Endothelial Blood-brain Barrier Research Articles

Progress in drug delivery to the central nervous system by the prodrug approach.

This review describes specific strategies for targeting to the central nervous system (CNS). Systemically administered drugs can reach the brain by crossing one of two physiological barriers resistant to free diffusion of most molecules from blood to CNS: the endothelial blood-brain barrier or the epithelial blood-cerebrospinal fluid barrier. These tissues constitute both transport and enzymatic barriers. The most common strategy for designing effective prodrugs relies on the increase of parent drug lipophilicity. However, increasing lipophilicity without a concomitant increase in rate and selectivity of prodrug bioconversion in the brain will result in failure. In these regards, consideration of the enzymes present in brain tissue and in the barriers is essential for a successful approach. Nasal administration of lipophilic prodrugs can be a promising alternative non-invasive route to improve brain targeting of the parent drugs due to fast absorption and rapid onset of drug action. The carrier-mediated absorption of drugs and prodrugs across epithelial and endothelial barriers is emerging as another novel trend in biotherapeutics. Several specific transporters have been identified in boundary tissues between blood and CNS compartments. Some of them are involved in the active supply of nutrients and have been used to explore prodrug approaches with improved brain delivery. The feasibility of CNS uptake of appropriately designed prodrugs via these transporters is described in detail.

MyD88 signaling in brain endothelial cells is essential for the neuronal activity and glucocorticoid release during systemic inflammation

Activation of neuronal circuits involved in the control of autonomic responses is critical for the host survival to immune threats. The brain vascular system plays a key role in such immune-CNS communication, but the signaling pathway and exact type of cells within the blood-brain barrier (BBB) mediating these functions have yet to be uncovered. To elucidate this issue we used myeloid differentiation factor 88 (MyD88)-deficient mice, because these animals do not show any responses to the cytokine interleukin-1beta (IL-1beta). We created chimeric mice with competent MyD88 signaling in either the BBB endothelium or perivascular microglia of bone marrow origin and challenged them with IL-1beta. Systemic treatment with the cytokine caused a robust transcriptional activation of genes involved in the prostaglandin E(2) (PGE(2)) production by vascular cells of the brain. Upregulation of these genes is dependent on a functional MyD88 signaling in the endothelium, because MyD88-deficient mice that received bone marrow stem cells from wild-type animals (for example, functional perivascular microglia) exhibited no response to systemic IL-1beta administration. MyD88 competent endothelial cells also mediate neuronal activation and plasma release of glucocorticoids, whereas chimeric mice with MyD88-competent perivascular microglia did not show a significant increase of these functions. Moreover, competent endothelial cells for the gene encoding Toll-like receptor 4 (TLR4) are essential for the release of plasma corticosterone in response to low and high doses of lipopolysaccharide. Therefore, BBB endothelial cells and not perivascular microglia are the main target of circulating inflammatory mediators to activate the brain circuits and key autonomic functions during systemic immune challenges.

Effect of Tacrolimus on Activity and Expression of P-Glycoprotein and ATP-Binding Cassette Transporter A5 (ABCA5) Proteins in Hematoencephalic Barrier Cells

Tacrolimus is an agent used in clinical immunosuppressive drug therapies. A wide spectrum of adverse effects has been reported in association with this immunosuppressor, including neurotoxic effect. The upper limit of therapeutic blood concentrations of tacrolimus has been described as 30 ng/ml in immunosuppressed patients. We investigated the effect of this therapeutic dose of tacrolimus on the expression and activity of the multidrug resistance protein 1 (MDR1 or Pgp, P-glycoprotein) and ATP-binding cassette transporters A5 (ABCA5) in human brain microvascular endothelial cells (HBMEC), derived from Blood-Brain Barrier (BBB) endothelium, these being the most predominantly expressed transcripts in these cells. The expression and activity of MDR1 transporter decreased with 30 ng/ml tacrolimus. The cell viability was not changed with the therapeutic dose used. By contrast, ABCA5 transcripts, of unknown role as yet, increased their expression at this concentration. We propose that the secondary cytotoxic effects of this immunosuppressor on CSN, besides the functional blockade related to multidrug resistance proteins, such as MDR1, and probably ABCA5, could be linked to variations in the expression levels of these proteins at the BBB.

Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system

Adhesion molecules of the immunoglobulin superfamily are crucial effectors of leukocyte trafficking into the central nervous system. Using a lipid raft-based proteomic approach, we identified ALCAM as an adhesion molecule involved in leukocyte migration across the blood-brain barrier (BBB). ALCAM expressed on BBB endothelium localized together with CD6 on leukocytes and with BBB endothelium transmigratory cups. ALCAM expression on BBB cells was upregulated in active multiple sclerosis and experimental autoimmune encephalomyelitis lesions. Moreover, ALCAM blockade restricted the transmigration of CD4+ lymphocytes and monocytes across BBB endothelium in vitro and in vivo and reduced the severity and delayed the time of onset of experimental autoimmune encephalomyelitis. Our findings indicate an important function for ALCAM in the recruitment of leukocytes into the brain and identify ALCAM as a potential target for the therapeutic dampening of neuroinflammation.

Localization of S100A8 and S100A9 expressing neutrophils to spinal cord during peripheral tissue inflammation

Investigation of hyperalgesia at the spinal transcriptome level indicated that carrageenan-induced inflammation of rat hind paws leads to a rapid but sustained increase in S100A8 and S100A9 expression, two genes implicated in the pathology of numerous inflammatory diseases including rheumatoid arthritis and gout. In situ hybridization revealed that the elevation occurred in neutrophils that migrate to the spinal cord vasculature during peripheral inflammation, not in spinal neurons or glial cells. Immunohistochemical analysis suggests, but does not prove, that these neutrophils abundantly release S100A8 and S100A9. Consistent with this, we detected an increase in ICAM and VCAM, both indicators of endothelial activation, a known trigger for secretion of S100A8 and S100A9. Migration of S100A8- and S100A9-expressing neutrophils to spinal cord is selective, since MCP-1- and CD68-expressing leukocytes do not increase in spinal cord vasculature during hind paw inflammation. Examination of many neutrophil granule mediators in spinal cord indicated that they are not regulated to the same degree as S100A8 and S100A9. Neutrophil migration also occurs in the vasculature of brain and pituitary gland during peripheral inflammation. Together, these findings suggest an interaction between a subpopulation of leukocytes and the CNS during peripheral tissue inflammation, as implied by an apparent release and possible diffusion of S100A8 and S100A9 through the endothelial blood–brain barrier. Although the present findings do not establish the neurophysiological or behavioral relevance of these observations to nociceptive processing, the data raise the possibility that selective populations of leukocytes may communicate the presence of disease or tissue damage from the periphery to cells in the central nervous system.

E- and P-Selectin Are Not Required for the Development of Experimental Autoimmune Encephalomyelitis in C57BL/6 and SJL Mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.

Therapeutic Targeting of Leukocyte Trafficking Across the Blood-Brain Barrier

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS not to disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses are mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly regulating immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier control immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis (MS), immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of our current knowledge on the molecular mechanisms involved in immune cell entry into the CNS has been derived from studies performed in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Thus, a large part of our current knowledge on immune cell entry across the BBBs is based on the results obtained in this animal model. Similarly, knowledge on the benefits and potential risks associated with therapeutic targeting of immune cell recruitment across the BBB in human diseases are mostly derived from such treatment regimen in MS. Other mechanisms of immune cell entry into the CNS might therefore apply under different pathological conditions such as bacterial meningitis or stroke and need to be considered.

Plasmodium falciparum–Infected Erythrocytes Decrease the Integrity of Human Blood‐Brain Barrier Endothelial Cell Monolayers

