Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins.

It is known that many cells produce extracellular vesicles, and this includes a range of different cancer cell types. Here we demonstrate the profound effects of large vesicular-like bodies produced by melanoma cells on the barrier integrity of human brain endothelial cells. These vesicular-bodies have not been fully characterised but range in size from ~500 nm to >10 µm, are surrounded by membrane and are enzymatically active based on cell-tracker incorporation. Their size is consistent with previously reported large oncosomes and apoptotic bodies. We demonstrate that these melanoma-derived vesicular-bodies rapidly affect brain endothelial barrier integrity, measured using ECIS biosensor technology, where the disruption is evident within ~60 min. This disruption involves acquisition of the vesicles through transcellular uptake into the endothelial cells. We also observed extensive actin-rearrangement, actin removal from the paracellular boundary of the endothelial cells and envelopment of the vesicular-bodies by actin. This was concordant with widespread changes in CD144 localisation, which was consistent with the loss of junctional strength. High-resolution confocal imaging revealed proximity of the melanoma vesicular-bodies juxtaposed to the endothelial nucleus, often containing fragmented DNA themselves, raising speculation over this association and potential delivery of nuclear material into the brain endothelial cells. The disruption of the endothelial cells occurs in a manner that is faster and completely distinct to that of invasion by intact melanoma cells. Given the clinical observation of large vesicles in the circulation of melanoma patients by others, we hypothesize their involvement in weakening or priming the brain vasculature for melanoma invasion.

Vmeasur: A software package for experimental and clinical measurement of mesenteric lymphatic contractile function over an extended vessel length.

OBJECTIVE: Conventionally, in vivo mesenteric lymphatic contractile function is measured using a high magnification transmission microscope (field of view 0.3-1.5 mm), which precludes visualization of extended lengths of vessels embedded in mesenteric fat. Existing software is not optimized for imaging at a low magnification using a contrast agent. We aimed to develop a simple and clinically transferable method for in situ visualization, image analysis, and quantitative assessment of mesenteric lymphatic contractile function over an extended area. METHODS: Subserosal injection of various blue dyes was taken up by mesenteric lymphatics and visualized under a stereomicroscope (25×), allowing for video recording of 1.4 × 1.1 cm of intact mesentery. A new R package ("vmeasur") that combines multiple high-performance image analyses into a single workflow was developed. The edges of each vessel were determined by applying an automated threshold to each frame (with real-time manual verification). The vessel width at every point in each frame was plotted to provide contractile parameters over time and along the lymphatic vessel length. RESULTS: Contractile parameters and their differences along the length of the vessel were accurately calculated in a rodent model. In a human mesenteric lymphatic, the algorithm was also able to measure changes in diameter over length. CONCLUSION: This software offers a low cost, rapid, and accessible method to measure lymphatic contractile function over a wide area, showing differences in contractility along the length of a vessel. Because the presence of mesenteric fat has less of an impact on imaging, due to the use of an exogenous contrast agent, there is potential for clinical application.

Comprehensive Assessment of Secreted Immuno-Modulatory Cytokines by Serum-Differentiated and Stem-like Glioblastoma Cells Reveals Distinct Differences between Glioblastoma Phenotypes.

Glioblastoma is refractory to therapy and presents a significant oncological challenge. Promising immunotherapies have not shown the promise observed in other aggressive cancers. The reasons for this include the highly immuno-suppressive tumour microenvironment controlled by the glioblastoma cells and heterogeneous phenotype of the glioblastoma cells. Here, we wanted to better understand which glioblastoma phenotypes produced the regulatory cytokines, particularly those that are implicated in shaping the immune microenvironment. In this study, we employed nanoString analysis of the glioblastoma transcriptome, and proteomic analysis (proteome profiler arrays and cytokine profiling) of secreted cytokines by different glioblastoma phenotypes. These phenotypes were cultured to reflect a spectrum of glioblastoma cells present in tumours, by culturing an enhanced stem-like phenotype of glioblastoma cells or a more differentiated phenotype following culture with serum. Extensive secretome profiling reveals that there is considerable heterogeneity in secretion patterns between serum-derived and glioblastoma stem-like cells, as well as between individuals. Generally, however, the serum-derived phenotypes appear to be the primary producers of cytokines associated with immune cell recruitment into the tumour microenvironment. Therefore, these glioblastoma cells have considerable importance in shaping the immune landscape in glioblastoma and represent a valuable therapeutic target that should not be ignored.

