RESUMEN
Experimental autoimmune encephalomyelitis is a CD4+ T cell-mediated demyelinating disease of the CNS that serves as a model for multiple sclerosis. Cytokines and chemokines shape Th1 and Th17 effector responses as well as regulate migration of leukocytes to the CNS during disease. The CNS cellular infiltrate consists of Ag-specific and nonspecific CD4+ and CD8+ T cells, neutrophils, B cells, monocytes, macrophages, and dendritic cells. The mechanism of immune-mediated inflammation in experimental autoimmune encephalomyelitis has been extensively studied in an effort to develop therapeutic modalities for multiple sclerosis and, indeed, has provided insight in modern drug discovery. The present Brief Review highlights critical pathogenic aspects of cytokines and chemokines involved in generation of effector T cell responses and migration of inflammatory cells to the CNS. Select cytokines and chemokines are certainly important in the regulatory response, which involves T regulatory, B regulatory, and myeloid-derived suppressor cells. However, that discussion is beyond the scope of this brief review.
Asunto(s)
Quimiocinas/inmunología , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Sistema Nervioso Central/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , Médula Espinal/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Proteínas de Unión al Calcio , Separación Celular , Quimiotaxis de Leucocito/inmunología , Citocinas/análisis , Citocinas/biosíntesis , Citocinas/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/inmunología , Médula Espinal/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease that is mediated, in part, by proinflammatory factors produced by RA synovial tissue (ST) fibroblasts and macrophages, resulting in monocyte migration from the blood to the ST. To characterize the potential role of IL-17 in monocyte migration, RA synovial fibroblasts and macrophages were activated with IL-17 and examined for the expression of monocyte chemokines. The two potentially important monocyte chemoattractants identified were CCL20/MIP-3alpha and CCL2/MCP-1, which were significantly induced in RA synovial fibroblasts and macrophages. However, in vivo, only CCL2/MCP-1 was detectable following adenovirus IL-17 injection. We found that IL-17 induction of CCL2/MCP-1 was mediated by the PI3K, ERK, and JNK pathways in RA ST fibroblasts and by the PI3K and ERK pathways in macrophages. Further, we show that neutralization of CCL2/MCP-1 significantly reduced IL-17-mediated monocyte recruitment into the peritoneal cavity. We demonstrate that local expression of IL-17 in ankle joints was associated with significantly increased monocyte migration and CCL2/MCP-1 levels. Interestingly, we show that RA synovial fluids immunoneutralized for IL-17 and CCL2/MCP-1 have similar monocyte chemotaxis activity as those immunoneutralized for each factor alone. In short, CCL2/MCP-1 produced from cell types present in the RA joint, as well as in experimental arthritis, may be responsible, in part, for IL-17-induced monocyte migration; hence, these results suggest that CCL2/MCP-1 is a downstream target of IL-17 that may be important in RA.
Asunto(s)
Movimiento Celular/inmunología , Quimiocina CCL2/biosíntesis , Interleucina-17/fisiología , Monocitos/inmunología , Monocitos/metabolismo , Animales , Articulación del Tobillo , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Quimiocina CCL2/fisiología , Enfermedad Crónica , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Interleucina-17/biosíntesis , Interleucina-17/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Traslado Adoptivo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Glicoproteína Mielina-OligodendrócitoAsunto(s)
Plaquetas/citología , Células de la Médula Ósea/citología , Mastocitos/citología , Subgrupos de Linfocitos T/citología , Plaquetas/fisiología , Células de la Médula Ósea/fisiología , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Humanos , Mastocitos/fisiología , Subgrupos de Linfocitos T/fisiologíaRESUMEN
There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC - VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.
