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.
Highlights
The neurovascular unit physically separates most regions of the brain from the peripheral circulation to maintain the specialized micromilieu of the central nervous system [1]
We explored the effects of activated polymorphonuclear leukocytes (PMNL) and purified MPO on barrier function of primary porcine brain microvascular endothelial cells (BMVEC) and studied blood-brain barrier (BBB) permeability in wild-type and MPO2/2 mice in response to peripheral LPS administration
LPS from Escherichia coli (0111:B4), pentobarbital sodium salt, heparin sodium salt, Evans Blue (EB), sodium fluorescein (SF), bovine serum albumin (BSA), DMEM Ham’s F12, hydrocortisone (HC), sodium hypochlorite (NaOCl), H2O2, methionine, the MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH), N,N-dimethyl formamide (DMF), dimethyl sulfoxid (DMSO), phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, pepstatin and other protease inhibitors were from Sigma Aldrich (Vienna, Austria)
Summary
The neurovascular unit physically separates most regions of the brain from the peripheral circulation to maintain the specialized micromilieu of the central nervous system [1]. The presence of TJ prevents paracellular transport of molecules and cells and maintains brain homeostasis via elaborately regulated transport mechanisms In addition to this ‘sealing’ function, TJ complexes physically separate a luminal and abluminal compartment thereby sustaining the polarized phenotype of BMVEC [3]. We could demonstrate significantly elevated MPO protein levels in brains of mice that received a single, peripheral lipopolysaccharide (LPS) injection [16] This was accompanied by a significant decrease of the brain plasmalogen concentration and concomitant formation of 2-chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde generated from HOCl-mediated attack of plasmalogens (ether phospholipids) [16]. It is conceivable that oxidative modification of BMVEC plasmalogens might have detrimental effects on BBB function because i) plasmalogens are important constituents of lipid rafts [17], and ii) barrier and fence function of TJ complexes depend on membrane scaffolding and transporter lipid rafts [18]
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