RESUMO
BACKGROUND: Efficacy testing of drugs in seasonal allergic rhinitis (SAR) is often disturbed by seasonal variations of environmental allergens, and assessment of onset and duration of action is hardly possible under natural conditions. Allergen provocation in an environmental challenge chamber (ECC) can be of added value in this respect. However, the specificity, sensitivity, and reproducibility of outcome measures under both settings are unclear. OBJECTIVE: The aim of this study was to investigate and compare the diagnostic value (specificity, sensitivity, and reproducibility) of clinical end-points and biomarkers both following allergen provocation in an ECC and under natural conditions. METHODS: Sixty adult patients with SAR to grass and 60 healthy subjects were exposed twice to grass pollen in an ECC and observed twice during the pollen season. Symptoms, nasal flow, as well as exhaled and nasal nitric oxide (NO) were investigated. RESULTS: The total nasal symptom score (TNSS) in the ECC had the best reproducibility (intraclass correlation coefficient ICC=0.86) and sensitivity/specificity [area under receiver operating characteristic curve (AUC)=0.99] of all measures. Symptoms in season also had good sensitivity/specificity but were far less reproducible. Nasal flow in the ECC had good sensitivity/specificity but reproducibility was limited. NO measurements showed good reproducibility but sensitivity/specificity were limited, except for exhaled NO in season (AUC=0.75). CONCLUSION: The high reproducibility and sensitivity/specificity in the ECC suggests that TNSS is a valuable outcome measure. While exhaled NO can be considered to monitor airway inflammation, nasal NO appears to be unspecific.
Assuntos
Biomarcadores/análise , Óxido Nítrico/análise , Poaceae/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/diagnóstico , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Interleukin-12 (IL-12) is a potent inducer of interferon-gamma production by T cells and is a major factor for the development of T-helper 1 (Th1) cells. It exerts its biological effects through binding to the IL-12 receptor (IL-12R), a heterodimer composed of a 1 and a beta2 subunits. The signaling beta2 chain is expressed on Th1 cells and to a lesser extent on Th0 cells, but not on Th2 cells, rendering these latter cells unresponsive to IL-12. Polymorphisms in the coding region of the IL-12Rbeta2 gene were shown to be associated with atopic disease. Here, we analyzed the 5'-regulatory region of the human IL-12Rbeta2 gene by denaturing high-performance liquid chromatography (Transgenomic WAVE system, San Jose, CA). We found five novel single-nucleotide polymorphisms (SNPs) in the proximal 1.2 kb IL-12Rbeta2 promoter region, i.e. -237C/T, -465A/G, -1023A/G, -1033T/C, and -1035A/G. SNP -465A/G is of particular interest as it determines the integrity of a GATA consensus site. By functional comparison of both -465 alleles in transient transfection assays, we show that promoter activity is increased in case of the -465G allele, disrupting the intact GATA site. Comparison of the prevalence of -465A/G SNP alleles in small cohorts of allergic asthmatic and healthy control individuals provided no evidence for an altered distribution in the asthmatic population. In conclusion, we have identified a novel polymorphic GATA site that may affect transciptional activity of the human IL-12Rbeta2 gene under GATA3-mediated, Th2-polarizing conditions.
