Amyloid-beta-induced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation.

Vascular dysfunction is emerging as a key pathological hallmark in Alzheimer's disease (AD). A leaky blood-brain barrier (BBB) has been described in AD patient tissue and in vivo AD mouse models. Brain endothelial cells (BECs) are linked together by tight junctional (TJ) proteins, which are a key determinant in restricting the permeability of the BBB. The amyloid beta (Abeta) peptides of 1-40 and 1-42 amino acids are believed to be pivotal in AD pathogenesis. We therefore decided to investigate the effect of Abeta 1-40, the Abeta variant found at the highest concentration in human plasma, on the permeability of an immortalized human BEC line, hCMEC/D3. Abeta 1-40 induced a marked increase in hCMEC/D3 cell permeability to the paracellular tracer 70 kD FITC-dextran when compared with cells incubated with the scrambled Abeta 1-40 peptide. Increased permeability was associated with a specific decrease, both at the protein and mRNA level, in the TJ protein occludin, whereas claudin-5 and ZO-1 were unaffected. JNK and p38MAPK inhibition prevented both Abeta 1-40-mediated down-regulation of occludin and the increase in paracellular permeability in hCMEC/D3 cells. Our findings suggest that the JNK and p38MAPK pathways might represent attractive therapeutic targets for preventing BBB dysfunction in AD.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis.

Microglia are mononuclear phagocytes of the central nervous system and are considered to derive from circulating bone marrow progenitors that colonize the developing human nervous system in the second trimester. They first appear as ameboid forms and progressively differentiate to process-bearing "ramified" forms with maturation. Signals driving this transformation are known to be partly derived from astrocytes. In this investigation we have used cocultures of astrocytes and microglia to demonstrate the relationship between motility and morphology of microglia associated with signals derived from astrocytes. Analysis of progressive cultures using time-lapse video microscopy clearly demonstrates the dynamic nature of microglia. We observe that ameboid microglial cells progressively ramify when cocultured with astrocytes, mirroring the "differentiation" of microglia in situ during development. We further demonstrate that individual cells undergo morphological transformations from "ramified" to "bipolar" to "tripolar" and "ameboid" states in accordance with local environmental cues associated with astrocytes in subconfluent cultures. Remarkably, cells are still capable of migration at velocities of 20-35 microm/h in a fully ramified state overlying confluent astrocytes, as determined by image analysis of motility. This is in keeping with the capacity of microglia for a rapid response to inflammatory cues in the CNS. We also demonstrate selective expression of the chemokines MIP-1alpha and MCP-1 by confluent human fetal astrocytes in cocultures and propose a role for these chemotactic cytokines as regulators of microglial motility and differentiation. The interchangeable morphological continuum of microglia supports the view that these cells represent a single heterogeneous population of resident mononuclear phagocytes capable of marked plasticity.

Expression of beta-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS.

Chemokines play specific roles in directing the recruitment of leukocyte subsets into inflammatory foci within the central nervous system (CNS). The involvement of these cytokines as mediators of inflammation is widely accepted. Recently, it has become evident that cells of the CNS (astrocytes, microglia, and neurons) not only synthesize, but also respond functionally or chemotactically to chemokines. We previously reported developmental events associated with colonization of the human fetal CNS by mononuclear phagocytes (microglial precursors), which essentially takes place within the first two trimesters of life. As part of the array of signals driving colonization, we noted specific anatomical distribution of chemokines and chemokine receptors expressed during this period. In order to further characterize expression of these molecules, we have isolated and cultured material from human fetal CNS. We demonstrate that unstimulated subconfluent human fetal glial cultures express high levels of CCR2 and CXCR4 receptors in cytoplasmic vesicles. Type I astrocytes, and associated ameboid microglia in particular, express high levels of surface and cytoplasmic CXCR4. Of the chemokines tested (MIP-1alpha, MIP-1beta, MCP-1, MCP-3, RANTES, SDF-1, IL-8, IP-10), only MIP-1alpha, detected specifically on microglia, was expressed both constitutively and consistently. Low variable levels of MCP-1, MIP-1alpha, and RANTES were also noted in unstimulated glial cultures. Recombinant human chemokines rhMCP-1 and rhMIP-1alpha also displayed proliferative effects on glial cultures at [10 ng/ml], but displayed variable effects on CCR2 levels on these cells. rhMCP-1 specifically upregulated CCR2 expression on cultured glia at [50 ng/ml]. It is gradually becoming evident that chemokines are important in embryonic development. The observation that human fetal glial cells and their progenitors express specific receptors for chemokines and can be stimulated to produce MCP-1, as well as proliferate in response to chemokines, supports a role for these cytokines as regulatory factors during development.