Central to the pathologic progression of human cerebral malaria (CM) is sequestration of Plasmodium falciparum-infected red blood cells (Pf-IRBCs) to the blood-brain barrier (BBB) endothelium. The molecular interactions between Pf-IRBCs and the BBB endothelium and their implications for barrier function are unclear. The effects of Pf-IRBCs on the integrity of the BBB were assessed by electrical cell substrate sensing and by transendothelial electrical resistance measurements in an in vitro human BBB model. In addition, Pf-IRBCs were subfractionated and treated with trypsin, artemisinin, or brefeldin A. Pf-IRBCs, but not normal red blood cells, significantly decreased BBB resistance. Subfractionation showed that both membrane-associated and soluble Pf-IRBC factors mediate the decrease in BBB resistance. Trypsin treatment significantly reduced Pf-IRBC binding but not their ability to decrease electrical resistance. Likewise, P. falciparum isolates with increased binding to human brain microvascular endothelial cells did not alter the electrical resistance response. Soluble factors from Pf-IRBC culture supernatant decreased resistance by 50%-70% and precipitated with 40% ammonium sulfate saturation. Brefeldin-A partially blocked the ability of Pf-IRBCs to reduce resistance. The results suggest that, in CM, trypsin-resistant membrane components and soluble factors of Pf-IRBCs contribute to the impedance of BBB integrity in a multistep and multifactorial process.

Persistent endothelial abnormalities and blood–brain barrier leak in primary and secondary progressive multiple sclerosis

Epithelial and endothelial tight junctions are pathologically altered in infectious, inflammatory, neoplastic and other diseases. Previously, we described such abnormalities, associated with serum protein leak, in tight junctions of the blood-brain barrier endothelium, in lesional and normal-appearing white matter (NAWM) in secondary progressive (SP) and acute multiple sclerosis (MS). This work is extended here to lesions and NAWM in primary progressive multiple sclerosis (PPMS) and to cortical grey matter in PPMS and SPMS. Immunocytochemistry and semiquantitative confocal microscopy for the tight junction protein zonula occludens 1 (ZO-1) was performed on snap-frozen sections from PPMS (n = 6) and controls (n = 5). Data on 2103 blood vessels were acquired from active lesions (n = 10), inactive lesions (n = 15), NAWM (n = 42) and controls (n = 20). Data on 1218 vessels were acquired from normal-appearing grey matter (PPMS, 5; SPMS, 6; controls, 5). In PPMS abnormal ZO-1 expression in active white matter lesions and NAWM, was found in 42% and 13% of blood vessels, respectively, comparable to previous data from acute and SPMS. In chronic white matter plaques, however, abnormalities were considerably more frequent (37%) in PPMS than in SPMS. Abnormality was also more frequent in normal-appearing grey matter in SPMS (23%) than in PPMS (10%). In summary, abnormal tight junctions in both SPMS and PPMS are most frequent in active white matter lesions but persist in inactive lesions, particularly in PPMS. Abnormal tight junctions are also common in normal-appearing grey matter in SPMS. Persistent endothelial abnormality with leak (PEAL) is therefore widespread but variably expressed in MS and may contribute to disease progression.

Gp120-mediated cytotoxicity of human brain microvascular endothelial cells is dependent on p38 mitogen-activated protein kinase activation

Breakdown of the blood-brain barrier has been shown to contribute to neurological disorders that are prevalent in human immunodeficiency virus type 1 (HIV-1)-infected individuals, but the mechanisms involved in HIV-1-associated blood-brain barrier dysfunction remain incompletely understood. Using human brain microvascular endothelial cells (HBMECs) that constitute the blood-brain barrier, the authors determined the cytotoxic effects of gp120 on HBMECs. The authors showed that gp120 induced cytotoxicity of HBMECs derived from children, which required cotreatment with interferon (IFN)-gamma. IFN-gamma treatment exhibited up-regulation of the chemokine receptors CCR3 and CCR5 in children's HBMECs. In contrast, HBMECs isolated from adults were not responsive to gp120-mediated cytotoxicity. Peptides of gp120 representing binding regions for CD4 and chemokine receptors as well as CD4 antibody inhibited gp120-mediated cytotoxicity of HBMECs. RANTES, as expected, inhibited M-tropic gp120-mediated HBMEC cytotoxicity, whereas stromal cell-derived factor (SDF)-1alpha failed to inhibit T-tropic gp120-mediated cytotoxicity. Of interest, gp120 peptides representing non-CD4/non-chemokine receptor binding regions inhibited gp120-mediated HBMEC cytotoxicity. In addition, the authors showed that gp120-mediated HBMEC cytotoxicity involved p38 mitogen-activated protein kinase pathway. Taken together, these findings showed that gp120, in the presence of IFN-gamma, can cause dysfunction of the blood-brain barrier endothelium via MAPK pathways involving several gp120-HBMEC interactions.

