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. 2015 Jul 15;146(1):130–143. doi: 10.1111/imm.12489

C5a alters blood–brain barrier integrity in a human in vitro model of systemic lupus erythematosus

Supriya D Mahajan 1, Neil U Parikh 1, Trent M Woodruff 2, James N Jarvis 3, Molly Lopez 3, Teresa Hennon 1, Patrick Cunningham 4, Richard J Quigg 1, Stanley A Schwartz 1, Jessy J Alexander 1,✉
PMCID: PMC4552508  PMID: 26059553

Abstract

The blood–brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5–50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor-κB translocation into nucleus and regulates the expression of the tight junction proteins, claudin-5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

Keywords: anaphylatoxins, blood–brain barrier, endothelium, neurodegeneration, systemic lupus erythematosus

Introduction

Systemic lupus erythematosus (SLE) is a complex, multiorgan disease affecting 1–2 million Americans with neurological complications in 5–50%.1 It is associated with significant morbidity and mortality and the culprit (or culprits) causing the disease, especially the neuropsychiatric symptoms, remain an enigma, with few therapeutic options.2–4 Complement activation is a key event in this disease and patients with SLE have significantly elevated levels of the complement activation products C5a and C3a, which correlate with a poor outcome.5,6 Our studies using rodent models have shown that these proteins could play an important role in cerebral lupus.7–9 Inhibition of the complement cascade by over-expression of the pan complement inhibitor, Crry, inhibition of the alternative pathway by deletion of factor B, and elimination of anaphylatoxin receptor activity using antagonists alleviated the central nervous system lupus pathology in experimental models by reducing oedema, and generating inflammatory mediators such as reactive oxygen species and inducible nitric oxide synthase.

The complement (C) cascade is an important arm of the innate immune system.10–13 Normally protective, when excessively activated, its beneficial effects can become detrimental to the host. Complement activation can be triggered through four different pathways consisting of > 40 circulating and membrane-bound proteins that ultimately converge to form the terminal membrane attack complex along with the generation of the activation peptides C3a and C5a.14,15 These peptides are small (molecular weight approximately 10,000 daltons) cleavage fragments that act as cell activators with nanomolar affinity, exerting their functions through binding to specific receptors [C3aR and C5aR1 (CD88) or C5aR2 (C5L2), respectively].16,17 These receptors have a nearly ubiquitous expression present on epithelial, endothelial and myeloid cells. In brain, they are present on microglia, astrocytes, neurons and endothelial cells.18 With the exception of C5aR2, they are pertussis toxin sensitive, G-protein coupled, transmembrane spanning receptors. Their activation can mediate both pro-inflammatory and anti-inflammatory functions.19,20 In experimental lupus, inhibition of the receptors C5aR1 and C3aR alleviated or significantly reduced the disease pathology.20,21 Using a monolayer of rodent brain endothelial cells our results showed that C5a alters the integrity of the blood–brain barrier (BBB) through the nuclear factor-κB (NF-κB) pathway.22,23

The BBB represents a dynamic interface between the circulatory system and the brain. The vascular endothelium regulates cellular metabolite levels, vascular tone and haemostasis as well as the ingress and egress of leucocytes to and from the brain.24–26 The brain endothelium is damaged and may be an important contributing factor in the pathogenesis of SLE. Vascular damage could lead to secondary neuronal disorders, where autoantibodies gain access to the brain and cause neuronal dysfunction.27,28

The goal of this study was to determine the translational potential of the studies identifying C5a/C5aR1 signalling as one of the key culprits inducing loss of BBB integrity in the lupus setting. The focus was C5a/C5aR signalling in endothelial cells and astrocytes, so we did not assess C5aR2, the more recently identified receptor of C5a. We studied the role of C5a in SLE brain using a novel two-dimensional in vitro set up that emulates the in vivo system, allowing the assessment of human brain microvascular endothelial cells (HBMVECs) and astroglial cells (normal human astrocytes; NHAs) involved in the formation of the BBB and their response to serum obtained from patients with SLE. The results of this study demonstrate that C5a/C5aR1 signalling regulates the BBB integrity in the human two-dimensional in vitro system in a similar way to results obtained in rodent endothelial monolayers. The results of this study demonstrate that C5a/C5aR1 signalling alters NF-κB translocation into the nucleus. The pro-inflammatory response anticipated in SLE could involve both transcription factors cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) mediated NF-κB activation via the NF-κB-inducing kinase/IκB kinase/I-κ-B-α (NIK/IKK/IKB) cascade, which regulates actin cytoskeletal reorganization leading to increased BBB permeability.

Materials and methods

In vitro model

To determine BBB integrity in lupus and the role of C5a/C5aR1 signalling we used the BBB model that closely mimics and accurately reflects the characteristics and functional properties of the in vivo BBB. It is a well validated two-dimensional BBB in vitro co-culture system transwell model.29,30 The two cell types that are known to constitute the human BBB, HBMVECs (Cat# ACBRI-376) and NHAs (Cat# ACBRI-371) obtained from Applied Cell Biology Research Institute (ACBRI, Kirkland, WA) were used in this system. The NHAs were cultured on the underside of a PET insert (basal end represents Brain side) and HBMVECs were cultured on the inside of the PET insert (apical end represents Blood side) and allowed to form monolayers and differentiate where the astrocytic feet extend to the HBMVEC monolayer and together they form a tight barrier that effectively mimics the in vivo BBB. This two-dimensional in vitro BBB model has been well validated by several investigators and established in our laboratory.30–39

HBMVECs and NHAs were seeded until confluence on 1% gelatine-coated 25-cm2 tissue-culture flasks. Cells were grown in RPMI-1640 medium Hyclone (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum Gibco- Life technologies, Grand Island, NY, USA, heparin (100 μg/ml), endothelial cell growth factor supplement (50 μg/ml), sodium pyruvate (2 mm), l-glutamine (2 mm), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma- Aldrich, St Loius, MO, USA) at 37° in a humidified 5% CO2 incubator. Cultured cells were identified as endothelial by their morphology and von Willebrand factor antibody and glial acidic fibrillary protein binding.

