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. 2009 Aug 29;30(2):247–253. doi: 10.1007/s10571-009-9446-7

Lipopolysaccharide-Activated Microglia Induce Dysfunction of the Blood–Brain Barrier in Rat Microvascular Endothelial Cells Co-Cultured with Microglia

Noriko Sumi 1, Tsuyoshi Nishioku 1, Fuyuko Takata 1,2, Junichi Matsumoto 1, Takuya Watanabe 1, Hideki Shuto 1, Atsushi Yamauchi 1, Shinya Dohgu 1, Yasufumi Kataoka 1,2,✉
PMCID: PMC11498813  PMID: 19728078

Abstract

The blood–brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.

Keywords: Blood–brain barrier, Microglia, Brain microvascular endothelial cells, NADPH oxidase, Lipopolysaccharide

Introduction

The blood–brain barrier (BBB) is primarily formed by brain capillary endothelial cells, which are sealed closely by tight junctions (TJ) (Abbott et al. 2006; Hawkins and Davis 2005; Zlokovic 2008), and diffuse the transport of plasma components, blood cells, and xenobiotics from the systemic circulation into the central nervous system (CNS). The main machinery underlying the barrier function of the BBB is TJ and efflux transporters. TJ primarily maintain the low paracellular permeability and high electrical resistance of the BBB (Bazzoni and Dejana 2004). Efflux transporters, such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins, are mainly localized on the luminal surfaces of brain microvascular endothelial cells, and limit the accumulation of brain-permeable xenobiotics and endogenous neurotoxic substances in the brain. These barrier properties of the BBB are maintained and regulated by the dynamic and continuous cross-talk among the cellular elements of the neurovascular unit (for example, brain endothelial cells, pericytes, astrocytes, microglia, and neurons).

The BBB is involved in various neuroinflammatory processes. The neuroinflammatory substances induce alterations of BBB functions, including disruption and alteration of TJ architecture and dysfunction of transporters. These events enhance transport of immune cells across the BBB, and subsequent secretion of inflammatory mediators (for example, cytokines) from the cells in the CNS. Microglia, the resident immune cells in the CNS, play an essential role in the immune response and become activated in response to brain injury and immunological stimuli (Kreutzberg 1996). Under conditions of microglial activation, microglia undergo several transformations from the resting state to the reactive state, and lipopolysaccharide (LPS) activates microglia to produce pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, reactive oxygen species (ROS), and reactive nitrogen species (Lee et al. 1993; Qin et al. 2004, 2005). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, one of the major enzymes generating ROS, is known to be a crucial molecule for inflammation-related neurotoxicity (Block et al. 2007). Microglial activation and subsequent neuroinflammation are considered to be responsible for the progression of neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease (McGeer and McGeer 1995; Liu and Hong 2003; Block et al. 2007). In these neurodegenerative diseases, structural and functional impairments of the BBB have been observed (Kortekaas et al. 2005; Zipser et al. 2007). We recently reported that mice with LPS-induced septic encephalopathy showed hyperpermeability of brain microvascular endothelial cells in parallel with microglial activation (Nishioku et al. 2008). These findings suggest that microglial activation may be related to BBB disruption. However, a direct interaction between activated microglia and brain microvascular endothelial cells in the mediation of BBB dysfunction has not yet been clarified.

In the present study, we provide direct evidence demonstrating that LPS activates microglia to induce functional impairments of the BBB by stimulating NADPH oxidase in an in vitro BBB model in which rat brain endothelial cells (RBEC) were co-cultured with microglia.

Methods

Materials

LPS (Escherichia coli O55:B5) and diphenyleneiodonium chloride (DPI) were purchased from Sigma (St. Louis, MO, USA). Wistar rats were obtained from Kyudo (Kumamoto, Japan). All procedures involving experimental animals adhered to the law (No. 105) and notification (No. 6) of the Japanese Government, and were approved by the Laboratory Animal Care and Use Committee of Fukuoka University.

Rat Microglia

Primary microglia cultures were prepared from the whole brains of 1- to 3-day-old Wistar rats, as previously described (Nishioku et al. 2002). Briefly, after removing the meninges and blood vessels, the forebrains were minced and gently dissociated by repeated pipetting in Dulbecco’s modified Eagle’ medium (DMEM) (WAKO, Osaka, Japan) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin, and filtered through a 70-μm cell strainer. Cells were collected by centrifugation (800×g, 6 min), resuspended in 10% FBS DMEM and cultured on 75-cm2 flasks (Corning, Acton, MA, USA) in a humidified atmosphere of 5% CO2/95% air at 37°C. Cells were fed every 2–3 day by changing medium. After 10–14 days, microglia were isolated by shaking the mixed glia-containing flasks for 15 min at 200 rpm. The floating cells were seeded onto plastic tissue culture dishes and incubated at 37°C for 30 min. The attached cells were harvested and seeded onto new plates for further studies. The purity of microglia was consistently >95%.

