RESUMO
The bloodbrain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.
Assuntos
Encéfalo/patologia , Movimento Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Pulmão/patologia , Linfócitos T/patologia , Transferência Adotiva , Animais , Autoimunidade/imunologia , Barreira Hematoencefálica/imunologia , Encéfalo/citologia , Encéfalo/imunologia , Moléculas de Adesão Celular Neuronais/metabolismo , Circulação Cerebrovascular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Memória Imunológica , Pulmão/citologia , Pulmão/imunologia , Ativação Linfocitária , Bainha de Mielina/imunologia , Fatores de Crescimento Neural/metabolismo , Ratos , Ratos Endogâmicos Lew , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.
Assuntos
Doenças do Sistema Nervoso Central/imunologia , Doenças do Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Meninges/irrigação sanguínea , Meninges/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Movimento Celular , Células Cultivadas , Meninges/patologia , Camundongos , Ovalbumina/imunologia , Fagócitos/imunologia , Ratos , Ratos Endogâmicos LewRESUMO
Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.
Assuntos
Citomegalovirus/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células-Tronco Embrionárias/citologia , Epigênese Genética , Proteínas Nucleares/química , Regiões Promotoras Genéticas , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cricetinae , Cricetulus , Metilação de DNA , DNA Metiltransferase 3A , Inativação Gênica , Humanos , Metiltransferases/química , Camundongos , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases , DNA Metiltransferase 3BRESUMO
Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate/calcium signalling pathway is essential for the recruitment and the activation of autoaggressive effector T cells within their target organ. Interference with this signalling pathway suppresses the formation of autoimmune inflammatory lesions and thus might qualify as a novel strategy for the treatment of T cell mediated autoimmune diseases.
Assuntos
Sinalização do Cálcio/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , NADP/análogos & derivados , Subpopulações de Linfócitos T/patologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Encefalomielite Autoimune Experimental/metabolismo , NADP/antagonistas & inibidores , NADP/fisiologia , Ácidos Nicotínicos/farmacologia , Ratos , Ratos Endogâmicos Lew , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismoRESUMO
The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)-specific T cells in Lewis rats, whereas transfer of S100beta- or myelin oligodendrocyte glycoprotein (MOG)-specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein(+) T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoantígenos/metabolismo , Linhagem Celular , Sistema Nervoso Central/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/etiologia , Proteínas de Fluorescência Verde , Mediadores da Inflamação/metabolismo , Proteínas Luminescentes/genética , Ativação Linfocitária , Especificidade de Órgãos , Fenótipo , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes/genéticaRESUMO
Treatment with glatiramer acetate (GA) is thought to induce an in vivo change of the cytokine secretion pattern and the effector function of GA-reactive T helper (TH) cells (TH1-TH2-shift). Current theories propose that GA-reactive TH2 cells can penetrate the CNS, since they are activated by daily immunization. Inside the CNS, GA-reactive T cells may cross-react with products of the local myelin turnover presented by local antigen-presenting cells (APCs). Thus, some of the GA-specific TH2 cells may be stimulated to release anti-inflammatory cytokines inhibiting neighbouring inflammatory cells by a mechanism called bystander suppression. We demonstrate that both GA-specific TH2 and TH1 cells produce the neurotrophin brain-derived neurotrophic factor (BDNF). To demonstrate that GA-reactive T cells produce BDNF, we analyzed GA-specific, long-term T-cell lines (TCLs) and used a combination of reverse-transcription PCR and two specially designed techniques for BDNF protein detection: one was based on ELISA of supernatants from co-cultures of GA-specific TCLs plus GA-pulsed antigen-presenting cells, and the other, on the direct intracellular staining of BDNF in individual T cells and flow-cytometric analysis. The different assays and different TCLs yielded similar, consistent results. All GA-specific TH1, TH2 and TH0 lines could be stimulated to produce BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Peptídeos/farmacologia , Células Th2/efeitos dos fármacos , Northern Blotting/métodos , Fator Neurotrófico Derivado do Encéfalo/genética , Ensaio de Imunoadsorção Enzimática/métodos , Acetato de Glatiramer , Humanos , Modelos Biológicos , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Th2/metabolismoRESUMO
OBJECTIVES: To characterize phenotypes of T cells that accumulated in multiple sclerosis (MS) lesions, to compare the lesional T-cell receptor (TCR) repertoire of T-cell subsets to peripheral blood, and to identify paired α and ß chains from single CD8(+) T cells from an index patient who we followed for 18 years. METHODS: We combined immunohistochemistry, laser microdissection, and single-cell multiplex PCR to characterize T-cell subtypes and identify paired TCRα and TCRß chains from individual brain-infiltrating T cells in frozen brain sections. The lesional and peripheral TCR repertoires were analyzed by pyrosequencing. RESULTS: We found that a TCR Vß1(+) T-cell population that was strikingly expanded in active brain lesions at clinical onset comprises several subclones expressing distinct yet closely related Vα7.2(+) α chains, including a canonical Vα7.2-Jα33 chain of mucosal-associated invariant T (MAIT) cells. Three other α chains bear striking similarities in their antigen-recognizing, hypervariable complementarity determining region 3. Longitudinal repertoire studies revealed that the TCR chains that were massively expanded in brain at onset persisted for several years in blood or CSF but subsequently disappeared except for the canonical Vα7.2(+) MAIT cell and a few other TCR sequences that were still detectable in blood after 18 years. CONCLUSIONS: Our observation that a massively expanded TCR Vß1-Jß2.3 chain paired with distinct yet closely related canonical or atypical MAIT cell-related α chains strongly points to an antigen-driven process in early active MS brain lesions.
