RESUMEN
BACKGROUND: Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). METHODS: Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. RESULTS: Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complex class II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. CONCLUSIONS: These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease.
Asunto(s)
Astrocitos/patología , Quimiocina CCL2/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Endotelio/patología , Imagenología Tridimensional , Microscopía Confocal , Animales , Sistema Nervioso Central/patología , Quimiocina CCL2/deficiencia , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Ratones , Ratones Noqueados , Microvasos/patología , Glicoproteína Mielina-Oligodendrócito , Fragmentos de PéptidosRESUMEN
BACKGROUND: There is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood-brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process. METHODS: To discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund's adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts. RESULTS: The CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments. CONCLUSIONS: MOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.
RESUMEN
Anterior Chamber-Associated Immune Deviation (ACAID) induced by an intracameral injection of antigen generates antigen-specific regulatory splenic T cells that suppress specifically cell-mediated immunity specific for the injected antigen. Circulating F4/80(+) cells recovered from mice receiving an intracameral injection of antigen are thought to be ocular in origin and induce the development of thymic and splenic regulatory T cells. We have shown previously that after the intracameral injection of antigen there is a CCR2/CCL2-dependent infiltration of circulating F4/80(+) cells into the anterior chamber associated with the generation of circulating, ACAID-inducing F4/80(+) monocytes. Here we tested the hypothesis that the intracameral injection of antigen induces events in the anterior chamber that are associated with the induction of circulating immunoregulatory monocytes that induce the suppression of cell-mediated immunity. The intracameral injection of antigen resulted in aqueous humor (i) a time- dependent increase of CCL2 and CCL7, (ii) a transient increase in TNF-α, and (iii) an infiltration of CD11b(hi), Gr1(hi) and F4/80(+) as well as F4/80(-) and Gr1(hi) peripheral blood cells into the anterior chamber. Further characterization of these F4/80(+) cells revealed that they are Ly 6C(hi), LY6G(lo) or negative, 7/4 (LY6B)(hi), CD115(+), CD45(+), CD49B(+), and CD62 L(+). Antibody-mediated neutralization of TGF-ß in situ in the anterior chamber prevented the induction of circulating, ACAID-inducing monocytes and ACAID. These cells did not increase in the irides of ACAID-refractory CCR2-/- and CCL2-/- mice that received an intracameral injection of antigen. Our results extend our suggestion that ACAID is initiated as the result of a mild proinflammatory response to intracameral injection that results in the infiltration of a CCR2(+) subset of monocytes into the anterior chamber where there is a TGF-ß-dependent induction of an immunosuppressive phenotype in the infiltrated monocytes that recirculate to induce antigen-specific regulatory T cells.
Asunto(s)
Cámara Anterior/inmunología , Antígenos de Diferenciación/inmunología , Hipersensibilidad Tardía/inmunología , Monocitos/inmunología , Análisis de Varianza , Animales , Cámara Anterior/citología , Humor Acuoso/citología , Humor Acuoso/metabolismo , Quimiocina CCL2/inmunología , Quimiocina CCL7/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BACKGROUND: CD8(+) suppressor T cells exert antigen-specific suppression of the expression of hypersensitivity by activated T cells. Therefore, CD8(+) suppressor T cells serve a major regulatory role for the control of active immunity. Accordingly, the number and/or activity of CD8(+) suppressor T cells should be influenced by an immune response to the antigen. To test this hypothesis we used an adoptive transfer assay that measures the suppression of the expression of delayed-type hypersensitivity (DTH) by CD8(+) suppressor T cells to quantify the antigen-specific suppression of DTH by these suppressor T cells. METHODS: Suppressor T cells were induced in the spleens of mice by the injection of antigen into the anterior chamber of an eye. Following this injection, the mice were immunized by the same antigen injected into the anterior chamber. Spleen cells recovered from these mice (AC-SPL cells) were titrated in an adoptive transfer assay to determine the number of AC-SPL cells required to effect a 50% reduction of antigen-induced swelling (Sw50) in the footpad of immunized mice challenged by antigen. RESULTS: Suppression of the expression of DTH is proportional to the number of AC-SPL cells injected into the site challenged by antigen. The number of AC-SPL cells required for a 50% reduction in DTH-induced swelling is reduced by injecting a cell population enriched for CD8(+) AC-SPL cells. Immunizing the mice receiving intracameral antigen to the same antigen decreases the RSw50 of AC-SPL cells required to inhibit the expression of DTH. CONCLUSIONS: The results provide the first quantitative demonstration that the numbers of antigen-specific splenic CD8(+) suppressor T cells are specifically amplified by antigen during an immune response.