Asunto(s)
Encéfalo/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Linfangiogénesis/inmunología , Vasos Linfáticos/inmunología , Linfocitos T/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos/inmunología , Antígenos/metabolismo , Encéfalo/diagnóstico por imagen , Proliferación Celular , Líquido Cefalorraquídeo/inmunología , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Hueso Etmoides , Azul de Evans/administración & dosificación , Femenino , Humanos , Vigilancia Inmunológica/inmunología , Vasos Linfáticos/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Glicoproteína Mielina-Oligodendrócito/inmunología , Toxina del Pertussis/administración & dosificación , Toxina del Pertussis/inmunología , Factor C de Crecimiento Endotelial Vascular/inmunología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: An endotoxin insult mimics a severe peripheral infection and recent evidence suggests that a single exposure can cause long-term cognitive deficits. A peripheral injection of LPS results in production of pro-inflammatory cytokines, such as IL-1beta and TNF-alpha, in the brain and periphery and these cytokines mediate many effects of the acute phase response including activation of the HPA axis. The chemokine MCP-1 is highly expressed during endotoxemia and although much is known about the importance of MCP-1 in peripheral inflammatory responses to LPS, information about MCP-1 and CNS responses to peripheral LPS is lacking. METHODS: C57Bl/6 mice were administered LPS by intraperitoneal (i.p.) injection, serum and brains were collected at several time points, and the time course of MCP-1 protein up-regulation was measured. To examine the role of MCP-1 in activation of the brain during acute systemic inflammation, we injected MCP-1 knockout (MCP-1-/-) or control C57Bl/6 (MCP-1+/+) mice with LPS i.p. and measured the levels of selected cytokines and chemokines in serum and brain extracts 6 hours later. Activated microglia were examined by CD45 immunohistochemistry, and serum corticosterone and ACTH levels were measured by enzyme immunoassay. RESULTS: We report that LPS injection induces a robust increase in MCP-1 protein levels in serum and brain, with peak brain levels reached at 6 hrs after LPS administration. MCP-1-/- mice injected with LPS showed higher levels of serum IL-1beta and TNF-alpha compared to LPS-treated MCP-1+/+ mice. In contrast, these MCP-1-/- mice showed significantly lower inductions of brain pro-inflammatory cytokines and chemokines, fewer activated microglia, and a reduction in serum corticosterone levels. CONCLUSION: MCP-1-/- mice have decreased brain inflammation after a peripheral LPS insult, despite an exaggerated peripheral response. These data demonstrate an important role for MCP-1 in regulation of brain inflammation after peripheral endotoxemia.
Asunto(s)
Quimiocina CCL2/metabolismo , Inflamación , Lipopolisacáridos , Hormona Adrenocorticotrópica/sangre , Animales , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/genética , Quimiocinas CC/inmunología , Corticosterona/inmunología , Femenino , Inflamación/inducido químicamente , Inflamación/inmunología , Interleucina-1beta/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated, autoimmune disease of the central nervous system (CNS) that serves as a model for various cellular and molecular aspects of the human disease, multiple sclerosis (MS). Although EAE has long been considered a T cell-mediated disease, macrophages play a role in disease pathogenesis and are known to accumulate in the CNS under the control of chemokines. In the present report we demonstrate that mice induced to develop EAE were treated with a small molecular weight molecule that suppresses proinflammatory cytokine production which resulted in significantly decreased clinical EAE, CNS CCL2 expression and CNS macrophage accumulation. These results demonstrate the efficacy of a novel class of therapeutic molecules for CNS demyelinating disease.
Asunto(s)
Quimiocina CCL2/antagonistas & inhibidores , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Piridazinas/farmacología , Pirimidinas/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Movimiento Celular/inmunología , Quimiocina CCL2/química , Femenino , Macrófagos/patología , Ratones , Peso Molecular , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Índice de Severidad de la EnfermedadRESUMEN
Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease, a murine model for multiple sclerosis, involves recruitment of T cells and macrophages to the CNS after infection. We hypothesized that CCR2, the only known receptor for CCL2, would be required for TMEV-induced demyelinating disease development because of its role in macrophage recruitment. TMEV-infected SJL CCR2 knockout (KO) mice showed decreased long-term clinical disease severity and less demyelination compared with controls. Flow cytometric data indicated that macrophages (CD45(high) CD11b(+) ) in the CNS of TMEV-infected CCR2 KO mice were decreased compared with control mice throughout disease. CD4(+) and CD8(+) T cell percentages in the CNS of TMEV-infected control and CCR2 KO mice were similar over the course of disease. There were no apparent differences between CCR2 KO and control peripheral immune responses. The frequency of interferon-gamma-producing T cells in response to proteolipid protein 139-151 in the CNS was also similar during the autoimmunity stage of TMEV-induced demyelinating disease. These data suggest that CCR2 is important for development of clinical disease by regulating macrophage accumulation after TMEV infection.
