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BACKGROUND: New therapies are necessary to address inadequate asthma control in many patients. This study sets out to investigate whether hypoxia-inducible factor (HIF) is essential for development of allergic airway inflammation (AAI) and therefore a potential novel target for asthma treatment. METHODS: Mice conditionally knocked out for HIF-1ß were examined for their ability to mount an allergic inflammatory response in the lung after intratracheal exposure to ovalbumin. The effects of treating wild-type mice with either ethyl-3,4-dihydroxybenzoate (EDHB) or 2-methoxyestradiol (2ME), which upregulate and downregulate HIF, respectively, were determined. HIF-1α levels were also measured in endobronchial biopsies and bronchial fluid of patients with asthma and nasal fluid of patients with rhinitis after challenge. RESULTS: Deletion of HIF-1ß resulted in diminished AAI and diminished production of ovalbumin-specific IgE and IgG(1) . EDHB enhanced the inflammatory response, which was muted upon simultaneous inhibition of vascular endothelial growth factor (VEGF). EDHB and 2ME antagonized each other with regard to their effects on airway inflammation and mucus production. The levels of HIF-1α and VEGF increased in lung tissue and bronchial fluid of patients with asthma and in the nasal fluid of patients with rhinitis after challenge. CONCLUSIONS: Our results support the notion that HIF is directly involved in the development of AAI. Most importantly, we demonstrate for the first time that HIF-1α is increased after challenge in patients with asthma and rhinitis. Therefore, we propose that HIF may be a potential therapeutic target for asthma and possibly for other inflammatory diseases.
Assuntos
Asma/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipersensibilidade Respiratória/fisiopatologia , Rinite/metabolismo , Adolescente , Adulto , Alérgenos/imunologia , Animais , Asma/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/imunologia , Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Rinite/imunologia , Regulação para Cima , Adulto JovemRESUMO
We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.
Assuntos
Brônquios/cirurgia , Metilação de DNA/efeitos dos fármacos , Ozônio/farmacologia , Proteínas de Transferência de Fosfolipídeos/genética , RNA Longo não Codificante/genética , Receptores Acoplados a Proteínas G/genética , Adulto , Brônquios/química , Brônquios/efeitos dos fármacos , Broncoscopia , Estudos Cross-Over , Epigênese Genética , Feminino , Voluntários Saudáveis , Humanos , Masculino , Adulto JovemRESUMO
Smoke Sense is a citizen science project with investigative, educational, and action-oriented objectives at the intersection of wildland fire smoke and public health. Participants engage with a smartphone application to explore current and forecast visualizations of air quality, learn about how to protect health from wildfire smoke, and record their smoke experiences, health symptoms, and behaviors taken to reduce their exposures to smoke. Through participation in the project, individuals engage in observing changes in their environment and recording changes in their health, thus facilitating progression on awareness of health effects of air pollution and adoption of desired health-promoting behaviors. Participants can also view what others are reporting. Data from the pilot season (1 August 2017 to 7 January 2018; 5,598 downloads) suggest that there is a clear demand for personally relevant data during wildfire episodes motivated by recognition of environmental hazard and the personal concern for health. However, while participants shared clear perceptions of the environmental hazard and health risks in general, they did not consistently recognize their own personal health risk. The engagement in health protective behavior was driven in response to symptoms rather than as preventive courses of action. We also observed clear differences in the adoption likelihood of various health protective behaviors attributed to barriers and perceived benefits of these actions. As users experience a greater number and severity of symptoms, the perceived benefits of taking health protective actions exceeded the costs associated with the barriers and thus increased adoption of those actions. Based on pilot season data, we summarize key insights which may improve current health risk communications in nudging individuals toward health protective behavior; there is a need to increase personal awareness of risk and compelling evidence that health protective behaviors are beneficial.