A critical role of LFA-1 in the development of Th17 cells and induction of experimental autoimmune encephalomyelytis

The αLβ2 integrin adhesion molecule LFA-1 is believed to be involved in the migration of autoreactive T cells to the central nervous system across the endothelial blood–brain barrier in experimental autoimmune encephalomyelitis (EAE). Here, we demonstrate that the incidence and clinical scores of EAE in LFA-1 −/− mice induced by the immunization with the myelin oligodendrocyte glycoprotein (MOG)-peptide antigen were significantly lower than those in wild type mice. Further studies demonstrated that lymphocytes recruitment to the draining lymph nodes (dLN) after the immunization with the MOG-peptide was severely suppressed in LFA-1 −/− mice. Moreover, generation of the MOG-specific IL-17-producing helper T (Th17) cells in the dLN was impaired in LFA-1 −/− mice. These results suggest that LFA-1 may play an important role for the generation of MOG-specific Th17 cells in the dLN as well as the immigration of MOG-specific naïve CD4 + T cells to the dLN.

HIV‐1 gp120 as Well as Alcohol Affect Blood–Brain Barrier Permeability and Stress Fiber Formation: Involvement of Reactive Oxygen Species

HIV-1 infection commonly leads to serious HIV-1-associated neurological disorders, such as HIV-1-associated encephalopathy and dementia. In addition, alcohol is commonly used and/or abused among AIDS patients, but it is unclear whether alcohol affects the disease progression and if it affects it, how this occurs. We hypothesized that alcohol could affect the blood-brain barrier (BBB) integrity and thus could affect the onset and/or progression of HIV-associated neurological disorders. Human brain microvascular endothelial cells (HBMEC) in a BBB model system were pretreated with alcohol (17 and 68 mM) and subsequently coexposed with HIV-1 gp120. Expression of chemokine receptors CCR3, CCR5, and CXCR4 was assessed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Changes in the permeability of the HBMEC monolayer were assessed using paracellular markers [(3)H]inulin or propidium iodide. Actin rearrangements in HBMEC were visualized by fluorescence microscopy and viability assessed using Live/Dead stain. Both gp120 and alcohol increased the permeability of the BBB model by up to 141%, without affecting HBMEC viability. Cotreatment with alcohol and gp120 did not result in a significant synergistic effect. Gp120 permeability involved chemokine receptor CCR5. Alcohol did not affect chemokine receptor expression on brain endothelial cells. Both gp120 and alcohol reorganized the cytoskeleton and induced stress fiber formation. Inhibition of reactive oxygen species (ROS) formation through NADPH blocked the effects of both gp120 and alcohol on permeability and stress fiber formation. These results indicate that both HIV-1 gp120 and alcohol induce stress fibers, causing increased permeability of the human BBB endothelium. Alcohol (68 mM)-mediated permeability increase was linked to ROS formation. The alcohol-mediated physiological changes in the HBMEC monolayers may increase diffusion of plasma components and viral penetration across the BBB. This suggests that alcohol, especially at levels attained in heavy drinkers, can potentially contribute in a negative fashion to HIV-1 neuropathogenesis.

Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific

Endothelial cells (ECs) from different vascular beds display certain common qualities, but each subtype is uniquely adapted to meet the demands of the underlying tissues. The structural peculiarities of intercellular junctions are, for instance, considered to account for the differences in permeability displayed by various vascular beds: strong occludin expression is unique to cerebral ECs and considered to account for the high electrical resistance and low paracellular permeability of brain microvessels which constitute the blood-brain barrier (BBB). The integrity of the BBB is compromised in many disorders of the human CNS; therapeutic strategies include treatment with glucocorticoids (GCs), which improve barrier properties of the BBB. In contrast, positive effects of GCs on peripheral vascular permeability could not be demonstrated clearly, while side-effects of prolonged GC treatment are considerable. In an effort to elucidate this difference, we analysed the expression of occludin and the glucocorticoid receptor (GR) in BBB and non-BBB (myocardium) endothelial cells. Our results demonstrate complete GR downregulation by GCs in murine non-BBB endothelial cells in vivo, whereas GC administration led to nuclear concentration of GRs in BBB endothelium. In correlation with these in vivo data, the use of cerebral and myocardial endothelial cell lines proved GR downregulation in non-BBB cells in vitro in response to GC treatment. Divergent transactivating activity of GRs in the BBB and non-BBB endothelial cellular context could be demonstrated after transfection of endothelial cells with a model GC-responsive test promoter plasmid in the presence and absence of dexamethasone. Our results thus suggest differential signalling mechanisms involved in endothelial barrier regulation, arguing for the development of tissue-specific drugs for therapeutic applications.