Asunto(s)
Infecciones por Cardiovirus/complicaciones , Enfermedades Desmielinizantes/etiología , Receptores de Quimiocina/fisiología , Theilovirus , Animales , Encéfalo/patología , Encéfalo/virología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/virología , Interferón gamma/biosíntesis , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR2 , Theilovirus/inmunología , Carga ViralRESUMEN
CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, a member of the CC chemokine family, is a chemoattractant for monocytes and T cells through interaction with its receptor CCR2. In the present study, we examined a T helper cell type 1 (Th1)-dependent disease, proteolipid protein-induced experimental autoimmune encephalomyelitis, in a transgenic mouse line that constitutively expressed low levels of CCL2 in the central nervous system (CNS) under control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed significantly milder clinical disease than littermate controls. As determined by flow cytometry, mononuclear cell infiltrates in the CNS tissues of CCL2 transgenic and littermate-control mice contained equal numbers of CD4+ and CD8+ T cells, and the CCL2 transgenic mice showed an enhanced number of CNS-infiltrating monocytes. CNS antigen-specific T cells from CCL2 transgenic mice produced markedly less interferon-gamma. Overexpression of CCL2 in the CNS resulted in decreased interleukin-12 receptor expression by antigen-specific T cells. Collectively, these results indicate that sustained, tissue-specific expression of CCL2 in vivo down-regulates the Th1 autoimmune response, culminating in milder clinical disease.
Asunto(s)
Sistema Nervioso Central/inmunología , Quimiocina CCL2/genética , Encefalomielitis Autoinmune Experimental/prevención & control , Animales , Sistema Nervioso Central/metabolismo , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/inmunología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Transgénicos , Proteína Proteolipídica de la Mielina/química , Proteína Proteolipídica de la Mielina/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated demyelinating disease of the central nervous system (CNS) that serves as a model for multiple sclerosis (MS). Insights into the pathogenesis of this model may help scientists understand the human disease and aid in rational drug discovery. In this review we summarize the role of chemokines and chemokine receptors in disease pathogenesis and suggest a pathway of events that leads to demyelination and subsequent clinical disease manifestation.
Asunto(s)
Quimiocinas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Receptores de Quimiocina/inmunología , Animales , Encefalomielitis Autoinmune Experimental/patología , HumanosRESUMEN
This article focuses on the distinct role of chemokines and chemokine receptors during CNS inflammation in experimental autoimmune encephalomyelitis (EAE) as an animal model for multiple sclerosis (MS). We review the evidence that chemokines and chemokine receptors have an intrinsic role in regulating and amplifying the inflammatory reactions in EAE or MS leading to disease outcome. A variety of studies examining temporal chemokine expression patterns, using chemokine and chemokine receptor knockout mice as well as administering passive anti-chemokine antibodies indicates that these molecules are critical regulatory components for leukocyte recruitment and/or leukocyte retention in the CNS. Therefore, chemokine and chemokine receptor expression is tightly interrelated to composition of inflammatory cells in CNS lesions and the onset of clinical diseases and provide viable targets for therapeutic intervention.
