RESUMO
Here we show that activated Th1 and Th2 cells have distinct patterns of membrane compartmentalization into lipid rafts. TCR complex members are recruited efficiently to rafts and aggregate with rafts at the site of MHC/peptide contact in Th1 cells but not Th2 cells. TCR/raft association in Th1 cells is deficient in the absence of CD4, suggesting that CD4 aids recruitment of the TCR to rafts. We show differential utilization of rafts in Th1 and Th2 cells by cholesterol depletion studies, which alters calcium signaling in Th1 but not Th2 cells. Furthermore, Th2 cells have a decreased ability to respond to low-affinity peptide stimulation. These studies indicate that components of membrane microdomains are differentially regulated in functionally distinct CD4 T cells.
Assuntos
Células Th1/imunologia , Células Th2/imunologia , Animais , Antígenos CD4/química , Antígenos CD4/imunologia , Ativação Linfocitária , Lipídeos de Membrana/química , Lipídeos de Membrana/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Células Th1/química , Células Th2/químicaRESUMO
Initiation and propagation of T cell receptor signaling pathways involves the mobilization and aggregation of a variety of signaling intermediates with the T cell receptor and associated molecules into specialized signaling complexes. Accumulating evidence suggests that differential regulation of the formation and composition of the T cell receptor macromolecular signaling complex may affect the different biological consequences of T cell activation. The regulatory mechanisms involved in the assembly of these complexes remains poorly understood, but in part is affected by the avidity of the T cell receptor ligand, co-stimulatory signals, and by the differentiation state of the T cell.
Assuntos
Ativação Linfocitária/imunologia , Microdomínios da Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Antígenos CD4/imunologia , Diferenciação Celular , Humanos , Antígenos Comuns de Leucócito/imunologia , Substâncias Macromoleculares , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Linfócitos T/citologiaRESUMO
In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.
Assuntos
Linfócitos B/citologia , Linfócitos B/virologia , Linfócitos T CD4-Positivos/imunologia , Divisão Celular , Herpesvirus Humano 4/fisiologia , Adulto , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD8-Positivos/imunologia , Divisão Celular/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Contagem de Linfócitos , Receptores de IgE/metabolismo , Sorologia , Subpopulações de Linfócitos T/imunologia , Tacrolimo/farmacologiaRESUMO
Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4(-/-) Th2 cells were transferred into IFN-gammaR(-/-) mice, indicating that IFN-gamma was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-gamma to mice after IL-4(+/+) Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-gamma. IFN-gamma reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-gamma, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.
Assuntos
Brônquios/patologia , Regulação para Baixo/imunologia , Interferon gama/antagonistas & inibidores , Interferon gama/fisiologia , Interleucina-4/fisiologia , Eosinofilia Pulmonar/imunologia , Hipersensibilidade Respiratória/imunologia , Administração Cutânea , Administração Intranasal , Transferência Adotiva , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Movimento Celular/genética , Movimento Celular/imunologia , Regulação para Baixo/genética , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/prevenção & controle , Interferon gama/biossíntese , Interferon gama/metabolismo , Interleucina-13/biossíntese , Interleucina-4/deficiência , Interleucina-4/genética , Interleucina-5/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/patologia , Eosinofilia Pulmonar/prevenção & controle , Receptores de Interferon/fisiologia , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Hipersensibilidade Respiratória/prevenção & controle , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/transplante , Regulação para Cima/genética , Regulação para Cima/imunologia , Receptor de Interferon gamaRESUMO
This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL-2), and interleukin 4 (IL-4), and of human IL-2 and IL-4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The Basic Protocol describes the use of the CTLL-2 line to detect murine IL-2 and IL-4 in supernatants. One alternate protocol describes the detection of IL-2 in samples of human serum or supernatants using CTLL-2 cells, while other alternate procedures describe the detection and quantitation of murine IL-4 using a mutagenized subline of CTLL-2, CT.4S, and the detection of human IL-4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL-2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3-(4,5-dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Support Protocol 1). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL-2, CT.4S, and CT.h4S cell lines.
