Evaluation of an in vitro coculture model for the blood-brain barrier: Comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources

Summary Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.     

The ultrastructure of cerebral blood capillaries in the raffish,Chimaera monstrosa

Summary Sharks and skates (Chondrichthyes: Elasmobranchii) have a glial blood-brain barrier, while all other vertebrates examined so far have an endothelial barrier. For comparative reasons it is desirable to examine the blood-brain barrier in species from the other subclass of cartilaginous fish, the holocephalans. The ultrastructure of cerebral capillaries in the chimaera (Chondrichthyes: Holocephali) is described in the present study. The endothelial cells are remarkably thick. Fenestrae and transendothelial channels were not observed. The endothelial cells are joined by elaborate tight junctions. The perivascular glial processes are separated by wide spaces (15–60nm) without obliterating junctional complexes. These findings indicate that the chimaera has an endothelial blood-brain barrier.     

Innate response to focal necrotic injury inside the blood-brain barrier

We have studied the initial innate immune response to focal necrotic injury on different sides of the mouse blood-brain barrier by two-photon intravital microscopy. Transgenic mice in which the promoter of the myeloid isoform of lysozyme drives GFP were used to track granulocytes and monocytes. Necrotic injury in the meninges, but not the brain parenchyma, recruited GFP+ cells within minutes that fully surrounded the necrotic site within a day. Recently, it has been suggested that microglial cells and astrocytes cooperate to mount a distinct response to laser injury behind the blood-brain barrier. We followed the microglial response in heterozygous knockin mice in which GFP replaces CX3CR1 coding sequence. Prior to injury, microglial cell bodies were immobile over days, but moved to the laser injury site within 1 day. We followed astrocytes, which have been proposed to cooperate with microglial cells in response to focal injury, using transgenic mice in which glial fibrillary acidic protein promoter drives GFP expression. Before injury fine astrocyte processes permeate the parenchyma. Astrocytes polarized toward the injury in an ATP, connexin hemichannels, and intracellular Ca2+ -dependent process. The astrocytes network established a cytoplasmic Ca2+ gradient that preceded the microglial response. This is consistent with astrocyte-microglial collaboration to mount this innate response that excludes blood leukocytes.     

The glial blood-brain barrier of crustacea and cephalopods: a review

1. The glial blood-brain barrier of invertebrates is an accessible, polarised glial layer that permits study of glial cells in their normal relations with neurons. Crayfish 2. The glial "perineurium" forms the blood-brain interface in crayfish, and acts as a barrier to horseradish peroxidase (HRP) and ionic lanthanum. By contrast, the perineurium of the peripheral nervous system is relatively permeable. 3. The ionic permeability of the blood-brain interface can be studied in a sucrose gap chamber, using an extra-cellular microelectrode to monitor the potential across the perineurium following changes in the bathing medium. Subtraction of the microelectrode trace from the sucrose gap records gives the change in the axonal membrane potential. 4. Raised [K+] in the bath causes a complex change in perineurial potential, with the initial transient indicating that the outer (basal) glial membrane is highly K+ selective. The axonal response shows that the time constant for K+ uptake (tau u) and efflux (tau E) across the perineurium of the order of 3-4 min, but the interstitial [K+] in the steady state, [K+] infinity is always less than in the bathing medium. The results are explained by a model incorporating a K+ sink, which may be glial. 5. Strophanthidin and ethacrynic acid have little effect on tau u or K infinity, but cause a rise of tau E. Cold temperature pulses causes changes in the perineurial potential compatible with depolarisation of the inner (apical) membrane. A model is proposed with a Na+-K+-2 Cl co-transporter on the perineurial basal membrane, and an electrogenic Na+-K+-ATPase on the apical.membrane, consistent with results from vertebrate glial/ependymal epithelia. Cephalopods 6. The brain of the cuttlefish Sepia has an extensive system of microvessels. In the vertical and optic lobes studied, a perivascular glial layer forms a barrier to HRP. The occluding structure appears not to be a classical tight junction but may involve condensation of extracellular material. There is no barrier between retinal axons and blood. 7. Studies with radiolabelled polyethylene glycol (PEG4000) and EDTA show that the Sepia blood-brain barrier is as tight as the endothelial barrier of mammals. 8. A modification of the Oldendorf arterial injection technique is used to show that glucose transport at the Sepia barrier is mediated by a Na+-independent hexose carrier resembling that of mammalian red cells and blood-brain barrier. 9. The blood-axon interface fo mantle nerves in the squid Alloteuthis is relatively impermeable to small ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

Cutting edge: cross-presented intracranial antigen primes CD8+ T cells

The CNS is considered immune privileged due to the blood-brain barrier and the absence of conventional lymphatics. Nonetheless, T cell immune responses specific for CNS Ag have been documented. Where these events are initiated and what cellular mechanisms are involved remain unknown. In this study, we established an experimental mouse model to evaluate the requirements for priming CD8+ T cells following the cross-presentation of intracranial Ag. Surprisingly, we find that even with a damaged blood-brain barrier, Ag presentation occurs in regional lymph nodes and not within the CNS itself. Only once the responding cells have expanded can they traffic to the site of CNS injury. Cross-presentation of intracranial Ag is efficient and the subsequent priming of CD8+ T cells is dependent on CD4+ T cell help and CD40 signaling in host APCs. Our findings have important implications for the initiation of T cell immune responses toward CNS Ags.     