MTT assay

Viability of HBMVECs and NHAs in culture was assessed using the MTT assay.40 The assay measures the ability of an active mitochondrial enzyme to reduce the MTT substrate (yellow to blue) in live cells. Isolated cells were plated in serum-free conditions on 48-well plates pre-coated with laminin. After 24 or 48 hr of culture, 0·5 mg/ml MTT substrate (Thiazolyl Blue Tertrazolium Bromide) was added and cells were incubated for an additional 4 hr, and then solubilized with 10% SDS (0·01 M HCI) overnight. Absorbance was measured at 595 nm.

Treatment

Cells were treated with serum isolated from control patients, patients wth SLE, human C5a (R&D Systems, Minneapolis, MN) (0·1 μg/ml)41,42 or C5aR1 antagonist (PMX205)41,42 for a period of 24 hr.

RNA extraction

Cytoplasmic RNA was extracted by an acid guanidinium–thiocyanate–phenol–chloroform method as described using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). The amount of RNA was quantified using a Nano-Drop ND-1000 spectrophotometer (Nano-Drop™, Wilmington, DE) and isolated RNA was stored at −80° until used.

Real-time, quantitative PCR

Quantitative PCR is used to quantify C5aR1, zonula occludens 1 (ZO-1), Claudin-5, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) gene expression in HBMVEC and NHA cultures. Approximately 1 × 106 HBMVECs and NHAs were treated with serum isolated from control patients, patients with SLE, human C5a (0·1 μg/ml) or C5aR1 antagonist (PMX205) (1 μm) for a period of 24 hr and RNA was extracted as described above. The RNA was then reverse transcribed to cDNA using a reverse transcriptase kit (Promega Inc., Madison, WI). Relative abundance of each mRNA species is quantified by quantitative PCR using specific primers and the Brilliant® SYBR® green Q-PCR master mix (Stratagene Inc, La Jolla, CA). The sequences of the primers for real-time PCR are given in Table1. The housekeeping gene β-actin was used as the internal control. To provide precise quantification of the initial target in each PCR, the amplification plot was examined and the data were calculated as described. Relative expression of mRNA species was calculated using the comparative threshold cycle number (CT) method. Briefly, for each sample, a difference in CT values (ΔCT) is calculated for each mRNA by taking the mean CT of duplicate tubes and subtracting the mean CT of the duplicate tubes for the reference RNA (β-actin) measured on an aliquot from the same reverse transcription reaction. The ΔCT for the treated sample is then subtracted from the ΔCT for the untreated control sample to generate a ΔΔCT. The mean of these ΔΔCT measurements is then used to calculate the levels in the targeted cytoplasmic RNA relative to the reference gene and normalized to the control as follows: Relative levels or Transcript Accumulation Index (TAI) = Inline graphic. This calculation assumes that all PCR reactions are working with 100% efficiency. All PCR efficiencies were found to be > 95%; therefore, this assumption introduces minimal error into the calculations. All data were controlled for quantity of RNA input and by performing measurements on an endogenous reference gene, β-actin.

Table 1.

The sequences of primers used in quantitative real time-PCR were

Gene Forward primer Reverse primer
5′ → 3′ 5′ → 3′
GAPDH 5′-GCAAATTCAACGGCACAGT-3 5′-AGATGGTGATGGGCTTCCC-3′
C5aR1 5′-GATGCCACCGCCTGTATAGT-3′ 5′-ACGAAGGATGGAATGGTGAG-3′
ZO-1 5′-GGGGCCTACACTGATCAAGA-3′ 5′-GGTCTCTGCTGGCTTGTTTC-3′
Claudin-5 5′-AAGGTGTACGACTCGCTGCT-3′ 5′-AGTCCCGGATAATGGTGTTG-3′
ICAM-1 5′-CGCAAGTCCAATTCACACTGA-3′ 5′-CAGAGCGGCAGAGCAAAAG-3′
VCAM-1 5′-ATCCAGGTGGAGATCTACTC-3′ 5′-TCTCAAAACTCACAGGGCTC-3′
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GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ICAM, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; ZO, zonula occludens 1.

Patients

Clinical samples were obtained from children attending the paediatric rheumatology clinics at the Women and Children’s Hospital of Buffalo. Samples were obtained from two boys and six girls who ranged in age from 7 to 15 years and processed identically along with healthy controls. Approval to acquire and use clinical materials was in accordance with the University at Buffalo Children and Youth Institutional Review Board. The patients with SLE fulfilled four or more of the revised classification criteria for SLE from the American College of Rheumatology 1997 were included in this study. In line with the focus of this study, concentration of circulating C5a was assessed in the samples.

ELISA for C5a

C5a ELISA was performed in human serum using a C5a ELISA kit (HK349; Hycult Biotech, Inc. Plymouth Meeting, PA, USA). Blood samples obtained from patients were kept at room temperature for 60 min and centrifuged to isolate serum. Samples were diluted four times with the dilution buffer provided and subjected to ELISA according to the manufacturer’s instructions. Assays were developed with tetramethylbenzidine conductivity substrate (BioFX Laboratories, Owings Mills, MD), and the OD value was measured at 450 nm. The ELISA measures both C5a and C5a-desArg. However, since C5a-desArg is a breakdown product of C5a, it was used as a measure of C5a in this study.

