Published online before print October 4, 2005
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,1
* Departments of Pharmacology and Experimental Neuroscience, Center for Neurovirology and Neurodegenerative Disorders, and
Pathology/Microbiology, University of Nebraska Medical Center, Omaha
1 Correspondence: Center for Neurovirology and Neurodegenerative Disorders, Departments of Pathology and Microbiology and Pharmacology and Experimental Neuroscience, 985215 Nebraska Medical Center, Omaha, NE 68198-5215. E-mail: ypersids{at}unmc.edu
ABSTRACT
Brain microvascular endothelial cells (BMVEC) connected by tight junctions (TJ) form a tight monolayer at the blood-brain barrier (BBB). We investigated the idea that BBB dysfunction seen in alcohol abuse is associated with oxidative stress stemming from ethanol (EtOH) metabolism in BMVEC. Exposure to EtOH induced catalytic activity/expression of EtOH-metabolizing enzymes, which paralleled enhanced generation of reactive oxygen species (ROS). EtOH-mediated oxidative stress led to activation of myosin light chain (MLC) kinase, phosphorylation of MLC and TJ proteins, decreased BBB integrity, and enhanced monocyte migration across BBB. Acetaldehyde or ROS donors mimicked changes induced by EtOH in BMVEC. Thus, oxidative stress resulting from alcohol metabolism in BMVEC can lead to BBB breakdown in alcohol abuse, serving as an aggravating factor in neuroinflammatory disorders.
Key Words: tight junctions • monocyte migration
INTRODUCTION
Blood-brain barrier (BBB) composed of brain microvascular endothelial cells (BMVEC) serves as a protective shield between blood and the central nervous system (CNS). BMVEC possess unique barrier functional properties as compared with microvascular endothelium of other organs and are connected by tight junctions (TJ), which are composed of transmembrane proteins occludin/claudin-5 and intracellular zonula occludens (ZO-1, -2, and -3). TJ ensure structural integrity and low permeability of monolayer [1 ]. Alcohol abuse and neuroinflammatory conditions [such as CNS human immunodeficiency virus type 1 (HIV-1) infection] are associated with BBB impairment. Diffusion-imaging studies in patients with chronic alcoholism suggest that white matter abnormalities are related to BBB dysfunction [2 ]. Similarly, neuroimaging indicated existence of a BBB leak in white matter during HIV-1 encephalopathy, which was reversed by suppression of virus replication and correlated with cognitive improvement after initiation of antiretroviral therapy [3 , 4 ]. Monocytes/macrophages egress across BBB seen in HIV-1 encephalitis (HIVE), the neuropathologic correlate of HIV-1 encephalopathy, paralleled TJ impairment of BMVEC documented by diminished immunostaining for occludin and ZO-1 [5 , 6 ]. It has been shown that the CNS suffers the additive effects of alcohol abuse and HIV-1 in infected patients [7 ] by imaging studies.
Previous studies have clearly indicated that long-term alcohol abuse leads to profound functional and morphological changes in the CNS, regardless of nutritional status. Neuropathologic examination of brain tissue from chronic alcoholics indicated the presence of neurodegeneration, ranging from minor dendritic and synaptic changes to neuronal cell death [8 ]. Thus, multiple lines of evidence suggest that chronic and excessive ethanol (EtOH) consumption may enhance oxidative injury of neural cells. By mechanisms not well understood, EtOH induces activity of the EtOH-metabolizing enzyme cytochrome P450-2E1 (CYP2E1) and enhances reactive oxygen species (ROS) generation in the brain (for review, see ref. [9 ]). It is interesting that expression of CYP2E1, the enzyme that catalyzes EtOH to ROS and acetaldehyde (AA), was prominent in the white matter of animals chronically exposed to alcohol [10 ]. Oxidative stress, associated with inflammatory factors secreted by activated macrophages or viral proteins, was suggested as an underlying cause of BBB disruption in HIV-1 CNS infection [11 ]. Overall, little is known about the mechanisms involved in BBB compromise during HIV-1 CNS infection and excessive alcohol consumption, conditions promoting neuroinflammation and oxidative stress [6 , 9 , 12 13 14 ].
