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.

Cannabinoid Receptor 2 (CB2) Signals via G-alpha-s and Induces IL-6 and IL-10 Cytokine Secretion in Human Primary Leukocytes.

Cannabinoid receptor 2 (CB2) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB2 signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB2-selective ligand (HU308). We observed a "lag" phase of unchanged cAMP concentration prior to development of classically expected Gαi-mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gαi effects by concurrent Gαs activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gαi, whereas ERK1/2 and Akt phosphorylation were mediated by Gαi-coupled ßγ. Activation of CREB integrated multiple components; Gαs and ßγ mediated ∼85% of the response, while ∼15% was attributed to Gαi. Responses to HU308 had an important functional outcome-secretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB2. Additionally, signaling and functional effects were completely blocked by a CB2-selective inverse agonist, giving additional evidence for CB2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB2 ligands as immunomodulatory therapeutics.

In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Zθ Technology.

Electric Cell-Substrate Impedance Sensing (ECIS) can produce reproducible wounding models by mechanically disrupting a cell monolayer. This study compared in vitro wound-healing using human cerebral microvascular endothelial cells (hCMVEC) with both single electrode (8W1E) and multiple electrodes (8W10E+) arrays. Measurements of hCMVEC migration and barrier functions were conducted, revealing variable levels of barrier disruption could be achieved by altering the duration and magnitude of the applied current. In all scenarios, the barrier (Rb) did not recover the strength observed prior to injury. Localization of junctional proteins following wounding were analyzed by immunocytochemistry. Following wounding, cell migration was generally faster on the 8W10E+ than the 8W1E array. Immunohistochemical analysis revealed non-viable cells remained on the 8W1E electrodes but not the 8W10E+ electrodes. However, viable cells partially remained on the 8W10E+ electrodes following wounding. In addition, the 8W10E+ electrodes demonstrated variation in cell loss across electrodes within the same well. This suggests the type of wounding is different on the two array types. However, our data show both arrays can be used to model incomplete barrier recovery and therefore both have potential for testing of drugs to improve endothelial barrier function. This is the first time that the possibility of using the 8W10E+ array as a wounding model is addressed. We highlight the differences in wounding produced between the two arrays, and can be used to study the underlying causes for impaired barrier function following CNS injuries.

The Importance of Multifrequency Impedance Sensing of Endothelial Barrier Formation Using ECIS Technology for the Generation of a Strong and Durable Paracellular Barrier.

In this paper, we demonstrate the application of electrical cell-substrate impedance sensing (ECIS) technology for measuring differences in the formation of a strong and durable endothelial barrier model. In addition, we highlight the capacity of ECIS technology to model the parameters of the physical barrier associated with (I) the paracellular space (referred to as Rb) and (II) the basal adhesion of the endothelial cells (α, alpha). Physiologically, both parameters are very important for the correct formation of endothelial barriers. ECIS technology is the only commercially available technology that can measure and model these parameters independently of each other, which is important in the context of ascertaining whether a change in overall barrier resistance (R) occurs because of molecular changes in the paracellular junctional molecules or changes in the basal adhesion molecules. Finally, we show that the temporal changes observed in the paracellular Rb can be associated with changes in specific junctional proteins (CD144, ZO-1, and catenins), which have major roles in governing the overall strength of the junctional communication between neighbouring endothelial 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.

The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release.

BACKGROUND: Connexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease. METHODS: Retinal pigment epithelial cells were exposed to HG, 10ng/mL pro-inflammatory cytokines IL-1ß and TNF-α, or a combination of both. Quantitative Cytometric Bead Array analysis was used to measure the release of inflammatory cytokines IL-6, IL-8, MCP-1, and sICAM-1, as well as VEGF and ATP. To determine the role of connexin43 hemichannels in the disease process, changes in cytokine and ATP release were evaluated following treatment with Peptide5, a connexin43 hemichannel blocker. Immunohistochemistry was used to compare NLRP3 inflammasome assembly under control and treatment conditions. RESULTS: Co-application of HG and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF and ATP, to significantly higher levels compared to cytokines alone. Peptide5 prevented cytokine release and prevented the increase in ATP release following co-application of HG and cytokines. Adding exogenous ATP negated Peptide5-mediated protection against inflammatory cytokine release in injury conditions. CONCLUSIONS: Our findings show that connexin43 hemichannels play an important role in the amplification and perpetuation of inflammation by mediating an ATP autocrine feedback loop in the inflammasome/inflammation cycle. GENERAL SIGNIFICANCE: Targeting connexin43 hemichannels offers a potential therapeutic strategy to break the inflammatory cycle in diseases such as DR, but also other chronic inflammatory indications.

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.

Is the Cannabinoid CB2 Receptor a Major Regulator of the Neuroinflammatory Axis of the Neurovascular Unit in Humans?