RESUMO
Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.
Assuntos
Neoplasias Encefálicas/secundário , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/ultraestrutura , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Melanoma/patologia , Melanoma/ultraestrutura , Camundongos , Camundongos Nus , Microscopia Confocal/métodos , Metástase Neoplásica/ultraestrutura , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
i.v. infusion of native autoantigen or its altered peptide variants is an important therapeutic option for the treatment of autoimmune diseases, because it selectively targets the disease-inducing T cells. To learn more about the mechanisms and kinetics of this approach, we visualized the crucial initial effects of i.v. infusion of peptides or intact protein on GFP-tagged autoaggressive CD4(+) effector T cells using live-video and two-photon in situ imaging of spleens in living animals. We found that the time interval between i.v. injection of intact protein to first changes in T cell behavior was extremely short; within 10 min after protein application, the motility of the T cells changed drastically. They slowed down and became tethered to local sessile stromal cells. A part of the cells aggregated to form clusters. Within the following 20 min, IFN-gamma mRNA was massively (>100-fold) up-regulated; surface IL-2 receptor and OX-40 (CD 134) increased 1.5 h later. These processes depleted autoimmune T cells in the blood circulation, trapping the cells in the peripheral lymphoid organs and thus preventing them from invading the CNS. This specific blockage almost completely abrogated CNS inflammation and clinical disease. These findings highlight the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity.
Assuntos
Autoantígenos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Imunoterapia , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Autoantígenos/administração & dosagem , Autoantígenos/uso terapêutico , Movimento Celular , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/patologia , Regulação da Expressão Gênica , Injeções Intravenosas , Cinética , Ativação Linfocitária/imunologia , Proteína Básica da Mielina/administração & dosagem , Proteína Básica da Mielina/imunologia , Proteína Básica da Mielina/uso terapêutico , Ratos , Solubilidade , Linfócitos T/citologia , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
RhoH is a small GTPase expressed only in the hematopoietic system. With the use of mice with targeted disruption of the RhoH gene, we demonstrated that RhoH is crucial for thymocyte maturation during DN3 to DN4 transition and during positive selection. Furthermore, the differentiation and expansion of DN3 and DN4 thymocytes in vitro were severely impaired. These defects corresponded to defective TCR signaling. Although RhoH is not required for TCR-induced activation of ZAP70 and ZAP70-mediated activation of p38, it is crucial for the tyrosine phosphorylation of LAT, PLCgamma1, and Vav1 and for the activation of Erk and calcium influx. These data suggest that RhoH is important for pre-TCR and TCR signaling because it allows the efficient interaction of ZAP70 with the LAT signalosome, thus regulating thymocyte development.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Biomarcadores , Antígenos CD18/metabolismo , Cálcio/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Hematopoese , Camundongos , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/imunologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The clinical effects of glatiramer acetate (GA), an approved therapy for multiple sclerosis, are thought to be largely mediated by a T-helper 1 (TH1) to T-helper 2 (TH2) shift of GA-reactive T-lymphocytes. Current theories propose that activated GA-reactive TH2 cells penetrate the CNS, release anti-inflammatory cytokines such as interleukin (IL)-4, IL-5 and IL-10, and thus inhibit neighbouring inflammatory cells by a mechanism termed 'bystander suppression'. We demonstrate that both GA-specific TH2 and TH1 cells produce the neurotrophin brain-derived neurotrophic factor (BDNF). As the signal-transducing receptor for BDNF, the full-length 145 tyrosine kinase receptor (trk) B, is expressed in multiple sclerosis lesions, it is likely that the BDNF secreted by GA-reactive TH2 and TH1 has neurotrophic effects in the multiple sclerosis target tissue. This may be an additional mechanism of action of GA, and may be relevant for therapies with altered peptide ligands in general. To demonstrate that GA-reactive T cells produce BDNF, we selected four GA-specific, long-term T-cell lines (TCLs), which were characterized according to their cytokine profile by intracellular double-fluorescence flow cytometry. Three TCLs (isolated from a normal subject) had the phenotypes TH1, TH1/TH0, and TH0; the fourth, derived from a GA-treated patient, had the phenotype TH2. To demonstrate BDNF production, we used a combination of RT-PCR (reverse transcription-polymerase chain reaction) and two specially designed techniques for BDNF protein detection: one was based on ELISA (enzyme-linked immunosorbent assay) of supernatants from co-cultures of GA-specific TCLs plus GA-pulsed antigen-presenting cells, and the other on the direct intracellular staining of BDNF in individual T cells and flow cytometric analysis. The different assays and different TCLs yielded similar, consistent results. All four GA-specific T-cell lines, representing the major different TH phenotypes, could be stimulated to produce BDNF.