RESUMO
Diesel exhaust particles (DEP) have been implicated in the increased incidence of allergic airway disorders. We investigated the effects of DEP on localized immunoglobulin production by performing nasal challenges with varying doses of DEP and analyzing the local immune response in nasal lavages obtained before and after. A significant rise in nasal IgE but not IgG, IgA, IgM, or albumin was observed in subjects 4 d after challenge with 0.30 mg DEP, equivalent to exposure on an average Los Angeles day. Direct evidence for DEP-enhanced local production of IgE was that challenge increased the number of IgE-secreting cells in lavage fluid from < 1 in 2,000,000 to > 1 in 100,000 but did not alter the number of IgA-secreting cells. There was a concomitant increase in epsilon mRNA production in the lavage cells. Additionally, DEP altered the relative amounts of five different epsilon mRNAs generated by alternative splicing, mRNAs that code for different IgE proteins. These results show that DEP exposure in vivo causes both quantitative and qualitative changes in local IgE production. The implication is that natural exposure to DEP may result in increased expression of respiratory allergic disease.
Assuntos
Imunoglobulina E/biossíntese , Mucosa Nasal/imunologia , RNA Mensageiro/biossíntese , Emissões de Veículos/toxicidade , Adulto , Processamento Alternativo , Sequência de Bases , Feminino , Humanos , Imunoglobulina E/genética , Imunoglobulinas/biossíntese , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mucosa Nasal/citologia , Nasofaringe/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Albumina Sérica/biossíntese , Método Simples-CegoRESUMO
Germ line C transcripts can be induced by IL-4 in the human B cell line, BL-2. Utilizing a IFN-gamma activation site-like DNA sequence element located upstream of the I epsilon exon, we demonstrated by gel mobility shift assays that IL-4 induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated IL-4 NAF (IL-4-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon IL-4 treatment. Because these are the characteristics of a signal transducer and activator of transcription (Stat) protein, we determined whether antibodies to Stat proteins will interfere with gel mobility shift and found that antibodies to IL-4 Stat, also known as Stat6, but not antibodies to other Stat proteins, interfere with the formation of the IL-4 NAF complex. Congruous with the involvement of a Stat protein, IL-4 induced robust Janus kinase 3 (JAK3) activity in BL-2 cells. Cotransfection of JAK3 with IL-4 Stat into COS-7 cells produced an intracellular activity which bound the same IFN-gamma activation site-like sequence and comigrated with IL-4 NAF in electrophoretic mobility shift assay. These results show that IL-4 NAF is IL-4 Stat, which is activated by JAK3 in response to IL-4 receptor engagement.
Assuntos
Linfócitos B/metabolismo , Éxons , Imunoglobulina E/genética , Interferon gama/metabolismo , Interleucina-4/farmacologia , Proteínas Tirosina Quinases/metabolismo , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular Transformada , Chlorocebus aethiops , Rearranjo Gênico do Linfócito B , Humanos , Janus Quinase 3 , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT6 , TransfecçãoRESUMO
The increase in allergic airway disease has paralleled the increase in the use of fossil fuels. Studies were undertaken to examine whether extracts of polyaromatic hydrocarbons (PAH) from diesel exhaust particles (DEP) (PAH-DEP) acted as mucosal adjuvants to help initiate or enhance immunoglobulin E (IgE) production in response to common inhaled allergens. In vitro studies demonstrated that PAH-DEP enhanced IgE production by tonsillar B-cells in the presence of interleukin-4 (IL-4) and CD40 monoclonal antibody, and altered the nature of the IgE produced, i.e. a decrease in the CH4'-CHe5 variant, a marker for differentiation of IgE-producing B-cells, and an increase in the M2' variant. In vivo nasal provocation studies using 0.30 mg DEP in saline also showed enhanced IgE production in the human upper respiratory mucosa, accompanied by a reduced CH4'-CHe5 mRNA splice variant. The effects of DEP were also isotype-specific, with no effect on IgG, IgA, IgM, or albumin, but it produced a small increase in the IgG4 subclass. The ability of DEP to act as an adjuvant to the ragweed allergen Amb a I was examined by nasal provocation in ragweed allergic subjects using 0.3 mg DEP, Amb a I, or both. Although allergen and DEP each enhanced ragweed-specific IgE, DEP plus allergen promoted a 16-times greater antigen-specific IgE production. Nasal challenge with DEP also influenced cytokine production. Ragweed challenge resulted in a weak response, DEP challenge caused a strong but non-specific response, while allergen plus DEP caused a significant increase in the expression of mRNA for TH0 and TH2-type cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) with a pronounced inhibitory effect on IFN-gamma gene expression. These studies suggest that DEP can enhance B-cell differentiation, and by initiating and elevating IgE production, may play an important role in the increased incidence of allergic airway disease.