Comprehensive analysis of inhibitory checkpoint ligand expression by glioblastoma cells.

Glioblastoma is a highly aggressive brain malignancy commonly refractory to classical and novel chemo-, radio- and immunotherapies, with median survival times of ~15 months following diagnosis. Poor immunological responses exemplified by the downregulation of T-cell activity, and upregulation of immunosuppressive cells within the tumor microenvironment have limited the effectiveness of immunotherapy in glioblastoma to date. Here we show that glioblastoma cells express a large repertoire of inhibitory checkpoint ligands known to control effector T cell responses. Furthermore, flow cytometry analysis reveals that glioblastoma cells with an enhanced stem cell-like phenotype express several investigated ligands at significant levels on their cell surface. This reveals that glioblastoma stem-like cells express suppressive ligands with the potential of suppressing major T cell checkpoint receptors. With this information, it is now essential that we understand the relevance of this extensive repertoire of immune checkpoint ligands and their functional consequence on immune evasion in glioblastoma. This is necessary to develop effective immunotherapeutics and to be able to match treatment to patient, especially in the light of CheckMate 143.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Analysis of Melanoma Secretome for Factors That Directly Disrupt the Barrier Integrity of Brain Endothelial Cells.

We have recently demonstrated that invasive melanoma cells are capable of disrupting the brain endothelial barrier integrity. This was shown using ECIS biosensor technology, which revealed rapid disruption via the paracellular junctions. In this paper, we demonstrate that melanoma cells secrete factors (e.g., cytokines) that weaken the endothelial barrier integrity. Through proteome profiling, we attempt to identify the barrier-disrupting cytokines. Melanoma conditioned media were collected from three New Zealand melanoma lines. ECIS technology was used to assess if the conditioned media disrupted the endothelial barrier independent of the melanoma cells. The melanoma cell secretome was assessed using cytometric bead array (CBA), Luminex immunoassay and multiplex Proteome Profilers, to detect the expression of secretory proteins, which may facilitate metastasis. Finally, ECIS technology was used to assess the direct effects of secreted proteins identified as candidates from the proteome screens. We show that melanoma-conditioned media significantly disrupted the brain endothelial barrier, however, to a much lesser extent than the cells from which they were collected. Cytokine and proteome profiling of the conditioned media showed evidence of high concentrations of approximately 15 secreted proteins (including osteopontin, IL-8, GDF-15, MIF and VEGF). These 15 secreted proteins were expressed variably across the melanoma lines. Surprisingly, the addition of these individually to the brain endothelial cells did not substantially affect the barrier integrity. ANGPTL-4 and TGFß were also produced by the melanoma cells. Whilst TGFß-1 had a pronounced effect on the barrier integrity, surprisingly ANGPTL-4 did not. However, its C-terminal fragment did and within a very similar period to the conditioned media, albeit not to the same extent. Herein we show that melanoma cells produce a wide-range of soluble factors at high concentrations, which most likely favour support or survival of the cancer cells. Most of these, except for TGFß-1 and the C-terminal fragment of ANGPTL-4, did not have an impact on the integrity of the brain endothelial cells.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

Pro-inflammatory TNFα and IL-1ß differentially regulate the inflammatory phenotype of brain microvascular endothelial cells.

