Pro-inflammatory TNFα and IL-1β differentially regulate the inflammatory phenotype of brain microvascular endothelial cells
© O’Carroll et al. 2015
Received: 21 December 2014
Accepted: 17 June 2015
Published: 8 July 2015
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-1β 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.
We used xCELLigence biosensor technology, which revealed global differences in the endothelial response between IL-1β 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.
Extensive cytokine-secretion profiling and cell-surface immune phenotyping confirmed that the immune response of the hCMVEC to IL-1β 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-1β or TNFα. Cell-surface expression of ICAM-1 and VCAM-1 were also differentially regulated by IL-1β 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-1β or TNFα.
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.
The vascular endothelium in the brain is the cellular interface between the blood and the central nervous system. These cells along with pericytes and vascular astrocytes form an essential physical barrier to immune cells, bacterial and viral infections (reviewed by [1, 2]). In addition, the nutrient homeostasis of the CNS is governed by the vascular endothelium, which is selective to or permeable to a range of small molecules and drugs . Collectively, this is often referred to as the blood-brain barrier (BBB). Rather than being an impenetrable fortress, the BBB serves a range of critical functions including maintaining the brain’s immune privileged status (low frequency of leukocytes) and tightly regulating the transfer of nutrients and waste products [4, 5].
The main cellular component of the BBB is an uninterrupted layer of microvascular endothelial cells that are joined by tight-junctions (reviewed by ). These cells are highly polarised, with their basolateral aspect juxtaposed to a continuous protein-containing basement membrane (basal lamina). The presence of tight-junction complexes (e.g. claudin-5, occludin, ZO-1 (see ) seals adjacent endothelial cells and restricts paracellular transfer of large molecules and cells . The apical aspect of the vascular endothelium is in direct contact with the blood and is therefore exposed to a range of insults including infections and systemic inflammatory responses.
It has become clear that the BBB is affected or changed in a wide variety of neurological diseases (e.g. multiple sclerosis [8, 9] and HIV infection [10, 11]), and in turn may modulate the disease process or clinical outcome [10, 12–14]. Although endothelial cells are not regarded as classical immune cells, extensive literature demonstrates that endothelial cells are inextricably involved in the inflammatory responses in many tissues. This is most likely very true in the human brain; however, most of what we believe about human brain endothelial (BBB) function has been inferred from observations using non-human or non-CNS-derived endothelial cells. This includes endothelial cells from other species [15–17] or from other tissues, such as the umbilical vein . Understandably, the number of studies that have actually used human brain endothelial cells (either primary or immortalised) represents a minor proportion of the literature.
Inflammatory cytokines regulate the function of brain microvascular endothelial cells leading to changes in endothelial activation , inflammatory phenotype  and extravasation of immune cells [19–21]. Clinically, this likely contributes to the severity of disease where BBB dysfunction is evident. However, the temporal profile of these changes has not been studied in detail using human endothelial cells from the brain (or of brain origin). Here we compare the temporal response profiles of brain endothelial cells to several major pro-inflammatory regulators (IL-1β and TNFα) and reveal that the nature of the inflammatory response differs substantially. Initially, we used xCELLigence biosensor technology, which revealed that the global temporal response of the brain endothelial cells to IL-1β and TNFα was different. This observation was corroborated where expression or secretion of a number of key pro-inflammatory mediators were differentially regulated by IL-1β and TNFα. Using an extensive cytokine cytometric bead array (CBA) panel and flow-cytometry panel, we also advance our understanding of which cytokines, chemokines and adhesion molecules are expressed by brain endothelial cells. IL-1β or TNFα also induced changes in endothelial barrier strength measured as transendothelial electrical resistance (TEER) using electric cell substrate impedance-sensing (ECIS) technology. We observed complex differences in the changes in TEER induced by IL-1β and TNFα, where there were differences in the magnitude of the response and temporal nature of the response.