Assuntos
Bioensaio/métodos , Interleucina-2/análise , Interleucina-4/análise , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , CamundongosRESUMO
Diisocyanates are asthma-causing chemicals used in the commercial production of polyurethane. We have previously shown that human lung epithelial cell proteins can become conjugated with hexamethylene diisocyanate (HDI) and may be biologically important in diisocyanate-induced asthma. The objective of this study was to identify specific human lung and skin proteins that become conjugated with diisocyanate after in vitro and in vivo exposure. Following in vitro exposure of human airway epithelial cells (A549), keratin 18, the 78-kD glucose-regulated protein, trans-1, 2-dihyrobenzene-1,2-diol dehydrogenase, and actin were identified as prominent diisocyanate-conjugated proteins through use of a combination of immunocytochemical and mass spectrometric techniques. Following in vivo inhalation of an HDI aerosol, keratin 18 was also identified as the predominant diisocyanate-conjugated protein in human endobronchial biopsy samples, whereas albumin was the predominant diisocyanate-conjugated protein in bronchoalveolar lavage fluid. Keratin was also identified as a predominant diisocyanate-conjugated protein in human skin biopsy samples after epicutaneous exposure to liquid-phase HDI, although the major skin diisocyanate-conjugated protein (56-kD) differed from the predominant lung diisocyanate-conjugated keratin (47-kD). The data from this study identify keratin and other proteins as potential "carriers" for diisocyanates in vivo, and suggest that HDI conjugation of these proteins may play a role in the pathogenesis of diisocyanate-induced asthma.
Assuntos
Poluentes Ocupacionais do Ar/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Cianatos/farmacologia , Pulmão/efeitos dos fármacos , Proteínas/efeitos dos fármacos , Pele/efeitos dos fármacos , Adulto , Idoso , Poluentes Ocupacionais do Ar/efeitos adversos , Western Blotting/métodos , Western Blotting/estatística & dados numéricos , Células Cultivadas , Reagentes de Ligações Cruzadas/efeitos adversos , Cianatos/efeitos adversos , Eletroforese em Gel de Poliacrilamida/métodos , Eletroforese em Gel de Poliacrilamida/estatística & dados numéricos , Células Epiteliais/química , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Isocianatos , Pulmão/química , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/análise , Ligação Proteica/efeitos dos fármacos , Proteínas/análise , Proteínas/metabolismo , Pele/química , Pele/metabolismoRESUMO
By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO(+), but not CD45ABC(+), T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO(+) expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO(+) T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC(+) T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.
Assuntos
Proteínas de Ligação a DNA , Antígenos Comuns de Leucócito/metabolismo , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Ativação Linfocitária , Modelos Biológicos , Isoformas de Proteínas , Proteínas Proto-Oncogênicas , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Proteínas Elk-1 do Domínio etsRESUMO
To study whether changes in the structure of a T cell receptor (TCR) at a single peptide-contacting residue could affect T cell priming with antigenic peptide, we made transgenic mice with a point mutation in the TCR alpha chain of the D10.G4.1 (D10) TCR and bred them to D10 beta chain transgenic mice. The mutation consisted of a leucine to serine substitution at position 51 (L51S), which we had already established contacted the second amino acid of the peptide such that the response to the reference peptide was reduced by approximately 100-fold. A mutation in the reference peptide CA134-146 (CA-WT) from the arginine at peptide position 2 to glycine (R2G) restored full response to this altered TCR. When we examined in vitro priming of naive CD4 T cells, we observed that the response to doses of CA-WT that induced T helper cell type 1 (Th1) responses in naive CD4 T cells from mice transgenic for the D10 TCR gave only Th2 responses in naive CD4 T cells derived from the L51S. However, when we primed the same T cells with the R2G peptide, we observed Th1 priming in both D10 and L51S naive CD4 T cells. We conclude from these data that a mutation in the TCR at a key position that contacts major histocompatibility complex-bound peptide is associated with a shift in T cell differentiation from Th1 to Th2.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Imunidade Celular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Células Th1/imunologia , Células Th2/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Interferon gama/biossíntese , Interleucina-4/biossíntese , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação , Peptídeos/imunologia , Células Th1/citologia , Células Th2/citologiaRESUMO