B7-H3 participates in the development of experimental pneumococcal meningitis by augmentation of the inflammatory response via a TLR2-dependent mechanism

In addition to a well-documented role in regulating T cell-mediated immune responses, B7-H3, a newly discovered member of the B7 superfamily, has been recently identified as a costimulator in the innate immunity-mediated inflammatory response. In this study, we further report that B7-H3 participates in the development of pneumococcal meningitis in a murine model. Exogenous administration of B7-H3 strongly amplified the inflammatory response, exacerbated blood-brain barrier disruption, and aggravated the clinical disease status in Streptococcus pneumoniae-infected C3H/HeN wild-type mice. Consistent with the in vivo findings, B7-H3 substantially augmented proinflammatory cytokine and chemokine production, upregulated NF-κB p65 and MAPK p38 phosphorylation, and enhanced the nuclear transactivation of NF-κB p65 at both TNF-α and IL-6 promoters in S. pneumoniae-stimulated primary murine microglia cells. These B7-H3-associated in vitro and in vivo effects appeared to be dependent on TLR2 signaling, as B7-H3 almost completely lost its amplifying actions in both TLR2-deficient microglial cells and TLR2-deficient mice. Furthermore, administration of the anti-B7-H3 mAb (MIH35) attenuated the inflammatory response and ameliorated blood-brain barrier disruption in S. pneumoniae-infected wild-type mice. Collectively, our results indicate that B7-H3 plays a contributory role in the development of S. pneumoniae infection-induced bacterial meningitis.     

B cell-derived IL-15 enhances CD8 T cell cytotoxicity and is increased in multiple sclerosis patients

Multiple lines of evidence suggest that CD8 T cells contribute to the pathogenesis of multiple sclerosis (MS). However, the sources and involvement of cytokines such as IL-15 in activating these cells is still unresolved. To investigate the role of IL-15 in enhancing the activation of CD8 T cells in the context of MS, we determined cell types expressing the bioactive surface IL-15 in the peripheral blood of patients and evaluated the impact of this cytokine on CD8 T cell cytotoxicity and migration. Flow cytometric analysis showed a significantly greater proportion of B cells and monocytes from MS patients expressing IL-15 relative to controls. We established that CD40L activation of B cells from healthy donors increased their IL-15 levels, reaching those of MS patients. We also demonstrated an enhanced cytotoxic profile in CD8 T cells from MS patients upon stimulation with IL-15. Furthermore, we showed that IL-15 expressed by B cells and monocytes is sufficient and functional, enhancing granzyme B production by CD8 T cells upon coculture. Exposure of CD8 T cells to this cytokine enhanced their ability to kill glial cells as well as to migrate across an in vitro inflamed human blood-brain barrier. The elevated levels of IL-15 in patients relative to controls, the greater susceptibility of CD8 T cells from patients to IL-15, in addition to the enhanced cytotoxic responses by IL-15-exposed CD8 T cells, stresses the potential of therapeutic strategies to reduce peripheral sources of IL-15 in MS.     

Comparative ultrastructure and blood-brain barrier in diapause and nondiapause larvae of the European corn borer Ostrinia nubilalis (Hübner)

Ultrastructural examination of diapause and nondiapause larval brains of the European corn borer disclosed anatomical differences that may be related to the insect's "blood-barrier". The perineurial type I cells are quite closely appressed in the diapause brain, but thrown into extensive folds with large intercellular spaces in the nondiapause brain. The perineurial type II cells of diapause and nondiapause larvae are basically similar in general ultrastructure, and most likely form the basis for the "blood-brain barrier." Horseradish peroxidase penetration studies indicated that the outer margin of the perineurial type II cells constitute the limits of infiltration into the brain. An enzymatic component of the "blood-brain barrier" is postulated in this insect. The localization of ATPase in the perineurial type II cells indicates that energy-requiring regulatory mechanisms may be localized here. Metabolic studies with isolated brains, coupled with recent evidence from mammalian systems, suggest that glial cells may be of importance in an enzymatic "blood-brain barrier."     

Expression of receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin in the inner blood-retinal barrier of rats

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.     