Cell culture of hCMVECs
The human cerebral microvascular endothelial cell (hCMVEC) line was purchased from ABMGood (USA see http://www.abmgood.com). Cells were maintained in growth media containing M199 media (Gibco) supplemented with 10 % FBS, 1 μg/mL hydrocortisone (Sigma), 3 μg/mL human FGF (Peprotech), 10 μg/mL human EGF (Peprotech), 10 μg/mL heparin (Sigma), 2 mM Glutamax (Gibco) and 80 μM butyryl cAMP (Sigma) referred to as M199 10 % growth media. The hCMVECs experiments were typically conducted using low-serum plating media containing M199 media, 2 % FBS, 110 nM hydrocortisone, 1 μM insulin and 80 μM butyryl cAMP (M199 2 % plating media).
The xCELLigence real-time cell analyser (RTCA) measures cellular adhesion in real-time using custom E-plates, which are coated with high-density gold arrays for measuring electrical impedance (ACEA Biosciences, USA) [22, 23]. The xCELLigence biosensor measures cellular adhesion, which is converted to Cell Index (arbitrary units) by the xCELLigence software (version 1.2.1). Cells were seeded into E96 well plates at 54,000 cells per well in M199 2 % plating media and allowed to recover until the cells have attained a stable cell index. This was typically 24 h after seeding. Cytokine treatments are detailed in the corresponding figure legends.
Immunocytochemistry for endothelial tight and adherens-junction proteins
For confocal imaging, the cells were grown to confluence on collagen-coated 8-chamber glass slides (BD Biosciences. Cat, no: 354108). Cells were fixed for immunocytochemical staining using 4 % PFA at room temperature for 10 min. Excess PFA was removed with several washes using PBS.
Primary antibody against VE-cadherin/CD144 (Santa Cruz Biotechnology Inc, sc-6458) was prepared in PBS containing 1 % donkey serum and used at 1:200 dilution. Primary antibody to zonula occludin-1 (ZO-1; Invitrogen, 339100) was prepared in PBS containing 1 % goat serum and used at 1:500 dilution. Primary antibodies were incubated at 4 °C overnight. Unbound antibodies were removed by washing in PBS containing 0.2 % triton-X100 (PBST). Fluorophore conjugated secondary detection was used using the species-specific anti-IgG Alexafluor 488 antibodies from Life Technologies (A-21206 and A11055). These secondaries were used at 1:400 and incubated with cells for 2 h at ambient. Again, unbound antibodies were removed with gentle agitation with PBST washes. Nuclei were counter stained using Hoescht (1:500 dilution in PBST), and cells were mounted using AF1 mountant. Confocal imaging was conducted using an Olympus FV100 microscope. Images were merged using ImageJ software.
Stimulation of hCMVEC for cytokines production
The hCMVEC cultures were seeded into 24-well plates coated with 1 μg/cm2 collagen I (Gibco) at the density of 80,000 cells per well. Cells were grown to confluence in M199 10 % growth media. On the day of stimulation, the growth media was changed to M199 2 % plating media. In the first series of experiments, the stimulatory cytokines IL-1β and TNFα were each added to corresponding wells to the final concentration of 5 ng/mL. Control group received media only. 100 μl of conditioned media was collected at 4, 24 and 72 h post-treatment. In the second series of experiments, the cytokines were added across a range of concentrations (50 ng/mL to 50 pg/mL) for 24 h. The collected media were centrifuged at 400×g for 5 min at 4 °C to remove cellular debris. 80 μl of the clarified media was recovered and stored in several single-use aliquots for cytokine profiling. Media samples were stored at −20 °C.