RBEC

Primary cultures of RBEC were prepared from 3-week-old Wistar rats, as previously described (Deli et al. 1993; Nakagawa et al. 2007). Meninges were carefully removed from forebrains and gray matter was minced into small pieces of approximately 1 mm3 in ice-cold DMEM, then dissociated by 25 up- and down-strokes with a 5-ml pipette, in DMEM containing collagenase type 2 (1 mg/ml, Worthington, USA), DNase I (15 μg/ml), and gentamicin (50 μg/ml), and then digested in a shaker for 1.5 h at 37°C. The cell pellet was separated by centrifugation in 20% bovine serum albumin (BSA)-DMEM (1,000×g, 20 min). The microvessels obtained in the pellet were further digested with collagenase dispase (1 mg/ml, Roche, Switzerland) and DNase I (6.7 μg/ml in DMEM for 1 h at 37°C). Microvessel endothelial cell clusters were separated on a 33% continuous Percoll (GE Healthcare, Buckinghamshire, UK) gradient, collected and washed twice in DMEM before plating on 100-mm plastic dishes coated with collagen type IV and fibronectin (both 0.1 mg/ml). RBEC cultures were maintained in DMEM/F12 supplemented with 10% plasma-derived serum (Animal Technologies, USA), basic fibroblast growth factor (Roche, 1.5 ng/ml), heparin (100 μg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml) (insulin–transferrin–sodium selenite media supplement), gentamicin (50 μg/ml), and puromycin (4 μg/ml) (Perriere et al. 2005) (RBEC medium I) at 37°C in a humidified atmosphere of 5% CO2/95% air, for 2 days. On the third day, the cells received new medium, which contained all the components of RBEC medium I except for puromycin (RBEC medium II). When the cultures reached confluency, the purified endothelial cells were passaged and used to construct in vitro BBB models.

Preparation of In Vitro BBB Models

In the RBEC/microglia co-culture system, rat microglia cells (1–10 × 104 cells/well) were first cultured in the wells of the 12-well culture plate (Corning, Midland, MI, USA). After 5–8 h, RBEC (15 × 104 cells/well) were seeded on the inside of a fibronectin-collagen IV (0.1 mg/ml, respectively)-coated polyester membrane of a Transwell-Clear inserts (diameter 12 mm, 0.4-μm pore size; Corning) placed in the well of a 12-well culture plate. A monolayer system was also made with RBEC alone (RBEC monolayer). Cells were cultured in RBEC medium II supplemented with 500 nM hydrocortisone (Sigma) (RBEC medium III) at 37°C with a humidified atmosphere of 5% CO2/95% air until the in vitro BBB models reached confluency.

Measurement of Transendothelial Electrical Resistance

Transendothelial electrical resistance (TEER) across the monolayers grown on the filter membranes was measured by an EVOM resistance meter (World Precision Instruments, Sarasota, FL, USA) using Endohme (World Precision Instruments), and the values are shown as Ω × cm2 based on culture inserts. The TEER of cell-free inserts was subtracted from those of filters with cells. The TEER of RBEC was measured before and after treatment with LPS.

