Yukio Takeshita

Inflammatory cell trafficking across the blood-brain barrier: chemokine regulation and in vitro models.

Summary:  The blood–brain barrier (BBB) is the brain‐specific capillary barrier that is critical for preventing toxic substances from entering the central nervous system (CNS). In contrast to vessels of peripheral organs, the BBB limits the exchange of inflammatory cells and mediators under physiological and pathological conditions. Clarifying these limitations and the role of chemokines in regulating the BBB would provide new insights into neuroprotective strategies in neuroinflammatory diseases. Because there is a paucity of in vitro BBB models, however, some mechanistic aspects of transmigration across the BBB still remain largely unknown. In this review, we summarize current knowledge of BBB cellular components, the multistep process of inflammatory cells crossing the BBB, functions of CNS‐derived chemokines, and in vitro BBB models for transmigration, with a particular focus on new and recent findings.

CXCL12-Induced Monocyte-Endothelial Interactions Promote Lymphocyte Transmigration Across an in Vitro Blood-Brain Barrier

An in vitro model of the human blood-brain barrier provides insights into how chemokine receptors regulate the transmigration of leukocytes into brain tissue. Going with the Flow White blood cells traffic ceaselessly throughout the body, using blood vessels as their conduits. They also migrate into inflamed tissues to defend the host against microbes or to repair damaged tissue. However, in many human diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, or type 1 diabetes, white blood cells invade apparently healthy uninfected tissues and cause injury. Recent research has identified the molecular regulators (chemokine receptors and their chemokine ligands; adhesion molecules) of white blood cell transmigration out of the blood vessels and into tissues. Because there are 100 or so such molecules (used selectively when specific types of white blood cells transmigrate), it is important to identify those molecules that are most directly involved in harmful inflammation. Chemokine receptors are molecules on white blood cell surfaces that receive signals to guide cells into tissue, and they change as white blood cells transmigrate across different vessel walls. In a new study, Man and colleagues have devised an in vitro model of a specialized vessel wall of the human brain termed the blood-brain barrier (BBB). They use this elegant model to study how chemokine receptors influence and are influenced by the transmigration of white blood cells across a human BBB-like endothelial cell layer. First, the authors coaxed a special type of human endothelial cell to form a BBB-like layer in a dual perfusion chamber. Then, they allowed human white blood cells to flow across the layer at a flow rate approximating that found in brain capillaries. Some white blood cells flowed across the layer and out of the device, whereas others transmigrated across the BBB-like endothelial layer into the lower chamber of the device. The researchers wanted to establish how chemokine receptor expression by white blood cells would alter as the cells transmigrated across the BBB. They studied a chemokine receptor termed CXCR4, which is expressed on almost all white blood cells. When they added the triggering molecule for CXCR4 to their in vitro system, unexpectedly, they found that only one cell type, monocytes, showed altered CXCR4 expression. CXCR4 appeared to deliver signals to monocytes, which empowered these cells to assist other white blood cells such as T and B cells to cross the BBB. This surprising result opens up new vistas for understanding how white blood cells and vessel wall endothelial cells “talk” to each other in inflamed tissues and should spur progress for identifying the best targets for blocking harmful inflammation in the brain. The accumulation of inflammatory cells in the brain parenchyma is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Chemokines and adhesion molecules orchestrate leukocyte transmigration across the blood-brain barrier (BBB), but the dynamics of chemokine receptor expression during leukocyte transmigration are unclear. We describe an in vitro BBB model system using human brain microvascular endothelial cells that incorporates shear forces mimicking blood flow to elucidate how chemokine receptor expression is modulated during leukocyte transmigration. In the presence of the chemokine CXCL12, we examined modulation of its receptor CXCR4 on human T cells, B cells, and monocytes transmigrating across the BBB under flow conditions. CXCL12 stimulated transmigration of CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes. Transmigration was blocked by CXCR4-neutralizing antibodies. Unexpectedly, CXCL12 selectively down-regulated CXCR4 on transmigrating monocytes, but not T cells. Monocytes underwent preferential CXCL12-mediated adhesion to the BBB in vitro compared with lymphocytes. These findings provide new insights into leukocyte-endothelial interactions at the BBB under conditions mimicking blood flow and suggest that in vitro BBB models may be useful for identifying chemokine receptors that could be modulated therapeutically to reduce neuroinflammation in diseases such as MS.

Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

The ability of the Blood Brain Barrier (BBB) to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS) activity and regulating leukocytes’ access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS): fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF) that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

Effects of neuromyelitis optica–IgG at the blood–brain barrier in vitro

Objective: To address the hypothesis that physiologic interactions between astrocytes and endothelial cells (EC) at the blood–brain barrier (BBB) are afflicted by pathogenic inflammatory signaling when astrocytes are exposed to aquaporin-4 (AQP4) antibodies present in the immunoglobulin G (IgG) fraction of serum from patients with neuromyelitis optica (NMO), referred to as NMO-IgG. Methods: We established static and flow-based in vitro BBB models incorporating co-cultures of conditionally immortalized human brain microvascular endothelial cells and human astrocyte cell lines with or without AQP4 expression. Results: In astrocyte–EC co-cultures, exposure of astrocytes to NMO-IgG decreased barrier function, induced CCL2 and CXCL8 expression by EC, and promoted leukocyte migration under flow, contingent on astrocyte expression of AQP4. NMO-IgG selectively induced interleukin (IL)-6 production by AQP4-positive astrocytes. When EC were exposed to IL-6, we observed decreased barrier function, increased CCL2 and CXCL8 expression, and enhanced leukocyte transmigration under flow. These effects were reversed after application of IL–6 neutralizing antibody. Conclusions: Our results indicate that NMO-IgG induces IL-6 production by AQP4-positive astrocytes and that IL-6 signaling to EC decreases barrier function, increases chemokine production, and enhances leukocyte transmigration under flow.

An in vitro blood–brain barrier model combining shear stress and endothelial cell/astrocyte co-culture

BACKGROUND In vitro blood-brain barrier (BBB) models can be useful for understanding leukocyte-endothelial interactions at this unique vascular-tissue interface. Desirable features of such a model include shear stress, non-transformed cells and co-cultures of brain microvascular endothelial cells with astrocytes. Recovery of transmigrated leukocytes for further analysis is also appealing. NEW METHODS We report an in vitro BBB model for leukocyte transmigration incorporating shear stress with co-culture of conditionally immortalized human endothelial cell line (hBMVEC) and human astrocyte cell line (hAST). Transmigrated leukocytes can be recovered for comparison with input and non-transmigrated cells. RESULT hBMVEC and hAST exhibited physiological and morphological BBB properties when cocultured back-to-back on membranes. In particular, astrocyte processes protruded through 3 μm membrane pores, terminating in close proximity to the hBMVEC with a morphology reminiscent of end-feet. Co-culture with hAST also decreased the permeability of hBMVEC. In our model, astrocytes promoted transendothelial leukocyte transmigration. COMPARISON WITH EXISTING METHODS This model offers the opportunity to evaluate whether BBB properties and leukocyte transmigration across cytokine-activated hBMVEC are influenced by human astrocytes. CONCLUSIONS We present a BBB model for leukocyte transmigration incorporating shear stress with co-culture of hBMVEC and hAST. We demonstrate that hAST promoted leukocyte transmigration and also increased certain barrier functions of hBMVEC. This model provides reproducible assays for leukocyte transmigration with robust results, which will enable further defining the relationships among leukocytes and the cellular elements of the BBB.

Fingolimod prevents blood-brain barrier disruption induced by the sera from patients with multiple sclerosis.