Assuntos
Regiões Promotoras Genéticas , Receptores de Interleucina/genética , Região 5'-Flanqueadora , Asma/genética , Sequência de Bases , Sequência Consenso , Humanos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-12RESUMO
In vitro data suggest that salmeterol, contrary to formoterol, can partly antagonise the effect of short-acting beta(2)-agonist rescue medication. To explore whether this occurs in vivo, we compared the effects of increasing doses (200-3200 microg) of fenoterol on the recovery of methacholine induced bronchoconstriction as well as PD(20) methacholine in 23 asthmatic patients, during two-week treatment periods with placebo, and standard doses of salmeterol or formoterol in a double blind, double-dummy, crossover study. Salmeterol showed a slightly higher propensity for the development of bronchodilator tolerance. The recovery of methacholine induced bronchoconstriction was more complete during regular use of formoterol relative to salmeterol. During regular use of both long-acting beta(2)-agonists the bronchoprotective efficacy of fenoterol was attenuated, but this was more pronounced during salmeterol than during formoterol. The mean maximum increase in PD(20) metacholine after the highest dose of fenoterol was 3.97 DD during placebo, 2.47 DD during formoterol (p<0.001) and 1.81 DD during salmeterol treatment (p<0.001). We conclude that in asthmatic patients the efficacy of short-acting beta(2)-adrenoceptor agonists can be significantly attenuated during regular use of long-acting beta(2)-agonists. In this respect, differences were observed between salmeterol and formoterol that may represent the expression of partial antagonism by salmeterol.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Albuterol/análogos & derivados , Albuterol/uso terapêutico , Asma/tratamento farmacológico , Broncoconstritores/antagonistas & inibidores , Broncodilatadores/uso terapêutico , Etanolaminas/uso terapêutico , Cloreto de Metacolina/antagonistas & inibidores , Adulto , Idoso , Broncoconstritores/efeitos adversos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Fumarato de Formoterol , Humanos , Masculino , Cloreto de Metacolina/efeitos adversos , Pessoa de Meia-Idade , Testes de Função Respiratória , Xinafoato de SalmeterolRESUMO
The central effector cells in the pathogenesis of atopic allergic diseases are type 2 T helper (Th2) cells, which display an aberrant cytokine profile dominated by type 2 cytokines. Initial reports from mouse studies indicated that established and committed Th2 cells are stable and unsusceptible to modulation. However, there is a growing awareness that in humans, established effector Th2 cells are more flexible and can be reverted to predominant Th1 phenotypes. In fact, the Th1-driving cytokine interleukin (IL)-12 is the crucial factor in this respect. IL-12 is mainly produced by dendritic cells (DC), which can be primed for high or low IL-12 production, depending on inflammatory and/or microbial signals they encounter during their residence in the peripheral tissues. Accordingly, both the regulation of and the priming for IL-12 production in DC form ideal targets for therapeutic intervention. The development of new therapies for atopic allergy now focuses on local IL-12-promoting substances to target both the development of new Th2 cells and the persistent population of established allergen-specific Th2 cells.
Assuntos
Células Dendríticas/fisiologia , Hipersensibilidade Imediata/imunologia , Interleucina-12/metabolismo , Células Th2/fisiologia , Animais , Diferenciação Celular , Humanos , Interleucina-12/farmacologia , Camundongos , Receptores de Interleucina/metabolismo , Células Th1/imunologia , Células Th1/fisiologia , Células Th2/imunologiaRESUMO
PURPOSE: The mechanisms involved in reactivations of latent ocular Toxoplasma gondii (Tg) infections in immunocompetent patients are poorly understood. In view of the possible role of T cells in the immunopathogenesis of the disease, ocular infiltrating T cells obtained from patients with recurrent ocular toxoplasmosis were characterized phenotypically and functionally. METHODS: Ocular infiltrating T cells were recovered from vitreous fluid (VF) samples of 10 patients with active recurrent ocular toxoplasmosis. Two patients with uveitis of other origins were included as control subjects. T-cell lines (TCLs) were generated by mitogenic stimulation and tested for reactivity to Tg and human retinal protein extracts. The TCLs of three patients were cloned by limiting dilution. Tg-reactive T-cell clones (TCCs) were characterized with respect to their phenotype, T-cell receptor variable (TCR V)-beta gene usage, HLA restriction, and cytokine secretion profile. RESULTS: Reactivity to Tg could be detected only in the TCLs of patients with ocular toxoplasmosis. None of the TCLs showed reactivity to human retinal antigens. All tested intraocular Tg-specific TCCs (n = 23) were CD3+CD4+ and displayed differential TCR Vbeta usage. Twenty-one TCCs were HLA-DR restricted and two TCCs were restricted by HLA-DP. The majority of the intraocular Tg-specific TCCs showed a bias toward a T-helper (Th)0-Th2 cytokine profile. CONCLUSIONS: The data indicate that T cells specific for the triggering microorganism infiltrate the eye of patients with recurrent ocular toxoplasmosis. The functional characteristics of the VF-derived Tg-specific T cells and their presence at the site of inflammation suggest their involvement in the local inflammatory response of ocular toxoplasmosis.