Measurement of Paracellular Transport of Sodium-Fluorescein

To initiate the transport experiments, the medium was removed and RBEC were washed three times with physiological buffer (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM HEPES, and 10 mM d-glucose, and pH 7.4). Physiological buffer (1.5 ml) was added to the outside of the insert (abluminal side). Physiological buffer (0.5 ml) containing 100 μg/ml of sodium-fluorescein (Na–F) (MW 376) (Sigma) was loaded on the luminal side of the insert. Samples (0.5 ml) were removed from the abluminal chamber at 30, 60, 90, and 120 min and immediately replaced with fresh physiological buffer. Aliquots (5 μl) of the abluminal medium were mixed with 200 μl of physiological buffer and then the concentration of Na–F was determined with a CytoFluor Series 4000 fluorescence multiwell plate reader (PerSeptive Biosystems, Framingham, MA, USA) using a fluorescein filter pair (Ex(λ) 485 ± 10 nm; Em(λ) 530 ± 12.5 nm). The permeability coefficient and clearance were calculated according to the method described by Dehouck et al. (1992). Clearance was expressed as microliters (μl) of tracer diffusing from the luminal to the abluminal chamber and was calculated from the initial concentration of tracer in the luminal chamber and the final concentration in the abluminal chamber: clearance (μl) = [C]A × V A/[C]L, where [C]L is the initial luminal tracer concentration, [C]A is the abluminal tracer concentration, and V A is the volume of the abluminal chamber. During the 120-min period of the experiment, the clearance volume increased linearly with time. The average volume cleared was plotted versus time, and the slope was estimated by linear regression analysis. The slope of clearance curves was denoted by PSapp, where PS is the permeability-surface area product (in μl/min). The slope of the clearance curve with a control membrane was denoted by PSmembrane. The real PS value (PStrans) was calculated from 1/PSapp = 1/PSmembrane + 1/PStrans. The PStrans values were divided by the surface area of the Transwell inserts to generate the permeability coefficient (Ptrans, in cm/min).

Immunostaining

Microglia (1.2 × 105 cells/well) plated on 8-well chamber slides were fixed with 4% formaldehyde for 10 min at room temperature. Cells were incubated with rabbit anti-Iba1 antibody (1:500) (WAKO) overnight at 4°C and then incubated with FITC-labeled anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. RBEC on Transwell inserts were fixed with 95% ethanol–5% acetic acid for 10 min at −20°C (ZO-1) or with ethanol for 1 min at room temperature (claudin-5 and occludin). Cells were blocked with blocking reagent, Blocking One (Nacalai Tesque, Inc., Kyoto, Japan), and incubated with primary antibodies against ZO-1 (1:50), claudin-5 (1:200), and occludin (1:200) overnight at 4°C (all purchased from Zymed, CA, USA). Cells were then incubated with FITC-labeled anti-rabbit IgG for ZO-1 (1:200) or Cy3-labeled anti-mouse IgG for claudin-5 and occludin (Jackson ImmunoResearch) for 1 h at room temperature. Cells were mounted in VECTASHIELD mounting medium with DAPI (VECTOR Laboratories, Burlingame, CA, USA) and then inspected using a fluorescence microscopy (KEYENCE BZ-8000, KEYENCE Corporation, Osaka, Japan).

Statistical Analysis

Values are expressed as means ± SEM. Statistical analysis was performed using Student’s unpaired t-test. One-way analyses of variance (ANOVA) followed by Dunnett’s test and Tukey–Kramer’s test were applied to multiple comparisons. The differences between means were considered to be significant when P values were less than 0.05.

Results

To test whether LPS-induced microglial activation influences TEER and Na–F permeability of RBEC, LPS (1 ng/ml) was added to the abluminal sides of both a RBEC monolayer and a RBEC/microglia co-culture. LPS (1 ng/ml) showed no effect on TEER in the RBEC monolayer (Fig. 1a). LPS (10 ng/ml) significantly decreased TEER in the RBEC monolayer by 38.5 ± 3.3%. Then, the low concentration of LPS (1 ng/ml) was employed in the present experiment. Treatment with LPS (1 ng/ml) decreased TEER, the decrease being dependent on the density of microglia in the well (Fig. 1b). This effect reached a peak at 10 h after LPS treatment, and recovered to the control level within 24 h of treatment (Fig. 1b). The maximum effect of LPS-lowered TEER (about 44% of inhibition) was obtained in the density ranging from 5 × 105 to 10 × 105 cells/well in RBEC/microglia co-culture. Thus 5 × 105 microglial cells/well was employed in the following co-culture experiments. As shown in Fig. 1c, treatment with LPS changed microglial morphology with time from round shapes (before, 0 h) to bipolar rod shapes (1–6 h after treatment). More than half of the cells changed to bipolar rod shapes at 3 h after treatment. Almost all cells changed to bipolar rod shapes at 6 h after treatment.

Fig. 1.

