RESUMO
Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine-producing cells. Neutrophils store â¼0.29 pg/cell and release â¼50% of the histamine content in an antigen-dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate-limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001-0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.
Assuntos
Liberação de Histamina/imunologia , Histamina/imunologia , Histidina Descarboxilase/imunologia , Neutrófilos/imunologia , Adulto , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Histamina/biossíntese , Liberação de Histamina/efeitos dos fármacos , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Humanos , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Allergen-specific immunotherapy (IT) is widely used to treat allergic diseases. The molecular mechanisms have not been clarified yet completely. The present work was undertaken to analyze the effect of IT in the activation of NF-κB. METHODS: Neutrophils from 15 pollen-allergic IT-treated patients, 10 untreated pollen-allergic patients, and 10 healthy donors were in vitro stimulated with LPS. NF-κB activation (p65/p52) was measured in their nuclear extracts by enzyme-linked immunosorbent assay (ELISA). IκBα phosphorylation, NF-κB-repressing factor (NRF) activation, and thromboxane A2 (TXA2 ) and Interleukin-8 (IL-8) release were measured by ELISA. RESULTS: There was a positive correlation between the score of symptoms and NF-κB activation in human neutrophils. IT significantly decreased NF-κB activation levels in neutrophils compared with neutrophils from untreated patients. IκBα phosphorylation and NRF activation levels were, respectively, significantly lower and higher in neutrophils from IT-treated patients than from untreated patients. IL-8 and TXA2 release were significantly lower in neutrophils from IT-treated patients than from untreated patients. CONCLUSIONS: IT positive effects are at least in part mediated by the negative regulation of NF-κB activation in human neutrophils. These observations represent a novel view of neutrophils as possible cell target to treat IgE-dependent diseases through NF-κB downmodulation.
Assuntos
Alérgenos/uso terapêutico , Dactylis/imunologia , Dessensibilização Imunológica/métodos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Pólen/imunologia , Rinite Alérgica Sazonal/terapia , Adolescente , Estudos de Casos e Controles , Células Cultivadas , Regulação para Baixo , Feminino , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-8/metabolismo , Masculino , Inibidor de NF-kappaB alfa , Subunidade p52 de NF-kappa B/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fosforilação , Proteínas Repressoras/metabolismo , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/imunologia , Transdução de Sinais/efeitos dos fármacos , Tromboxano A2/metabolismo , Fator de Transcrição RelA/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: Despite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question. METHODS: Neutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed. RESULTS: Lf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients.
Assuntos
Alérgenos/imunologia , Asma/imunologia , Lactoferrina/imunologia , Neutrófilos/imunologia , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Galectina 3/imunologia , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de IgE/imunologia , Adulto JovemRESUMO
UNLABELLED: CD66b is a member of the carcinoembryonic antigen family, which mediates the adhesion between neutrophils and to endothelial cells. Allergen-specific immunotherapy is widely used to treat allergic diseases, and the molecular mechanisms underlying this therapy are poorly understood. The present work was undertaken to analyze A) the in vitro effect of allergens and immunotherapy on cell-surface CD66b expression of neutrophils from patients with allergic asthma and rhinitis and B) the in vivo effect of immunotherapy on cell-surface CD66b expression of neutrophils from nasal lavage fluid during the spring season. Myeloperoxidase expression and activity was also analyzed in nasal lavage fluid as a general marker of neutrophil activation. RESULTS: CD66b cell-surface expression is upregulated in vitro in response to allergens, and significantly reduced by immunotherapy (p<0.001). Myeloperoxidase activity in nasal lavage fluid was also significantly reduced by immunotherapy, as were the neutrophil cell-surface expression of CD66b and myeloperoxidase (p<0.001). Interestingly, CD66b expression was higher in neutrophils from nasal lavage fluid than those from peripheral blood, and immunotherapy reduced the number of CD66+MPO+ cells in nasal lavage fluid. Thus, immunotherapy positive effects might, at least in part, be mediated by the negative regulation of the CD66b and myeloperoxidase activity in human neutrophils.