Assuntos
Linfócitos T/imunologia , Toxoplasma/imunologia , Toxoplasmose Ocular/imunologia , Corpo Vítreo/imunologia , Adulto , Idoso , Animais , Complexo CD3/análise , Antígenos CD4/análise , Citocinas/metabolismo , Feminino , Antígenos HLA/análise , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Linfócitos T/fisiologia , Toxoplasmose Ocular/patologia , Toxoplasmose Ocular/fisiopatologia , Uveíte/imunologia , Uveíte/patologia , Uveíte/fisiopatologia , Corpo Vítreo/patologiaRESUMO
CD4(+) T cells play a major role in the host defense against viruses and intracellular microbes. During the natural course of such an infection, specific CD4(+) T cells are exposed to a wide range of antigen concentrations depending on the body compartment and the stage of disease. While epitope variants trigger only subsets of T-cell effector functions, the response of virus-specific CD4(+) T cells to various concentrations of the wild-type antigen has not been systematically studied. We stimulated hepatitis B virus core- and hepatitis C virus NS3-specific CD4(+) T-cell clones which had been isolated from patients with acute hepatitis during viral clearance with a wide range of specific antigen concentrations and determined the phenotypic changes and the induction of T-cell effector functions in relation to T-cell receptor internalization. A low antigen concentration induced the expression of T-cell activation markers and adhesion molecules in CD4(+) T-cell clones in the absence of cytokine secretion and proliferation. The expression of CD25, HLA-DR, CD69, and intercellular cell adhesion molecule 1 increased as soon as T-cell receptor internalization became detectable. A 30- to 100-fold-higher antigen concentration, corresponding to the internalization of 20 to 30% of T-cell receptor molecules, however, was required for the induction of proliferation as well as for gamma interferon and interleukin-4 secretion. These data indicate that virus-specific CD4(+) T cells can respond to specific antigen in a graded manner depending on the antigen concentration, which may have implications for a coordinate regulation of specific CD4(+) T-cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Hepacivirus/imunologia , Vírus da Hepatite B/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Clonais , Hepatite B/imunologia , Hepatite B/virologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Memória Imunológica , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/metabolismo , Proteínas não Estruturais Virais/imunologiaRESUMO
Interleukin 12 (IL-12) is a potent enhancer of interferon gamma production by activated T cells. The high-affinity IL-12 receptor (IL-12R) is a heterodimer of a beta1 and a beta2 subunit. Expression of the signaling IL-12Rbeta2 chain is usually low, as compared with the more abundant beta1 chain, and may be rate-limiting for IL-12 sensitivity. Little is known about the mechanisms controlling IL-12Rbeta2 gene expression. Reporter gene assays in IL-12Rbeta2-expressing Jurkat cells showed that truncation of the region from -151 to -61 abrogated promoter activity. The proximal promoter region does not contain a typical TATA box, suggesting a role for SP-1. Indeed, mutagenesis of the -63 SP-1 consensus site decreased transcription by 50%. Electrophoretic mobility shift experiments confirmed the binding of SP-1 and SP-3 at this site. In contrast, truncation of -252 to -192 increased promoter activity. Likewise, mutagenesis of the consensus nuclear factor of activated T cells site at -206 increased promoter activity by 70%, suggesting silencer activity of this element. Electrophoretic mobility shift experiments with primary Th (T helper) cells showed the formation of a specific, T-cell receptor-inducible complex at this site that is sensitive to cyclosporin A and supershifted with anti-NFATc2 in both Th1 and Th2 cells. Accordingly, cyclosporin A dose-dependently increased IL-12Rbeta2 mRNA expression. These first data on IL-12Rbeta2 gene regulation indicate a TATA-less promoter, depending on SP-1/SP-3 transcription factors, and a negative regulatory NFAT element at -206. This element may contribute to the overall low level of IL-12Rbeta2 expression on Th cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Inativação Gênica , Proteínas Nucleares , Podofilina/análogos & derivados , Regiões Promotoras Genéticas , Receptores de Interleucina/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica , Genes Reguladores , Humanos , Células Jurkat , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Podofilina/metabolismo , Podofilotoxina/análogos & derivados , Subunidades Proteicas , RNA Mensageiro/análise , Receptores de Interleucina-12RESUMO