Using our previously developed BBB models and primary BMVEC, we demonstrated that EtOH or its metabolite AA in nontoxic concentrations decreased BBB tightness via activation of myosin light chain (MLC) kinase (MLCK), leading to phosphorylation of MLC/TJ proteins and promoted monocyte migration across BBB models [15 ]. Antioxidant treatment or suppression of EtOH metabolism in BMVEC prevented these phenomena, suggesting that alcohol-associated BBB impairment is mediated, in part, by oxidative stress [15 ]. The current study addresses the role of oxidative stress in BBB impairment caused by alcohol exposure and whether these mechanisms exacerbate BBB dysfunction associated with HIVE. Here, we provide evidence that EtOH metabolites AA and ROS activate MLCK, leading to phosporylation of MLC/TJ proteins and resulting in decreased BBB integrity and enhanced monocyte migration across the BBB. All of these effects were reproduced in the BBB by mediators of oxidative stress. Our data demonstrate a clear link amongst BMVEC alcohol metabolism, oxidative stress, and BBB impairment, with potential implications in neuroinflammation and alcohol abuse.
MATERIALS AND METHODS
Cell isolation and culture
Primary human BMVEC were isolated from the temporal cortex of brain tissue obtained during surgical removal of epileptogenic foci in adult patients by the procedure described previously [16
] and supplied by Dr. Marlys Witte (University of Arizona, Tucson). Routine evaluation for von Willebrand factor, Ulex europeus lectin, and CD31 demonstrated that cells were >99% pure. Primary bovine BMVEC were isolated from the gray matter of fresh bovine cerebral cortices through enzymatic digestion and subsequent gradient density centrifugation [17
]. As described before [15
], freshly isolated cells were cultured in 96-well plates, 24-well tissue-culture inserts (Cyclopore® polyethylene terephthalate membrane, pore diameter 0.4 µm or 3 µm, Collaborative Biochemical Products, Becton Dickinson, San Jose, CA), or in 75 cm2 flasks. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activity as described previously [18
]. The following concentrations were used in our experiments: 50 mM EtOH (corresponding to 0.23%); 1 mM 4-methylpyrazole (4-MP), an inhibitor of EtOH metabolizing enzymes CYP2E1 and alcohol dehydrogenase (ADH); 50 µM uric acid (UA) antioxidant; 100 µM AA; 10 µM ML-7 (a specific inhibitor of MLCK); 100 µM H2O2, superoxide donor; 50 µM S-nitroso-N-acetyl-penicillamine (SNAP) nitric oxide (NO) donor; and 50 µM 3-morpholinylsydnoneimine (SIN-1; peroxynitrite donor). All compounds and inhibitors were used in nontoxic concentrations determined by cell viability assay, MTT (96 h exposure).
Dichlorofluorescein-diacetate (DCF-DA) assay
DCF-DA is converted to highly fluorescent DCF by ROS. The fluorescence was detected at excitation 488 nm and at 525 nm emission spectra using a fluorescence plate reader as described previously [19
]. Results were expressed as specific mean fluorescence intensity (MFI) per mg protein.
CYP2E1 and ADH activity
For ADH and CYP2E1 activity assays, extracted protein lysates were centrifuged at 35,000 rpm (105,000 g) for 1 h at 4°C. High-speed cytosolic fractions (supernatants) were used for ADH activity assay, whereas the microsomes (pellets) were resuspended in 1x phosphate-buffered saline for CYP2E1 activity assay. CYP2E1 activity was determined by hydroxylation of p-nitrophenol to 4-nitrocatechol mediated by CYP2E1 as described before [19
]. ADH catalytic activity was assayed by formation of reduced nicotinamide adenine dinucleotide (NADH) from NAD oxidation following the method described previously [20
]. Briefly, 50 µl high-speed cytosolic samples were added to 430 µl reaction mixture consisting of 0.5 M Tris-HCl, 0.01 M dithiothreitol, and 0.5 M EtOH in 1.5 ml disposable cuvette, prewarmed for 5 min, followed by addition of 20 µl 90 mM NAD, mixed well. After 30 s, the change in optical density was monitored at 340 nm using the time-drive kinetics for 10 min at 30 s intervals, and the activity was expressed as nmoles/min/mg total cellular protein.