Expression of C5aR1 on the cells in the BBB model

C5aR1 expression was evaluated on native HBMVECs and NHAs in the presence of control or lupus serum. The cells were harvested, blocked with 3% BSA for 20 min and exposed to RPE anti-CD88 monoclonal antibody (clone 38-13; final dilution 1 : 100; Immunotech, Laboratories, Inc. Monrovia, CA, USA) for 45 min at 4° along with cell markers, CD31 and glial acidic fibrillary protein for HBMVECs and NHA, respectively. Immunostained cells were then washed and immediately analysed by confocal microscopy. Isotype-matched control immunostaining was performed in parallel.

BBB integrity by transendothelial electrical resistance

Resistance is inversely proportional to permeability. Therefore the integrity and permeability of the BBB in this model was quantified by measuring the transendothelial electrical resistance (TEER) across the layers. In this BBB model, the TEER directly correlates with BBB tightness and permeability. Formation of an intact BBB typically takes 5 days when using primary human brain microvascular endothelial cells and astrocytes.28,29 Measurement of TEER across the barrier enables monitoring of BBB integrity. The typical TEER values for the intact stabilized tight two-dimensional BBB range between 200 and 300 ohm/cm2 and were measured using the Millicell® ERS-2 Voltammeter system (EMD Millipore, Billerica, MA, USA). Cells were treated with 5% control serum, 5% lupus serum and control serum + 0·1 μg/ml C5a or and lupus serum + C5aR1 antagonist (1 μm) for 24 hr. TEER values were obtained before treatment (baseline) and after treatment, and are expressed as relative changes in TEER values as a percentage of the baseline value.

Tight junctions

The BBB model is characterized by high expression of tight junctions, appropriately segregated luminal (blood-facing)/abluminal (brain-facing) transporters (i.e. potassium, amino acids, glucose GLUT-1), reflecting the biochemical characteristics of functional BBB.43–48 We assessed the expression of the tight junction proteins, ZO-1, Claudin-5 in lupus and in response to C5a using real-time PCR. The gene-specific primers used are given in Table1. Additionally, we used immunofluorescent staining to quantify the expression of tight junction proteins in HBMVECs. HBMVECs were treated with 5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or C5aR1 antagonist (1 μm) + 5% lupus serum for 24 hr and immunofluorescent staining of Claudin-5 and ZO-1 was carried out using anti-human primary antibodies raised in goat obtained from Santa Cruz Biotech [Santa Cruz, CA; Cat # sc-17667 (G-15) goat polyclonal IgG and Cat # sc-8147 (N-19) goat polyclonal IgG, respectively]. The secondary antibody was a fluorescence labelled Alexa Fluor 647 rabbit anti-goat IgG (H+L) (Cat # A-21446; Molecular Probes Inc., Life Technologies, Grand Island, NY). DAPI (Cat #D1306; Molecular Probes Inc., Life Technologies) was used to stain the nuclei. The protein expression levels of Claudin-5 and ZO-1 were quantified based on the intensity of the fluorescence signal analysed using the computer image analysis image J software (National Institutes of Health, Bethesda, MA).

Structural integrity

After the cells reached confluency, the medium was replaced with Dulbecco’s modified Eagle’s medium (Gibco BRL, Chagrin Falls, OH) with 2% fetal bovine serum for synchronization. Cells were subjected to the different treatments (5% control serum, 5% lupus serum, or 0·1 μg/ml C5a) for 3 hr. So the cytoskeletal rearrangement could be observed, cells were fixed with 1% paraformaldehyde in PBS for 20 min then washed and permeabilized with 0·25% Triton X-100 as described earlier.45 F actin was visualized using FITC-conjugated phalloidin. Cells were mounted with ProLong Gold Antifade (Molecular Probes, Eugene, OR). F-actin distribution was evaluated by confocal microscopy using the above equipment by considering maximum fluorescence intensity projections along the whole Z-stack.

Evaluation of cell adhesion molecules

To evaluate HBMVEC activation, mRNA expression of ICAM-1 and VCAM-1 was determined by real-time PCR. Gene-specific primers used are given in Table1. HBMVECs were stimulated with C5a and lupus serum. The adhesion molecule expression was also determined on astrocytes that contribute to the BBB.

Quantification of ERK and CREB levels in cell lysates by ELISA

Both HBMVECs and NHAs were treated with control serum, C5a, lupus serum and/or C5aR1 antagonist for 96 hr. Cells were lysed and protein was extracted using a commercially available M-PER reagent (Thermo Fisher Scientific, Inc. Waltham, MA USA), a mammalian protein extraction reagent. The cell lysates were analysed for the levels of total ERK and CREB protein using commercially available InstantOneTM ELISA kits (eBioscience, San Diego, CA): Cat #85-86151 to measure total CREB and Cat # #85-86011 to measure total ERK in cell lysates as per the manufacturers’ instruction.

Statistical analysis

Statistical differences among treatments were determined using paired t-test for the two-group comparisons and paired one-way analysis of variance followed by Bonferroni post hoc test for multiple comparison. A P-value < 0·05 was considered significant. All tests were carried out using Prism 5·0 (Graph Pad Software, La Jolla, CA). Potential correlations among variables were determined by calculating Pearson product moment correlation coefficients and their P values. Significance was determined as P < 0·05.

Results

C5a concentration is increased in patients with SLE

Sera samples from five patients with SLE (females, 11–15 years old) and five healthy controls were obtained with informed consent and processed identically for use in in vitro experiments. In line with our study, we assessed circulating C5a levels by ELISA. The concentration of C5a was significantly elevated in the patients with SLE compared with controls (36·78 ± 5·13 ng/ml versus 7·15 ± 1·56 ng/ml; P < 0·05).

