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. 2013 May 14;8(5):e64034.
doi: 10.1371/journal.pone.0064034. Print 2013.

Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo

Andreas Üllen  1 , Evelin Singewald, Viktoria Konya, Günter Fauler, Helga Reicher, Christoph Nusshold, Astrid Hammer, Dagmar Kratky, Akos Heinemann, Peter Holzer, Ernst Malle, Wolfgang Sattler
Affiliations

Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo

Andreas Üllen et al. PLoS One. .

Abstract

Peripheral leukocytes can exacerbate brain damage by release of cytotoxic mediators that disrupt blood-brain barrier (BBB) function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) formed via the myeloperoxidase (MPO)-H2O2-Cl(-) system. In the present study we examined the role of leukocyte activation, leukocyte-derived MPO and MPO-generated oxidants on BBB function in vitro and in vivo. In a mouse model of lipopolysaccharide (LPS)-induced systemic inflammation, neutrophils that had become adherent released MPO into the cerebrovasculature. In vivo, LPS-induced BBB dysfunction was significantly lower in MPO-deficient mice as compared to wild-type littermates. Both, fMLP-activated leukocytes and the MPO-H2O2-Cl(-) system inflicted barrier dysfunction of primary brain microvascular endothelial cells (BMVEC) that was partially rescued with the MPO inhibitor 4-aminobenzoic acid hydrazide. BMVEC treatment with the MPO-H2O2-Cl(-) system or activated neutrophils resulted in the formation of plasmalogen-derived chlorinated fatty aldehydes. 2-chlorohexadecanal (2-ClHDA) severely compromised BMVEC barrier function and induced morphological alterations in tight and adherens junctions. In situ perfusion of rat brain with 2-ClHDA increased BBB permeability in vivo. 2-ClHDA potently activated the MAPK cascade at physiological concentrations. An ERK1/2 and JNK antagonist (PD098059 and SP600125, respectively) protected against 2-ClHDA-induced barrier dysfunction in vitro. The current data provide evidence that interference with the MPO pathway could protect against BBB dysfunction under (neuro)inflammatory conditions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Systemic inflammation induces cell-associated and extracellular (secreted) MPO.
C57BL/6 mice received (A, D) PBS or (B, C, E, F) a single systemic LPS injection (250 µg LPS/30 g body weight; i.p.). After 6 h, animals were killed by cervical dislocation, brains were removed and snap frozen in liquid nitrogen. (A-C) Immunostaining of MPO was performed on sagittal cryosections (5 µm) of brain tissue using rabbit anti-human MPO IgG (1∶500) and HRP-labeled goat-anti rabbit IgG (1∶300) as primary and secondary antibodies. MPO (red) was visualized using the AEC system. Staining in a venule (B) and a smaller vessel (C) is shown. (D-F) Negative controls using rabbit non-immune IgG as primary antibody. Sections were counterstained with Mayer's hemalum. Arrows indicate cell-associated MPO, arrowheads indicate secreted MPO located at the abluminal side of vessels. Scale bars: 50 µm.
Figure 2
Figure 2. Systemic inflammation induces neutrophil activation and MPO deposition at the cerebrovasculature.
C57BL/6 mice received a single systemic LPS injection (250 µg/30 g; i.p.). After 6 h, animals were killed by cervical dislocation, brains were removed and snap frozen in liquid nitrogen. Immunofluorescence staining of (A) endothelial cells, (B) neutrophils and (C) MPO was performed on sagittal cryosections (5 µm) of brain tissue using DyLight 650-labeled rabbit anti-human vWF (1∶75), FITC-labeled rat anti-mouse neutrophil IgG (1∶50) and rabbit anti-human MPO IgG (1∶500) as primary antibodies. Triple immunofluorescence of DyLight 650-labeled anti-human vWF IgG, Cy-3-labeled anti-rabbit IgG and Cy-2-labeled anti-rat IgG was performed by confocal laser scanning microscopy using a Leica SP2. Overlays of blue (vWF), green (neutrophils), and red (MPO) channels are shown in (D). Scale bars: 20 µm.
Figure 3
Figure 3. BMVEC barrier function is compromised by activated PMNL.
BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of hydrocortisone-induced endothelial monolayers (7.5×104 cells) was continuously monitored by impedance sensing at 4 kHz. (A) After stabilization of BMVEC monolayers, fMLP (1), unprimed PMNL (2; 2.5×106), or TNFα-primed/fMLP-activated PMNL (3; 2.5×106) were added to BMVEC monolayers at the indicated time points (arrows). (B) Statistical evaluation of impedance values from (A) after 2 h. Impedance was normalized to baseline recorded before applying the treatments and represents mean values ± SD of four independent experiments (ns = not significant, ***p<0.001, one-way ANOVA).(C) PMNL (2.3×106 cells) were incubated in the presence of a confluent BMVEC (1.5×105) monolayer and stimulated with PMA (250 nM), LPS (1 µg/ml) and fMLP (10 µM) with or without priming with TNFα (10 ng/ml) in the absence or presence of 4-ABAH (100 µM). After 3 h released MPO was determined. Results were normalized to controls (no addition; 100%) and represent means ± SD of triplicate (absence of 4-ABAH) or quadruplicate (presence of 4-ABAH) experiments (**p<0.01, ***p<0.001, one-way ANOVA).(D) After stabilization of BMVEC monolayers induction medium was changed to slightly acidic conditions (HBSS, pH 6). Afterwards, TNFα-primed PMNL were added in the absence or presence of 4-ABAH (100 µM) and activated with fMLP (10 µM). Impedance was normalized to baseline recorded prior to treatments and represents mean values ± SD of four independent experiments (*p<0.05, **p<0.01; Student’s t-test).
Figure 4
Figure 4. BMVEC barrier function is compromised by the MPO-H2O2-Cl− system.
BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of hydrocortisone-induced endothelial monolayers (7.5×104 cells) was continuously monitored by impedance sensing at 4 kHz. (A) After stabilization of BMVEC monolayers induction medium was changed (arrow) to slightly acidic conditions (HBSS, pH 6). After a 30 min pre-conditioning period, BMVEC were incubated (arrow) with MPO (120 nM) and methionine (1; ‘Met’; 5 mM), H2O2 (2; 4×125 µM every 3 min), MPO and H2O2 (3), or MPO, H2O2 and Met (4). (B) Statistical evaluation of impedance values after 2 h from (A). Impedance was normalized to baseline and represents mean values ± SD of 4 independent experiments (ns = not significant; **p<0.01, ***p<0.001, one-way ANOVA). (C) After stabilization of BMVEC monolayers induction medium was changed (arrow) to slightly acidic conditions (HBSS, pH 6). After a 70 min pre-conditioning period, BMVEC were incubated (arrow) with 4-ABAH (1; 100 µM), MPO (2; 120 nM), MPO and H2O2 (3; 4×125 µM), or MPO, H2O2 and 4-ABAH (4). (D) Statistical evaluation of impedance values after 4 h from (C). Impedance was normalized to baseline and represents mean values ± SD of 4 independent experiments (**p<0.01, ***p<0.001, one-way ANOVA).
Figure 5
Figure 5. BBB permeability in response to LPS is attenuated in MPO−/− mice.
Control animals (‘control’; n = 8) were injected with Evans Blue (EB) in PBS. Wt (n = 12) and MPO−/− mice (MPO−/−; n = 13) received a single injection (i.p.) of LPS (250 µg LPS/30 g body weight) and EB. Twelve hours post LPS treatment animals were anaesthetized with pentobarbital (150 mg/kg body weight) and transcardially perfused with 25 ml PBS. Thereafter brains were removed, frozen in liquid nitrogen and homogenized. EB was quantitated spectrophotometrically using an external EB calibration curve. Results shown represent mean values ± SD (*p<0.05; ***p<0.001, one way ANOVA).
Figure 6
Figure 6. Modification of endogenous BMVEC plasmalogens by the MPO-H2O2-Cl− system or TNFα-primed/fMLP-activated neutrophils generate chlorinated aldehydes.
(A) Adherent BMVEC were hydrolyzed (0.5 M HCl, 37°C, 2 h) in the presence of 1 µg [13C8]HDA as internal standard. Following extraction, PFB oximes were prepared, and total FALD concentrations (hexadecanal, HDA; octadecanal, ODA; octadecenal, ODEA) were quantitated by NICI-GC-MS analysis. FALD content was converted to plasmalogen concentrations (molar ratio = 1∶1). Results represent mean values ± SD from 3 independent experiments. (B) BMVEC were washed with HBSS (pH 6) and incubated in the presence of MPO (120 nM) and H2O2 (500 µM). One µg 2-Cl[13C8]HDA) was added as internal standard at treatment start. 2-chlorohexadecanal (2-ClHDA), 2-chlorooctadecanal (2-ClODA), and 2-chlorooctadecenal (2-ClODEA) were analyzed as PFB-oximes by NICI-GC-MS. Results are shown as mean values ± SD of 2-ClFALD concentrations from 4 independent experiments. (C) BMVEC were washed with HBSS (pH 6) and incubated with PMA or primed with TNFα (10 ng/ml) followed by activation with LPS (1 µg/ml) or fMLP (10 µM) for 3 h. 2-Cl[13C8]HDA (250 ng) were added as internal standard after treatment start. Lipids were extracted, converted to PFB oximes, and 2-ClHDA concentrations were quantitated by NICI-GC-MS. Results represent mean values ± SD from 3 independent experiments.