Objective Effect of fingolimod in multiple sclerosis (MS) is thought to involve the prevention of lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system across blood-brain barrier (BBB). However, brain microvascular endothelial cells (BMECs) represent a possible additional target for fingolimod in MS patients by directly repairing the function of BBB, as S1P receptors are also expressed by BMECs. In this study, we evaluated the effects of fingolimod on BMECs and clarified whether fingolimod-phosphate restores the BBB function after exposure to MS sera. Methods Changes in tight junction proteins, adhesion molecules and transendothelial electrical resistance (TEER) in BMECs were evaluated following incubation in conditioned medium with or without fingolimod/fingolimod-phosphate. In addition, the effects of sera derived from MS patients, including those in the relapse phase of relapse-remitting (RR) MS, stable phase of RRMS and secondary progressive MS (SPMS), on the function of BBB in the presence of fingolimod-phosphate were assessed. Results Incubation with fingolimod-phosphate increased the claudin-5 protein levels and TEER values in BMECs, although it did not change the amount of occludin, ICAM-1 or MelCAM proteins. Pretreatment with fingolimod-phosphate restored the changes in the claudin-5 and VCAM-1 protein/mRNA levels and TEER values in BMECs after exposure to MS sera. Conclusions Pretreatment with fingolimod-phosphate prevents BBB disruption caused by both RRMS and SPMS sera via the upregulation of claudin-5 and downregulation of VCAM-1 in BMECs, suggesting that fingolimod-phosphate is capable of directly modifying the BBB. BMECs represent a possible therapeutic target for fingolimod in MS patients.

Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica

An autoantibody target on brain microvascular endothelial cells in patients with neuromyelitis optica may be exploited to manipulate blood-brain barrier permeability. Bringing down the blood-brain barrier Patients afflicted by neuromyelitis optica suffer from disruption of the blood-brain barrier. Shimizu et al. generated recombinant antibodies from patient cerebral spinal fluid and demonstrated that some antibodies targeting glucose-regulated protein 78 were able activate brain microvascular endothelial cells and induced protein extravasation in cell lines and in mice. These findings suggest that glucose-regulated protein 78–targeted antibodies could be instigating blood-brain barrier breakdown and development of hallmark anti–aquaporin-4 autoantibody pathology. Not only that, the application of these antibodies could help open up the blood-brain barrier for transit of treatments for many central nervous system diseases. Neuromyelitis optica (NMO) is an inflammatory disorder mediated by antibodies to aquaporin-4 (AQP4) with prominent blood-brain barrier (BBB) breakdown in the acute phase of the disease. Anti-AQP4 antibodies are produced mainly in the periphery, yet they target the astrocyte perivascular end feet behind the BBB. We reasoned that an endothelial cell–targeted autoantibody might promote BBB transit of AQP4 antibodies and facilitate NMO attacks. Using monoclonal recombinant antibodies (rAbs) from patients with NMO, we identified two that strongly bound to the brain microvascular endothelial cells (BMECs). Exposure of BMECs to these rAbs resulted in nuclear translocation of nuclear factor κB p65, decreased claudin-5 protein expression, and enhanced transit of macromolecules. Unbiased membrane proteomics identified glucose-regulated protein 78 (GRP78) as the rAb target. Using immobilized GRP78 to deplete GRP78 antibodies from pooled total immunoglobulin G (IgG) of 50 NMO patients (NMO-IgG) reduced the biological effect of NMO-IgG on BMECs. GRP78 was expressed on the surface of murine BMECs in vivo, and repeated administration of a GRP78-specific rAb caused extravasation of serum albumin, IgG, and fibrinogen into mouse brains. Our results identify GRP78 antibodies as a potential component of NMO pathogenesis and GRP78 as a candidate target for promoting central nervous system transit of therapeutic antibodies.

Interaction of ataxin-3 with huntingtin-associated protein 1 through Josephin domain.

