Joel S. Pachter

Inflammation and Alzheimer’s disease

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimers disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Where is the blood–brain barrier … really?

Few terms in the biomedical lexicon are as widely recognized as the phrase blood–brain barrier (BBB). Indeed, it immediately conjures up a “barricade” between the blood and the brain, a feature often considered more obstacle than safeguard. In truth, the BBB performs in both capacities, and it is precisely this duality that imparts such a vital role to the BBB in influencing physiological and pathophysiological processes in the CNS. Although the concept is more than a century old, the BBB continues to remain enigmatic in both substance and idea, with seemingly resolved issues once again beckoning for clarification. In this regard, recent technological advancements, such as sequencing of the human genome and development of microarray analysis, have illuminated novel aspects of vascular gene expression and provoked reconsideration of the cellular and biochemical makeup of the BBB. In light of the critical impact of the BBB in the realms of science and medicine, this Mini‐Review will revisit the topic of the composition of the BBB, specifically highlighting how recent developments in endothelial biology have prompted a reevaluation of its precise vascular location. We have intentionally avoided discussing generalized features of the BBB, as these have been skillfully described elsewhere as noted.

CULTURE OF MURINE BRAIN MICROVASCULAR ENDOTHELIAL CELLS THAT MAINTAIN EXPRESSION AND CYTOSKELETAL ASSOCIATION OF TIGHT JUNCTION-ASSOCIATED PROTEINS

SummaryA readily obtainable in vitro paradigm of the blood-brain barrier (BBB) would offer considerable benefits. Toward this end, in this study, we describe a novel method for purifying murine brain microvascular endothelial cells (BMEC) for culture. The method uses limited collagenase-dispase digestion of enriched brain microvessels, followed by immunoisolation of digested, microvascular fragments by magnetic beads coated with antibody to platelet-endothelial cell adhesion molecule-1. When plated onto collagen IV-coated surfaces, these fragments elaborated confluent monolayers of BMEC that expressed, as judged by immunocytochemistry, the adherens junction-associated proteins, VE-cadherin and β-catenin, as well as the tight junction (TJ)-associated proteins, claudin-5, occludin, and zonula occludin-1 (ZO-1), in concentrated fashion along intercellular borders. In contrast, cultures of an immortalized and transformed line of murine brain capillary-derived endothelial cells, bEND.3, displayed diffuse cytoplasmic localization of occludin and ZO-1. This difference in occludin and ZO-1 staining between the two endothelial cell types was also reflected in the extent of association of these proteins with the detergent-resistant cytoskeletal framework (CSK). Although both occludin and ZO-1 largely partitioned with the CSK fraction in BMEC, they were found predominantly in the soluble fraction of bEND.3 cells, and claudin-5 was found associated equally with both fractions in BMEC and bEND.3 cells. Moreover, detergent-extracted cultures of the BMEC retained pronounced immunostaining of occludin and ZO-1, but not claudin-5, along intercellular borders. Because both occludin and ZO-1 are thought to be functionally coupled to the detergent-resistant CSK and high expression of TJs is considered a seminal characteristic of the BBB, these results impart that this method of purifying murine BMEC provides a suitable platform to investigate BBB properties in vitro.

Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro

A simplified protocol for isolating brain microvessel endothelial cells (BMEC) from human cortex and culturing them on a thick collagen plug is described. This method results in the establishment of monolayers of BMEC that retain numerous properties indicative of the blood-brain barrier (BBB) phenotype, such as elevated transendothelial electrical resistance, attenuated paracellular flux of sucrose, peripheral actin filament distribution and asymmetric localization of the efflux peptide, P-glycoprotein, to the apical (luminal) BMEC surface. The novel 3-dimensional nature of this model system renders it ideally suitable for assaying such varied aspects of BBB physiology as solute transport, pathogen penetrance, leukocyte infiltration and tumor metastasis into the brain. Moreover, the fact that the system is derived from human brain allows for the study of pathogenetic mechanisms that may only be operative in humans.

Macrophages/microglial cells in human central nervous system during development : an immunohistochemical study

