David Male

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.

Expression of the β-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.

Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues.

Neuropathologically, human immunodeficiency virus (HIV) is associated with a range of inflammatory disorders, extensive cortical neuronal loss, and dendritic and synaptic damage. Although the mechanisms resulting in these abnormalities are still unclear, the neurotoxic effects are thought to be due in part to viral products including the tat gene product. We have previously shown that Tat when presented to neurons extracellularly interacts with neuronal cell membranes to cause neuronal excitation and toxicity in fmole amounts. To determine the role of Tat in mediating HIV encephalitis (HIVE), we detected tat mRNA and protein in tissue extracts of nine patients with HIVE and seven patients without HIVE. Despite long autopsy times and significant degradation, tat mRNA was detected in 4/9 patients with HIVE but not in any of the seven patients without dementia. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with (5/9) or without (2/7) HIVE. Using protein extracts from the brains of the same groups of patients we were unable to detect Tat by enzyme linked immunosorbant assay (ELISA) (sensitivity of 2 ng Tat/ml of brain tissue). However, Tat could be detected immunohistochemically and in protein extracts from the brains of rhesus macaques with encephalitis due to a chimeric strain of HIV and simian immunodeficiency virus (SHIV). Our observations support the role of Tat in the neuropathogenesis of HIV and SHIV encephalitis.

Regulation of chemokine receptor expression in human microglia and astrocytes

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.

Colonisation of the developing human brain and spinal cord by microglia: a review

Microglia are the immune effector cells of the nervous system. The prevailing view is that microglia are derived from circulating precursors in the blood, which originate from the bone‐marrow. Colonisation of the central nervous system (CNS) by microglia is an orchestrated response during human fetal development related to the maturation of the nervous system. It coincides with vascularisation, formation of radial glia, neuronal migration and myelination primarily in the 4th–5th months and beyond. Microglial influx generally conforms to a route following white matter tracts to gray areas. We have observed that colonisation of the spinal cord begins around 9 weeks, with the major influx and distribution of microglia commencing around 16 weeks. In the cerebrum, colonisation is in progress during the second trimester, and ramified microglial forms are widely distributed within the intermediate zone by the first half of intra‐uterine life (20–22 weeks). A distinct pattern of migration occurs along radial glia, white matter tracts and vasculature. The distribution of these cells is likely to be co‐ordinated by spatially and temporally regulated, anatomical expression of chemokines including RANTES and MCP‐1 in the cortex; by ICAM‐2 and PECAM on radiating cerebral vessels and on capillaries within the germinal layer, and apoptotic cell death overlying this region. The phenotype and functional characteristics of fetal microglia are also outlined in this review. The need for specific cellular interactions and targeting is greater within the central nervous system than in other tissues. In this respect, microglia may additionally contribute towards CNS histogenesis. Microsc. Res. Tech. 45:359–382, 1999.

Chemokines, chemokine receptors and adhesion molecules on different human endothelia: discriminating the tissue-specific functions that affect leucocyte migration

The selective accumulation of different leucocyte populations during inflammation is regulated by adhesion molecules and chemokines expressed by vascular endothelium. This study examined how chemokine production and the expression of adhesion molecules and chemokine receptors vary between endothelia from different vascular beds. Human saphenous vein endothelium was compared with lung and dermal microvascular endothelia and with umbilical vein endothelium and a bone‐marrow endothelial cell line. All endothelia produced CCL2 and CXCL8 constitutively, whereas CXCL10 and CCL5 were only secreted after tumour necrosis factor (TNF)‐α or interferon (IFN)‐γ stimulation. In combination with TNF‐α, IFN‐γ suppressed CXCL8 but enhanced CCL5 and CXCL10, whereas transforming growth factor (TGF)‐β reduced secretion of all chemokines. Basal chemokine secretion was higher from umbilical vein than other endothelial cells. Chemokine receptors, CXCR1, CXCR3 and CCR3, were present on all endothelia but highest on saphenous vein. CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 were also detected at variable levels on different endothelia. The variation between endothelia in chemokine secretion was much greater than the variations in adhesion molecules, both on resting cells and following cytokine stimulation. These results indicate that it is the tissue‐specific variations in endothelial chemokine secretion rather than variations in adhesion molecules that can explain the different patterns of inflammation and leucocyte traffic seen in non‐lymphoid tissues.

Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response.