Asunto(s)
Quimiocinas/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Receptores de Quimiocina/fisiología , Animales , Sistema Nervioso Central/inmunología , Quimiocinas/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Esclerosis Múltiple/inmunología , Receptores de Quimiocina/genéticaRESUMEN
BACKGROUND: The classic immunophenotype for chronic lymphocytic leukemia (CLL) is CD19(+), restricted dim surface expression of kappa or lambda light chain, CD5(+), CD23(+), dim CD20(+), negative FMC7, and negative CD79b. However, the necessity of assaying for all 3 pan B-cell markers (CD20, FMC7, and CD79b) by flow cytometry has not been definitively documented for CLL. METHODS: Qualitative patterns and semi-quantitative assessment of staining intensity for CD20, FMC7 and CD79b were performed in 70 cases with a current or prior diagnosis of CLL or CLL with increased prolymphocytes leukemia (CLL/PL). The concurrent morphology in 66 of 70 specimens was classified as typical CLL in 53 cases, CLL/PL in 10 cases, and large cell lymphoma in 3 cases. RESULTS: Forty percent of the cases varied from the characteristic immunophenotype by having moderate or bright staining of CD20 (36%), FMC7 (7%), and/or CD79b (18%). Discrepant qualitative staining patterns were found between FMC7 and CD20 (21%), CD20 and CD79b (15%), and CD79b and FMC7 (10%). Semiquantitative measurement of staining intensity showed little correlation between CD79b and CD20 or FMC7. Moderate correlation was seen between CD20 and FMC7. No correlation was observed between morphology and intensity of marker expression. CONCLUSIONS: Variable patterns and intensity of staining were seen for FMC7, CD20, and CD79b in this cohort of CLL samples. Dim or negative staining was most consistently seen for FMC7 (93% of specimens). Although FMC7 staining intensity was moderately correlated with CD20, CD79b intensity was poorly correlated with either CD20 or FMC7, and thus, may provide some independent information.
Asunto(s)
Linfocitos B/inmunología , Biomarcadores de Tumor/inmunología , Inmunofenotipificación , Leucemia Linfocítica Crónica de Células B/clasificación , Leucemia Linfocítica Crónica de Células B/inmunología , Antígenos CD/análisis , Antígenos CD/inmunología , Antígenos CD20/análisis , Antígenos CD20/inmunología , Linfocitos B/patología , Biomarcadores de Tumor/análisis , Antígenos CD79 , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glicoproteínas/análisis , Glicoproteínas/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Reproducibilidad de los ResultadosRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a CD4 T cell-mediated demyelinating disease of the central nervous system (CNS) where macrophages are the end-stage effector cell. EAE serves as a model for multiple sclerosis where it has been instructive in delineating the autoimmune cellular response in the CNS for the purpose of developing more effective therapies. Understanding the nature of how cytokine and chemokine networks regulate the migration of leukocytes to the CNS requires the ability to track subpopulations of those cells in vivo. We describe a flow cytometric technique to monitor the migration of macrophages during EAE development.
Asunto(s)
Quimiocinas/metabolismo , Quimiotaxis , Encefalomielitis Autoinmune Experimental/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Receptores de Quimiocina/metabolismo , Traslado Adoptivo , Animales , Separación Celular/métodos , Rastreo Celular/métodos , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Citometría de Flujo , Macrófagos/inmunología , Macrófagos/trasplante , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Transducción de SeñalRESUMEN
PIMT (also known as PIPMT/NCOA6IP/Tgs1), first isolated as a transcription coactivator PRIP (NCOA6)-interacting 96-kDa protein with RNA-binding property, possesses RNA methyltransferase activity. As a transcription coactivator binding protein, PIMT enhances the nuclear receptor transcriptional activity and its methyltransferase property is involved in the formation of the 2,2,7-trimethylguanosine cap of non-coding small RNAs, but the in vivo functions of this gene have not been fully explored. To elucidate the biological functions, we used gene targeting to generate mice with a disrupted PIMT/Tgs1 gene. Disruption of PIMT gene results in early embryonic lethality due to impairment of development around the blastocyst and uterine implantation stages. We show that PIMT is expressed in all cells of the E3.5day blastocyst in the mouse. PIMT null mutation abolished PIMT expression in all cells of the blastocyst and caused a reduction in the expression of Oct4 and Nanog transcription factor proteins in the E3.5 blastocyst resulting in the near failure to form inner cell mass (ICM). With conditional deletion of PIMT gene, mouse embryonic fibroblasts (MEFs) exhibit defective wound healing in the scratch assay and a reduction in cell proliferation due to decreased G0/G1 transition and G2/M phase cell cycle arrest. We conclude that PIMT/NCOA6IP, which is expressed in all cells of the 3.5 day stage blastocyst, is indispensable for early embryonic development.
