential expressions of NOS isoforms in HCMC to the changes in NOS expressions in asthma models suggest that the mast cell is a source of NO in asthmatic airways.
Assuntos
Citocinas/farmacologia , Imunoglobulina E/farmacologia , Mastócitos/enzimologia , Óxido Nítrico Sintase/biossíntese , Antígenos CD34/metabolismo , Células Cultivadas , Primers do DNA/farmacologia , Indução Enzimática/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases , Humanos , Isoenzimas/biossíntese , Mastócitos/efeitos dos fármacos , Monócitos/metabolismo , Óxido Nítrico Sintase Tipo I/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo III/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismoRESUMO
Mast cells are important effector cells of allergy and techniques for culturing human mast cells have been developed in recent years. In the current investigation, we studied the phenotypic and functional characteristics of mast cells cultured from adult human peripheral blood mononuclear cells. Mature human mast cells were obtained by first culturing mononuclear cells in methylcellulose containing stem cell factor (SCF), IL-3 and IL-6 for six weeks and subsequently in liquid medium containing SCF and IL-6 for another six weeks. These cells expressed numerous basophilic cytoplasmic granules that were predominantly tryptase positive but chymase negative. Following sensitization with human IgE, these cells released histamine and synthesized prostaglandin D2 and cysteinyl-leukotrienes dose-dependently upon activation by anti-IgE and calcium ionophores. Compound 48/80 and substance P were ineffective. When the effects of anti-asthmatic agents on anti-IgE induced mediator release from these cells were compared, only the beta2-adrenoceptor agonists and phosphodiesterase inhibitors produced dose-dependent inhibition but not cromolyn. In total, mast cells cultured from human peripheral blood shared similar morphological, immunocytochemical and functional properties of enzymatically dispersed human lung mast cells. These cultured mast cells can be a convenient substitute for the in vitro studies of human lung mast cell reactions and may be useful for investigating the roles of mast cells in allergic diseases.
Assuntos
Leucócitos Mononucleares/citologia , Mastócitos/citologia , Mastócitos/imunologia , Agonistas Adrenérgicos beta/farmacologia , Adulto , Albuminas/imunologia , Tamanho Celular , Células Cultivadas , Cisteína/imunologia , Liberação de Histamina/efeitos dos fármacos , Humanos , Fatores Imunológicos/imunologia , Leucotrienos/imunologia , Mastócitos/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Prostaglandina D2/imunologiaRESUMO
Cross-linkage of the high-affinity immunoglobulin E (IgE) receptor (FcÉRI) on mast cells by antigen ligation has a critical role in the pathology of IgE-dependent allergic disorders, such as anaphylaxis and asthma. Restraint of intracellular signal transduction pathways that promote release of mast cell-derived pro-inflammatory mediators is necessary to dampen activation and restore homoeostasis. Here we show that the ligase Nedd4-2 and the adaptor Ndfip1 (Nedd4 family interacting protein 1) limit the intensity and duration of IgE-FcÉRI-induced positive signal transduction by ubiquitinating phosphorylated Syk, a tyrosine kinase that is indispensable for downstream FcÉRI signalosome activity. Importantly, loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo. Our findings reveal an important negative regulatory function for Nedd4-2 and Ndfip1 in IgE-dependent mast cell activity.
Assuntos
Proteínas de Transporte/imunologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , Proteínas de Membrana/imunologia , Ubiquitina-Proteína Ligases Nedd4/imunologia , Transferência Adotiva , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Imunoglobulina E/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Mastócitos/metabolismo , Mastócitos/transplante , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Anafilaxia Cutânea Passiva/genética , Anafilaxia Cutânea Passiva/imunologia , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Quinase Syk/imunologia , Quinase Syk/metabolismoRESUMO
Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Lipopeptídeos/farmacologia , Peptidoglicano/farmacologia , Receptor 2 Toll-Like/agonistas , Calcineurina/química , Calcineurina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Imunidade Inata , Interleucina-8/metabolismo , Ligantes , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismoRESUMO
OBJECTIVES: Mast cells are believed to contribute to the pathogenesis of osteoporosis as their number is increased in osteoporotic bones. Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae are three Chinese herbs traditionally for tonifying the 'kidney system' and a herbal formula (ELP) containing the respective herbs at the weight ratio of 5 : 4 : 1 was shown to prevent osteoporosis. This study evaluated if suppression of mast cell accumulation and activity contribute to the anti-osteoporotic action of ELP. METHODS: The herbs were boiled under reflux to produce the aqueous extract that was further concentrated under reduced pressure and lyophilized. An in-vivo rat osteoporosis model using hind limb unloading was employed for studying the accumulation of mast cells. The human mast cell line, LAD2, was employed to evaluate the mast cell modulating action of ELP. KEY FINDINGS: Mast cell number in the tibiae of hind limb unloaded rats increased significantly during the course of osteoporosis. ELP treatment (10 g/kg/day) prevented both osteoporosis and mast cell accumulation in these rats. Furthermore, ELP significantly inhibited histamine and tumour necrosis factor-α release from LAD2 cells. CONCLUSION: Mast cells contributed to hormone independent osteoporosis. The suppression of mast cell accumulation and activation may contribute to the anti-osteoporotic action of ELP.