The use of progenitors and stem cells for neural grafting is promising, as these not only have the potential to be maintained in vitro until use, but may also prove less likely to evoke an immunogenic response in the host, when compared to primary (fetal) grafts. We investigated whether the short-term survival of a grafted conditionally immortalised murine neuroepithelial stem cell line (MHP36) (2 weeks post-implantation, 4 weeks post-ischaemia) is influenced by: (i) immunosuppression (cyclosporin A (CSA) vs. no CSA), (ii) the local (intact vs. lesioned hemisphere), or (iii) global (lesioned vs. sham) brain environment. MHP36 cells were transplanted ipsi- and contralateral to the lesion in rats with middle cerebral artery occlusion (MCAo) or sham controls. Animals were either administered CSA or received no immunosuppressive treatment. A proliferation assay of lymphocytes dissociated from cervical lymph nodes, grading of the survival of the grafted cells, and histological evaluation of the immune response revealed no significant difference between animals treated with or without CSA. There was no difference in survival or immunological response to cells grafted ipsi- or contralateral to the lesion. Although a local upregulation of immunological markers (MHC class I, MHC class II, CD45, CD11b) was detected around the injection site and the ischaemic lesion, these were not specifically upregulated in response to transplanted cells. These results provide evidence for the low immunogenic properties of MHP36 cells during the initial period following implantation, known to be associated with an acute host immune response and ensuing graft rejection.

Expression of β-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‐1α, MIP‐1β, MCP‐1, MCP‐3, RANTES, SDF‐1, IL‐8, IP‐10), only MIP‐1α, detected specifically on microglia, was expressed both constitutively and consistently. Low variable levels of MCP‐1, MIP‐1α, and RANTES were also noted in unstimulated glial cultures. Recombinant human chemokines rhMCP‐1 and rhMIP‐1α 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. GLIA 37:64–75, 2002.

MicroRNA-155 negatively affects blood–brain barrier function during neuroinflammation

Blood–brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR‐155 as a critical miRNA in neuroinflammation at the BBB. miR‐155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR‐155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR‐155 was strongly and rapidly upregulated by inflammatory cytokines. miR‐155 up‐regulation mimicked cytokine‐induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR‐155 partially prevented a cytokine‐induced increase in permeability. Furthermore, miR‐155 modulated brain endothelial barrier function by targeting not only cell–cell complex molecules such as annexin‐2 and claudin‐1, but also focal adhesion components such as DOCK‐1 and syntenin‐1. We propose that brain endothelial miR‐155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.—Lopez‐Ramirez, M. A., Wu, D., Pryce, G., Simpson, J. E., Reijerkerk, A., King‐Robson, J., Kay, O, de Vries, H. E., Hirst, M. C., Sharrack, B., Baker D., Male, D. K., Michael, G. J., Romero, I. A. MicroRNA‐155 negatively affects blood–brain barrier function during neuroinflammation. FASEB J. 28, 2551–2565 (2014). www.fasebj.org

Mesoglia & microglia: a historical review of the concept of mononuclear phagocytes within the central nervous system

More than a century and a half has elapsed since the first accounts of mesodermal phagocytic elements were proposed within the central nervous system. Over the intervening decades, body and substance were added to this concept through the advancement of histological techniques at the disposal of the researcher and the acute and keen-minded skills of the pathologist. Notable among these pioneering efforts were the contributions of W. Ford Robertson, Santiago Ramon y Cajal, Pio del Rio-Hortega and Wilder Penfield amongst an entire cavalcade of other noteworthy figures. The term ‘mesoglia’ and ‘third element of the nervous system’ was bestowed upon these cells towards the beginning of the twentieth century to account for their separate origins from neurons and macroglia. It was later amended by del Rio-Hortega in 1919, to ‘microglia’ in order to further discriminate between true mesodermal elements and oligodendrocytes, previously regarded as a component of ‘mesoglia’. This particular contention sparked much controversy among del Rio-Hortega’s peers and resulted in an escalation of fruitful research throughout Europe that eventually declined up to the outbreak of the Second World War. The post-war years were a period of the ‘dark ages’ that cast doubt on the very existence and nature of microglia, until the ‘renaissance’ of research was once again rejuvenated in the 1960s, by a new cohort of intrigued minds: Cammermeyer, Blinzinger, Kreutzberg and others who saw in the ‘third element’ the potential that is now commonly ascribed to microglia: the intrinsic immune effector cells of the CNS. It is now universally accepted that microglia are involved as the first line of rapid defence in any pathology of the nervous system, and as such, present a diagnostic tool for the neuropathologist. Although our knowledge of microglia stems from an extensive body of work conducted over the last two decades, much of the earlier work (pre-1960s) has remained somewhat obscure. This is partly accountable due to the limited availability of translated works, and additionally to the lack of a compendium of these articles. This paper will present a comprehensive overview of the pioneering research on mononuclear phagocytes within the central nervous system, which has direct bearing on our present-day understanding of the concept of microglia.