IL-12 is a potent inducer of IFN-gamma production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling beta2-subunit of the IL-12R and, therefore, do not signal in response to IL-12. The question was raised as to what extent the loss of the IL-12Rbeta2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL-12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA-3 expression and induction of T-bet expression. These reversed cells are further characterized by a marked IL-12Rbeta2 chain expression and fully restored IL-12-inducible STAT4 activation. The IL-12-induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL-4 and in the absence of IL-12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen-specific Th2 cell clones. These findings suggest the possibility of restoring IL-12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL-12, and that IL-12-promoting immunotherapy can be beneficial for Th2-mediated immune disorders, targeting both naive and memory effector T cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interleucina-12/farmacologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Transativadores/metabolismo , Alérgenos/imunologia , Anticorpos Monoclonais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais/efeitos dos fármacos , Células Clonais/imunologia , Células Clonais/metabolismo , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Fator de Transcrição GATA3 , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipersensibilidade/imunologia , Imuno-Histoquímica , Imunoterapia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-4/imunologia , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Ativação Linfocitária , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Transativadores/genética , Fatores de Transcrição/genéticaAssuntos
Células Apresentadoras de Antígenos/metabolismo , Polaridade Celular/imunologia , Hipersensibilidade Imediata/fisiopatologia , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Humanos , Hipersensibilidade Imediata/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Linfócitos T Auxiliares-Indutores/imunologia , Células Th2/metabolismoAssuntos
Hipersensibilidade Imediata/fisiopatologia , Interleucina-4/genética , Transcrição Gênica/imunologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Humanos , Hipersensibilidade Imediata/genética , Hipersensibilidade Imediata/imunologia , Interleucina-4/biossíntese , Camundongos , Polimorfismo Genético , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Genetic lack of interleukin 12 receptor beta1 (IL-12Rbeta1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-gamma production. To study IL-12Rbeta1-independent residual IFN-gamma production, we have generated mycobacterium-specific T cell clones (TCCs) from IL-12Rbeta1-deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-gamma production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rbeta2 was found to be normally expressed in the absence of IL-12Rbeta1, and could be upregulated by IFN-alpha. Expression of IL-12Rbeta2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-alpha/IFN-alphaR ligation resulted in Stat4 activation in both control and IL-12Rbeta1-deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin alpha6, and IL-12Rbeta2 on IL-12Rbeta1-deficient cells, whereas this was normal on control cells. IL-12-induced IFN-gamma production in IL-12Rbeta1-deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway.Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rbeta1. Moreover, the results reveal the presence of a novel IL-12Rbeta1/Stat4-independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rbeta1 deficiency.