The central nervous system (CNS) is an immune privileged site where the neurovascular unit (NVU) and the blood-brain barrier (BBB) act as a selectively permeable interface to control the passage of nutrients and inflammatory cells into the brain parenchyma. However, in response to injury, infection, or disease, CNS cells become activated, and release inflammatory mediators to recruit immune cells to the site of inflammation. Increasing evidence suggests that cannabinoids may have a neuroprotective role in CNS inflammatory conditions. For many years, it was widely accepted that cannabinoid receptor type 1 (CB1) modulates neurological function centrally, while peripheral cannabinoid receptor type 2 (CB2) modulates immune function. As knowledge about the physiology and pharmacology of the endocannabinoid system advances, there is increasing interest in targeting CB2 as a potential treatment for inflammation-dependent CNS diseases (Ashton & Glass, 2007), where recent rodent and human studies have implicated intervention at the level of the NVU and BBB. These are incredibly important in brain health and disease. Therefore, this review begins by explaining the cellular and molecular components of these systems, highlighting important molecules potentially regulated by cannabinoid ligands and then takes an unbiased look at the evidence in support (or otherwise) of cannabinoid receptor expression and control of the NVU and BBB function in humans.

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.

Statins Inhibit Fibrillary ß-Amyloid Induced Inflammation in a Model of the Human Blood Brain Barrier.

BACKGROUND: Astrocytes and cerebral endothelial cells are important components of the blood-brain barrier (BBB). Disruption to this barrier through inflammation is a major contributor to Alzheimer's disease (AD) pathology. The amyloid beta (Aß) protein is known to exist in several forms and is a key modulator of AD that is known to cause inflammation and changes to BBB function. While one of these forms, fibrillary Aß (fAß), is known to cause endothelial cell death at the BBB, no studies have looked specifically at its role on inflammation in a model of the human BBB. AIMS: To determine if fAß is inflammatory to the human BBB. As statins have been shown to be anti-inflammatory and protective in AD, we also tested if these could inhibit the inflammatory effect of fAß. METHODS: Using cultured cerebral endothelial cells and astrocytes we determined changes in cytokine release, cell toxicity and barrier function in response to fibrillary ß-amyloid1-42 (fAß1-42) alone and in combination with statins. RESULTS: fAß1-42 induced inflammatory cytokine release from endothelial cells in the absence of cell toxicity. It also induced astrocyte cytokine release and cell death and caused a loss of barrier integrity. Statin treatment inhibited all of these effects. CONCLUSIONS: We conclude that fAß1-42 has both inflammatory and cytotoxic effects on the BBB and the protective effect of statins in AD may in part be through inhibiting these effects.

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.

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.

An anti-inflammatory role for C/EBPδ in human brain pericytes.

Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1ß (IL-1ß). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1ß-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1ß-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1ß, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

Application of xCELLigence RTCA Biosensor Technology for Revealing the Profile and Window of Drug Responsiveness in Real Time.

The xCELLigence technology is a real-time cellular biosensor, which measures the net adhesion of cells to high-density gold electrode arrays printed on custom-designed E-plates. The strength of cellular adhesion is influenced by a myriad of factors that include cell type, cell viability, growth, migration, spreading and proliferation. We therefore hypothesised that xCELLigence biosensor technology would provide a valuable platform for the measurement of drug responses in a multitude of different experimental, clinical or pharmacological contexts. In this manuscript, we demonstrate how xCELLigence technology has been invaluable in the identification of (1) not only if cells respond to a particular drug, but (2) the window of drug responsiveness. The latter aspect is often left to educated guess work in classical end-point assays, whereas biosensor technology reveals the temporal profile of the response in real time, which enables both acute responses and longer term responses to be profiled within the same assay. In our experience, the xCELLigence biosensor technology is suitable for highly targeted drug assessment and also low to medium throughput drug screening, which produces high content temporal data in real time.

Exposure to inflammatory cytokines IL-1ß and TNFα induces compromise and death of astrocytes; implications for chronic neuroinflammation.

BACKGROUND: Astrocytes have critical roles in the human CNS in health and disease. They provide trophic support to neurons and are innate-immune cells with keys roles during states-of-inflammation. In addition, they have integral functions associated with maintaining the integrity of the blood-brain barrier. METHODS: We have used cytometric bead arrays and xCELLigence technology to monitor the to monitor the inflammatory response profiles and astrocyte compromise in real-time under various inflammatory conditions. Responses were compared to a variety of inflammatory cytokines known to be released in the CNS during neuroinflammation. Astrocyte compromise measured by xCELLigence was confirmed using ATP measurements, cleaved caspase 3 expression, assessment of nuclear morphology and cell death. RESULTS: Inflammatory activation (IL-1ß or TNFα) of astrocytes results in the transient production of key inflammatory mediators including IL-6, cell surface adhesion molecules, and various leukocyte chemoattractants. Following this phase, the NT2-astrocytes progressively become compromised, which is indicated by a loss of adhesion, appearance of apoptotic nuclei and reduction in ATP levels, followed by DEATH. The earliest signs of astrocyte compromise were observed between 24-48 h post cytokine treatment. However, significant cell loss was not observed until at least 72 h, where there was also an increase in the expression of cleaved-caspase 3. By 96 hours approximately 50% of the astrocytes were dead, with many of the remaining showing signs of compromise too. Numerous other inflammatory factors were tested, however these effects were only observed with IL-1ß or TNFα treatment. CONCLUSIONS: Here we reveal direct sensitivity to mediators of the inflammatory milieu. We highlight the power of xCELLigence technology for revealing the early progressive compromise of the astrocytes, which occurs 24-48 hours prior to substantive cell loss. Death induced by IL-1ß or TNFα is relevant clinically as these two cytokines are produced by various peripheral tissues and by resident brain cells.