C5aR1 expression in human HBMVECs and NHAs is increased in lupus

HBMVECs and NHAs were cultured as described in the Materials and methods. The cells were viable (95%) using MTT assay. The expression of the canonical C5a receptor, C5aR1, was assessed at the transcriptional and translational levels. C5aR1 mRNAs were detected by quantitative RT-PCR (primers given in Table1) and the cellular presence of the receptor was analysed by immunofluorescence. C5aR1 is present along the membrane in HBMVECs with a more ubiquitous staining in the NHAs. C5aR1 expression in both HBMVECs and NHAs were significantly increased [HBMVECs, (TAI = 1·60 ± 0·15); 60% increase versus control (TAI = 1·00 ± 0·06); P < 0·001, and NHAs, (TAI = 1·91 ± 0·18); 91% increase versus control (TAI = 1·00 ± 0·06), P < 0·0001] in response to treatment with lupus serum compared with normal serum controls (Fig.1).

Figure 1.

Figure 1

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Expression of C5aR1 is up-regulated in lupus serum treated human brain microvascular endothelial cells (HBMVECs) and normal human astrocytes (NHAs). Expression of C5aR1 was assessed by immunofluorescence and quantitative RT-PCR. C5aR1 expression was localized to the membrane in HBMVECs (a) whereas it had ubiquitous expression in the NHAs (b). Lupus serum treatment significantly up-regulated C5aR1 expression in both cell types at the mRNA level as determined by real-time quantitative PCR (c), mRNA expression was normalized to GAPDH. Values are expressed as mean ± SEM. *P < 0·05, **P < 0·01. A P value of < 0·05 is considered a statistically significant difference.

Effect of C5a/C5aR1 signalling on actin reorganization in BBB HBMVECs and NHAs

The actin cytoskeleton maintains the structural integrity of the cell, and its reorganization could lead to increased vascular permeability. FITC-phalloidin staining of cells fixed with 4% paraformaldehyde and permeabilized with 0·2% Triton X-100 was performed to permit visualization of the actin filaments (Fig.2). The addition of lupus serum caused increased F-actin stress fibre formation in the endothelial cell layer. In endothelial cells treated with control serum, F-actin staining was orderly. Addition of lupus serum altered the F-actin appearance to an aggregated form with increase in intercellular gaps, which could lead to altered BBB permeability. C5a treatment showed similar changes with increased actin polymerization and intercellular gaps suggesting that it could potentially be an important protein contributing to lupus pathology.

Figure 2.

Figure 2

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C5a reduces stress fibre formation in human brain microvascular endothelial cells (HBMVECs) and normal human astrocytes (NHAs). Monolayers of HBMVECs and NHA cells were treated with 5% control serum (left), lupus serum (centre) or C5a (right) for 3 hr. Typical patterns of FITC-phalloidin staining indicating actin rearrangement are seen in cells treated with lupus serum compared with cells treated with control serum. Treatment of cells with C5a resulted in actin rearrangement and increased intercellular gaps, similar to that observed with lupus serum. Representative images from three independent experiments are shown. Nuclei were stained with DAPI.

C5a regulates BBB permeability in lupus

To analyse BBB integrity, TEER was measured across human HBMVECs and NHAs in vitro, after challenge with the conditions described in Materials and methods (Fig.3). Before treatment of the BBB, the TEER values at baseline were similar and decreased significantly when treated with lupus serum and C5a (Table2). The TEER values before and after C5aR1 antagonist (1 μm) treatment were similar to the untreated control or the BBB treated with control serum (Fig.3). The BBB was then treated with 5% control serum, 5% lupus serum, and control serum + 0·1 μg/ml C5a or and lupus serum + C5aR1 antagonist (1 μm) for 24 hr after which TEER values were measured post treatment. TEER values dramatically decreased in lupus serum-treated cells compared with cells treated with control serum (56% decrease versus control, P < 0·0001). Treatment with C5a reduced TEER compared to controls (35% decrease versus control, P < 0·006). Increased concentration of C5a in lupus serum reduced TEER even further compared with lupus serum alone (data not shown). Pre-treatment of the BBB with the C5aR1 antagonist before lupus serum treatment, resulted in TEER values that approached normal control levels (11% decrease versus control, P < 0·009). These results indicate the key role of C5a in endothelial layer integrity, signalling through its receptor C5aR1.

Figure 3.

Figure 3

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C5a/C5aR1 signaling regulates transendothelial electrical resistance (TEER) of blood–brain barrier (BBB) in vitro. Human brain microvascular endothelial cells (HBMVECs) and normal human astrocytes (NHAs) were grown as described in the Materials and methods, and after 3 hr of treatment, TEER was monitored over time. Changes in TEER (%) are expressed as means ± SE. Pooled data from three independent experiments are shown. Effects of different treatments [5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or 1 μm C5aR1 antagonist +5% lupus serum] were recorded. Increased permeability was observed after treatment with lupus serum or C5a compared with control serum, which was prevented by C5aR1 antagonism.

Table 2.