Transendothelial electrical resistance (TEER) and monocyte migration
The BBB models were constructed [16
] by placing the BMVEC (200 µl containing 20,000 cells) in the upper chamber of 24-well tissue-culture inserts (Cyclopore® polyethylene terephthalate membrane, pore diameter 0.4 µm for TEER or 3 µm for monocyte migration). The cells were cultured for at least 4 days before use. Using this in vitro BBB model [16
], the TEER and monocyte migration across the BBB of endothelial cell monolayers were studied as described previously [15
]. Electrical resistance of the blank inserts (150 
cm2) with media alone was subtracted from the TEER readings obtained from inserts with confluent endothelial cell monolayers. The resulting TEER values (200–250 cm2) represented the resistance ("tightness") of the endothelial cell monolayers. For migration studies across the BBB constructs, peripheral blood monocytes obtained from HIV- and hepatitis B-seronegative donors by leukopheresis were purified by counter-current centrifugal elutriation [21
] and applied to upper chambers of BBB models (105 cells/insert). Migrated monocytes were counted on the lower chamber after immunostaining with the CD68 antibody (macrophage marker at 1:100 dilution, Dako, Carpenteria, CA).
Immunoconjugation, immunoprecipitation, and Western blot
Immunoconjugation, immunoprecipitation, and Western blot analyses were performed as described [15
]. Antibodies to MLC, MLCK (Sigma Chemical Co., St. Louis, MO), phospho-MLC-2 (serine 19/threonine18; Cell Signaling, Inc., Beverly, MA), occludin, ZO-1, claudin-5, and antiphosphoserine (Zymed, San Francisco, CA) were used for immunoconjugation, immunoprecipitation, and Western blot analyses. Results were expressed as ratio of relative intensity of target protein to that of the internal standard, 
-actin.
Statistical analysis
Results were expressed as mean values (±SEM), and a value of P < 0.05 was considered significant. The statistical significance effects of EtOH or inhibitor effects on TEER, monocyte migration, and expression of proteins were assessed by two-way ANOVA analyses with Newman-Keuls post-test for multiple comparisons.
RESULTS
EtOH induces ROS generation and ROS-nitrated protein accumulation in BMVEC
Our previous experiments demonstrated that EtOH-induced activation of MLCK led to phosphorylation of MLC and TJ proteins. As a result, BMVEC monolayers demonstrated redistributed TJ staining, gap formation, decreased BBB integrity, and enhanced monocyte migration [15
]. AA mimicked EtOH-induced effects, and inhibition of EtOH metabolism in endothelial cells reversed these changes. We hypothesized that oxidative stress could be the cause of BBB impairment. To explore this idea, we first examined whether EtOH could lead to oxidative stress in endothelial cells using the DCF-DA assay. H2O2 (100 µM), a ROS-donating agent, and SNAP (50 µM), a NO donor, were used as positive controls. EtOH-exposed BMVEC showed a 38% increase in reactive species levels compared with control (P<0.01), and this increase in ROS formation was abrogated by the metabolic EtOH inhibitor (4-MP) or antioxidant (UA; Fig. 1A
). As expected, treatment of BMVEC with H2O2 or SNAP, individually or in combination, showed significant increases in reactive species production as compared with control (38%, 34%, and 42%, respectively; P<0.01; Fig. 1A
), and antioxidant treatment reverted ROS production to control levels (P>0.05).
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Figure 1. EtOH metabolism in BMVEC leads to oxidative stress, which is reversible by antioxidant. BMVEC were treated with EtOH (50 mM) or Ros donors for 2 h, with or without inhibitor of EtOH metabolism (4-MP) or antioxidant (50 µM UA). (A) DCF fluorescence was measured as specific MFI per milligram protein. (B) Representative immunoreactive bands for 210 kDa nitrated proteins and -actin. (C) Changes in nitrotyrosine (210 kDa) content, expressed as relative intensity ratio of 210 kDa nitrated protein to that of -actin. Results were expressed as mean values (±SEM; n=4) and represented by the bar graph. *, Statistical differences (P<0.01) as compared with untreated control cells.
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EtOH metabolism in endothelial cells
As EtOH metabolism inhibitor blocked ROS and nitrotyrosine formation in EtOH-treated BMVEC, we next investigated functional activity and expression of two putative EtOH-metabolizing enzymes, CYP2E1 and ADH, in BMVEC. ADH metabolizes EtOH to AA, and CYP2E1-mediated EtOH metabolism leads to production of AA and ROS.