Strain specific variation in cytokine regulated ICAM-1 expression by rat brain-endothelial cells.

Cytokine induced levels of ICAM-1 expressed by rat brain-endothelial cells were quantitated by enzyme immunoassay in response to stimulation by TNF-alpha in the presence or absence of IFN-gamma. The rat strains investigated differ in their susceptibility to experimental allergic encephalomyelitis; significantly less ICAM-1 was induced by BEC derived from the resistant PVG strain as compared to the susceptible LEW strain with both cytokine combinations. In contrast, despite the difference in disease susceptibility, equivalent levels of ICAM-1 were induced between the LEW and BN strain. Furthermore, evidence for a synergistic interaction of both TNF-alpha and IFN-gamma was observed in the BN strain. The results are discussed with relevance to the disease profile of each strain.

Microglia in the human fetal spinal cord--patterns of distribution, morphology and phenotype.

Microglia, the intrinsic macrophages of the nervous system, colonise the cerebrum around the second trimester in man. In order to determine the extent of microglial influx into the nervous system, we have examined their distribution within the human fetal spinal cord in relation to astrocytic and vascular development between 9 and 16 weeks of gestation, using conventional immunohistochemistry [CD11b; CD45; CD64; CD68; ICAM-1; ICAM-2; VCAM-1; PECAM; GFAP; vimentin] and lectin histochemistry [RCA-1]. Microglia are identifiable by 9 weeks, within the ventricular/sub-ventricular zones. Human fetal microglia display heterogeneity in phenotype and are more readily identified by CD68 in the spinal cord. There is a marked influx of cells dorsal and ventral to the neural cavity, from the marginal layer [meninges/connective tissue] with advancing gestational age, with greatest cell densities towards the end of the time period in this study. This inward migration is associated with progressive vascularisation, ICAM-2 expression and co-localises with GFAP and vimentin positive radial glia. The patterns of microglial migration in human fetal cord differ from that within the cerebrum, but generally conform to a route following white to gray matter.

Strain specific variation in IFN-gamma inducible lymphocyte adhesion to rat brain endothelial cells.

We have examined the interferon-gamma (IFN-gamma) induced increase in lymphocyte adhesion to rat brain endothelial cells (BEC) in the experimental allergic encephalomyelitis (EAE) susceptible LEW and resistant PVG strain. A significant increase in adhesion of mitogen activated lymphocytes could be demonstrated by stimulating LEW BEC with 50 U/ml IFN-gamma for 24 h. In contrast the same treatment failed to induce a significant increase in lymphocyte adhesion in the PVG strain. Flow cytometric analysis of lymphocyte integrin expression indicated a marked increase in the number of cells expressing both LFA-1 and VLA-4 following mitogen activation and was similar between the LEW and PVG strain. Depletion of LFA-1 and VLA-4 positive lymphocytes resulted in an equivalent inhibition of adhesion to BBB-EnC indicating that the adherent population was comprised predominantly of the VLA-4 + /LFA-1 + phenotype. We conclude that this strain variation in the IFN-gamma activation of BEC may be related to the disease phenotypes of these strains.

Expression of adhesion molecules on human fetal cerebral vessels: relationship to microglial colonisation during development.

Microglia represent the primary immune effector cells of the adult central nervous system (CNS). The origin of these cells has been a subject of intense debate over the last century. However, immunohistochemical and chimera developmental studies in rodents support the hypothesis that microglia are monocytic in origin. There have been relatively few studies to date on microglia in human fetal development, and the mechanisms by which microglial precursors enter the developing CNS are as yet unknown. It is possible that microglial precursors use combinations of adhesion molecules on cerebral endothelium to gain entry into the developing CNS. In the present study, we have shown the distribution of microglia within human fetal cerebral cortex between 16 and 22 weeks of gestation using RCA-1 lectin histochemistry. We have also demonstrated dual anti-macrophage antibody labelling of these cells in conjunction with adhesion molecules ICAM-1, ICAM-2 and PECAM on cerebral endothelium throughout this period. We conclude that fetal microglia usually occur at highly vascularised sites within the developing human fetal brain and are more specifically associated with the expression of ICAM-2 on cerebral endothelium.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Control of lymphocyte adhesion to brain and aortic endothelium: ICAM-1, VCAM-1 and negative charge.