Transendothelial electrical resistance (TEER) measurements in the in vitro blood–brain barrier in response to treatment withC5a/lupus serum and C5a antagonist

Treatments TEER pre-treatment TEER post-treatment P value
Blank 140 ± 2·0 123·33 ± 10·26 NS
Untreated control 282·60 ± 3·06 285·33 ± 3·06 NS
C5a 0·1 μg/ml 292·66 ± 7·57 186·66 ± 8·33 P < 0·006
Lupus serum 288·67 ± 4·62 128·66 ± 4·16 P < 0·0001
C5a antagonist + lupus serum 288 ± 4·0 254 ± 4·0 P < 0·015
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C5aR1 signalling affects tight junction/intercellular protein expression

Tight junctions located in the BBB are an important facet of regulation of transport into and out of the brain, and their destruction causes barrier hyperpermeability. ZO-1 is a scaffolding protein that links tight junction transmembrane proteins to cytoskeletal filaments and disrupting the expression or distribution of ZO-1 leads to disruption of tight junction assembly.46–51 Therefore, we examined the effect of C5a/C5aR1 signalling on the expression of tight junction proteins claudin and ZO-1 in HBMVECs and NHAs treated with lupus serum, C5a or the C5aR1 antagonist (Fig.4). Messenger RNA was extracted and subjected to quantitative RT-PCR using specific primers to ZO-1 and Claudin-5. Claudin-5 (Fig.4a) mRNA expression was significantly reduced in cells treated with lupus serum compared with those treated with control serum [35% decrease in HBMVECs (TAI = 0·65 ± 0·09) versus control (1·00 ± 0·07), P < 0·018; and 24% decrease in NHAs (TAI = 0·76 ± 0·14) versus control (1·00 ± 0·11), P < 0·027, respectively]. When the cells were pre-treated with the C5aR1 antagonist and subsequently with lupus serum, Claudin-5 expression was significantly increased compared with those treated with normal serum [33% increase in HBMVECs (TAI = 1·33 ± 0·14) versus control, (TAI = 1·00 ± 0·14) P < 0·003 and 51% increase in NHAs (TAI = 1·51 ± 0·08) versus control (TAI = 1·00 ± 0·04), P < 0·003, respectively] indicating that C5a/C5aR1 signalling is an important aspect of BBB integrity in lupus. Claudin-5 gene expression levels in HBMVEC and NHA cells treated with the C5aR1 antagonist alone were 0·92 ± 0·07 and 1·01 ± 0·02, respectively, and were comparable to the Claudin-5 gene expression levels in the untreated media control (0·99 ± 0·03 and 1·03 ± 0·05) for the two cell types, respectively. When the cells were treated with lupus serum, expression of ZO-1 (Fig.4b) was significantly decreased [28% decrease in HBMVECs (TAI = 0·72 ± 0·076) versus control, (TAI = 1·00 ± 0·13) P < 0·031 and 35% decrease in NHAs (TAI = 0·65 ± 0·13) versus control, (TAI = 1·00 ± 0·010) P < 0·018 respectively], similarly when cells were treated with C5a, expression of ZO-1 was significantly decreased [43% decrease in HBMVECs (TAI = 0·57 ± 0·11) versus control, (TAI = 1·00 ± 0·04) P < 0·001 and 24% decrease in NHAs (TAI = 0·76 ± 0·16) versus control, (TAI = 1·00 ± 0·09) P < 0·02 respectively]. When cells were pre-treated with the C5aR1 antagonist, ZO-1 expression returned to those equivalent to control levels, in both HBMVECs (TAI = 0·90 ± 0·06) and NHA (TAI = 1·14 ± 0·18) indicating a C5a/C5aR1 dependent alteration of tight junctions. ZO-1 gene expression levels in HBMVEC and NHA cells treated with the C5aR1 antagonist alone were 0·99 ± 0·01 and 0·99 ± 0·04, respectively and were comparable to the ZO-1 gene expression levels in the untreated medium control (0·94 ± 0·03 and 0·97 ± 0·04) for the two cell types, respectively. We also performed immunofluorescent staining of tight junction proteins, Claudin-5 and ZO-1 in HBMVEC cell cultures and our data shows concordance with our gene expression data. Figs5 and 6 show representative images of the protein expression levels for Claudin-5 and ZO-1, respectively, and the intensity of the fluorescent signal analysed using the computer image analysis image J software (National Institutes of Health, Bethesda, MA) and represented in mean pixel units.

Figure 4.

Figure 4

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Inhibition of C5aR1 prevents changes in tight junction proteins. Human brain microvascular endothelial cell (HBMVEC) and normal human astrocyte (NHA) cultures were treated with 5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or C5aR1 antagonist (1 μg/ml) +5% lupus serum for 24 hr, RNA was extracted, reverse transcribed and the gene expression levels of Claudin-5 and zonula occludens 1 (ZO-1) were measured using real-time quantitative PCR. Our data show that treatment with lupus serum significantly decreased Claudin-5 (P = 0·018) and ZO-1 (P = 0·03) gene expression in HBMVECs and significantly decreased Claudin-5 (P = 0·027) and ZO-1 (P = 0·018) gene expression in NHAs. Treatments of both these cell types with C5a also decreased both Claudin-5 and ZO-1 gene expression. The decrease induced by lupus serum and C5a in Claudin-5 and ZO-1 gene expression was reversed in both HBMVECs and NHAs when these cells were treated with C5aR1 antagonist. Data shown are means ± SEM from three experiments. A P-value of < 0·05 is considered a statistically significant difference. All statistical comparisons were made to the control group and between groups as indicated by the lines above the bars.

Figure 5.

Figure 5

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Immunofluorescent staining of tight junction protein, Claudin-5. human brain microvascular endothelial cell (HBMVEC) cultures were treated with 5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or C5aR1 antagonist (1 μm) +5% lupus serum for 24 hr and immunofluorescent staining of Claudin-5 was carried out using anti-human primary antibodies raised in goats obtained from Santacruz Biotech [Santa Cruz, CA; Cat # sc-17667 (G-15) goat polyclonal IgG]. The secondary antibody was a fluoresence labelled [Alexa Fluor 647 rabbit anti-goat IgG (H+L) Cat # A-21446, Molecular Probes Inc -Life Technologies, Grand Island,NY]. DAPI (Cat #D1306 Molecular Probes Inc -Life Technologies, Grand Island,NY) was nuclear to stain the nuclei. Representative images show the protein expression levels of Claudin-5 which were quantified based on the intensity of the fluorescence signal analysed using the computer image analysis image J software (National Institutes of Health, Bethesda, MA).