Assuntos
Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Hipersensibilidade Respiratória/induzido quimicamente , Emissões de Veículos/toxicidade , Animais , Células Cultivadas , Humanos , Injeções Intraperitoneais , Hidrocarbonetos Policíclicos Aromáticos/administração & dosagem , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/imunologiaRESUMO
In a previous paper we described how the toxin ricin stimulates IgE but not IgG antibody responses in rats. In this study we have examined the cellular basis for this observation. The proportion of CD4- and CD8-positive cells present in the spleen at the peak of the IgE response was determined. Those animals injected with both ricin and antigen produced a substantial IgE response (50-fold increase). Their CD4+/CD8+ ratio was also markedly increased (P less than 0.001) compared with animals given toxin or antigen alone. In addition, mitogen-stimulated proliferation of mononuclear cells from spleens of the IgE-producing rats was enhanced nearly five-fold compared with cells from animals given toxin or allergen alone. The sensitivity of CD4- and CD8-positive rat spleen cells from unexposed animals to ricin in vitro was also studied. Spleen cells from untreated rats were co-cultured with optimal doses of mitogen and varying amounts of ricin. Mitogen-driven proliferation was inhibited at 10(-3) - 10(-6) mg/ml ricin. This effect was abrogated by the addition of as little as 0.01 M lactose but not by as much as 10 mg/ml mannan to the culture. Cultures depleted of CD4+ cells by rosetting were approximately 100 times more sensitive to ricin (P less than 0.01). Furthermore, the proportion of CD8+ to CD4+ cells present after culture of untreated cells with mitogen and ricin was significantly reduced. These results show (i) that the ability of ricin to increase the IgE response depends on the administration of antigen together with the toxin; (ii) that CD8+ spleen cells are more sensitive to ricin than CD4+ cells; (iii) that increased IgE responsiveness is associated with a reduction in the proportion of CD8- relative to CD4-positive cells in the spleen and increased responsiveness to mitogen. We believe that enhancement of the IgE responses by ricin may be due to inactivation of IgE-specific T-suppressor cells generated by immunization with antigen and speculate that these may be some or all of those bearing the CD8 marker.
Assuntos
Antígenos CD/análise , Imunoglobulina E/biossíntese , Ricina/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Células Produtoras de Anticorpos/imunologia , Divisão Celular/fisiologia , Imunoglobulina G/biossíntese , Fosfolipases A/imunologia , Ratos , Ratos Endogâmicos , Ricina/antagonistas & inibidoresRESUMO
The role of the cytokines interleukin-4 and interferon-gamma in the regulation of IgE responses in the mouse and man have focused on the role of CD4 T cells. In the rat, antigen-specific CD8 T cells, generated following inhalation of antigen, have been shown to be capable of suppressing IgE responses. Repeated intraperitoneal injections of 1 ng ricin and 1 microgram antigen established a long-lived IgE response in both low- and high-IgE responder rat strains (Wistar and Brown Norway). The duration of the IgE antibody response was 204 and 248 days, respectively. Total IgE levels rose from 30 +/- 20 to 39,000 +/- 7,500 ng/ml in the Wistar rat and from 120 +/- 100 to 47,000 +/- 8,000 ng/ml in the Brown Norway rat. An even greater (10(4)-fold) increase was seen in antigen-specific IgE antibody levels. Ricin alone had no effect and concomitant or prior stimulation with antigen was required. The proportion of CD4+ and CD8+ cells present in the spleen at the peak of the IgE response was markedly increased compared with animals given ricin or antigen alone. Furthermore, CD8 T cells were approximately 100 times more sensitive to ricin than CD4 T cells. These data suggest that enhancement of IgE responses in ricin-treated animals results from the selective deletion of T cells which suppress IgE and are of the CD8 phenotype.