Fig. 1

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The time-course of TEER in a RBEC monolayer (a) and in RBEC/microglia co-culture (b) after exposure to LPS. (a) In the RBEC monolayer, RBEC were treated with LPS (1.0 ng/ml) for 24 h. TEER was expressed as a percent of the basal value at 0 h (84.9 ± 2.2 Ω cm2). Values are the means ± SEM (n = 12). (b) RBEC were co-cultured with microglia (0–10 × 104 cells/well) exposed to LPS (1.0 ng/ml). TEER were expressed as a percent of the corresponding basal values at 0 h (0 × 104 cells/well; 74.3 ± 4.5, 1 × 104 cells/well; 74.7 ± 10.7, 2 × 104 cells/well; 73.3 ± 12.4, 5 × 104 cells/well; 85.9 ± 6.8, 10 × 104 cells/well; 75.2 ± 8.3 Ω cm2, respectively). Values are the means ± SEM (n = 5–12). * P < 0.05, ** P < 0.01, significant differences from 0 × 104 cells/well. (c) A series of photographs showing microglia were taken at 0 (before), 1 and 6 h after LPS treatment. Microglia were immunostained with anti-Iba-1 antibody. Scale bars = 50 μm

LPS (1 ng/ml) had no effect on the permeability coefficient of Na–F in RBEC monolayer (102.1 ± 3.3% of control) (Fig. 2a). In RBEC/microglia co-culture, treatment with LPS for 6 h increased the Na–F permeability by 38.7 ± 5.1% (Fig. 2b). An NADPH oxidase inhibitor, DPI at concentrations of 0.5 and 1 μM completely reversed LPS-induced Na–F hyperpermeability (138.7 ± 5.1%) to 89.2 ± 4.2 and 88.0 ± 5.5% of vehicle, respectively, in RBEC/microglia co-culture (Fig. 2b). DPI (0.1, 0.5, and 1 μM) had no effect on the Na–F permeability in RBEC/microglia co-culture without LPS treatment (inset of Fig. 2b). LPS-decreased TEER was also restored from 46.4 ± 4.0% to 95.5 ± 3.7 and 115.4 ± 5.3% of vehicle by 0.5 and 1 μM DPI, respectively. DPI (0.5 μM) failed to block LPS-induced morphological changes in microglia (data not shown).

Fig. 2.

Fig. 2

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Changes in RBEC permeability to Na–F in a RBEC monolayer (a) and in RBEC/microglia co-culture (b). (a) In the RBEC monolayer, RBEC were treated with LPS (1 ng/ml) for 6 h. The control value of permeability coefficients for Na–F was 4.99 ± 0.29 × 10−5 cm/min. Values are the means ± SEM (n = 9). (b) RBEC were co-cultured with microglia exposed to LPS for 6 h in the presence or absence of DPI (0.1, 0.5, and 1 μM). The inset shows effect of DPI on RBEC permeability to Na–F in RBEC/microglia co-culture without LPS treatment. RBEC were co-cultured with microglia exposed to DPI (0.1, 0.5, and 1 μM) for 6 h. The vehicle values of permeability coefficients for Na–F in (b) and the inset of (b) were 5.19 ± 0.54 × 10−5 and 5.93 ± 0.59 × 10−5 cm/min, respectively. Values are the means ± SEM (n = 6–9). ** P < 0.01, significant difference from vehicle, ## P < 0.01, significant difference from LPS (1 ng/ml)

Immunostaining for the TJ proteins zonula occludens-1 (ZO-1), claudin-5, and occludin revealed a continuous distribution of these proteins along the cell border in vehicle-treated RBEC/microglia co-culture (Fig. 3, left panels). This linear shape along the junctions between cells was changed to a zipper-like or zigzag shape by LPS treatment (Fig. 3, middle panels). These fragmented immunocytochemical staining patterns of TJ proteins along the cell border were restored to a linear shape by DPI (0.5 μM) (Fig. 3, right panels). Western blot analysis of TJ proteins showed no significant changes in the co-culture of RBECs and LPS-activated microglia (data not shown).

Fig. 3.

Fig. 3

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Immunofluorescent staining for TJ proteins (ZO-1, occludin, and claudin-5) in RBEC co-cultured with microglia exposed to LPS or LPS plus DPI for 6 h. Left panel: vehicle-treated RBEC co-cultured with microglia, middle panel: LPS (1.0 ng/ml)-treated RBEC co-cultured with microglia, right panel: LPS (1.0 ng/ml) plus DPI (0.5 μM)-treated RBEC co-cultured with microglia. Scale bars: 10 μm

Discussion

In the present study, addition of LPS to the abluminal side of a RBEC/microglia co-culture, but not to that of a RBEC monolayer, lowered TEER, increased Na–F permeability. Immunofluorescence staining for TJ proteins demonstrated that ZO-1, claudin-5, and occludin immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes in RBEC were blocked by DPI. The present findings suggest that LPS-activated microglia produce ROS through NADPH oxidase to impair BBB function.