Assuntos
Interferon gama/biossíntese , Interleucina-12/imunologia , Infecção por Mycobacterium avium-intracellulare/imunologia , Receptores de Interleucina/fisiologia , Infecções por Salmonella/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Antígenos CD/metabolismo , Butadienos/farmacologia , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/farmacologia , Lactente , Integrina alfa6 , Interferon-alfa/metabolismo , Interleucina-18/metabolismo , Subunidade alfa de Receptor de Interleucina-18 , Interleucina-4/metabolismo , Masculino , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Receptor de Interferon alfa e beta , Receptores de Interferon/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Receptores de Interleucina-18 , Fator de Transcrição STAT4 , Transdução de Sinais , Células Th1/imunologia , Transativadores/genética , Transativadores/metabolismoRESUMO
IL-12 is a key inducer of Th1-associated inflammatory responses, protective against intracellular infections and cancer, but also involved in autoimmune tissue destruction. We report that human Th2 cells interacting with monocyte-derived dendritic cells (DC) effectively induce bioactive IL-12p70 and revert to Th0/Th1 phenotype. In contrast, the interaction with B cells preserves polarized Th2 phenotype. The induction of IL-12p70 in Th2 cell-DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2 cell-specific cofactor of IL-12p70 induction. Like IFN-gamma, IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-gamma at low concentrations of both cytokines. However, in contrast to IFN-gamma, IL-4 inhibits the CD40 ligand-induced production of inactive IL-12p40 and the production of either form of IL-12 induced by LPS, which may explain the view of IL-4 as an IL-12 inhibitor. The presently described ability of IL-4 to act as a cofactor of Th cell-mediated IL-12p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis.
Assuntos
Adjuvantes Imunológicos/fisiologia , Células Dendríticas/imunologia , Interleucina-12/biossíntese , Interleucina-4/fisiologia , Células Th2/imunologia , Células Th2/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40 , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Humanos , Imunofenotipagem , Interferon gama/fisiologia , Interleucina-12/farmacologia , Ligantes , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/imunologiaRESUMO
Dendritic cells (DC) are key initiators of primary immune responses. Myeloid DC can secrete IL-12, a potent Th1-driving factor, and are often viewed as Th1-promoting APC. Here we show that neither a Th1- nor a Th2-inducing function is an intrinsic attribute of human myeloid DC, but both depend on environmental instruction. Uncommitted immature DC require exposure to IFN-gamma, at the moment of induction of their maturation or shortly thereafter, to develop the capacity to produce high levels of IL-12p70 upon subsequent contact with naive Th cells. This effect is specific for IFN-gamma and is not shared by other IL-12-inducing factors. Type 1-polarized effector DC, matured in the presence of IFN-gamma, induce Th1 responses, in contrast to type 2-polarized DC matured in the presence of PGE2 that induce Th2 responses. Type 1-polarized effector DC are resistant to further modulation, which may facilitate their potential use in immunotherapy.
Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Monócitos/imunologia , Células Th1/citologia , Células Th1/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Dinoprostona/fisiologia , Humanos , Interferon gama/biossíntese , Interferon gama/fisiologia , Interleucina-12/biossíntese , Ativação Linfocitária , Monócitos/citologia , Monócitos/metabolismo , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The interleukin-12 receptor (IL-12R) is composed of two subunits, referred to as beta1 and beta2. Both chains are necessary for high-affinity IL-12 binding and signalling, although only the IL-12Rbeta2 chain contains the intracellular tyrosine residues responsible for STAT4 activation. This study presents the intron-exon organization of the human IL-12Rbeta2-chain gene. Polymerase chain reaction (PCR) primers designed across the cDNA (U46198) were used to trace introns, by comparing PCR product sizes obtained using cDNA and genomic DNA as templates. PCR products spanning introns were sequenced to determine the exact splice sites and flanking regions. The coding region of the gene was found to consist of 15 exons and 14 introns. All intron-exon boundaries are consistent with the consensus sequence for splice junctions (5' GT/AG 3'). Comparison of the intron-exon organization with the human GCSFR gene indicated a remarkably well conserved genomic organization between these two class I cytokine receptors. Interestingly, we identified an alternatively spliced mRNA, encoding a putative, truncated protein, lacking all signalling potential.