Cytokine measurements using cytometric bead array (CBA)
Details of the cytokines measured in this study and whether they were secreted by the hCMVEC cultures
CBA flex set
Secreted by hCMVECsa
Human Basic FGF Flex Set
Human CD106 (VCAM-1) Flex Set
Human CD54 (ICAM-1) Flex Set
Human CD62E (E-Selectin) Flex Set
Human CD62P (P-Selectin) Flex Set
Human Soluble CD121a (IL-1 RI) Flex Set
Human CD121b (IL-1 RII) Flex Set
Human Eotaxin Flex Set
Human CD178 (Fas Ligand) Flex Set
Human Fractalkine Flex Set
Human Granzyme B Flex Set
Human GCSF Flex Set
Human IFN-γ Flex Set
Human IL-1β Flex Set
Human IL-2 Flex Set
Human IL-4 Flex Set
Human IL-6 Flex Set
Human IL-7 Flex Set
Human IL-8 Flex Set
Human IL-9 Flex Set
Human IL-10 Flex Set
Human IL-13 Flex Set
Human IL-17A Flex Set
Human IL-17 F Flex Set
Human IL-12/IL-23p40 Flex Set
Human IL-21 Flex Set
Human I-TAC Flex Set
Human IP-10 Flex Set
Human MCP-1 Flex Set
Human MIG Flex Set
Human MIP-1α Flex Set
Human MIP-1β Flex Set
Human RANTES Flex Set
Human Soluble TNFRI Flex Set
Human Soluble TNFRII Flex Set
Human TNFα Flex Set
Human VEGF Flex Set
Cell-surface flow-cytometry analysis of hCMVEC
Details of the cell-surface adhesion molecules investigated in this study
Alexa Fluor 488
Alexa Fluor 647
Alexa Fluor 648
Alexa Fluor 488
Measurement of TEER using ECIS technology
ECIS 96W20idf plates were coated with 10-mM L-cysteine to clean and modify the electrode surface so as to provide highly reproducible electrode capacitance. This was followed by coating the wells with 1 μg/cm2 collagen I. Then 50 μl of media was added to each well, and well stabilisation was carried out on the ECIS ZΘ to clean the electrodes prior to plating of cells. The hCMVEC cells were seeded at 20,000 cells/well and cultured for 48 h prior to treatment with cytokines as detailed in the corresponding figure legends. Multi frequency/time data was collected. Data were plotted at 4000 Hz, which is the optimal frequency to measure barrier formation and resistance .
Analysis of hCMVEC response profile using xCELLigence biosensor technology
Characterisation of cytokine and chemokine secretion under basal and activated conditions
Multiplex cytokine analysis provides a powerful tool to assess complex secretory profiles of human immune cells [23, 25, 26]. This was employed to assess the basal secretory output of the hCMVECs and measure any changes following treatment with TNFα and IL-1β over a time course of 3 days. This period is highlighted by the red arrows in Fig. 1a.
The phorbol ester PMA, was also used to potentially activate a wider signaling network than that induced by TNFα or IL-1β. PMA increased secretion of most of those regulated by TNFα and IL-1β. The amount secreted was generally lower in comparison to the cytokine-induced responses. That said, this clearly shows that activation of the PKC-signalling network results in a pro-inflammatory response in brain endothelial cells. There were, however, no additional cytokines present in our panel of 38 (see Table 1) specifically regulated by PMA.
It is worth noting that under these conditions, the hCMVEC did not secrete the MIP1α or MIP1β, which are potent pro-inflammatory chemokines nor did we detect secretion of the anti-inflammatory cytokines IL-4 or IL-10 from the endothelial cells under any conditions. The temporal profile of secretion, although expensive to conduct, reveals that IL-6 and GCSF levels at 72 h post IL-1β activation are lower than the levels observed earlier at 24 h. This is an interesting observation which suggests that the endothelial cells have the capacity to assimilate or degrade some of the cytokines they secrete. This is also an important observation in terms of future experiments, as the timing of media collection has to be carefully considered in this regard to maximise sensitivity of the response, but avoiding this later period, where additional mechanism are at play.