We detected high levels of CYP2E1 catalytic activity (129 nmoles/mg protein) and protein expression in BMVEC. Exposure of BMVEC to EtOH (50 mM) resulted in a 1.6-fold increase of CYP2E1 catalytic activity (up to 200 nmoles/hr/mg protein; P<0.01) and augmented protein level (49%; P<0.01) when compared with the control, unexposed cells (Fig. 2A 2C and 2D ). EtOH-induced increase in CYP2E1 activity and protein content were diminished to control level either by 4-MP or by withdrawal of EtOH from the culture media. Similar results were obtained for ADH activity and protein content in brain endothelial cells. EtOH (50 mM) treatment of BMVEC resulted in a 1.6-fold increase in ADH catalytic activity (from 28 to 44 nmoles/min/mg total cellular protein; P<0.01) and a 43% increase in ADH protein level (P<0.01) as compared with unexposed controls. Addition of 4-MP (ADH inhibitor) or withdrawal of EtOH decreased ADH activity and protein content to basal levels. Using liver microsomal or cytosolic fractions as positive controls (generous gift of Dr. Terrence Donohue, Alcohol Research Center, VA Medical Center, Omaha, NE), derived from control or EtOH pair-fed rats for 5 weeks on a liquid diet, we assayed CYP2E1 and ADH activity in human BMVEC (50 mM EtOH) and monocyte-derived macrophages (MDM; 25 mM EtOH) after 2 h EtOH exposure. Our results indicate that CYP2E1 levels following EtOH exposure increased from 43 to 168 nmoles/hr/mg protein in liver microsomes, from 126 to 164 nmoles/hr/mg protein in human BMVEC, and from 61 to 109 nmoles/hr/mg protein in human MDM. Similarly, ADH activity increased from 41 to 59 nmoles/min/mg protein in liver microsomes, from 20 to 37 nmoles/min/mg protein in human BMVEC, and from 27 to 42 nmoles/min/mg protein. Our data indicate that ADH and CYP2E1 are highly expressed and are functionally active in bovine as well as in human BMVEC, resulting in EtOH metabolism to AA and reactive species.
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Figure 2. EtOH effects on CYP2E1 and ADH activity and expression in BMVEC. (A) CYP2E1 activity (nmole/hr/mg protein) and (B) ADH activity (nmole/min/mg protein) in primary bovine BMVEC after 2 h EtOH (50 mM) treatment with or without 4-MP or 2 h after EtOH withdrawal. (C) represents immunoreactive bands for CYP2E1, ADH, and internal standard -actin. (D) CYP2E1 and (E) ADH contents were expressed as ratio of densitometric intensity of them to -actin. Results were expressed as mean values ± SEM (n=4). *, Statistical differences (P<0.01) as compared with untreated control cells.
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Figure 3. EtOH and oxidative stress induce activation of MLCK and MLC phosphorylation. BMVEC were treated for 2 h with EtOH, AA, H2O2, SNAP, and SIN-1, or cells were preincubated with 4-MP, ML-7, or UA for 0.5 h prior to application of test compounds. Immunoprecipitation and Western blot analyses were performed to determine the expression of p-MLC, MLC, and MLCK. (A) Representative immunoreactive bands of MLC, MLCK, p-MLC, and -actin. Relative intensity was expressed as the ratio of the target protein to that of -actin. Results were expressed as mean values (±SEM; n=4) and were represented by the bar graph for (B) p-MLC, (C) MLC, and (D) MLCK. Antibodies to antiphosphoserine-MLC were used to measure p-MLC. *, Statistical differences (P<0.01) as compared with untreated control cells.
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Figure 4. EtOH exposure and oxidative stress lead to phosphorylation of TJ proteins. BMVEC were treated for 2 h treatment with EtOH, AA, H2O2, SNAP, and SIN-1, or cells were pre-incubated with 4-MP, ML-7, or UA for 0.5 h prior to application of test compounds. Immunoprecipitation and Western blot analyses were performed to determine the expression of TJ proteins. Results were expressed as mean values (±SEM; n=4). (A) Representative immunoreactive bands of phosphorylated and total occludin, (B) ratio of phosphorylated/total occludin, (C) representative immunoreactive bands of phosphorylated and total claudin-5, (D) ratio of phosphorylated/total claudin-5, (E) representative immunoreactive bands of phosphorylated and total ZO-1, (F) ratio of phosphorylated/total ZO-1. Phosphorylated forms of TJ proteins were determined by probing the total immunoprecipitated proteins with antiphosphoserine antibody. *, Statistical differences (P<0.01) as compared with untreated control cells.