Asunto(s)
Blastocisto/metabolismo , Implantación del Embrión/genética , Desarrollo Embrionario/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Factores de Transcripción/genética , Útero/embriología , Secuencia de Aminoácidos , Animales , Masa Celular Interna del Blastocisto/metabolismo , Puntos de Control del Ciclo Celular/genética , Proliferación Celular , Femenino , Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Útero/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Chemokines and their receptors play a critical role in orchestrating the immune response during experimental autoimmune encephalomyelitis (EAE). Expression of CCR4 and its ligand CCL22 has been observed in ongoing disease. Here we describe a role for CCR4 in EAE, illustrating delayed and decreased disease incidence in CCR4(-/-) mice corresponding with diminished CNS infiltrate. Peripheral T cell responses were unaltered in CCR4(-/-) mice; rather, disease reduction was related to reduced CD11b(+)Ly6C(hi) inflammatory macrophage (iMÏ) numbers and function. These results provide evidence that CCR4 regulates EAE development and further supports the involvement of CCR4 in iMÏ effector function.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Mediadores de Inflamación/fisiología , Macrófagos/patología , Receptores CCR4/fisiología , Secuencia de Aminoácidos , Animales , Encefalomielitis Autoinmune Experimental/etiología , Femenino , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia MolecularRESUMEN
EAE is a demyelinating disease of the CNS and serves as a mouse model of MS. Expression of CCL22 in the draining LNs and spinal cord correlated with the onset of clinical EAE development and remained elevated. Administration of anti-CCL22 at the time of autoantigen immunization delayed the initiation of clinical disease and dampened the severity of peak initial disease and relapses. Reduced EAE severity correlated with the reduction of pathology and leukocytes in the CNS, particularly, activated CD11b+Ly6C(hi) macrophages. There were no differences in effector T cell-proliferative responses or effector T cell IFN-γ or IL-17 responses. However, treatment at the onset of disease did not reduce disease progression. Treatment of adoptive T cell transfer recipient mice with anti-CCL22 resulted in decreased clinical disease development accompanied by a decrease in CNS accumulation of CD11b+Ly6C(hi) macrophages. Neutralization of CCL22 resulted in a macrophage population whose effector cytokine expression consisted of decreased TNF and increased IL-10, a phenotype more consistent with M2 macrophages. This was corroborated by in vitro cultures of macrophages with CCL22. These results suggest that CCL22 functions to regulate development of EAE through macrophage chemoattraction and effector function.
Asunto(s)
Quimiocina CCL22/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Traslado Adoptivo , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Quimiocina CCL22/genética , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Epítopos/inmunología , Femenino , Inflamación/genética , Recuento de Linfocitos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. A protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.
Asunto(s)
Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/etiología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/inmunología , Proteolípidos/inmunologíaRESUMEN
Chemokines are a superfamily of chemotactic cytokines that play an important role in leukocyte trafficking and have been implicated as functional mediators of immunopathology in experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the role of the CCL20 receptor, CCR6, in chronic EAE. After immunization with myelin oligodendrocyte glycoprotein 35-55 in CFA, CCR6(-/-) mice developed a significantly more severe chronic EAE as compared to wild type immunized animals. CCR6 expression was not required by T cells to induce EAE. Measurement of peripheral T cell responses showed differences in IFN-gamma and IL-17 responses between CCR6(-/-) and wild type mice. At the time when CCR6(-/-) mice showed significantly more severe chronic EAE there was a significant decrease in PD-L1-expressing mDC in the spleens and no differences in Foxp3 Treg. Furthermore, add back of mDC with increased PD-L1 expression to CCR6(-/-) mice reduced the severe chronic EAE disease phase to that of wild type controls. The results suggest a role for CCR6-expressing PDL1(+) mDC in regulating EAE progression.