Delivery of foreign antigens to mucosal surfaces, such as the pulmonary airways, has been shown to preferentially induce Th2-mediated responses in humans and in mice. What is not clear from these studies is whether this preferential skewing in responses is the result of the limited types of antigen being administered and/or a bias towards using particular genetic strains of mice, or whether the lung environment in itself provides a favored site for the priming of Th2-type cells. We have addressed this issue using an antigen/mouse strain combination that, under typical conditions of immunization, is strongly biased towards priming for Th1 CD4+ T cells. We show that Leishmania major parasites delivered to C57BL/6 mice via an intranasal route fail to induce the expected Th1-dominated responses and instead preferentially prime for Th2 responses. These included an influx in lymphocytes and eosinophils into alveoli, as well as the induction of Th2-type foci of inflammation around pulmonary blood vessels and airways. Moreover, high levels of Th2-associated cytokines (IL-4 and IL-5) were generated when lung-draining lymph node and tissue cells were restimulated with L. major lysate. These data suggest that the lung environment per se favors Th differentiation towards the Th2 phenotype.
Assuntos
Antígenos/administração & dosagem , Pulmão/citologia , Pulmão/imunologia , Linfócitos T/imunologia , Administração Intranasal , Animais , Antígenos de Protozoários/administração & dosagem , Diferenciação Celular , Citocinas/metabolismo , Humanos , Injeções Subcutâneas , Leishmania major/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Linfócitos T/citologia , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologiaRESUMO
Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL-4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization.
Assuntos
Hipersensibilidade Imediata/imunologia , Interleucina-4/fisiologia , Ovalbumina/administração & dosagem , Células Th2/imunologia , Administração Cutânea , Administração por Inalação , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Eosinófilos/imunologia , Feminino , Imunização , Interleucina-13/antagonistas & inibidores , Interleucina-13/fisiologia , Interleucina-4/deficiência , Interleucina-4/genética , Pulmão/metabolismo , Pulmão/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologiaRESUMO
CD4 T helper (Th) type 1 and Th2 cells have been identified in the airways of asthmatic patients. Th2 cells are believed to contribute to pathogenesis of the disease, but the role of Th1 cells is not well defined. In a mouse model, we previously reported that transferred T cell receptor-transgenic Th2 cells activated in the respiratory tract led to airway inflammation with many of the pathologic features of asthma, including airway eosinophilia and mucus production. Th1 cells caused inflammation with none of the pathology associated with asthma. In this report, we investigate the role of Th1 cells in regulating airway inflammation. When Th1 and Th2 cells are transferred together into recipient mice, there is a marked reduction in airway eosinophilia and mucus staining. To address the precise role of Th1 cells, we asked (i), Are Th2-induced responses inhibited by interferon (IFN)-gamma? and (ii) Can Th1 cells induce eosinophilia and mucus in the absence of IFN-gamma? In IFN-gamma receptor(-/-) recipient mice exposed to inhaled antigen, the inhibitory effects of Th1 cells on both airway eosinophilia and mucus production were abolished. In the absence of IFN-gamma receptor signaling, Th1 cells induced mucus but not eosinophilia. Thus, we have identified new regulatory pathways for mucus production; mucus can be induced by Th2 and non-Th2 inflammatory responses in the lung, both of which are inhibited by IFN-gamma. The blockade of eosinophilia and mucus production by IFN-gamma likely occurs through different inhibitory pathways that are activated downstream of Th2 cytokine secretion and require IFN-gamma signaling in tissue of recipient mice.