Lymphocyte adhesion to CNS endothelium is low by comparison with non-CNS endothelium. It has been proposed that this could be due to the high surface charge of brain endothelium, or a low constitutive expression of adhesion molecules. In this study we compared the influence of these factors on lymphocyte adhesion to BEC and aortic endothelium (AEC) in culture. Brain endothelium expresses very low levels of VCAM-1, and lower levels of ICAM-1 than aortic endothelium. The negative charge differed between the endothelia, but this had a minimal effect on lymphocyte adhesion. The anionic sites were, however, more stable on brain endothelium, remaining unchanged after endothelial cell activation with cytokines, while redistribution was observed on cytokine-activated aortic endothelium.

Distribution and analysis of surface charge on brain endothelium in vitro and in situ.

Vascular endothelial cells are associated with a number of anionic molecules. These anions are important in endothelial function, particularly in regulating permeability, haemostasis and cellular traffic. To explore the nature and distribution of anions on the brain endothelial cell (BEC) surface, we have examined rat brain endothelium in culture, and in situ. The anionic sites were probed with cationic colloidal gold and cationised ferritin, and visualised by light microscopy. Additionally we compared the distribution of the anionic sites on BEC with that present on other endothelial cell types in culture. The predominant anion detected on BEC was heparan sulphate (HS). This was distributed throughout the cell membrane, but was most densely associated with intercellular junctions. This pattern was distinct from the anionic locations observed in endothelia from aorta and epididymal fat microvessels. The distribution of anions was dependent on the age of cultured cells, with only minimal levels of HS seen at the periphery of younger cells. The nature and distribution of negative charge was different in situ. Here, sialic acid was the major surface anion, with only a small contribution from HS. The significance of these findings are discussed in relation to endothelial function in normal tissue and in pathological conditions.

Strain-specific variation in constitutive and inducible expression of MHC class II, class I and ICAM-1 on rat cerebral endothelium.

Strain variation in levels of inducible major histocompatibility complex (MHC) class II expression by rat cerebral endothelium has previously been reported. Using primary cell cultures of rat cerebral endothelium from PVG (RT1c), LEW (RT1l), PVG.LEW (RT1l) and PVG.AGUS (RT1lv?) strains it was determined that variation in levels of inducible MHC class II expression between strains can be accounted for by both cis-acting elements within the MHC region and by trans-acting elements outside the MHC. In addition it was determined that levels of constitutive MHC class I expression varied between PVG (RT1c) and LEW (RT1l) strains which can be attributed to cis-acting elements within the MHC region. Furthermore, while levels of constitutive class I expression vary between PVG (RT1c) and LEW (RT1l) endothelium we could find no difference in the interferon-gamma (IFN-gamma) inducible expression of class I between these two strains. In contrast the inducibility of intercellular adhesion molecule-1 (ICAM-1) in response to IFN-gamma was found to differ between PVG and LEW endothelium. Significant levels of ICAM-1 are induced on LEW cerebral endothelium after 24 hr exposure to 50 U/ml IFN-gamma. However, no significant induction of ICAM-1 could be demonstrated on PVG, or BN cerebral endothelium after the same exposure to IFN-gamma. Induction of ICAM-1 by IFN-gamma precedes MHC class II by at least 24 hr and its persistence is proportional to the concentration of IFN-gamma used. We suggest that the rat MHC region (RT1) contains elements which control the levels of constitutive class I expression and inducible class II expression in response to IFN-gamma, but that other non-RT1 genes influence the inducibility of MHC class II on rat cerebral endothelial cells. This observation, together with the finding that ICAM-1 expression is not significantly increased in response to IFN-gamma on PVG or BN endothelium, suggests that IFN-gamma responsiveness by these strains differs from LEW.

Control of lymphocyte migration into brain: selective interactions of lymphocyte subpopulations with brain endothelium.

We have determined whether particular lymphocyte populations bind preferentially to cerebral endothelium, using adhesion assays and a new method for in situ staining of adherent lymphocytes. B cells bind more strongly than T cells, an effect enhanced by lymphocyte activation or endothelial cell stimulation with interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha). This is not equated with levels of CD18 expression on the lymphocytes. CD8+ T cells bound more efficiently than CD4+ cells under all conditions. To determine whether there was a population of cells which selectively homes to the brain, we compared adhesion of cervical lymph nodes cells to brain endothelium, with adhesion of lymphocytes from other nodes. In 50% of the experiments there was significantly enhanced binding of activated cervical lymph cells to cerebral endothelium but not to control (aortic) endothelium. This effect was seen using both normal and IFN-gamma-activated endothelium. The explanation for this finding is that cervical lymph nodes frequently, but not invariably, contain higher proportions of CD8+ cells and B cells than other lymph nodes. These data imply that selective adhesion of lymphocytes to brain endothelium is related to the subpopulations involved and this may be reflected in the cell types seen in immunological lesions of the brain, and in the relative proportions of the subpopulations seen in cervical lymph nodes.

Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4+ T cell lines in vitro.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.

Adhesion of lymphocytes to cerebral microvascular cells: effects of interferon-gamma, tumour necrosis factor and interleukin-1.

Although lymphocyte traffic through the brain is normally low, this can increase dramatically in response to infection or an autoimmune reaction. We have studied the adhesion of lymphocytes to cerebral endothelium in vitro in an attempt to model the first step of the infiltration process--that is, the initial interaction between the lymphocytes and endothelial cells--by brief co-culture of lymph node cells with monolayers of cultured cerebral endothelium. In this system we find that the basal level of adhesion can be increased in a dose-dependent manner by pre-treatment of the endothelial cells with interferon-gamma (IFN-gamma) and/or tumour necrosis factor (TNF) but not with interleukin-1 (IL-1). This increased adhesion can be blocked by incubating the IFN-gamma-treated cells with an antibody that is thought to bind to the common beta-chain of the lymphocyte functional antigen-1 (LFA-1) family of molecules. This suggests that endothelial cells express either LFA-1 or a molecule sharing the beta-subunit (of which several have been described) and furthermore that this molecule is involved in the regulation of lymphocyte traffic into the brain.

Serological evidence for a defect in RT1.B (I-A) expression by the BDIX rat strain.

Astrocytes, astrocytic cell lines and endothelium from BDIX rats were stimulated with recombinant interferon-gamma (IFN-gamma) and the expression of MHC molecules quantified using an enzyme immunoassay (EIA). Using the two mouse anti-RT1.B monoclonal antibodies MRC OX4 and OX6, previously described as recognizing a monomorphic determinant on RT1.B, as well as polyvalent rabbit anti-rat class II antisera, we were unable to demonstrate any induction of RT1.B molecules on these cells under conditions that induced RT1.B expression in all other strains tested. In contrast, RT1.D locus class II molecules, detectable by the antibody MRC OX17, are more strongly expressed in BDIX than in other strains. In experiments using BDIX lymphocytes, this serologically detected defect in RT.1B expression was confirmed using four additional mouse anti-mouse I-Ak monoclonal antibodies, which cross-reacted on all rat strains tested except BDIX. It appears likely that BDIX rats lack either a structural or controlling gene required for RT1.B expression.

Antigen presentation in brain: MHC induction on brain endothelium and astrocytes compared.

Primary cultures of rat brain endothelium and astrocytes were cultured in vitro, stimulated with interferon-gamma (IFN gamma), and the levels of MHC expression were then measured by an enzyme immunoassay (EIA). Class I expression is enhanced on brain endothelium by Day 1 following stimulation, and attains a plateau level of expression. Class II is normally absent, but starts to appear at Day 2, and continues to increase until Day 5. Class II rat I-A homologue is induced much more strongly than I-E, and the dose-response curves show that I-A expression is dependent on interferon dose within the range 2-500 units/ml, whereas class I enhancement is uniform over this range. The endothelium was compared with astrocytes, in regard to MHC induction. The surface density of class I and class II molecules was lower on the astrocytes in all conditions, and both class I enhancement and class II induction were in some cases slower to appear than on endothelium treated similarly. We also describe a pre-astrocytic cell line, C9, which shows strong I-E expression when stimulated with IFN gamma, but is apparently unable to express I-A. The implications of these findings for the development of immune reactions in the brain are discussed.

Antigenic determinants of human thyroglobulin differentiated using antigen fragments.

Human thyroglobulin (Tg) was digested with V8 protease and the fragments separated by high performance liquid chromatography (HPLC). The antigenic relationship of the fragments was investigated using mouse monoclonal antibodies to human Tg. The binding of Hashimoto's disease autoantibodies to the fragments was measured by radioimmunoassay. This demonstrated that a minority of the patients recognize an epitope on Tg which others do not. The epitopes identified by the autoantibodies were substantially destroyed in the smaller fragments tested, but these smaller fragments were more efficient stimulators of Tg-specific T-cell lines than the larger fragments which carry the antibody binding determinants. This suggests that the parts of the Tg molecule which stimulate autoimmune B cells differ from those which stimulate T cells.