Figure 7
Figure 7. 2-ClHDA impairs barrier function in vitro and in vivo.
(A) BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of HC-induced BMVEC was continuously monitored by impedance sensing at 4 kHz in the presence of the indicated 2-ClHDA concentrations or vehicle (DMSO). (B) BMVEC were cultured on coverslips until confluence. After incubation for 3 h in the presence of vehicle (DMSO; upper panel) or 15 µM 2-ClHDA (lower panel) immunofluorescence labeling of ZO-1 (red) and VE-cadherin (green) was performed. Sites of nuclear ZO-1 redistribution (arrows) and frizzy-like structures (arrowheads) are indicated. Scale bars: 20 µm. (C, D) The left common carotid artery of anesthetized rats was exposed and cannulated. After sectioning of jugular veins animals were perfused (3 ml/min) with Ringer solution for 5 min. Subsequently, perfusion was switched for 90 min to Ringer solution containing vehicle (DMSO) or 25 µM 2-ClHDA. This was followed by perfusion with Ringer solution supplemented with EB and SF for 5 min and a washout with Ringer solution (without dyes) for 7 min. Animals were decapitated and the brains were immediately removed. (C) Macroscopic evaluation of EB extravasation, and (D) determination of SF fluorescence intensity in brain homogenates are shown. Results represent mean values ± SD from 4 animals (**p<0.01, Students t-test).
Figure 8
Figure 8. 2-ClHDA activates MAPK pathways.
(A) Concentration-dependent activation of the MAPK cascade. BMVEC were incubated with NaOCl or 2-ClHDA or DMSO (used as vehicle for 2-ClHDA delivery) at the indicated concentrations for 3 h. (B) Time-dependent activation of the MAPK cascade in response to 2-ClHDA. BMVEC were incubated with 2-ClHDA (25 µM) for the indicated time periods. After treatment, cells were lysed, aliquots of protein lysates were subjected to SDS-PAGE and transferred to PVDF membranes. Pan- or phospho-specific polyclonal antibodies against p38, JNK1/2, or ERK1/2 were used as primary antibodies. Immunoreactive bands were visualized with peroxidase-conjugated secondary antibodies using the ECL-system. Bar graphs in the right panels show the ratio of optical densities of immunoreactive phosphorylated normalized to non-phosphorylated proteins.
Figure 9
Figure 9. Inhibition of ERK and JNK provides partial rescue against 2-ClHDA-induced barrier dysfunction.
BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of endothelial monolayers was continuously monitored by impedance sensing at 4 kHz. After stabilization, cells were challenged (arrow) with 2-ClHDA in the absence or presence of (A) 100 µM PD098059, (C) 25 µM SP600125, or (E) 25 µM SB203580. Results represent mean values ± SD from 4 independent experiments. 2-ClHDA concentrations were 5 (A) and 10 (C, E) µM. (B, D, and F) Statistical evaluation of relative barrier function at the indicated time periods post 2-ClHDA treatment in the absence or presence of the respective antagonist. Impedance was normalized to baseline and represent mean values ± SD of 4 independent experiments (**p<0.01; ***p<0.001; two-way ANOVA). (G) BMVEC were incubated with 2-ClHDA (25 µM) in the absence or presence of PD098059 (100 µM) for the times indicated. Cells were lysed, aliquots of protein lysates were subjected to SDS-PAGE and transferred to PVDF membranes. (Phospho)Specific polyclonal antibodies against ERK1/2, p38, or JNK1/2 were used as primary antibodies. Immunoreactive bands were visualized with peroxidase-conjugated secondary antibodies using the ECL-system. (H) Bar graphs represent the ratio of optical densities of immunoreactive phosphorylated proteins normalized to non-phosphorylated proteins.
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