BACKGROUND: The vasculature of the brain is composed of endothelial cells, pericytes and astrocytic processes. The endothelial cells are the critical interface between the blood and the CNS parenchyma and are a critical component of the blood-brain barrier (BBB). These cells are innately programmed to respond to a myriad of inflammatory cytokines or other danger signals. IL-1ß and TNFα are well recognised pro-inflammatory mediators, and here, we provide compelling evidence that they regulate the function and immune response profile of human cerebral microvascular endothelial cells (hCMVECs) differentially. METHODS: We used xCELLigence biosensor technology, which revealed global differences in the endothelial response between IL-1ß and TNFα. xCELLigence is a label-free impedance-based biosensor, which is ideal for acute or long-term comparison of drug effects on cell behaviour. In addition, flow cytometry and multiplex cytokine arrays were used to show differences in the inflammatory responses from the endothelial cells. RESULTS: Extensive cytokine-secretion profiling and cell-surface immune phenotyping confirmed that the immune response of the hCMVEC to IL-1ß was different to that of TNFα. Interestingly, of the 38 cytokines, chemokines and growth factors measured by cytometric bead array, the endothelial cells secreted only 13. Of importance was the observation that the majority of these cytokines were differentially regulated by either IL-1ß or TNFα. Cell-surface expression of ICAM-1 and VCAM-1 were also differentially regulated by IL-1ß or TNFα, where TNFα induced a substantially higher level of expression of both key leukocyte-adhesion molecules. A range of other cell-surface cellular and junctional adhesion molecules were basally expressed by the hCMVEC but were unaffected by IL-1ß or TNFα. CONCLUSIONS: To our knowledge, this is the most comprehensive analysis of the immunological profile of brain endothelial cells and the first direct evidence that human brain endothelial cells are differentially regulated by these two key pro-inflammatory mediators.

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines.

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells. The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Melanoma Mediated Disruption of Brain Endothelial Barrier Integrity Is Not Prevented by the Inhibition of Matrix Metalloproteinases and Proteases.

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium.

Distinctive localization of antigen-presenting cells in human lymph nodes.

Professional antigen-presenting cells (APCs) are sentinel cells of the immune system that present antigen to T lymphocytes and mediate an appropriate immune response. It is therefore surprising that knowledge of the professional APCs in human lymph nodes is limited. Using 3-color immunohistochemistry, we have identified APCs in human lymph nodes, excluding plasmacytoid APCs, that fall into 2 nonoverlapping classes: (1) CD209+ APCs, coexpressing combinations of CD206, CD14, and CD68, that occupied the medullary cords, lined the capsule and trabeculae and were also scattered throughout the diffuse T-lymphocyte areas of the paracortex; and (2) APCs expressing combinations of CD1a, CD207, and CD208, that were always restricted to the paracortex. Surprisingly, this second class of APCs was almost entirely absent from many lymph nodes. Our data suggest that most CD208+ cells, often referred to as "interdigitating cells," derive from migratory APCs, and that the major APC subset consistently resident in the paracortex of human lymph nodes is the CD209+ subset. All APC subsets were demonstrated to be in close contact with the fibroreticular network. The identification of 2 distinct APC populations in the paracortex of human lymph nodes has important implications for understanding T-lymphocyte responses and optimizing vaccine design.

Targeting antigen to MHC class II molecules promotes efficient cross-presentation and enhances immunotherapy.

An efficient pathway of cross-presentation common to a range of dendritic cell (DC) populations was identified by targeting Ag to MHC class II molecules. This finding was achieved by conjugating Ag to M1, which is a modified version of the superantigen streptococcal mitogenic exotoxin Z-2 that binds to MHC class II molecules but cannot directly stimulate T cells. M1 conjugates were efficiently presented to CD4(+) and CD8(+) T cells by bone marrow-derived DC and Langerhans cells in vitro. Whereas nonconjugated Ag was preferentially cross-presented by splenic CD8alpha(+) DC in vivo, M1-conjugated Ag was cross-presented by all dendritic subtypes assessed. Potent effector T cell responses with antitumor activity were elicited when M1 conjugates were injected together with an adjuvant. This method of Ag delivery has significant potential in therapeutic applications.

Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1ß in Real-Time.

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1ß were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

Can ECIS Biosensor Technology Be Used to Measure the Cellular Responses of Glioblastoma Stem Cells?