Figure 6.

Figure 6

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Immunofluorescent staining of tight junction protein, zonula occludens 1 (ZO-1. Human brain microvascular endothelial cell (HBMVEC) cultures were treated with 5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or C5aR1 antagonist (1 μm) +5% lupus serum for 24 hr and immunofluorescent staining of ZO-1 was carried out using anti-human primary antibodies raised in goat obtained from Santacruz Biotech [Santa Cruz, CA; Cat # sc-8147 (N-19) goat polyclonal IgG]. The secondary antibody was a fluoresence labelled [Alexa Fluor 647 rabbit anti-goat IgG (H+L) Cat # A-21446, Molecular Probes Inc -Life Technologies, Grand Island,NY]. DAPI (Cat #D1306 Molecular Probes Inc -Life Technologies) was nuclear to stain the nuclei. Representative images show the protein expression levels of ZO-1 which were quantified based on the intensity of the fluorescent signal analysed using the computer image analysis image J software (National Institutes of Health, Bethesda, MA).

Differential regulation of adhesion molecule expression by C5a in HBMVECs and NHAs in an SLE setting

Adhesion molecules play an important role in the translocation of circulating cells in the brain, however, VCAM and ICAM were observed to have different effects in skin and brain.52 Here we assessed the effect of C5a/C5aR1 signalling on mRNA expression of VCAM (Fig.7a) and ICAM (Fig.7b) by quantitative RT-PCR in the astrocytes and endothelial cells that constitute the BBB. Treatment with lupus serum resulted in a 32% decrease (P < 0·016) in VCAM expression in HBMVECs (TAI = 0·68 ± 0·17) compared with the control (TAI = 1·00 ± 0·09). Similar to lupus serum (32% decrease versus control, P < 0·016), C5a treatment resulted in ∼20% decrease in VCAM expression in both brain HBMVECs (TAI = 0·79 ± 0·07, P < 0·001) and NHAs (TAI = 0·81 ± 0·18, P < 0·001). C5aR1 inhibition prevented the decrease in VCAM expression in both these cells and showed VCAM expression levels similar to control levels [HBMVEC’s (TAI = 1·10 ± 0·13) and NHAs (TAI = 1·09 ± 0·06)], indicating an important role for C5a/C5aR1 signalling in regulation of VCAM expression. VCAM-1 gene expression levels in HBMVECs and NHA cells treated with the C5aR1 antagonist alone were 0·98 ± 0·04 and 0·98 ± 0·01, respectively, and were comparable to the VCAM-1 gene expression levels in the untreated media control (0·95 ± 0·07 and 0·91 ± 0·06) for the two cell types, respectively. On the other hand, no significant change in ICAM expression was observed in both cell types on treatment with C5a with gene expression levels similar to the respective controls [HBMVECs (TAI = 0·82 ± 0·11) and NHAs (TAI = 1·06 ± 0·04)]. However, treatment of HBMVECs with lupus serum decreased ICAM gene expression by about 21% (TAI = 0·79 ± 0·03; P < 0·04) compared with the control (TAI = 1·00 ± 0·02). Interestingly, C5aR1 inhibition significantly reduced the expression of ICAM in NHAs showing a 35% decrease (TAI = 0·65 ± 0·011) in ICAM expression levels as compared with the control (TAI = 1·00 ± 0·08) and/or lupus-treated cells (TAI = 0·98 ± 0·013; P < 0·04). ICAM-1 gene expression levels in HBMVECs and NHA cells treated with the C5aR1 antagonist alone were 0·97 ± 0·01 and 0·99 ± 0·05, respectively, and were comparable to the ICAM-1 gene expression levels in the untreated media control (0·97 ± 0·03 and 1·08 ± 0·04) for the two cell types, respectively.

Figure 7.

Figure 7

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Differential regulation of adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) by C5a/C5aR1 signalling. As shown by real-time quantitative PCR, VCAM-1 mRNA expression was reduced in human brain microvascular endothelial cells (HBMVECs) by lupus serum (P < 0·05) and C5a treatment, which was prevented by C5aR1 antagonism. Both treatments reduced ICAM-1 in HBMVECs, but did not affect ICAM-1 in NHAs. Surprisingly, C5aR1 inhibition did not affect the change in HBMVECs and decreased the protein expression in NHAs. Data are means ± SEM from three experiments. A P-value of < 0·05 is considered a statistically significant difference. All statistical comparisons were made to the control group and between groups as indicated by the lines above the bars.

Aggregation and translocation of NF-κB in brain endothelial cells is significantly reduced by C5aR1 inhibition

On activation, the NF-κB heterodimer complex including the p65-subunit is translocated to the nucleus where it up-regulates a number of genes that affect cell survival. NF-κB regulates both immune and inflammatory responses and was altered in rodent endothelial cells treated with lupus serum.22 Therefore, we investigated the translocation of NF-κB in human endothelial cells in vitro, after stimulation with lupus serum. Controls included cells treated with normal serum. On treatment with lupus serum, the NF-κB staining changed from a diffuse cytoplasmic pattern to a prominent aggregated pattern. In addition, there was nuclear staining, suggesting that a redistribution of NF-κB from the cytoplasm to the nucleus had occurred compared with controls. Treatment of cells with the C5aR1 antagonist prevented the aggregation and translocation of NF-κB induced by lupus serum in these cells (Fig.5).