Molecular mechanisms involved in T cell migration across the blood–brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Development of the blood‐brain barrier: A historical point of view

Although there has been considerable controversy since the observation by Ehrlich more than 100 years ago that the brain did not take up dyes from the vascular system, the concept of an endothelial blood-brain barrier (BBB) was confirmed by the unequivocal demonstration that the passage of molecules from blood to brain and vice versa was prevented by endothelial tight junctions (TJs). There are three major functions implicated in the term "BBB": protection of the brain from the blood milieu, selective transport, and metabolism or modification of blood- or brain-borne substances. The BBB phenotype develops under the influence of associated brain cells, especially astrocytic glia, and consists of complex TJs and a number of specific transport and enzyme systems that regulate molecular traffic across the endothelial cells. The development of the BBB is a complex process that leads to endothelial cells with unique permeability characteristics due to high electrical resistance and the expression of specific transporters and metabolic pathways. This review article summarizes the historical background underlying our current knowledge of the cellular and molecular mechanisms involved in the development and maintenance of the BBB.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.

The circumventricular organs participate in the immunopathogenesis of experimental autoimmune encephalomyelitis

BackgroundDuring inflammatory conditions of the central nervous system (CNS), such as in multiple sclerosis or in its animal model, experimental autoimmune encephalomyelitis (EAE), immune cells migrate from the blood stream into the CNS parenchyma and into the cerebrospinal fluid (CSF) spaces. The endothelial blood-brain barrier (BBB) has been considered the most obvious entry site for circulating immune cells. Recently, the choroid plexus has been considered as an alternative entry site for circulating lymphocytes into the CSF. The choroid plexus, belongs to the circumventricular organs (CVOs) localized in the walls of the ventricles. Other CVOs, which similar to the choroid plexus lack an endothelial BBB, have not been considered as possible entry sites for immune cells into the CNS parenchyma or the CSF. Here we asked, whether CVOs are involved in the recruitment of inflammatory cells into the brain during EAE.MethodsWe performed an extensive immunohistological study on the area postrema (AP), the subfornical organ (SFO), the organum vasculosum of the lamina terminalis (OVLT) and the median eminence (ME) in frozen brain sections from healthy SJL mice and mice suffering from EAE. Expression of cell adhesion molecules, the presence of leukocyte subpopulations and the detection of major histocompatibility complex antigen expression was compared.ResultsSimilar changes were observed for all four CVOs included in this study. During EAE significantly increased numbers of CD45+ leukocytes were detected within the four CVOs investigated, the majority of which stained positive for the macrophage markers F4/80 and Mac-1. The adhesion molecules ICAM-1 and VCAM-1 were upregulated on the fenestrated capillaries within the CVOs. A considerable upregulation of MHC class I throughout the CVOs and positive immunostaining for MHC class II on perivascular cells additionally documented the immune activation of the CVOs during EAE. A significant enrichment of inflammatory infiltrates was observed in close vicinity to the CVOs.ConclusionOur data indicate that the CVOs are a site for the entry of immune cells into the CNS and CSF and consequently are involved in the inflammatory process in the CNS during EAE.

Blood-brain barrier invasion by group B Streptococcus depends upon proper cell-surface anchoring of lipoteichoic acid

Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS DeltaiagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the DeltaiagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the DeltaiagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the DeltaiagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

P-Selectin Glycoprotein Ligand 1 Is Not Required for the Development of Experimental Autoimmune Encephalomyelitis in SJL and C57BL/6 Mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.