Assuntos
Éxons/genética , Íntrons/genética , Receptores de Interleucina/genética , Processamento Alternativo/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Códon de Terminação/genética , Sequência Conservada/genética , Primers do DNA/genética , DNA Complementar/genética , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Interleucina/química , Receptores de Interleucina/fisiologia , Receptores de Interleucina-12 , Deleção de Sequência/genética , Linfócitos T/metabolismo , Tirosina/genéticaAssuntos
Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Apresentação de Antígeno , Antígenos/imunologia , Babesia/imunologia , Diferenciação Celular , Movimento Celular , Células Dendríticas/classificação , Inflamação/imunologia , Leishmania major/imunologia , Linfonodos/imunologia , Linfocinas/metabolismo , Microglia/imunologia , Infecções por Protozoários/imunologia , Schistosoma mansoni/imunologiaRESUMO
How the T-cell receptor (TCR)/CD3 complex mediates positive as well as negative signals for T-cell regulation is not fully understood. We have previously described the induction of anergy in resting human T lymphocytes after mitogenic, high-dose CD3 triggering with monoclonal antibodies. Here we report the concomitantly occurring cell death to be largely caused by apoptosis, which mainly affects the CD4+ T-helper population. Because cell death becomes detectable after initial cell proliferation, it appears that a high-dose CD3 stimulus constitutes a negative signal for resting T cells leading to an 'abortive proliferation' that is followed by anergy and/or apoptosis of the cells. In contrast, if initial proliferation is induced by a low dose of anti-CD3 in the presence of an accessory signal via the CD28 receptor, anergy and cell death are markedly reduced and 'productive proliferation' may occur. Productive proliferation is characterized by an increased secretion of various cytokines measured (interleukin (IL)-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma)). A low-dose submitogenic CD3 stimulus induced neither anergy nor cell death, supporting the view that negative CD3 signalling requires proliferation of resting cells.
Assuntos
Antígenos CD28/imunologia , Complexo CD3/imunologia , Linfócitos T/imunologia , Apoptose/imunologia , Relação CD4-CD8 , Técnicas de Cultura de Células , Morte Celular/imunologia , Divisão Celular/imunologia , Humanos , Linfocinas/biossínteseRESUMO
Optimal clearance of the various pathogen types encountered by the human body requires the selective activation of particular cellular and/or humoral immune responses. The orchestration of the types of effector responses is directed by Th cells through the production of type 1 (Th1 cell-associated) and type 2 (Th2 cell-associated) cytokines. The way in which the Th cell cytokine profile is matched to the type of invading pathogen, and why these profiles sometimes derail and lead to disease, is not well understood. Here, we will discuss the concept that antigen-presenting cells (APC) provide Th cells not only with antigen and costimulatory signals, but also with a polarizing signal (signal 3). This signal can be mediated by many APC-derived factors, but IL-12 and PGE2 seem to be of major importance. The Th2-biased responses in atopic allergy appeared to be associated with monocytes with a decreased IL-12/PGE2 ratio and, consequently, with the down-regulation of type 1 cytokine production in Th cells. As for Th cells, APC can be functionialy polarized. In vitro experiments with monocyte-derived dendritic cells (DC) showed that the presence of IFN-gamma during activation of immature DC primes for mature DC with the ability of high IL-12 production and, consequently, a Th1-driving capacity (APC1 or DC1). In contrast, PGE2 primes for a low IL-12 production ability and a Th2-driving capacity (APC2 or DC2). These findings suggest that pathogens provoke either Th1- or Th2-cell development by inducing the production of a certain pattern of inflammatory DC-polarizing mediators (e.g. IFN-gamma and PGE2) at the site of infection. The type of immune polarization will not only depend on the type of pathogen, but also varies with the type of infected tissue, i.e. that different tissues produce different mediators in response to the same pathogen. In the case of atopic allergy, this concept implies that the Th2-cell bias may be related to low levels of cross-regulatory infections, to Th1 cell-inducing pathogens, or to an aberrant function of stromal cells in peripheral tissues.