Microglia express toll-like receptors (TLRs). LPS is a well known exogenous ligand for TLR4, receptors mediating inflammatory neurodegeneration (Lehnardt et al. 2003). LPS stimulates TLR4 to activate the transcription factor NF-κB through MyD88-dependent and -independent pathways (Chow et al. 1999; Akira and Takeda 2004), resulting in the induction of inflammatory mediators, such as TNF-α. TNF-α-treated microglia showed an increase in ROS production, which was blocked by a NADPH oxidase inhibitor (Mander et al. 2006). These findings suggest that LPS stimulates NADPH oxidase through TLR4–TNF-α signaling to produce ROS in microglia.

The membrane-bound NADPH oxidase system is a critical source of ROS in microglia (Sankarapandi et al. 1998). NADPH oxidase consists of the cytosolic regulatory subunits p40phox, p47phox, p67phox, and Rac, and the membrane-associated subunits p22phox and gp91phox (Leusen et al. 1996). Activation of NADPH oxidase requires phosphorylation of the cytosolic component p47phox followed by the translocation of the cytosolic subunits to the plasma membrane (Groemping and Rittinger 2005). LPS induces phosphorylation of p47phox and translocation of p47phox to the membrane in microglia (Qian et al. 2006, 2008). There is no significant difference in ROS production by microglia between TLR4 knockout and wild-type mice (Qin et al. 2005). Based on these reports, LPS appears to stimulate microglial NADPH oxidase through a TLR4-independent pathway to produce ROS. Qin et al. (2004, 2005) also demonstrated that LPS-induced TNF-α production in mice microglia is lowered by a deficiency of NADPH oxidase. An NADPH oxidase inhibitor blocked activation of NF-κB induced by LPS and interferon-γ in microglia (Pawate et al. 2004). These findings suggest that LPS-induced ROS production via NADPH oxidase facilitates TNF-α expression by activating NF-κB. Therefore, LPS might stimulate NADPH oxidase through TNF-α-dependent and -independent pathways in microglia. The resulting ROS production is also involved in LPS-induced expression of TNF-α. Considering this notion together with our present findings that an NADPH oxidase inhibitor completely blocked functional and morphological damage to the BBB, activation of NADPH oxidase is considered to have the main causal role in BBB dysfunction owing to activated microglia.

The TJ proteins ZO-1, occludin, and claudin-5 are known to be responsible for the selective barrier function of TJ at the BBB (Hawkins and Davis 2005; Zlokovic 2008). The brain microvascular endothelial cells exposed to hydrogen peroxide exhibited changes in the subcellular localization of TJ proteins, including ZO-1 and occludin, leading to hyperpermeability of the BBB (Lee et al. 2004; Fischer et al. 2005). ROS induced cytoskeleton rearrangements of TJ and redistribution and disappearance of the TJ proteins claudin-5 and occludin by activating PI3 kinase and the protein kinase B pathway via RhoA, and consequently disrupted the integrity of the BBB (Schreibelt et al. 2007). Oxidative stress with various reagents caused BBB disruption owing to degradation of basement membrane proteins and enhanced tyrosine phosphorylation of TJ proteins (Haorah et al. 2007). These reports support the present findings. Here, we demonstrated that the presence of LPS-activated microglia showed fragmented immunoreactivities for TJ proteins at intercellular borders, suggesting a shift in the subcellular distribution of TJ proteins from intercellular borders to intracellular regions. This morphological disorganization of TJ proteins contributes to endothelial TJ dysfunction (hyperpermeability to Na–F and lowered TEER) probably because of ROS production induced by activated microglia.

In conclusion, we demonstrate that activated microglia impair BBB function by producing ROS through NADPH oxidase. Under pathophysiological conditions, including neurodegenerative disease and inflammation, activated microglia is attributable to the breakdown of BBB functions. The inhibition of NADPH oxidase may protect the BBB from damage.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research [(B) 17390159], Grants-in-Aid for Young Scientists [(Start-up) 18890227 and (Start-up) 20800066], and Grants-in-Aid for Young Scientists [(B) 19790199, (B) 21790102, (B) 21790255, (B) 21790257, and (B) 21790526] from JSPS, Japan, the Ministry of Health, Labor and Welfare of Japan (H19-nanchi-ippan-006), the Nakatomi Foundation, Research Foundation ITSUU Laboratory, and Kakihara Science and Technology Foundation.

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