Cell-surface immunophenotyping of human brain microvascular endothelial cells
Expression of cell-surface adhesion molecules by brain endothelial cells
Expressed by the hCMVECs
Flow-cytometry analysis using hCMEC/D3 cells 
Flow cytometry using BMVEC 
RT-PCR using BMVEC 
Yes; highly inducible
Rat brain and rat brain-derived endothelial cells 
IHC using human brain tissue (not expressed) 
Regional distribution in human brain 
IHC using human brain tissue 
MCAM; S-endo-1; MUC-18
Flow cytometry using BMVEC 
Functional assay using hCMEC/D3, and IHC-rat brain 
Flow cytometry using hCMEC/D3 
Flow cytometry and RT-PCR using BMVEC 
Flow cytometry and ICC using HBMEC 
IHC on human brain microvessels 
Neural cadherin (NCAD)
Flow cytometry using BMVEC 
Flow cytometry using hCMEC/D3 
Alpha 4 integrin
Expressed by leukocytes but not detected on endothelial cells in human brain by IHC 
Flow cytometry using hCMEC/D3 
Yes; highly inducible
ELISA detection using HCEC 
Yes; highly inducible
IHC using human brain ;
Yes; very low
IHC using human infarcted brain 
There was a pronounced “bimodal” change in VCAM-1 expression following 24-h IL-1β treatment, but not as evident with TNFα (Fig. 4). It is possible that the left peak (lower expression) is related to the sVCAM-1 liberation in a population of cells as measured during the cytokine profiling. It was therefore decided to conduct a longer time course for these key leukocyte-adhesion molecules to ascertain their profile more fully. Figure 6 reveals the temporal profile of (a) ICAM-1 and (b) VCAM-1 following cytokine activation of the hCMVECs over a 144-h time course. Of particular interest is that TNFα induces a markedly higher level of both ICAM-1 and VCAM-1 expression in comparison to IL-1β. This time course reveals that both ICAM-1 and VCAM-1 expression occurs quickly and, in the case of ICAM-1, is sustained at high levels for a number of days. In contrast, VCAM-1 expression is shorter lived and peaks around 24 h (highlighted by the red line). The temporal breakdown (right-hand plots) reveals that the increased VCAM-1 expression is bimodal for both IL-1β and TNFα treatments (Fig. 6).
The response to IL-1β was rather different, with the maximal increase in TEER observed with 500 pg/mL, and this relative increase in TEER was not as large as that seen with TNFα. Interestingly, at 5- and 50-ng/mL IL1β, there was a pronounced delay in the increase in TEER of approximately 20 h, which was not observed following TNFα treatment. These responses have been plotted against each other for direct comparison in Fig. 8 (see also Additional file 5: Figure S5). This reveals that the response profiles are quite different from each other, except at 500 pg/mL (Fig. 8).
To our knowledge this study represents the most comprehensive simultaneous analysis of cell-surface adhesion molecules and cytokine secretion following pro-inflammatory cytokine activation of human brain microvascular endothelial cells (hCMVECs). The goal was to ascertain the consequence of inflammatory activation on a broad range of immune mediators involved in BBB neuroinflammation, leukocyte communication and recruitment. The rationale was to advance our understanding of the repertoire of adhesion molecules, cytokines and chemokines expressed by brain endothelial cells and how these change in a temporal manner following activation by key inflammatory triggers (i.e. TNFα and IL-1β). Specific expression profiles for human brain endothelial cells is limited [27–30] (see Table 3), in comparison to endothelial cells derived from other tissues (e.g. HMEC-1 skin endothelial cells).
