A new angle on blood–CNS interfaces: A role for connexins?

Neuronal signaling in the CNS depends on the microenvironment around synapses and axons. To prevent fluctuations in blood composition affecting the interstitial fluid and CSF, two barriers, the blood–brain barrier (BBB) and blood–CSF barrier (BCSFB), are interposed between the blood and the brain/CSF compartment. Brain capillary endothelial cells (ECs) constitute the BBB whereas choroid plexus epithelial (CPE) cells form the BCSFB. The anatomical basis of these barriers is located at the level of an intercellular junctional complex that impedes paracellular diffusion. Tight and adherens junctions are known as the principal constituents of this junctional complex. Transmembrane connexins (Cxs) are the prime building blocks of plasma membrane hemichannels that combine to form intercellular gap junctions (GJ). Although Cxs co-exist within the junctional complex, their influence on tight/adherens junctions and their role in barrier function of BBB ECs and CPE has been mostly ignored. Here, we review current knowledge on the role of Cxs in the BBB, BCSFB and other interfaces that subside within the CNS. We conclude that Cxs are a rather unexplored but promising target for influencing CNS barrier function.     

Development of the blood-brain barrier

The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over the substances that leave or enter the brain. An elaborate network of complex tight junctions (TJ) between the endothelial cells forms the structural basis of the BBB and restricts the paracellular diffusion of hydrophilic molecules. Additonally, the lack of fenestrae and the extremely low pinocytotic activity of endothelial cells of the BBB inhibit the transcellular passage of molecules across the barrier. On the other hand, in order to meet the high metabolic needs of the tissue of the central nervous system (CNS), specific transport systems selectively expressed in the membranes of brain endothelial cells in capillaries mediate the directed transport of nutrients into the CNS or of toxic metabolites out of the CNS. Whereas the characteristics of the mature BBB endothelium are well described, the cellular and molecular mechanisms that control the development, differentiation and maintenance of the highly specialized endothelial cells of the BBB remain unknown to date, despite the recent explosion in our knowledge of the growth factors and their receptors specifically acting on vascular endothelium during development. This review summarizes our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.     

The blood–brain barrier in neurodegenerative disease: a rhetorical perspective

Recent studies suggest that the function of the blood–brain barrier (BBB) is not static under normal physiologic conditions and is likely altered in neurodegenerative disease. Prevailing thinking about CNS function, and neurodegenerative disease in particular, is neurocentric excluding the impact of factors outside the CNS. This review challenges this perspective and discusses recent reports suggesting the involvement of peripheral factors including toxins and elements of adaptive immunity that may not only play a role in pathogenesis, but also progression of neurodegenerative diseases. Central to this view is neuroinflammation. Several studies indicate that the neuroinflammatory changes that accompany neurodegeneration affect the BBB or its function by altering transport systems, enhancing immune cell entry, or influencing the BBB's role as a signaling interface. Such changes impair the BBB's normal homeostatic function and affect neural activity. Moreover, recent studies reveal that alterations in BBB and its transporters affect the entry of drugs used to treat neurodegenerative diseases. Incorporating BBB compromise and dysfunction into our view of neurodegenerative disease leads to the inclusion of peripheral mediators in its pathogenesis and progression. In addition, this changing view of the BBB raises interesting new therapeutic possibilities for drug delivery as well as treatment strategies designed to reinstate normal barrier function.     

How do immune cells overcome the blood–brain barrier in multiple sclerosis?

The presence of the blood-brain barrier (BBB) restricts the movement of soluble mediators and leukocytes from the periphery to the central nervous system (CNS). Leukocyte entry into the CNS is nonetheless an early event in multiple sclerosis (MS), an inflammatory disorder of the CNS. Whether BBB dysfunction precedes immune cell infiltration or is the consequence of perivascular leukocyte accumulation remains enigmatic, but leukocyte migration modifies BBB permeability. Immune cells of MS subjects express inflammatory cytokines, reactive oxygen species (ROS) and enzymes that can facilitate their migration to the CNS by influencing BBB function, either directly or indirectly. In this review, we describe how immune cells from the peripheral blood overcome the BBB and promote CNS inflammation in MS through BBB disruption.     