Assuntos
Antígenos CD8/análise , Imunoglobulina E/biossíntese , Linfócitos T/fisiologia , Animais , Relação CD4-CD8 , Ricinus communis/imunologia , Lectinas de Plantas , Plantas Tóxicas , Ratos , Ricina , Linfócitos T/efeitos dos fármacosRESUMO
Certain strains of rats infested with the nematode parasite Nippostrongulus brasiliensis mount vigorous, persistent immunoglobulin E (IgE) responses. In the absence of parasites, adjuvants such as Bordatella pertussis or Al(OH)3 are needed to produce IgE responses to soluble antigens. These are short-lived, even in high IgE responder strains. In this study we have produced long-lived IgE responses in both low (Wistar) and high (Brown Norway) IgE responder strains of rats by repeated injections of ricin, a toxic lectin from castor beans, and phospholipase A2 (PLA2), a bee venom protein. Total IgE levels rose from 30 +/- 20 ng/ml to 39,000 +/- 7500 ng/ml in the Wistar rats compared with an increase from 120 +/- 100 ng/ml to 47,000 +/- 8000 ng/ml in the Brown Norway rats. An even greater (10(4)-fold) increase was seen in PLA2-specific IgE antibody levels. total and PLA2-specific IgE started to fall 6 weeks after treatment was stopped in the Wistar and after 12 weeks in the Brown Norway rats. The duration of the response was 204 and 248 days, respectively. The IgE-enhancing properties of ricin were compared in low, mid (Hooded Lister) and high IgE responder rats. Total IgE and PLA2-specific IgE but not IgG antibody (Ab) responses were enhanced in all animals given ricin and PLA2 but not in animals given ricin or PLA2 alone. The increase was greater in Wistar rats (48-fold) than in Brown Norway rats (eightfold) and by Day 24 the levels of both total and PLA2-specific IgE in three different strains were indistinguishable. PLA2-specific IgE antibody-secreting cells were detected in the spleen at a frequency of 1:5000. These results show: (i) that repeated immunization of rats with antigen and ricin produce a very large IgE response which was long-lived; (ii) that this response was indistinguishable in different IgE responder strains of rat; and (iii) that the IgE response declines earlier in low IgE responder strains of rats.
Assuntos
Adjuvantes Imunológicos , Antígenos/imunologia , Imunoglobulina E/biossíntese , Ricina/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Imunoglobulina G/biossíntese , Fosfolipases A/imunologia , Fosfolipases A2 , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos , Baço/imunologia , Fatores de TempoRESUMO
We investigated the effects of diesel exhaust particulates on the human allergic response using in vivo human nasal challenges. Diesel particles and phenanthrene, one of their constituent polyaromatic hydrocarbons, were shown to enhance total allergic antibody (IgE) production, enhance allergen-specific IgE in the presence of allergen, and induce sensitization to a neoantigen.