The brain endothelial cells used in this study express all of the prototypic adhesion molecules expected of endothelial cells; including CD31 (PECAM), CD34, CD44, CD144 (VE-cadherin) and the tight-junction-associated protein ZO-1. In addition, the hCMVECs have very low levels of CD62E, ICAM-1 and VCAM-1 under basal non-inflamed conditions. This is consistent with the phenotype of brain endothelial cells in vivo  and confirms that our in vitro culture conditions maintain the cells in a non-activated state. This is extremely important for being able to induce inflammation. As anticipated, both IL-1β and TNFα increased cell-surface expression of these key leukocyte-adhesion molecules; however, the potency of the response and temporal nature was different for induction of ICAM-1 and VCAM-1. Both were up-regulated to a greater extent and for longer by IL-1β. Elevated expression levels of CD62E, ICAM-1 and VCAM-1 are reliable indicators that endothelial cells have been activated in some manner. However, the temporal window of their expression is very specific. Most of the other adhesion molecules appeared unaffected by activation with IL-1β or TNFα. However, it is worthy to note that this conclusion is drawn from flow-cytometry analysis. As such, it is possible that compartmentalised localisation may change, which could then affect the function of the adhesion molecule.
Brain endothelial cells are the cellular interface between the blood and CNS tissue and like most cells with innate immune functions can release a myriad of cytokines in response to inflammatory challenge or damage. Here, we measured the release of 38 different factors, which were comprised of cytokines, chemokines, growth factors and soluble forms of receptors/adhesion molecules (see Table 1). In total, 13 of these were secreted, with most only after activation by IL-1β or TNFα. Interestingly, we observed the selective secretion of two CCL-class chemokines, MCP-1 (CCL2) and RANTES (CCL5), but no secretion of Eotaxin (CCL11), MIP-1α (CCL3) or MIP1β (CCL4). These are, however, sufficient to target a broad spectrum of immune-cell subsets expressing CCR1, CCR2, CCR3 and CCR5. Fractalkine (CX3CL1), the only known CX3CL chemokine, was not secreted by the endothelial cells under basal or activated conditions.
The CXCL chemokines (in our panel) released by the hCMVECs were IL-8 (CXCL-8) and IP-10 (CXCL10), whereas there was no secretion of either MIG (CXCL9) or I-TAC (CXCL11). IL-8 is a potent chemoattractant of neutrophils, which signals through the CXCR1 and CXCR2 receptors, IP-10 signals through CXCR3, which is expressed by subsets of T cells and monocytes/macrophages. Of particular importance to note was that the secretion of IP-10, GCSF and GMCSF, were almost exclusively induced by IL-1β, whereas the effects of TNFα were negligible in comparison. The increased GCSF and GMCSF maybe associated with creation of an environment within the CNS to support monocyte/macrophage differentiation post extravasation. The endothelial cells secrete an abundance of MCP-1 following activation, which is a potent chemokine for monocyte/macrophage subsets. In this study, TNFα increased the secretion of IL-8 and RANTES to a much greater extent than that induced by IL-1β. These differences suggest that the leukocyte recruitment signals released by the brain endothelial cells are considerably different between TNFα and IL1β responses.
Truncated versions of certain receptors and adhesion molecules can be liberated (cleaved or secreted) from the cell surface and consequently detected in the conditioned media. Here we measured the soluble forms of the IL-1 receptors, TNF receptors and ICAM-1 and VCAM-1. Soluble IL-1 receptors were not detected under any conditions; however, soluble TNFα receptors (both TNFRI and TNFRII) were detected. Interestingly, the amount was greater following IL-1β treatment, rather than TNFα treatment. The relevance of this novel finding is unclear but may relate to the desensitisation of the inflammatory environment to restrict TNFα signaling.
It is very interesting to note that TNFα appears to induce a much greater level of ICAM-1 and VCAM-1 on the surface of the endothelial cells, in comparison to IL-1β. This could be due to the fact that the level of soluble ICAM-1 and VCAM-1 present in the culture media was greater following IL-1β activation. This demonstrates that more ICAM-1 and VCAM-1 are cleaved during the IL-1β response (compare the data in Figs. 3 and 6). These adhesion molecules are the interacting partners of CD11 family members (e.g. LFA-1) and CD49d (VLA-4), respectively, which are broadly expressed by a range of leukocyte subsets. This data series nicely provides the basis to conduct adhesion/diapedesis experiments, which will be the subject of a future study. At this stage, it is exciting to hypothesise that TNFα and IL-1β differentially regulate tethering/attachment of different leukocyte subsets to the apical surface of the brain endothelium.