The effects of an anti-mitotic drug,bleomycin, on glial repair in an insect central nervous system

Summary The DNA-binding drug, bleomycin, has a profound effect on neural repair following selective glial disruption by ethidium bromide. The contribution of the granule-containing cells (which normally appear in the early stages of repair) is greatly reduced, the restoration of the blood-brain barrier is delayed and the ultrastructural organization of the reorganising perineurium is dramatically changed. The aberrant perineurial structure and function observed in the presence of bleomycin are postulated to result from the effects of the drug on haemocytes which, together with endogenous reactive cells, contribute to the normal process of glial repair.     

Butyric acid mediated induction of enhanced transendothelial resistance in an in vitro model blood-brain barrier system.

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more differentiated endothelial cell phenotype which exhibits important properties of the blood-brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and differentiation of cells in culture we examined the effects of four differentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest effect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a beneficial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.     

Distribution of putative neurotransmitters in the brain of the European corn borer, Ostrinia nubilalis.

Radioautographic localization of three putative neurotransmitters in the brain of larvae of the European corn borer was studied by electron microscopy. Acetylcholine and dopamine were found to be accumulated in median neurosecretory cells. Both acetylcholine and dopamine were preferentially found in glial cells as compared to neuronal cells. This may be indicative of active participation of glial cells in a blood-brain barrier in the intact brain. Gamma-aminobutyric acid was not accumulated by any particular cell in the brain, but was apparently excluded to the periphery of the ganglion.It is suggested that dopamine, or more likely an immediate metabolite may be involved in an endogenous time measuring system and/or diapause induction-termination.     

Early immune events in the induction of allergic contact dermatitis

The skin is a barrier site that is exposed to a wide variety of potential pathogens. As in other organs, pathogens that invade the skin are recognized by pattern-recognition receptors (PRRs). Recently, it has been recognized that PRRs are also engaged by chemical contact allergens and, in susceptible individuals, this elicits an inappropriate immune response that results in allergic contact dermatitis. In this Review, we focus on how contact allergens promote inflammation by activating the innate immune system. We also examine how innate immune cells in the skin, including mast cells and dendritic cells, cooperate with each other and with T cells and keratinocytes to initiate and drive early responses to contact allergens.     

Neuroscience nanotechnology: progress, opportunities and challenges

Nanotechnologies exploit materials and devices with a functional organization that has been engineered at the nanometre scale. The application of nanotechnology in cell biology and physiology enables targeted interactions at a fundamental molecular level. In neuroscience, this entails specific interactions with neurons and glial cells. Examples of current research include technologies that are designed to better interact with neural cells, advanced molecular imaging technologies, materials and hybrid molecules used in neural regeneration, neuroprotection, and targeted delivery of drugs and small molecules across the blood-brain barrier.     

Immune responses to RNA-virus infections of the CNS

A successful outcome for the host of virus infection of the central nervous system (CNS) requires the elimination of the virus without damage to essential non-renewable cells, such as neurons. As a result, inflammatory responses must be tightly controlled, and many unique mechanisms seem to contribute to this control. In addition to being important causes of human disease, RNA viruses that infect the CNS provide useful models in which to study immune responses in the CNS. Recent work has shown the importance of innate immune responses in the CNS in controlling virus infection. And advances have been made in assessing the relative roles of cytotoxic T cells, antibodies and cytokines in the clearance of viruses from neurons, glial cells and meningeal cells.     

The effects of interferon-γ on the central nervous system

Interferon-gamma (IFN-γ) is a pleotropic cytokine released by T-lymphocytes and natural killer cells. Normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, IFN-γ is generally undetectable within the central nervous system (CNS). Nevertheless, in response to CNS infections, as well as during certain disorders in which the CNS is affected, T-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of IFN-γ. A large portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that IFN-γ plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE). Moreover, the biochemical and physiological effects of IFN-γ are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems.     

The role of hyperglycemia in mechanisms of exacerbated inflammatory responses within the oral cavity

Immune modulating factors are necessary for pathogen clearance, but also contribute to host tissues damage, as those seen in periodontal diseases. Many of these responses can be exacerbated by host conditions including type 2 diabetes [T2D], where toll-like receptor 4 [TLR4] and the receptor for advanced glycated end products [RAGE] play a significant role. Here we investigate causality associated with the increase in inflammatory markers observed in periodontally diseased patients with T2D using multi-variant correlation analysis. Inflammation associated with periodontal diseases, characterized by elevated pro-inflammatory cytokines, innate immune receptor expression, and cellular infiltrate was exacerbated in patients with T2D. In addition, a feed forward loop regulated by poor glycemic control was associated with a loss of mucosal barrier integrity and accumulation of innate immune receptor ligands resulting in an exacerbation of ongoing inflammation, where RAGE and TLR4 cooperated to induce responses in oral epithelial cells, which were exacerbated by hyperglycemia.