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Linfocitos T CD8-positivos/inmunología , Traslado Adoptivo , Animales , Cámara Anterior , Antígenos/administración & dosificación , Femenino , Hipersensibilidad Tardía/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/citología , Bazo/inmunologíaRESUMEN
To determine the origin of peripheral blood mononulclear cells (PBMC) that activate regulatory T cells in anterior chamber-associated immune deviation (ACAID), fluorescein-labeled PBMC were intravenously injected into mice before the mice received an intracameral injection of antigen. Six-24 hr after intracameral injection, fluorescein-labeled PBMC increased in the iris. Twenty-four-48 hr labeled cells decreased in the iris and increased in the thymus and spleen. The entry of the labeled PBMC into the anterior chamber and subsequent production of PBMC that transfer ACAID required the expression of CCR2 by the PBMC and the production of the chemokine CCL2 by the recipient of the PBMC. The results suggest that the intracameral injection of antigen induces i) the infiltration of F4/80(+) PBMC into the AC, ii) where these PBMC are converted to a regulatory phenotype, and iii) recirculate to activate T cells that suppress cell-mediated immunity.
RESUMEN
The introduction of antigen into the anterior chamber (AC) of the eye induces the production of antigen-specific splenic CD8(+) regulatory T cells (AC-SPL cells) that suppress a delayed-type hypersensitivity (DTH) reaction in immunized mice. Because the generation of these regulatory T cells is also induced by exposure to transforming growth factor (TGF)-beta and antigen or F4/80(+) cells exposed to TGF-beta and antigen in vitro, we investigated (i) whether these cells are produced in dominant negative receptor for transforming growth factor beta receptor type II (dnTGFbetaRII) or Cbl-b(-/-) mice whose T cells are resistant to TGF-beta, (ii) whether DTH is suppressed by wild type (WT) CD8(+) AC-SPL cells in Cbl-b(-/-) and dnTGFbetaRII mice and (iii) the effect of antibodies to TGF-beta on the suppression of DTH by CD8(+) AC-SPL cells. DnTGFbetaRII immunized and Cbl-b(-/-) mice produced splenic CD8(+) regulatory cells after the intracameral injection of antigen and immunization. The suppression of a DTH reaction by CD8(+) AC-SPL cells in WT mice was blocked by the local inclusion of antibodies to TGF-beta when WT splenic CD8(+) AC-SPL cells were injected into the DTH reaction site. Moreover, the DTH reaction in immunized dnTGFbetaRII and Cbl-b(-/-) mice was not suppressed by the transfer of WT CD8(+) AC-SPL cells to the site challenged with antigen. In aggregate, these observations suggest that T cell sensitivity to TGF-beta is not an obligate requirement for the in vivo induction of CD8(+) AC-SPL T cells but the suppression of an in vivo DTH reaction by CD8(+) AC-SPL cells is dependent on TGF-beta.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Hipersensibilidad Tardía/inmunología , Proteínas Proto-Oncogénicas c-cbl/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Cámara Anterior/inmunología , Hipersensibilidad Tardía/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Proteínas Proto-Oncogénicas c-cbl/genética , Albúmina Sérica Bovina/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The injection of antigen into the anterior chamber (AC, intracameral injection) of a murine eye induces the generation of splenic CD8(+) regulatory T cells (AC-SPL cells) that effect the antigen-specific suppression of a delayed-type hypersensitivity (DTH) reaction. Here we show (i) for the first time that the local antigen-specific suppression of DTH-induced swelling in immunized mice either by an intracameral injection of antigen or by the direct injection of CD8(+) AC-SPL cells into an antigen-challenged site is associated with an absence of infiltrated mononuclear cells, (ii) that the local antigen-specific suppression of the DTH reaction by CD8(+) AC-SPL cells requires compatibility between the Qa-1 but not H2 antigen haplotype of the immunized recipient and the injected AC-SPL regulatory T cells, (iii) that the suppression of the DTH reaction by CD8(+) AC-SPL cells requires the expression of Qa-1 but not H2 antigens and is not due to bystander suppression.