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

The blood-brain barrier (BBB), which protects the CNS from pathogens, is composed of specialized brain microvascular endothelial cells (BMECs) joined by tight junctions and ensheathed by pericytes and astrocyte endfeet. The stability of the BBB structure and function is of great significance for the maintenance of brain homeostasis. When a neurotropic virus invades the CNS via a hematogenous or non-hematogenous route, it may cause structural and functional disorders of the BBB, and also activate the BBB anti-inflammatory or pro-inflammatory innate immune response. This article focuses on the structural and functional changes that occur in the three main components of the BBB (endothelial cells, astrocytes, and pericytes) in response to infection with neurotropic viruses transmitted by hematogenous routes, and also briefly describes the supportive effect of three cells on the BBB under normal physiological conditions. For example, all three types of cells express several PRRs, which can quickly sense the virus and make corresponding immune responses. The pro-inflammatory immune response will exacerbate the destruction of the BBB, while the anti-inflammatory immune response, based on type I IFN, consolidates the stability of the BBB. Exploring the details of the interaction between the host and the pathogen at the BBB during neurotropic virus infection will help to propose new treatments for viral encephalitis. Enhancing the defense function of the BBB, maintaining the integrity of the BBB, and suppressing the pro-inflammatory immune response of the BBB provide more ideas for limiting the neuroinvasion of neurotropic viruses. In the future, these new treatments are expected to cooperate with traditional antiviral methods to improve the therapeutic effect of viral encephalitis.     

A peroxynitrite-dependent pathway is responsible for blood-brain barrier permeability changes during a central nervous system inflammatory response: TNF-alpha is neither necessary nor sufficient

Elevated blood-brain barrier (BBB) permeability is associated with both the protective and pathological invasion of immune and inflammatory cells into CNS tissues. Although a variety of processes have been implicated in the changes at the BBB that result in the loss of integrity, there has been no consensus as to their induction. TNF-alpha has often been proposed to be responsible for increased BBB permeability but there is accumulating evidence that peroxynitrite (ONOO(-))-dependent radicals may be the direct trigger. We demonstrate here that enhanced BBB permeability in mice, whether associated with rabies virus (RV) clearance or CNS autoimmunity, is unaltered in the absence of TNF-alpha. Moreover, the induction of TNF-alpha expression in CNS tissues by RV infection has no impact on BBB integrity in the absence of T cells. CD4 T cells are required to enhance BBB permeability in response to the CNS infection whereas CD8 T cells and B cells are not. Like CNS autoimmunity, elevated BBB permeability in response to RV infection is evidently mediated by ONOO(-). However, as opposed to the invading cells producing ONOO(-) that have been implicated in the pathogenesis of CNS inflammation, during virus clearance ONOO(-) is produced without pathological sequelae by IFN-gamma-stimulated neurovascular endothelial cells.     

Perforin Competent CD8 T Cells Are Sufficient to Cause Immune-Mediated Blood-Brain Barrier Disruption

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theiler''s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin^−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.     

Neurovascular Unit: a Focus on Pericytes

The blood–brain barrier (BBB) is a highly specialized system that controls the exchanges between the blood and the central nervous system (CNS). This barrier shields the CNS from toxic substances in the blood and provides nutrients to CNS, thus playing an essential role in the maintenance of homeostasis. The anatomical basis of the BBB is formed by the endothelial cells of brain microvasculature, with elaborated tight and adherens junctions, which together with pericytes, the basement membrane, and astrocytes, as well as neurons, microglia and oligodendrocytes form the neurovascular unit. The interaction between all these components guarantees a proper environment for neural function and a restricted permeability and transport. Pericytes were initially reported by Rouget in 1873 and since then they have been recognized as an important component of the BBB, despite the difficulty of their identification. Diverse functions have been assigned to pericytes, including a role in BBB properties, hemostasis, and angiogenesis, as well as a contractile, immune, and phagocytic function. These cells are also seen like multipotent cells and so with a great potential for therapy. Here, we review the neurovascular unit composition and the interplay between the diverse components, addressing pericytes with a particular detail.     