Relationship between complement C5a and CREB and ERK in endothelial cells in lupus

Members of the mitogen-activated protein kinase (MAPK) family are important mediators of signal transduction pathways that serve to coordinate the cellular response to a variety of extracellular stimuli. Activated MAPKs phosphorylate their specific substrates on serine and/or threonine residues, ultimately leading to activation of various transcription factors and control of a vast array of physiological processes, including cell survival and death.53 In this study we assessed the concentration of ERK, a member of the MAPK family and the transcription factor, CREB in cell lysates of endothelial cells and astrocytes treated with lupus serum, C5a or a C5aR1 antagonist (Fig.6). Both astrocytes and endothelial cells were treated with lupus serum, C5a and C5aR1 inhibitor. We did not observe any significant change in both ERK and CREB expression in NHA treated with C5a, lupus serum and/or C5aR1 antagonist. We observed a 20% increase (P < 0·05) in CREB expression and an 80% increase (P < 0·01) in ERK expression, respectively, in endothelial cells when compared with the control. C5a treatment of endothelial cells did not result in a significant change in CREB expression compared with controls; however, C5a did increase ERK expression by 35% (P < 0·05) compared with the control. C5aR1 antagonist treatment reversed the lupus-serum-induced increase in CREB expression significantly (P < 0·001) by 78% and the ERK expression by 34% (P < 0·05), indicating the involvement of the ERK/CREB signalling pathways in C5a/C5aR1-mediated regulation of endothelial cell function and consequently in BBB integrity.

Discussion

The BBB is the ‘gateway’ to the brain, and protects the brain from circulating toxins and inflammatory cells and maintains an optimal environment for neuronal function. BBB dysfunction occurs in several neuroinflammatory settings, including SLE.51 Our earlier studies using rodent models demonstrated that the anaphylatoxin C5a, generated during complement activation, was one of the culprits causing activation of the endothelium and actin rearrangement, thereby leading to BBB dysfunction. The present study takes this aspect a step further and using a three-dimensional in vitro system with HBMVECs and NHAs shows that similar to rodents, the human BBB responds to C5a and SLE serum leading to actin rearrangement rendering the BBB leaky.

Complement activation occurs during SLE and levels of the complement proteins C3 and C4 are part of the panel used to assess disease. The concentrations of several complement proteins are found to be altered in SLE54–56 and the circulating concentration of the anaphylatoxin C5a correlates with behavioural changes. C5a/C5aR1 signalling regulates BBB integrity in experimental models of SLE, through the NF-κb pathway.23 C5aR1 is present on both human HBMVECs and NHAs.18 In our studies, the patterns of localization in the HBMVECs and NHAs were different. This could be a result of the differences in receptor trafficking to the surface or the localization itself. The differences in distribution form an interesting aspect that will be addressed in the future. For the first time, the current study using a three-dimensional system allowed the assessment of factors causing BBB dysfunction. In line with the observation in rodents, lupus serum increased permeability through the BBB, as estimated by TEER. In addition, actin remodelling occurred in these cells on treatment, as visualized by rhodamine–phalloidin staining. C5a treatment produced similar changes, indicating its potential role in causing these changes.

The NF-κB pathway in endothelial cells regulates the expression of adhesion molecules such as ICAM-1 and VCAM-1.57,58 Together, these proteins mediate the capture and adhesion of leucocytes in the bloodstream in a well-known multistep process (reviewed in refs 57–60). ICAM expression is enhanced in the brain of both man and mouse in SLE.61 In the current study ICAM mRNA was differentially regulated: the expression was decreased in HBMVECs treated with lupus serum or C5a and remained unchanged in NHAs on treatment. Both ICAM-1 and VCAM-1 are important adhesion molecules for the interaction of endothelial cells of the BBB with leucocytes in a neuroinflammatory response resulting in BBB breakdown that is anticipated in SLE. Similar observations of decreased or no change/or lack in correlation in VCAM-1 and ICAM-1 levels in SLE patients have been reported.62,63 Yet another explanation for this aberration, could be that lupus serum or C5a increases interleukin-6 levels and that interlukin-6 trans-signalling indirectly modulates VCAM-1 expression by BBB endothelial cells. Interleukin-6 trans-signalling may play a role in autoimmune inflammation of the CNS mainly by regulating early expression of adhesion molecules, possibly via cellular networks at the BBB.64 Different studies show that ICAM is not involved in the leucocyte trafficking in the brain and affords protection through other mechanisms in this setting.61,62 Interestingly, pre-treatment with the peptide C5aR1 antagonist prevented these alterations, indicating the important role of C5a/C5aR1 signalling in causing the pathology. In contrast, VCAM-1 expression was reduced in both in HBMVECs and NHAs, which was prevented by the pre-treatment with the C5aR1 inhibitor. VCAM-1 is the more important adhesion molecule involved in leucocyte trafficking in brain. However, the decrease in expression is contrary to expectations and needs further investigation.

Tight junctions present between endothelial cells prevent the infiltration of circulating cells and toxins such as immune complexes into the brain. The presence of large proteins such as IgG and albumin in brain, and magnetic resonance imaging of the brains of patients with SLE indicate loss of BBB in the lupus setting.51,52 Our studies using experimental models showed this phenomenon occurred in MRL/lpr mice with lupus-like disease. Using human HBMVECs and NHAs our results in this study show that similar to the mouse models, the tight junction proteins Claudin-5 and ZO-1 were altered in lupus and C5a treatment (Figs6). Both our gene expression data and our immunofluorescence staining data concurred and showed a significant decrease in ZO-1 and Claudin-5 expression on treatment with lupus serum. However, although C5a treatment decreased Claudin-5 and ZO-1 expression compared with normal, it occurred to a lesser extent compared with the response to lupus serum. This could indicate the presence of other unidentified factors in the lupus serum that may enhance the effect of C5a and consequently increased Claudin-5 expression on antagonist treatment. Our results indicate that increased C5a/C5aR1 signalling is an important contributor to this event.