As well as being a comprehensive investigation of adhesion molecules important in the wider context of endothelial biology, we reveal expression of a number of adhesion molecules by hCMVEC not described previously for human brain endothelial cells. These include CD138, CD141, CD147, CD325 and CD50. This advances our understanding of their phenotype and adhesion-molecule expression profile, all of which are molecules important in endothelial functional biology.
A very important area clinically is the influence of neuroinflammatory events on vascular barrier function. Dysfunction or breakdown of the brain vascular integrity is a well-documented event in diseases such as relapsing remitting multiple sclerosis or in ischemic stroke patients, which can lead to vascular haemorrhage in some stroke patients. Surprisingly, there is a lack of studies comparing the effect of IL-1β and TNFα on barrier function using human brain endothelial cells (primary or cell line-derived) and advanced autonomous technologies such as ECIS. We therefore used ECIS-Ztheta system to monitor the effect of TNFα and IL1β on barrier function in a label-free temporal manner. Of specific importance for us was to determine whether there were any differences between the cytokine responses and to monitor the changes in barrier resistance either acutely (first few hours—24 h) or chronically over days. There was a small acute reduction in the endothelial barrier resistance in the first few hours with higher concentrations of TNFα (above 500 pg/mL) and IL-1β. This response was small, reflecting approximately 10–15 % difference in norm resistance. Reduced TEER over this period has been reported by others [32, 33]. After this acute period, the barrier resistance of the HCMVECs increased in a cytokine, concentration and time-dependant manner. This strengthening of the barrier occurs over a period, which is (surprisingly) omitted in other ECIS studies [32, 33]. The magnitude of the increase in barrier resistance is greater and occurs for longer for TNFα. This perhaps suggests that the brain endothelial cells are innately programmed to recognise elevated TNFα as a more dangerous signal than IL-1β and respond accordingly in terms of strengthening its integrity. This may also be the innate response to protect against any danger signals that may lead to neutrophil activation and invasion, which in terms of the brain vasculature, can be devastating. Our barrier resistance (ECIS) data for the hCMVECs highlight the importance of conducting acute and longer term analysis of barrier function, which we have achieved in a real-time autonomous manner using ECIS ZΘ. It is clear that this is an area which requires considerably more research to be conducted to understand the pathways leading to barrier protection.
This current study highlights the fact that these two potent inflammatory mediators induce different inflammatory responses from brain endothelial cells. This was originally suggested from the xCELLigence data, which measures the net changes in cellular focal adhesion. This therefore indicates that the brain endothelial cells are innately programmed to respond to different insults in specific ways. Therefore in vivo, certain diseases, infections or tissue injury may cause elevated levels of IL-1β, which may trigger different pathways and cytokine networks in the brain endothelium in comparison to conditions where TNFα is predominantly elevated (e.g. bacterial infection and autoimmune diseases).
The data obtained in this study will be invaluable for investigating the dynamics of leukocyte attachment under different inflamed conditions and whether IL-1β induces recruitment of different subsets of immune cells in comparison to TNFα. The xCELLigence technology provides a cutting-edge tool to investigate the real-time temporal effects of activation of brain endothelial cells. In addition, the temporal sensitivity of ECIS for investigating cytokine changes in barrier function is incredibly powerful. This is clearly a complex area in the field of neuroinflammation, where there is still much to be discovered in terms of the endothelial responses and roles in disease. This will be applied to a range of activation signals and for conducting poly-pharmacological (multiple cytokines) analysis of how brain endothelial cells are regulated under more complex inflamed conditions.
This research was conducted with funding from the New Zealand Lottery Health Fund (ESG; xCELLigence and Accuri C6 and SO/ESG ECIS ZΘ), the Health Research Council (ESG) and from the University of Auckland Faculty Research Development Fund (ESG, SO and CEA).
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