CD8 T cell-initiated blood-brain barrier disruption is independent of neutrophil support

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.     

Cellular Elements of the Blood-Brain Barrier

The Blood-brain-barrier (BBB) provides both anatomical and physiological protection for the central nervous system (CNS), shielding the brain for toxic substances in the blood, supplying brain tissues with nutrients and filtering harmful compounds from the brain back to the bloodstream. The BBB is composed of four main cellular elements: endothelial cells (ECs), astrocyte end-feet, microglial cells, and perycites. Transport across the BBB is limited by both physical and metabolic barriers (enzymes, and different transport systems). Tight junctions (TJs) present between ECs form an important barrier against diffusion, excluding most blood-borne substances for entering the brain.     

Friend or Foe? Resident Microglia vs Bone Marrow-Derived Microglia and Their Roles in the Retinal Degeneration

Microglia are immune cells in the central nervous system (CNS) that originate from the yolk sac in an embryo. The renewal of the microglia pool in the adult eye consists of two components. In addition to the self-proliferation of resident cells, microglia in the CNS also derive from the bone marrow (BM). BM-derived cells pass through the blood–brain barrier (BBB) or blood–retina barrier (BRB) and differentiate into microglia under specific conditions which involves a complex mechanism. Recent studies have widely investigated the role of resident microglia and BM-derived microglia in the retinal degenerative disease. Both two cell types play dual roles and share many similar functions. However, resident microglia tend to polarize to the M1 phenotype which is pro-inflammatory and neurotoxic, whereas BM-derived microglia mainly polarize to the neuroprotective M2 phenotype in retinal degeneration. The molecular mechanism that underlines the invasion of peripheral cells has led to extensive discussions. In addition to the BBB and BRB disruption, many signaling pathways are involved in this process. Based on these studies, we discuss the roles of these two types of microglia in retinal degeneration disease and the potential clinical application of BM-derived microglia, which may benefit future therapies.     

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and--when barrier integrity is impaired--in the pathology of many common CNS disorders such as Alzheimer's disease, Parkinson's disease and stroke.     

Induction of blood brain barrier tight junction protein alterations by CD8 T cells

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.     

Lethal silver-haired bat rabies virus infection can be prevented by opening the blood-brain barrier

Silver-haired bat rabies virus (SHBRV) infection induces a strong virus-specific immune response in the periphery of the host, but death is common due to the failure to open the blood-brain barrier (BBB) and deliver immune effectors to central nervous system (CNS) tissues. Mice with an SJL background are less susceptible to lethal infection with rabies viruses. In addition, these animals are known to have reduced hypothalamus-pituitary-adrenal (HPA) axis activity and an elevated capacity to mediate CNS inflammatory responses. We show here that approximately one-half of PLSJL mice survive an SHBRV infection that is invariably lethal for 129/SvEv mice. This difference is associated with the elevated capacity of PLSJL mice to mediate BBB permeability changes in response to the infection. The induction of more extensive BBB permeability and CNS inflammation in these animals results in greater virus clearance and improved survival. On the other hand, treatment of SHBRV-infected PLSJL mice with the steroid hormone dehydroepiandrosterone reduced BBB permeability changes and caused greater mortality. We conclude that the infiltration of immune effectors across the BBB is critical to surviving a rabies virus infection and that HPA axis activity may influence this process.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Brain Barrier Breakdown as a Cause and Consequence of Neuroinflammation in Sepsis

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) are important for the maintenance of brain homeostasis. During sepsis, peripheral production of proinflammatory cytokines and reactive oxygen species are responsible for structural alterations in those brain barriers. Thus, an increasing permeability of these barriers can lead to the activation of glial cells such as microglia and the production of cytotoxic mediators which in turn act on the brain barriers, damaging them further. Thereby, in this review, we try to highlight how the brain barrier’s permeability is not only a cause but a consequence of brain injury in sepsis.     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier

Leukocyte transmigration across the blood-brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

Ninjurin1 is expressed in myeloid cells and mediates endothelium adhesion in the brains of EAE rats

Ninjurin1 (nerve injury-induced protein, Ninj1) is an adhesion molecule that is essential for cell-to-cell interactions. However, little is known about the function of Ninj1 in the central nervous system (CNS). To address its role in the CNS, we analyzed the expression pattern of Ninj1 in normal rats and in an experimental autoimmune encephalomyelitis (EAE) model. Ninj1 was expressed in three major compartments of brains, meninges, the choroid plexus, and parenchymal perivascular spaces. In the EAE brains, Ninj1 was strongly expressed in myeloid cells (macrophages/monocytes and neutrophils) and partially expressed in endothelial cells (ECs). Furthermore, Ninj1 enhanced adhesion between BV2 cells (murine monocyte lineage microglia) and HBMECs (human brain microvascular endothelial cells). Collectively, our findings suggest that Ninj1 may mediate the entry of myeloid cells into the CNS in normal and EAE brains, and it is a potential therapeutic target for regulating myeloid cell trafficking across the blood-brain barrier (BBB) in CNS immune processes.     

Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood–brain barrier

Leukocyte transmigration across the blood–brain barrier (BBB) is a multistep process that can be mediated by chemokines. These low-molecular-weight chemoattractant proteins are secreted by cells within the central nervous system (CNS) in response to injury or on activation. Leukocytes transmigrate toward this chemokine gradient, crossing the BBB and gaining access to the CNS parenchyma. Depending on the chemokine, the nature of the insult, and the type of cell that transmigrates, the BBB integrity may be disrupted, leading to its increased permeability. Both the inflammation resulting from leukocyte transmigration and BBB perturbations contribute to CNS pathology. The mechanisms that mediate leukocyte transmigration and BBB disruption, as well as tissue culture models that are used to study leukocyte trafficking, are the focus of this review.     

Is Peripheral Immunity Regulated by Blood-Brain Barrier Permeability Changes?

S100B is a reporter of blood-brain barrier (BBB) integrity which appears in blood when the BBB is breached. Circulating S100B derives from either extracranial sources or release into circulation by normal fluctuations in BBB integrity or pathologic BBB disruption (BBBD). Elevated S100B matches the clinical presence of indices of BBBD (gadolinium enhancement or albumin coefficient). After repeated sub-concussive episodes, serum S100B triggers an antigen-driven production of anti-S100B autoantibodies. We tested the hypothesis that the presence of S100B in extracranial tissue is due to peripheral cellular uptake of serum S100B by antigen presenting cells, which may induce the production of auto antibodies against S100B. To test this hypothesis, we used animal models of seizures, enrolled patients undergoing repeated BBBD, and collected serum samples from epileptic patients. We employed a broad array of techniques, including immunohistochemistry, RNA analysis, tracer injection and serum analysis. mRNA for S100B was segregated to barrier organs (testis, kidney and brain) but S100B protein was detected in immunocompetent cells in spleen, thymus and lymph nodes, in resident immune cells (Langerhans, satellite cells in heart muscle, etc.) and BBB endothelium. Uptake of labeled S100B by rat spleen CD4+ or CD8+ and CD86+ dendritic cells was exacerbated by pilocarpine-induced status epilepticus which is accompanied by BBBD. Clinical seizures were preceded by a surge of serum S100B. In patients undergoing repeated therapeutic BBBD, an autoimmune response against S100B was measured. In addition to its role in the central nervous system and its diagnostic value as a BBBD reporter, S100B may integrate blood-brain barrier disruption to the control of systemic immunity by a mechanism involving the activation of immune cells. We propose a scenario where extravasated S100B may trigger a pathologic autoimmune reaction linking systemic and CNS immune responses.