Assuntos
Células Dendríticas/imunologia , Hipersensibilidade Imediata/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Citocinas/imunologia , HumanosRESUMO
Allergen-specific T cells in atopic patients are polarized IL-4-producing Th2 cells, promoting IgE synthesis by B cells. The molecular basis for increased IL-4 gene expression in atopy is not fully understood. IL-4 gene regulation in general involves the nuclear factor of activated T cells (NFAT) family of transcription factors, of which NFAT1 and NFAT2 are most prominent in peripheral T cells. Recently, a unique inhibitory role of NFAT1 in IL-4 gene control was shown in the mouse. In a series of electrophoretic mobility shift assays with protein extracts of highly polarized Th2 clones from atopics and Th1 clones from controls we compared DNA-binding activities at the two NFAT-binding elements P0 and P1 of the crucial proximal human IL-4 promoter. At the most proximal P0 site, NFAT-containing complexes devoid of NFAT2 were readily inducible in the Th1 clones, but hardly or not in the Th2 clones. In contrast, both in Th1 and Th2 clones NFAT-containing complexes were strongly inducible at the P1 site, consisting of NFAT2 and a P0-compatible NFAT activity, without apparent differences between Th1 and Th2 clones. Like in Th2 clones, suppressed NFAT-P0 complex formation was observed also at the polyclonal level in peripheral blood mononuclear cells (PBMC) of three of five severe atopic dermatitis patients with strongly elevated serum IgE levels, but not in control PBMC. These findings suggest that high-level IL-4 production in atopic Th2 cells is associated with selective reduction of suppressive NFAT1 activity at the IL-4 P0 element and that some patients with this multifactorial disease may have a putative systemic disorder at this level.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dermatite Atópica/imunologia , Regulação da Expressão Gênica , Interleucina-4/genética , Proteínas Nucleares , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Dermatite Atópica/genética , Poeira , Eletroforese/métodos , Humanos , Interleucina-4/metabolismo , Ativação Linfocitária , Camundongos , Ácaros/imunologia , Fatores de Transcrição NFATC , Regiões Promotoras Genéticas , Linfócitos T/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Transcrição GênicaRESUMO
Activation of immature CD83- dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1 beta and TNF-alpha-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-gamma. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-gamma, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-gamma R (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureus Cowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.
Assuntos
Antígenos de Bactérias/farmacologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Interferon gama/fisiologia , Interleucina-12/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Receptores de Interferon/antagonistas & inibidores , Receptores de Interferon/biossíntese , Staphylococcus aureus/imunologia , Receptor de Interferon gamaRESUMO
Glucocorticoids (GC) are known to affect the immune response at several stages. However, little is known about how GC influence the initiation of the specific immune response at the level of dendritic cells (DC), the highly professional APC for T cells. Therefore, we studied whether GC modulate the cytokine production and T cell stimulatory function of DC. In LPS-stimulated DC, GC strongly reduced the secretion of the Thl-skewing factor IL-12p70 and, to a lesser extent, the production of the proinflammatory cytokines IL-6 and TNF-alpha. Regarding the T cell stimulatory function of DC, GC did not influence the cell surface expression of HLA-DR or the costimulatory molecules CD40 and CD80 and did not influence the ability of DC to take up Ag. Consequently, GC pretreatment of DC indeed did not affect their ability to stimulate CD4+ Th cell proliferation in response to superantigen. However, as a result of their defective production of bioactive IL-12, GC-pretreated DC have a reduced ability to promote the production of IFN-gamma in CD4+ Th lymphocytes, as shown by the observation that IFN-gamma production could be restored by exogenous IL-12. In contrast, GC treatment of DC enhanced the secretion of the antiinflammatory cytokine IL-10 and the type 2 cytokine IL-5 by the T cells. It is concluded that, in addition to their role as potent inhibitors of inflammation via the direct suppression of cytokine production in T cells, GC may further inhibit T cell-mediated inflammation indirectly via the suppression of IL-12 production by DC.