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Figure 5. Effects of EtOH, AA, and oxidative stress on TEER and monocyte migration across BBB. (A) EtOH, AA, H2O2, SNAP, and SIN-1 significantly reduced TEER, and TEER measurement across the BMVEC monolayer assessed the BBB tightness. TEER values recorded in ohms ()/insert area were corrected to /cm2 from three experiments in triplicate. Results were then expressed as mean percent of controls at initial time-point (±SEM; n=3). (B) Monolayer treatment for 2 h with EtOH, AA, or exogenous reactive species donors significantly increased monocyte (Mo) migration across the BBB. 4-MP and UA inhibited monocyte migration across the BBB enhanced by EtOH or SIN-1 exposure. *, Statistical differences (P<0.01) as compared with untreated control cells.
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The present study demonstrates that EtOH-metabolizing enzymes ADH and CYP2E1 are expressed in BMVEC, and EtOH metabolism in BMVEC leads to oxidative stress, resulting in MLCK activation (Fig. 6 ). Activated MLCK phosphorylates MLC and TJ proteins, leading to cytoskeletal rearrangement and impairment of TJ integrity. These events cause BBB dysfunction and enhanced leukocyte migration across BBB. It is important that our data indicate that oxidative stress per se could result in similar BBB alterations via the same pathways.
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Figure 6. Schematic representation: effects of EtOH and oxidative stress on BBB. EtOH metabolism in brain endothelial cells results in production of AA and reactive species. These, in turn, activate MLCK, leading to phosphorylation of MLC and TJ proteins, functional alterations of TJ, and cytoskeletal rearrangement. Ultimately, these changes result in BBB compromise and enhanced leukocyte migration across BBB.
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Organ dysfunction stemming from alcohol abuse has significant clinical implications [37 ]. Oxidative stress associated with alcohol abuse exacerbated endothelial and epithelial cell injury in lungs seen during acute inflammation in trauma patients [38 ]. These pathologic changes were ameliorated by antioxidant treatment in an animal model with chronic alcohol exposure [39 ]. ROS-related damage of brain tissue was documented in alcoholics including cerebral edema, neuronal loss, and damage of BBB [40 ]. Other pathophysiologic conditions associated with oxidative stress may decrease BBB integrity and enhance monocyte migration. Increased expression of adhesion molecules on endothelial cells and their respective ligands on monocytes-macrophages occur shortly after exposure to oxidative stress, or lipid oxidation products can be modulated by antioxidants [41 42 43 ]. Leukocyte-endothelial cell interactions via adhesion molecules lead to ROS generation and disruption of junctional complexes [44 ]. ROS scavenging inhibits these effects on junctions and transendothelial leukocyte migration [45 ]. Leukocyte adhesion to endothelium induces ROS production, which changes the functional state of endothelium, facilitating leukocyte passage [46 ], and our data suggest that the EtOH metabolism in BMVEC can activate similar pathways, increasing monocyte migration.
By a yet-unknown mechanism, oxidative stress activated MLCK in BMVEC, which phosphorylated cytoskeletal (MLC) and TJ proteins, diminishing the integrity of the BMVEC monolayer. Exogenously supplied AA, peroxide, peroxynitrite, and NO mimicked EtOH-metabolic effects in BMVEC, leading to reduced TEER and increased monocyte migration across the BBB, further supporting the role of oxidative stress in BBB dysfunction. Similar to our findings, Zhao and Davis [47 ] reported the peroxide-induced activation of MLCK and subsequent MLC phosphorylation in primary pulmonary endothelial cells.
