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-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ 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.
Highlights
Disruption of the blood brain barrier (BBB) is a hallmark feature of immunemediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis [1,2,3,4,5,6]
We did not observe overt differences in the number of isolated immune cells or frequencies of CD8+ cells among the three genotypes. These results demonstrated that the expansion of central nervous system (CNS) infiltrating Db:VP2121– 130 epitope specific CD8 T cells was not markedly different between C57BL/6, C57BL/6 Prf12/2 and C57BL/6 FasL2/2 mice post Theiler’s murine encephalomyelitis virus (TMEV) infection
The observation that CD8 T cells contribute to vascular permeability and utilize class I molecules to traffic to the CNS extends beyond the TMEV model put forth in this study [26]
Summary
Disruption of the BBB is a hallmark feature of immunemediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis [1,2,3,4,5,6]. While immune-mediated CNS vascular permeability is a likely contributor to pathology in neurologic disease, the role of CD8 T cells in BBB breakdown under inflammatory conditions remains largely undefined. Studies using EM have determined that the healthy, intact neurovascular unit (NVU) consists of cerebral endothelial cells (CECs), basal lamina, astrocytic endfoot processes, pericytes and neurons [7]. Among these cell types, pericytes and astrocytes have the most direct interaction with vasculature. Consistent with in vitro models, CNS vascular permeability coincides with alteration of CEC tight junctions in rodent models of BBB breakdown [3,14,15,16]
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