The development of microglia and macrophages was studied in 14 human embryos and fetuses ranging in age from 4.5-13.5 gestational weeks (g.w.), using lectins, Ricinus communis agglutinin-1 [RCA-1], and Lycopersicon esculentum, tomato lectin (TL), which recognize macrophages and microglia, and antibodies for the macrophage antigen CD68. Lectin-positive (+) cells were observed at 4.5 g.w., the youngest age examined. They were detected in the leptomeninges around the neural tube, and only rarely were observed in the CNS parenchyma. At 5.5 g.w., lectin+ cells were present throughout the CNS parenchyma, and a portion of these cells could also be labeled with antibody to CD68. In subsequent weeks, both types of cells, lectin+ and CD68+/lectin+ cells co-existed and progressively developed typical microglial morphology. In addition, in double label experiments, an antibody that labels CD14 antigen present on monocytes, hematogenous precursors of tissue macrophages, did not label either lectin+ or CD68+/lectin+ cells in CNS parenchyma. Additional immunocytochemical studies with appropriate markers excluded the possibility that any of the cells described here were either astrocytes, oligodendrocytes, endothelial cells or neurons. Our finding that one class of cells can be labeled early only with lectins, while another can be labeled with both lectins and CD68 macrophage antibody, may reflect a different origin of microglia in the early embryonic CNS compared to the fetal stages. This subdivision appears to be maintained in the adult brains as well.

Human ESC-Derived MSCs Outperform Bone Marrow MSCs in the Treatment of an EAE Model of Multiple Sclerosis

Summary Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.

Transcellular transport of CCL2 across brain microvascular endothelial cells.

The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood–brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal‐to‐luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous 125I‐labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin‐1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double‐label immuno‐electronmicroscopy, which revealed co‐localization of caveolin‐1 with exogenous CCL2, during this chemokine’s transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.

Characterization of Binding Sites for Chemokines MCP‐1 and MIP‐1α on Human Brain Microvessels

Abstract: The presence of binding sites for the β chemokines monocyte chemoattractant protein‐1 (MCP‐1) and macrophage inflammatory protein‐1α (MIP‐1α) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP‐1 and MIP‐1α: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP‐1 binding to isolated brain microvessels was inhibited by unlabeled MCP‐1 and MCP‐3, the latter another CCR2 ligand, but not by MIP‐1α. Inhibition of labeled MIP‐1α binding was achieved with unlabeled MIP‐1α and RANTES, the latter a β chemokine that binds to both CCR1 and CCR5, but not by MCP‐1. Labeled MIP‐1α binding was also antagonized by unlabeled MCP‐3, which is also recognized by CCR1, and MIP‐1β, which is a ligand for CCR5. Labeled MCP‐1 and MIP‐1α were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37°C. Additionally, exposure of microvessels to unlabeled MCP‐1 or MIP‐1α was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand‐induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP‐1 and MIP‐1α with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.

Functional expression of CCR2 by human fetal astrocytes.

Astrocytes from different sources bind the chemokine monocyte chemoattractant factor (MCP‐1), yet functional expression in these cells of CCR2, the major receptor for this ligand, has been a matter of controversy. Here we show that cultured human fetal astrocytes express CCR2 at the mRNA and protein levels, and display chemotaxis and calcium flux in response to MCP‐1. Surface CCR2 protein expression and MCP‐1 binding activity were observed to undergo near parallel downmodulation and recovery following MCP‐1 exposure, supporting the argument that CCR2, and not another receptor, mediates MCP‐1 ligation in these cells. Downmodulation was further determined to occur via receptor internalization, and to apparently proceed via both clathrin‐coated vesicles and caveolae, the latter being a novel mode for the endocytosis of chemokine receptors. Insofar as MCP‐1 is thought to mediate inflammatory and developmental processes within the central nervous system (CNS), such astrocyte responses to this chemokine are likely to significantly impact physiological and pathophysiological events at the blood‐brain barrier and within the CNS parenchyma.

Endothelial cell heterogeneity of blood‐brain barrier gene expression along the cerebral microvasculature

The blood‐brain barrier (BBB) refers to the network of microvessels that selectively restricts the passage of substances between the circulation and the central nervous system (CNS). This microvascular network is comprised of arterioles, capillaries and venules, yet the respective contribution of each of these to the BBB awaits clarification. In this regard, it has been postulated that brain microvascular endothelial cells (BMEC) from these different tributaries might exhibit considerable heterogeneity in form and function, with such diversity underlying unique roles in physiological and pathophysiological processes. Means to begin exploring such endothelial differences in situ, free from caveats associated with cell isolation and culturing procedures, are crucial to comprehending the nature and treatment of CNS diseases with vascular involvement. Here, the recently validated approach of immuno‐laser capture microdissection (immuno‐LCM) coupled to quantitative real‐time PCR (qRT‐PCR) was used to analyze gene expression patterns of BMEC retrieved in situ from either capillaries or venules. From profiling 87 genes known to play a role in BBB function and/or be enriched in isolated brain microvessels, results imply that most BBB properties reside in both segments, but that capillaries preferentially express some genes related to solute transport, while venules tend toward higher expression of an assortment of genes involved in inflammatory‐related tasks. Fuller appreciation of such heterogeneity will be critical for efficient therapeutic targeting of the endothelium and the management of CNS disease.