Assuntos
Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Emissões de Veículos , Animais , Humanos , Emissões de Veículos/efeitos adversos , Xenobióticos/efeitos adversosRESUMO
Ricin, a toxic lectin from castor beans greatly enhances IgE responses to bee venom phospholipase A2 (PLA2) in high and low IgE responder strains of rat. The increase in IgE is accompanied by a 60% reduction in the number of CD8+ but not CD4+ T cells in the spleen. Optimal enhancement of IgE by ricin occurs when it is given at the same time as the antigen or 24 hr later, suggesting that it acts on cells which were activated as a consequence of immunization. Radio ligand-binding studies with 125I ricin were used to compare the number of ricin binding sites on CD4+ and CD8+ T cells. No difference was seen in either the affinity or the number of receptors for ricin on the CD4+ and CD8+ T cells of unimmunized rats. In contrast, CD8+ T cells taken from rats which had been immunized with 10 micrograms of PLA2 24 hr earlier demonstrated considerably more ricin receptors (3.9 x 10(7) +/- 2.2 x 10(6) binding sites/cell) than CD4+ T cells (3.19 x 10(6) +/- 1.08 x 10(6) binding sites/cell). However the affinity of the receptors for ricin was unchanged. Cytofluorographic analysis with fluorescein isothiocyanate (FITC)-labelled ricin confirmed these observations and indicated that increased ricin binding occurred on a subpopulation of CD8+ T cells. The effect of CD8+ T cells on IgE regulation was investigated by adoptive transfer. 1 x 10(8) highly purified (> 98%) splenic CD8+ T cells collected from Brown Norway rats 3 days after immunization with 10 micrograms of PLA2 were adoptively transferred to naive, syngeneic recipients. The IgE antibody response to PLA2 + A1(OH)3 seen in these animals was reduced by 91%. Adoptive transfer of CD4+ T cells from the same donor animals did not induce suppression and nor did adoptive transfer of CD8+ T cells from animals given both ricin and PLA2. However, when recipients of CD8+ T cells taken from rats immunized with PLA2 were immunized with a different antigen [ovalbumin (OVA)] and A1(OH)3 the IgE antibody response was also suppressed, although to a lesser extent (66%). These results show that co-administration of ricin and PLA2 depletes a subpopulation of ricin-sensitive, early activated CD8+ T cells and that these CD8+ T cells are potent suppressors of the primary IgE response.
Assuntos
Antígenos CD8/análise , Imunoglobulina E/biossíntese , Ricina/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Contagem de Leucócitos , Fosfolipases A/imunologia , Fosfolipases A2 , Ratos , Ratos Endogâmicos , Baço/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
We review the literature indicating that the adverse health effects of ambient particulate matter involve the generation of oxidative stress and inflammation, as well as immunomodulating effects by particle-associated chemicals. We discuss evidence that diesel exhaust particle organic extracts induce reactive oxygen species in macrophages and bronchial epithelial cells, two key cell types targeted by particulate matter in the lung. Reactive oxygen species activate the promoters of cytokines and chemokines involved in allergic inflammation through activator protein-1 and nuclear factor- kappaB signaling pathways, which may explain exacerbation of allergic inflammation. Organic diesel exhaust particle chemicals also induce apoptosis and necrosis in bronchial epithelial cells via a mitochondrial pathway. This may be responsible for epithelial shedding and bronchial hyperreactivity in asthma.
Assuntos
Asma/etiologia , Poluentes Ambientais/efeitos adversos , Compostos Orgânicos/efeitos adversos , Estresse Oxidativo , Pneumonia/etiologia , Pneumonia/fisiopatologia , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Asma/fisiopatologia , Monitoramento Ambiental , Feminino , Humanos , Masculino , Tamanho da Partícula , Prognóstico , Medição de Risco , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The ability of combustion products, such as diesel exhaust particles (DEPs), to modulate the immune system has now been firmly established. DEPs can synergize with allergen at the human upper respiratory mucosa to enhance allergen-specific IgE production, initiate a T(H)2 cytokine environment, and even promote primary allergic sensitization. Experiments suggest that these effects result from the initial activation of mast cells to produce IL-4. OBJECTIVE: We sought to demonstrate that in vivo mast cell activation by DEPs plus allergen will also affect the release of classic mast cell mediators and consequently enhance the immediate-phase response. METHODS: Dust mite-sensitive subjects were challenged intranasally with allergen, and symptom scores and histamine levels in nasal wash samples were compared after prechallenge with 0.3 mg of DEPs. RESULTS: If the subjects were first sprayed with DEPs, mean symptom scores rose from 3.7 to 9.9; additionally, only one fifth of the amount of intranasal dust mite allergen was required to induce clinical symptoms. DEPs alone had no effect. The changes in symptoms correlated with histamine levels measured in nasal lavage specimens from these subjects. Although challenge with DEPs alone did not induce histamine release, challenge with both DEPs and allergen resulted in 3-fold higher histamine concentrations than those seen with allergen alone. In contrast, carbon black particles (elemental carbon devoid of chemicals) had no effect. The role of chemicals was confirmed because degranulation of a murine mast cell line by FcepsilonRI cross-linking was increased significantly (by 72%) by the soluble organic chemicals extracted from DEPs. CONCLUSIONS: Overall, these results suggest that exposure to DEPs can enhance the severity of clinical symptoms to allergen by enhancing mast cell degranulation.