We demonstrated that MLC, claudin-5, and occludin were predominantly phosphorylated at serine and minimally at threonine residues after BMVEC treatment with EtOH [15 ]. Here, we present evidence that EtOH-induced oxidative stress via MLCK activation led to phosphorylation of MLC and TJ proteins. Mediators of oxidative stress (peroxide, peroxynitrite, and NO) elicited the same response in BMVEC. Phosphorylation of MLC changes BBB integrity, likely through altered actin-myosin interactions. In endothelial cells, MLC phosphorylation at serine/threonine residues is associated with cell contraction, cytoskeletal rearrangement, or loss of barrier integrity, and the MLCK inhibitor can prevent these changes [15 , 48 , 49 ].
Although occludin phosphorylation at serine/threonine residues altered the actin-cytoskeletal interactions [50 , 51 ], occludin phosphorylation at tyrosine residues attenuated the interactions with ZO-1, -2, and -3 [52 ]. Occludin phosphorylation at these sites also increased the permeability of endothelial monolayers [53 , 54 ], and dephosphorylation restored barrier tightness [55 ]. Diminished occludin expression, enhanced occludin phosphorylation at serine residues, and increased permeability were observed in endothelial cells exposed to H2O2 [56 ]. These studies concur with our findings of enhanced occludin phosphorylation and decreased content in BMVEC caused by oxidative stress.
Claudin-5 is believed to be a vital component of TJ in the BBB [57 , 58 ]. Thus, decreased claudin-5 content mediated by oxidative stress/EtOH metabolism observed here could be one of the important contributing factors for BBB breakdown. Increased ZO-1 phosphorylation at serine/threonine residues by EtOH, AA, or ROS donors correlated with augmented monocyte migration across the BBB, and these findings are supported by those of others that phosphorylation of ZO-1 at these sites was associated with an increase in paracellular permeability in BMVEC after hypoxia [59 ]. ZO-1 phosphorylation at tyrosine residues resulted in augmented permeability of endothelial monolayers [53 ]. Exposure of endothelial cells to H2O2 [60 ] resulted in decreased of TEER and ZO-1/occludin redistribution, similar to effects of EtOH in BMVEC [15 ].
A number of studies demonstrated inhibitory effects of EtOH on cytokine and chemokine production and reduction of leukocyte migration. In vivo, these effects are usually observed after acute short-term alcohol exposure [61 ], and more chronic studies pointed to existence of vascular injury promoting leakiness and leukocyte adhesion [62 ]. Similarly, chronic exposure to EtOH in vitro led to increased cytokine and NO production in glial cells [63 ]. Diffusion-imaging studies in patients with chronic alcoholism suggest that white matter abnormalities may be related to BBB dysfunction [2 ]. In our system, alcohol-induced leakiness of BBB and enhanced monocyte migration were highly reproducible phenomena. Differences between in vitro and in vivo systems and length of EtOH exposure in vivo could explain seemingly contradictory observations.
Cytokines, chemokines, other secretory inflammatory products and viral proteins, such as gp120 or Tat produced by virus-infected and/or immune-activated macrophages, are thought to be the ultimate cause of HAD [6 ]. Oxidative stress plays an important role in HIV-1 CNS infection [12 ]. It has been shown that HIV-1 Tat protein-induced oxidative stress altered ZO-1 protein in mouse brain [64 ] and claudin-5 protein in rat brain endothelial cells [11 ], leading to disruption of BBB. Thus, apart from proinflammatory products or viral proteins, oxidative stress could be the significant contributing factor in HIV-1-infected patients with a history of alcohol abuse, further worsening BBB dysfunction. Indeed, it has been shown that CNS suffers the additive effects of alcohol abuse and HIV-1 [7 ]. Additional studies will be necessary to address relevance of our in vitro findings and potential synergy amongst multiple deleterious factors such as viral proteins, proinflammatory cytokines, and oxidative stress and their net effects on BBB during HIV-1 CNS infection in the setting of alcohol abuse. This will be accomplished using an animal model for HIVE [65 ].
ACKNOWLEDGEMENTS
This work was supported in part by National Institutes of Health Grants R21 AA013846 and R01 MH65151 (to Y. P.). The authors of this manuscript appreciate excellent administrative support from Ms. Robin Taylor and Ms. Julie Ditter. We thank Dr. Mark Thomas for critical reading of the manuscript. Liver microsomal and cytosolic fractions, which served as positive controls, were provided Dr. Terrence Donohue (Alcohol Research Center, VA Medical Center, Omaha, NE).
Received June 23, 2005; revised August 1, 2005; accepted August 8, 2005.
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