Huntingtin-associated protein 1 (HAP1) is an essential component of the stigmoid body (STB) and known as a possible neuroprotective interactor with causative proteins for Huntingtons disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 17 (SCA17), and Joubert syndrome. To clarify what other causative molecules HAP1/STB could interact with, we cloned normal causative genes for several neural disorders from human brain RNA library and evaluated their subcellular interaction with HAP1/STB by immunocytochemistry and immunoprecipitation after cotransfection into Neuro2a cells. The results clearly showed that HAP1/STB interacts with the normal ataxin-3 through Josephin domain and polyglutamine-expanded mutants derived from SCA3 as well. The findings suggest that HAP1/STB could modify the physiological function of normal ataxin-3 and pathogenesis of SCA3 attributable to the mutant ataxin-3.

Anti-human placental antigen complex X-P2 (hPAX-P2) anti-serum recognizes C-terminus of huntingtin-associated protein 1A common to 1B as a determinant marker for the stigmoid body

The anti-serum against an unknown human placental antigen complex X-P2 (hPAX-P2) immunohistochemically recognizes three putative molecules (hPAX-P2S, hPAX-P2N, and hPAX-P2R), each of which is associated with the stigmoid bodies (STBs), necklace olfactory glomeruli (NOGs), or reticulo-filamentous structures (RFs) in the rat brain. The STBs also contain huntingtin-associated protein 1 (HAP1), and the HAP1-cDNA transfection induces STB-like inclusions in cultured cells. In order to clarify the relationship between hPAX-P2S and HAP1 isoforms (A/B), we performed Western blotting, immuno-histo/cytochemistry for light- and electron-microscopy and pre-adsorption tests with HAP1 deletion fragments. The results showed that the anti-hPAX-P2 anti-serum recognizes HAP1474–577 of HAP1A/B in Western blotting and strongly immunostains HAP1A-induced STB-like inclusions but far weakly detects HAP1B-induced diffuse structures in HAP1-transfected HEK 293 cells. In the rat brain, immunoreactivity of the anti-hPAX-P2 anti-serum for the STBs was eliminated by pre-adsorption with HAP1474–577, whereas no pre-adsorption with any different HAP1 fragments can suppress immunoreactivity for the NOGs and RFs, which were not immunoreactive to anti-HAP1 anti-serum. These findings indicate that hPAX-P2S, which is distinct from hPAX-P2N and hPAX-P2R, is identical with STB-constituted HAP1 and that the HAP1-induced/immunoreactive inclusions correspond to the hPAX-P2-immunoreactive STBs previously identified in the brain.

Markedly increased IP-10 production by blood-brain barrier in neuromyelitis optica.

Objective Severe damage to the blood-brain barrier (BBB) allows anti-aquaporin 4 (AQP4) antibodies to access the astrocytic endfeet in neuromyelitis optica (NMO). In the current study, we identified the pathogenic cytokines/chemokines that are responsible for the BBB malfunction induced by NMO sera. Methods We measured the levels of 27 cytokines/chemokines in human brain microvascular endothelial cells (BMECs) after exposure to sera obtained from patients with the acute and stable phases of anti-AQP4 antibody-positive NMO spectrum disorder (NMOSD), multiple sclerosis (MS) patients and healthy controls (HC) using a multiplexed fluorescent bead-based immunoassay system. Results The induced protein (IP)-10 level in the cells was markedly increased following exposure to acute phase NMOSD sera. Other cytokines/chemokines including interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 were also significantly increased in the acute NMOSD group compared to both the MS and HC groups. The up-regulation of the IP-10 levels in the cells after exposure to the acute-phase NMOSD sera was also observed using another specified ELISA, and this effect was significantly decreased during the remission phase in the individual NMOSD patients. Furthermore, the increase in the level of IP-10 after exposure to the sera was significantly correlated with the cerebrospinal fluid/serum albumin ratio. Conclusions Sera from the acute phase of NMO markedly increased the autocrine secretion of IP-10 by BMECs. The over-production of IP-10 in BMECs may play an important role in the pathogenesis of NMO and may therefore help to mediate the trafficking of T cells expressing its receptor across the BBB.