Assuntos
Histamina/metabolismo , Hipersensibilidade Imediata/metabolismo , Mastócitos/citologia , Emissões de Veículos , Adulto , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Provocação Nasal , Índice de Gravidade de DoençaRESUMO
We have previously shown that in vivo nasal challenge with diesel exhaust particles (DEP) induces both quantitative and qualitative changes in local IgE production and stimulates generalized local cytokine production. We have now investigated the combined effects of intranasal challenge with DEP plus ragweed allergen on local humoral immune responses. We collected nasal lavages from ragweed sensitized subjects at different times after nasal challenge. As compared with challenge with ragweed alone, challenge with both DEP and ragweed induced markedly higher ragweed-specific IgE but not total IgE levels or IgE-secreting cell numbers. Total and specific IgG4 levels also were enhanced, while total IgG levels were not. Synergy was also observed between the DEP and ragweed in altering the profile of epsilon mRNAs generated by alternative splicing, mRNAs that code for different expressed IgE proteins. Intranasal challenge with ragweed alone induced inconsistent and low levels of mucosal cytokine mRNAs. In contrast, challenge with both ragweed plus DEP resulted in decreased expression for Th1-type cytokines (IFN-gamma and IL-2) but elevated expression of mRNA for other cytokines (IL-4, -5, IL-6, IL-10, IL-13). This synergy between DEP and natural allergen exposure is suggested as a key feature in increasing allergen-induced respiratory allergic disease.
Assuntos
Alérgenos/administração & dosagem , Citocinas/biossíntese , Imunoglobulina E/biossíntese , Mucosa Nasal/imunologia , Proteínas de Plantas/administração & dosagem , Compostos Policíclicos/administração & dosagem , Células Th2/metabolismo , Emissões de Veículos , Adulto , Alérgenos/imunologia , Especificidade de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/metabolismo , Antígenos de Plantas , Feminino , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina E/genética , Cadeias épsilon de Imunoglobulina/biossíntese , Cadeias épsilon de Imunoglobulina/genética , Imunoglobulinas/biossíntese , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Testes de Provocação Nasal , Poaceae/imunologia , RNA Mensageiro/biossínteseRESUMO
The use of splice sites for human epsilon mRNAs is tightly regulated, as the potential number of splice products far exceeds that actually produced. Our studies show that use of the epsilon alternative splices is regulated by a limited number of stimuli, and the relative production of the epsilon mRNA variants follows a developmental profile. In addition, we have found disease-related changes in the pattern of epsilon mRNA variants encoding distinctive IgE isoforms. Analysis of the biological activity of expressed recombinant IgE proteins and measurement of their levels in disease conditions will directly answer questions as to the biological relevance of the changes observed in epsilon mRNA splicing. This information will then be able to be used for rational therapeutic design interventions in IgE-mediated disorders.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Imunoglobulina E/biossíntese , Linfócitos B/imunologia , Engenharia Genética/métodos , Variação Genética , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Imunoglobulina E/química , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Transcrição GênicaRESUMO
