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ABSTRACT: Blood-brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G-specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.
The Journal of Immunology 07/2012; 189(4):1937-45. · 5.79 Impact Factor
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ABSTRACT: The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS.
PIFS was induced by intravenous injection of VP2121-130 peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer.
C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2121-130 peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system.
C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.
Journal of Neuroinflammation 03/2012; 9:60. · 3.83 Impact Factor
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ABSTRACT: Recent evidence in multiple sclerosis (MS) suggests that active CMV infection may result in more benign clinical disease. The goal of this pilot study was to determine whether underlying murine CMV (MCMV) infection affects the course of the Theiler's murine encephalitis virus (TMEV) induced murine model of MS. A group of eight TMEV-infected mice were co-infected with MCMV at 2 weeks prior to TMEV infection while a second group of TMEV-infected mice received MCMV two weeks post TMEV. We also used 2 control groups, where at the above time points MCMV was replaced with PBS. Outcome measures included (1) monthly monitoring of disability via rotarod for 8 months; (2) in vivo MRI for brain atrophy studies and (3) FACS analysis of brain infiltrating lymphocytes at 8 months post TMEV infection. Co-infection with MCMV influenced the disease course in mice infected prior to TMEV infection. In this group, rotarod detectable motor performance was significantly improved starting 3 months post-infection and beyond (p≤0.024). In addition, their brain atrophy was close to 30% reduced at 8 months, but this was only present as a trend due to low power (p = 0.19). A significant reduction in the proportion of brain infiltrating CD3+ cells was detected in this group (p = 0.026), while the proportion of CD45+ Mac1+ cells significantly increased (p = 0.003). There was also a strong trend for a reduced proportion of CD4+ cells (p = 0.17) while CD8 and B220+ cell proportion did not change. These findings support an immunomodulatory effect of MCMV infection in this MS model. Future studies in this co-infection model will provide insight into mechanisms which modulate the development of demyelination and may be utilized for the development of novel therapeutic strategies.
PLoS ONE 01/2012; 7(2):e32767. · 4.09 Impact Factor
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Istvan Pirko, Yi Chen,
Anne K Lohrey,
Jeremiah McDole,
Jeffrey D Gamez,
Kathleen S Allen,
Kevin D Pavelko,
Diana M Lindquist,
R Scott Dunn,
Slobodan I Macura,
Aaron J Johnson
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ABSTRACT: MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.
PLoS ONE 01/2012; 7(2):e31459. · 4.09 Impact Factor
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ABSTRACT: A fundamental question in neuroimmunology is the extent to which CD8 T cells actively engage virus-infected neurons. In the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis, an effective central nervous system (CNS)-infiltrating antiviral CD8 T cell response offers protection from this demyelinating disease. However, the specific CNS cell types engaged by these protective CD8 T cells in TMEV-resistant strains remains unknown. We used confocal microscopy to visualize the morphology, migration, and specific cellular interactions between adoptively transferred CD8 T cells and specific CNS cell types. Adoptively transferred GFP+ CD8+ splenocytes migrated to the brain and became 93% specific for the immunodominant virus epitope D(b):VP2(121-130). These CD8 T cells also polarized T cell receptor, CD8 protein, and granzyme B toward target neurons. Furthermore, we observed CD8 T cells forming cytoplasmic processes up to 45 μm in length. Using live tissue imaging, we determined that these T cell-extended processes (TCEPs) could be rapidly formed and were associated with migratory behavior through CNS tissues. These studies provide evidence that antiviral CD8 T cells have the capacity to engage virus-infected neurons in vivo and are the first to document and measure the rapid formation of TCEPs on these brain-infiltrating lymphocytes using live tissue imaging.
American Journal Of Pathology 10/2010; 177(4):1823-33. · 4.89 Impact Factor
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ABSTRACT: Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.
The Journal of Immunology 12/2009; 184(2):1031-40. · 5.79 Impact Factor
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ABSTRACT: Advanced MRI studies demonstrated several diffuse non-lesional features in multiple sclerosis, including changes detectable in gray matter areas. Standard T2 weighted MRI scans of deep gray matter structures, including the thalamus, caudate, putamen, dentate nuclei often demonstrate hypointensity. T2 hypointensity has been shown to correlate with cognitive, neuropsychiatric and motor dysfunction. The exact pathogenesis of this MRI phenomenon remains unknown. In this manuscript, we demonstrate the first known MS animal model of deep gray matter T2 hypointensity. In TMEV infected SJL/J mice, gradual development of thalamic T2 hypointensity was noted over the disease course. Quantitative analysis of the hypointensity demonstrated a strong correlation between the degree of T2 hypointensity and rotarod detectable disability. We propose that this model will allow mechanistic studies investigating the pathogenesis and significance of deep gray matter T2 hypointensity in MS.
Journal of the neurological sciences 02/2009; 282(1-2):34-8. · 2.32 Impact Factor
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ABSTRACT: Defining the epitope specificity of CD8+ T cells is an important goal in autoimmune and immune-mediated disease research. We have developed a translational molecular approach to determine the epitope specificity of CD8+ T cells using the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS). TMEV-specific CD8+ T cells were isolated from brains and spleens of 7-day TMEV-infected C57BL/6J mice and stimulated by Cos-7 cells that were co-transfected with expression vectors encoding the D(b) class I molecule along with overlapping segments of the TMEV genome. Both brain-infiltrating and spleen-derived CD8+ T cells expressed IFN-gamma when Cos-7 cells were co-transfected with D(b) class I molecule and the TMEV genomic segment that encoded the immunodominant TMEV epitope. This demonstrated that peripheral and brain-infiltrating CD8+ T-cell responses were focused on peptide epitope(s) encoded by the same region of the TMEV genome. We propose that a similar molecular approach could also be used to determine the antigen specificity of suppressor CD8 T cells by the measurement of transforming growth factor-beta (TGF-beta) production. In addition, with a randomly generated library and peripheral blood or isolated CSF CD8+ T cells, this would be an effective method of predicting the epitope specificity of CD8+ T cells in human inflammatory CNS diseases, in animal models of MS or other organ-specific inflammatory diseases with a protective or pathogenic role of CD8 T cells.
Human Immunology 10/2008; 69(11):805-10. · 2.84 Impact Factor
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ABSTRACT: Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II(-/-) (CD4 T cell deficient), IFN-gammaR(-/-), TNF-alpha(-/-), TNFR1(-/-), TNFR2(-/-), and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTbeta activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability.
PLoS ONE 02/2008; 3(8):e3037. · 4.09 Impact Factor