Assuntos
Hipersensibilidade/imunologia , Inflamação/imunologia , Interferon gama/imunologia , Pulmão/imunologia , Receptores de Interferon/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Asma/imunologia , Modelos Animais de Doenças , Eosinofilia/imunologia , Citometria de Fluxo , Interferon gama/farmacologia , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Muco/imunologia , Ovalbumina/imunologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
The development of peripheral naive CD4 T cells is dependent on the success of positive selection of immature T cells in the thymus. Only thymocytes that express a T cell receptor (TCR) capable of recognizing self-MHC with low affinity are selected for survival and differentiation into mature naive T cells. Although the TCR of naive T cells has to maintain self-tolerance, it also propagates naive CD4 T cell proliferation on recognition of appropriate foreign peptide associated with MHC class II on antigen-presenting cells (APCs). Naive CD4 T cells that successfully engage foreign peptide undergo further differentiation that leads to the maturation of a select few into the memory T cell pool. Although the requirements that lead to memory T cell development are currently not known, functional changes have been described that are thought to be associated with the greater efficiency with which memory T cells respond to antigen. This article will discuss differences associated with signaling through the TCR of naive and memory CD4 T cells and describe unique control mechanisms imposed on memory CD4 T cells that are likely to have ari sen to counterbalance the altered TCR signaling.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunoconjugados , Memória Imunológica , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Abatacepte , Animais , Células Apresentadoras de Antígenos/fisiologia , Antígenos CD , Antígenos de Diferenciação/fisiologia , Antígeno CTLA-4 , Diferenciação Celular , Camundongos , Linfócitos T Citotóxicos/fisiologiaRESUMO
The regulation of naive T cell development into different effector cell subsets is mediated by a complex interplay between the cytokine microenvironment, receptor ligand interactions on the T cell and the antigen presenting cell, and the potency of T cell receptor (TCR) signaling. In this review we will focus on how alterations in the strength of TCR ligation initiate different signal transduction patterns which regulate the developmental fate of naive T cells. We propose a model in which specific signals are required to initiate Th2 differentiation, but that this pathway can be inhibited following a strong TCR stimulus.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/citologia , Animais , Humanos , Transdução de Sinais/imunologiaRESUMO
CD45 is the predominant transmembrane tyrosine phosphatase in lymphocytes and is required for the efficient induction of T cell receptor signaling and activation. However, the regulation of CD45 activity and substrate specificity are poorly understood. In the present study, we demonstrate a basal biochemical association of CD45 with the T cell receptor complex that is regulated in part by CD45 isoform expression. Further, maintenance of CD45/TCR association is differentially regulated following TCR ligation with peptide: a partial agonist peptide induces CD45/TCR dissociation while an agonist peptide promotes sustained association in a CD4-dependent manner. These data suggest that T cell receptor signaling pathways may be modulated by altering access of CD45 to TCR-associated substrates involved in T cell activation.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/química , Citoplasma , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/química , Ligantes , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/imunologia , Ligação Proteica/imunologia , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Células Tumorais CultivadasRESUMO
Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4-/- Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4-/- Th2 cells are transferred into IL-4Ralpha-/- mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5-/- Th2 cells were transferred into IL-5-/- recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Ralpha is critically important in Th2 cell stimulation of mucus production.