Suspended airborne particulate matter such as diesel exhaust particles (DEP) have been implicated in the increased incidence of respiratory allergic diseases that has occurred over the past century. Studies in vitro and in vivo have shown that DEP may enhance allergic antibody (IgE) expression. DEP contain a wide spectrum of polycyclic aromatic hydrocarbons (PAH) that have been reported to have direct effects on the immune system, including the modulation of IgE production using various human and murine cell populations. We investigated the effects of the organic extract of DEP (PAH-DEP) and particularly, phenanthrene, a major component of DEP, in vitro on IgE production by 2C4/F3, a human Epstein-Barr virus transformed isotype switched, IgE producing B cell line. Phenanthrene consistently enhanced 2C4/F3 IgE production two- to threefold. This in vitro enhancement was associated with an increased expression of total IgE mRNA. Furthermore, the pattern of mRNA's coding for distinct isoforms of the epsilon chain was altered by both DEP-PAH and phenanthrene. While phenanthrene increased the level of productive epsilon transcripts, it did not increase epsilon germ line transcription. These effects were not due to an alteration of the cell cycle. Unstimulated 2C4/F3 cells contained detectable mRNA for IL6, IL10, TNF-alpha, and interestingly IL4; however, addition of PAH-DEP or phenanthrene did not significantly alter the level of these cytokines and thus did not appear to account for our findings. Thus, we have used our in vitro model to dissect the mechanism of DEP-PAH on IgE production in postswitch IgE producing cells and shown that phenanthrene, an important component in DEP and other pollutants, can act in a similar manner.
Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulina E/biossíntese , Fenantrenos/toxicidade , Emissões de Veículos/toxicidade , Linhagem Celular Transformada , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Imunoglobulina E/efeitos dos fármacos , Imunoglobulina E/genética , Cadeias épsilon de Imunoglobulina/efeitos dos fármacos , Cadeias épsilon de Imunoglobulina/genética , Ativação Linfocitária , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Epidemiological studies have suggested increased prevalence of atopy in children of maternal smokers. Although secondhand smoke or environmental tobacco smoke (ETS) has been shown to augment allergic responses, its role in atopic sensitization is still controversial. We studied whether ETS could initiate a Th2 response and thus induce primary allergic sensitization. Mice were exposed for 10 consecutive days to either 1% aerosolized OVA, ETS (5 cigarettes), or both ETS and OVA. C57BL/6 mice receiving both ETS and OVA developed OVA-specific IgE and IgG1, 12, 14, and 25 days after the initial exposure, whereas those receiving OVA alone did not. Thirty days after the initial challenge (20 days after its completion), mice were re-exposed to OVA. Bronchoalveolar lavage performed 24 h later revealed an influx of eosinophils in the group initially challenged with both ETS and OVA, but not in those exposed to ETS alone or OVA alone. Increases in IL-5, GM-CSF, and IL-2 were observed in bronchoalveolar lavage from this OVA/ETS-exposed group, whereas IFN-gamma levels were significantly inhibited. These results suggest that ETS can induce allergic sensitization to a normally harmless Ag, and they may explain why secondhand smoke is a major risk factor for the development of allergy in children.