Our results (see Fig.8) suggest that both CREB and ERK are crucial signal kinases that mediates NF-κB activation in HBMVECs, resulting in modulation of BBB permeability. The transcription factor NF-κB plays a key role in the transcriptional regulation of many proteins involved in chronic inflammatory diseases such as lupus. CREB is a transcription factor that regulates diverse cellular responses and is induced by a variety of inflammatory signals and it mediates gene transcription. CREB has been proposed to directly inhibit NF-κB activation by blocking the binding of CREB-binding protein to the NF-κB complex, thereby limiting pro-inflammatory responses. Under normal baseline conditions, the NF-κB transcription factors are sequestered in the cytoplasm associated with inhibitor molecule IkB, which prevents NF-κB activation. When endothelial cells are treated with lupus serum, NF-κB is activated, followed by a cascade of signalling events that ultimately lead to IkB degradation, which allows the release of NF-κ and facilitates the translocation of NF-κB to the nucleus as we observed (Fig.9) through the activation of CREB. The NF-κB translocation to the nucleus promotes the transcription of genes involved in pro-inflammatory immune responses as anticipated in patients with lupus. Optimal NF-κB activity is mediated by direct interaction of a subunit of NF-κB called Rel A, with a CREB co-activator called CREB-binding protein (CBP). CREB interacts with its co-activator protein CBP to initiate transcription of CREB-responsive genes. The Rel A component of NF-κB interacts with CBP at the same region as phosphorylated CREB, and it is believed that NF-κB activity is inhibited by activated CREB through competition for limiting amounts of CBP.65–68 Based on this competitive binding it is speculated that the balance between CREB and CBP determines the modulation of NF-κ activity.

Figure 8.

Figure 8

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Cyclic cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) mediate nuclear factor-κB (NF-κB) activation in human brain microvascular endothelial cells (HBMVECs). HBMVEC and normal human astrocyte (NHA) cultures were treated with 5% control serum, 5% lupus serum, control serum + C5a (0·1 μg/ml), or C5aR1 antagonist (1 μg/ml) + 5% lupus serum for 96 hr. Cells were lysed, protein extracted and the amount of total CREB and ERK expression was quantified using the commercially available InstantOne™ ELISA kits (eBioscience, San Diego, CA). Treatment with lupus serum significantly increased both CREB (P = 0·05) and ERB (P = 0·01) expression in HBMVECs but no statistically significant difference in CREB and ERB expression was observed in NHAs. Treatments of HBMVEC with C5a increased ERK expression (P = 0·05) but no significant effect of C5a was observed with regards to CREB expression. The lupus serum and C5a induced increase in CREB and ERB expression was reversed in HBMVECs when these cells were treated with the C5aR1 antagonist. Data shown are means ± SEM from three experiments. A P-value of < 0·05 is considered a statistically significant difference. All statistical comparisons were made to the control group and between groups as indicated by the lines above the bars.

Figure 9.

Figure 9

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Signalling through C5aR1 induces translocation of nuclear factor-κB (NF-κB) into the nucleus. Human endothelial cells treated with control serum, lupus serum and C5aR1 antagonist + lupus serum were stained for NF-κB p65 (green) and DAPI (red). Cells treated with control serum had NF-κB in the cytoplasm (arrowhead) and none in the nucleus compared with the lupus serum where the NF-κB was found in the nucleus (arrow). In cells pre-treated with C5aR1 inhibitor, the NF-κB remained in the cytoplasm, indicating the important role of C5a in the process. Inset shows enlarged pictures of the cells demonstrating the C5a induced translocation of NF-κB into the nucleus (arrow).

Pharmacological studies have shown that ERK is essential for NF-κB transactivation in response to an inflammatory response. MAPKs and ERK are necessary and cooperative machinery for NF-κB transactivation; however, the mechanisms that underlie the ERK-induced NF-κB transactivation are not yet elucidated. Bergmann et al.69 suggested that an IkB degradation-independent mechanism may contribute to ERK-linked NF-κb activation. Pro-inflammatory cytokines induce NF-κB activation by virtue of ERK-induced phosphorylation of NF-κB subunit RelA before its nuclear translocation, thereby positively regulating NF-κB-dependent transcription.69–71 ERK may also play a regulatory role in the activation of IKK/IkBs/NF-κB signalling. Hence, both CREB and ERK signalling mediates NF-κB activation in HBMVECs resulting in BBB modulation.

In conclusion, these results indicate the translational potential of results obtained in mouse models and suggest that C5a/C5aR1 signalling plays a key role in disrupting the BBB integrity through different cascades including NF-κB translocation leading to altered tight junction proteins, Claudin-5 and ZO-1, and actin reorganization in the lupus setting. Therefore, as blocking of C5aR1 will retain the protective functions of the complement cascade yet alleviate the symptoms of SLE, our studies demonstrate that C5aR1 is a potentially important therapeutic target for SLE.

Acknowledgments

This work was supported by R01-AR-060604 from NIH (JNJ); an 1R21DA030108-01 CEBRA award from NIH (Mahajan SD) and the New York State Department of Health (DOH) funded Empire Clinical Research Investigator Program (ECRIP) (Schwartz SA).

Glossary

BBB

blood–brain barrier

CREB

cAMP response element-binding protein

ERK

extracellular signal-regulated kinase

HBMVECs

human brain microvascular endothelial cells

ICAM

intercellular adhesion molecule

MAPK

nitogen-activated protein kinase

NF-κB

nuclear factor-κB

NHAs

normal human astrocytes

SLE

systemic lupus erythematosus

TEER

transendothelial electrical resistance

VCAM

vascular cell adhesion molecule

Disclosures

The authors declare having no competing interests.

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