Assuntos
Asma/imunologia , Brônquios/imunologia , Muco/metabolismo , Receptores de Interleucina-4/metabolismo , Células Th2/imunologia , Administração por Inalação , Animais , Eosinofilia/imunologia , Eosinófilos/imunologia , Subunidade alfa1 de Receptor de Interleucina-13 , Interleucina-4/imunologia , Interleucina-5/metabolismo , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores de Interleucina/metabolismo , Receptores de Interleucina-13 , Células Th2/transplanteRESUMO
The diversity of naive CD4 T cells plays an important role in the adaptive immune response by ensuring the capability of responding to novel pathogens. In the past, it has been generally accepted that naive CD4 T cells are intrinsically long-lived; however, there have been studies suggesting some CD4 T cells are short-lived. In this report, we identify two populations of naive CD4 T cells: a long-lived population as well as a short-lived population. In addition, we identify two factors that contribute to the establishment of long-lived naive CD4 T cells. We confirm earlier findings that MHC class II interaction with the TCR on CD4 T cells is important for survival. Furthermore, we find that MHC class II alleles with the correct restriction element for Ag presentation mediate the peripheral survival of naive CD4 T cells more efficiently than other positively selecting alleles, regardless of the selecting MHC in the thymus. The second component contributing to the survival of naive CD4 T cells is contact with the cytokines IL-4 and IL-7. We find that the physiological levels of IL-4 and IL-7 serve to enhance the MHC class II-mediated survival of naive CD4 T cells in vivo.
Assuntos
Linfócitos T CD4-Positivos/citologia , Citocinas/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Adjuvantes Imunológicos/fisiologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular/imunologia , Deleção Clonal , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-4/fisiologia , Interleucina-7/fisiologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/imunologia , Peptídeos/metabolismo , TimectomiaAssuntos
Células Dendríticas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Citocinas/fisiologia , Células Dendríticas/citologia , Humanos , Interleucina-12/fisiologia , Interleucina-4/fisiologia , Ativação Linfocitária , Modelos Imunológicos , Células Th1/citologia , Células Th2/citologiaRESUMO
The role of STAT (signal transducer and activator of transcription) proteins in T cell receptor (TCR) signaling was analyzed. STAT5 became immediately and transiently phosphorylated on tyrosine 694 in response to TCR stimulation. Expression of the protein tyrosine kinase Lck, a key signaling protein in the TCR complex, activated DNA binding of transfected STAT5A and STAT5B to specific STAT inducible elements. The role of Lck in STAT5 activation was confirmed in a Lck-deficient T cell line in which the activation of STAT5 by TCR stimulation was abolished. Expression of Lck induced specific interaction of STAT5 with the subunits of the TCR, indicating that STAT5 may be directly involved in TCR signaling. Stimulation of T cell clones and primary T cell lines also induced the association of STAT5 with the TCR complex. Inhibition of STAT5 function by expression of a dominant negative mutant STAT5 reduced antigen-stimulated proliferation of T cells. Thus, TCR stimulation appears to directly activate STAT5, which may participate in the regulation of gene transcription and T cell proliferation during immunological responses.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Ativação Linfocitária , Proteínas do Leite , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Transativadores/metabolismo , Animais , Anticorpos , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Divisão Celular , Linhagem Celular , Proteínas de Ligação a DNA/genética , Interferon gama/farmacologia , Interleucina-2/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT5 , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Transativadores/genética , TransfecçãoRESUMO
The reliable identification of naive and memory CD4 T cells is critical to understanding the cellular basis of immunological memory. However, it has long been a controversial issue whether naive and memory phenotypes are stable among resting CD4 T cells in the absence of overt stimulation or whether the proposed memory phenotype is a transient, reversible one that represents recently activated cells. In this study, adoptively transferred, purified populations of naive or memory phenotype CD4 T cells are monitored over time to assess the stability of phenotypes and the functional capabilities of transferred cells. Studying both TCR transgenic and nontransgenic CD4 T cell populations allows one to control for the capacity to respond to environmental Ags in vivo. Several findings are reported. The first is that in the absence of Ag, both naive and memory phenotypes remain unchanged over time. Second, when changes are seen in populations of transferred naive phenotype CD4 T cells, they take place only when there is a potential for antigenic challenge, suggesting that it is an Ag-driven event. Furthermore, when a change from naive to memory phenotype is observed, these transferred donor cells also function as memory cells. Third, the ability of memory CD4 T cells to retain the memory phenotype is independent of specific Ag.