Assuntos
Hipersensibilidade/etiologia , Poluição por Fumaça de Tabaco/efeitos adversos , Alérgenos/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/metabolismo , Feminino , Imunização , Imunoglobulina E/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Eosinofilia Pulmonar/etiologia , Especificidade da EspécieRESUMO
Induction of an IgE response involves several discrete steps: 1) induction of epsilon germ line transcription, 2) DNA recombination, and 3) mature RNA transcription/translation. Here we show that ligation of B cell CD58 by CD2, its natural ligand on T cells, or by mAb, provides a novel IL-4-dependent signal for the latter two steps. Highly purified human B cells were induced to produce IgE by costimulation with IL-4 and CD58 mAb. Although CD58 ligation alone was unable to induce epsilon germ-line transcription, in concert with IL-4-stimulated epsilon germ-line transcription it induced the appearance of productive epsilon transcripts and IgE production. The direct involvement of CD2 was demonstrated: B cells cultured with IL-4 plus murine T hybridoma cells transfected with human CD2 produced IgE. A CD40 Fc fusion protein had no effect on CD58-driven IgE production while inhibiting CD40-dependent responses. Furthermore, cells from patients with common variable immunodeficiency produced IgE in response to IL-4 plus CD40 mAb but not to IL-4 plus CD58 mAb. CD58-driven IgE synthesis was IFN-gamma independent and was not enhanced by exogenous IL-6. Functional differences between CD40 and CD58 IgE stimulation were demonstrated. Thus, the CD2:CD58 ligand/counterligand system provides an alternative pathway by which cell contact signaling may regulate IgE. Given the relative importance of CD2 triggering on mucosal T cells and the mucosal location of IgE production, this may be especially true on mucosal surfaces.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Linfócitos B/imunologia , Switching de Imunoglobulina , Imunoglobulina E/biossíntese , Glicoproteínas de Membrana/fisiologia , Adulto , Antígenos de Diferenciação de Linfócitos T/fisiologia , Sequência de Bases , Antígenos CD2 , Antígenos CD40 , Antígenos CD58 , Imunodeficiência de Variável Comum/imunologia , Primers do DNA/química , Humanos , Isotipos de Imunoglobulinas/biossíntese , Interferon gama/fisiologia , Interleucina-4/farmacologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Agregação de Receptores , Receptores Imunológicos/fisiologia , Transdução de SinaisRESUMO
An early step in immunoglobulin isotype switching involves the initiation of active transcription of downstream heavy chain loci while they are still in germ-line configuration. This results in the production of 'sterile' germ-line RNA transcripts that do not contain VDJ sequences, the role of which in isotype switching is under intense scrutiny. We investigated whether such human epsilon germ-line transcripts employ the full complement of complex alternative 3' RNA splicing, splicing we have recently reported occurring with productive epsilon mRNA transcripts. Using a reverse transcriptase polymerase chain reaction (RT-PCR) strategy, in which the 5' primer was located in the I region of the epsilon gene, a region expressed in germ-line but not productive (VDJ containing) epsilon transcripts, we showed that the full range of alternative 3' epsilon splices occur with germ-line transcripts. These results demonstrate that epsilon 3' splicing events are independent of 5' isotype DNA switching. Additionally, we showed that, just as with mature epsilon mRNA, the relative production of the various epsilon germ-line mRNA isoforms was responsive to modulation by stimuli such as interleukin-10 (IL-10). Thus B cells, when stimulated to produce epsilon germ-line transcripts, generate a family of germ-line mRNA that differ not only in their initiation sites but, more importantly, also differ in their 3' sequences, sequences that could be important in regulation of the parent gene itself. Furthermore, by discontinuous or trans-splicing, cells could utilize these various epsilon germ-line transcripts to produce the full range of mature IgE proteins prior to undergoing deletional recombination of isotype switching.
Assuntos
Células Germinativas/imunologia , Switching de Imunoglobulina , Cadeias épsilon de Imunoglobulina/genética , Splicing de RNA , RNA Mensageiro/análise , Sequência de Bases , Southern Blotting , Primers do DNA , Humanos , Interleucina-10/farmacologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
In this study we undertook to provide evidence for local in vivo isotype switching to IgE following nasal challenges. Detection of deleted switch circular DNA (switch circles) by a novel nested polymerase chain reaction-based approach was employed as definitive molecular evidence of Ig isotype switching. Nasal challenge in humans with diesel exhaust particles (DEP) plus ragweed antigen has been shown to enhance local IgE production, stimulate local cytokine production, and markedly increase mucosal IgE antibody to ragweed. Four days after combined intranasal DEP plus ragweed challenge, we detected and characterized clones of deleted switch circular DNA (Sepsilon /Smu) representing switching from mu to epsilon from nasal lavage cells. No switch circular DNA was detected in nasal lavage cells following challenge with DEP alone nor with ragweed allergen alone. These results indicate that the combination of mucosal stimulation with DEP and ragweed allergen is capable of driving in vivo isotype switching to IgE in humans with ragweed allergy. These results are the first direct demonstration of in vivo IgE isotype switching in humans.