Glioblastoma is considered the most aggressive and lethal form of brain cancer. Glioblastoma tumours are complex, comprising a spectrum of oncogenically transformed cells displaying distinct phenotypes. These can be generated in culture and are called differentiated-glioblastoma cells and glioblastoma stem cells. These cells are phenotypically and functionally distinct, where the stem-like glioblastoma cells give rise to and perpetuate the tumour. Electric cell-substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method for the analysis of cellular behaviour, based on cellular adhesion. Therefore, we asked the question of whether ECIS was suitable for, and capable of measuring the adhesion of glioblastoma cells. The goal was to identify whether ECIS was capable of measuring glioblastoma cell adhesion, with a particular focus on the glioblastoma stem cells. We reveal that ECIS reliably measures adhesion of the differentiated glioblastoma cells on various array types. We also demonstrate the ability of ECIS to measure the migratory behaviour of differentiated glioblastoma cells onto ECIS electrodes post-ablation. Although the glioblastoma stem cells are adherent, ECIS is substantially less capable at reliably measuring their adhesion, compared with the differentiated counterparts. This means that ECIS has applicability for some glioblastoma cultures but much less utility for weakly adherent stem cell counterparts.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

Real-Time Measurement of Melanoma Cell-Mediated Human Brain Endothelial Barrier Disruption Using Electric Cell-Substrate Impedance Sensing Technology.

Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30-60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.

The functional and inflammatory response of brain endothelial cells to Toll-Like Receptor agonists.

Toll-Like receptors (TLRs) represent an important early warning mechanism for the immune system to detect infection or tissue damage. The focus of this research was to determine the neuroinflammatory responses to commercial TLR ligands and their effects on brain endothelial barrier strength. Using biosensor technology we screened TLR ligands to all human TLRs and found that the brain endothelial hCMVECs cell line only responded to Poly(I:C) (TLR3-ligand), LPS (TLR4-ligand) and Imiquimod (TLR7 ligand). Both Poly(I:C) and LPS induced pronounced pro-inflammatory cytokine secretion as expected, whereas Imiquimod did not induce secretion of any pro-inflammatory cytokines. Using ECIS technology to measure endothelial barrier function, LPS and Poly(I:C) both acutely reduced barrier-strength, whereas Imiquimod caused immediate and sustained strengthening of the barrier. Further cytokine and ECIS studies showed that Imiquimod could abrogate some of the pro-inflammatory responses to Poly(I:C) and LPS. Most surprisingly, PCR revealed that the hCMVECs lacked TLR7 but expressed both TLR3 and TLR4 and did not respond to other structurally different TLR7 ligands. These data demonstrate that brain endothelial cells can be regulated by TLR 3 and TLR4 ligands in a pro-inflammatory manner and have receptors to Imiquimod, distinct to the classical TLR7, that function in an anti-inflammatory manner.

Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1ß activated endothelium in comparison to TNFα.

BACKGROUND: We have previously shown that TNFα and IL-1ß differentially regulate the inflammatory phenotype of human brain endothelial cells (hCMVECs). In this regard, IL-1ß treatment was considerably more potent than TNFα at increasing expression of inflammatory chemokines and leukocyte adhesion molecules. We therefore hypothesised that interaction of the hCMVECs with human monocytes would also be dependent on the activation status of the endothelium. Therefore, the primary aim of this study was to assess whether brain endothelial cells activated by IL-1ß or TNFα differed in their interaction with monocytes. METHODS: Monocyte interaction was measured using the real time, label-free impedance based ECIS technology, to evaluate endothelial barrier integrity during monocyte attachment and transendothelial migration. RESULTS: ECIS technology revealed that there was a greater loss of barrier integrity with IL-1ß activation and this loss lasted for longer. This was expected and consistent with our hypothesis. However, more striking and concerning was the observation that the method of monocyte enrichment greatly influenced the extent of endothelial barrier compromise. Importantly, we observed that positively isolated monocytes (CD14+ve) caused greater reduction in barrier resistance, than the negatively selected monocytes (untouched). Analysis of the isolated monocyte populations revealed that the CD14+ve isolation consistently yields highly pure monocytes (>92%), whereas the untouched isolation was much more variable, yielding ~70% enrichment on average. These two enrichment methods were compared as it was thought that the presence of non-classical CD16hi monocytes in the untouched enrichment may mediate greater compromise than the classical CD14hi monocytes. This however, was not the case and these observations raise a number of important considerations pertaining to the enrichment strategy, which are essential for generating reliable and consistent data. CONCLUSIONS: We conclude that IL-1ß and TNFα differentially influence monocyte interaction with brain endothelial cells and moreover, the enrichment method also influences the monocyte response as revealed using ECIS technology.

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1ß. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.