1486 Research Article

IntroductionChemokines are a superfamily of structurally related small proteinswith strong chemotactic activity. By binding to their specific G-protein-coupled receptors, chemokines induce cell-specificmigration and activation of immune cells (Glabinski et al., 1996;Hulkower et al., 1993; Lahrtz et al., 1998; Miller and Meucci,1999). Monocyte chemoattractant protein-1 (MCP1; officiallyknown as C-C motif chemokine 2, CCL2) is one of the mosthighly and transiently expressed chemokines in many centralnervous system (CNS) injuries, including ischemia, hemorrhageand excitotoxic injury (Capoccia et al., 2008; Dimitrijevic et al.,2006; Frangogiannis et al., 2007; Hanisch, 2002; Kim et al., 2008;Morimoto et al., 2008; Sheehan et al., 2007; Yan et al., 2007). Therodent MCP1 differs from the human protein at the C-terminus,with the rodent protein having a longer highly glycosylated C-terminus. We have previously shown that mouse MCP1 can becleaved by plasmin at K104, and the truncated MCP1 is morepotent than the full-length MCP1 in inducing microglial migration(Sheehan et al., 2007; Yao and Tsirka, 2010) through its receptorCCR2.

Besides chemotaxis, MCP1 is also involved in blood–brainbarrier (BBB) compromise (Dimitrijevic et al., 2006; Stamatovicet al., 2006; Stamatovic et al., 2003; Stamatovic et al., 2005). Here,we investigated whether plasmin-mediated cleavage regulatesMCP1-induced BBB breakdown. It has been reported thatcomponents of the plasmin(ogen) system can regulate BBBintegrity. Specifically, tissue-type plasminogen activator (tPA),which converts inactive plasminogen into active plasmin, promotesBBB disruption and subsequent peripheral blood mononuclear cell(PBMC) infiltration (Reijerkerk et al., 2008). Additionally, BBB

compromise and PBMC infiltration have been found in micedeficient for plasminogen activator inhibitor 1 (PAI-1) (Kataoka etal., 2000), suggesting that plasmin-mediated cleavage of MCP1might play a role in BBB disruption.

The BBB, a specialized structure that prevents the infiltration ofcomponents from the peripheral blood into the CNS, mainlycomprises brain microvascular endothelial cells (BMECs), pericytesand astrocytes (Guillemin and Brew, 2004). BMECs form animpermeable monolayer through tight junctions, where bothtransmembrane and cytoplasmic tight junction proteins (TJPs) areexpressed. The transmembrane proteins, such as occludin andclaudin-1, -5 and -11, function to seal gaps between adjacent cells,whereas cytoplasmic accessory proteins, such as the zonulaoccluden proteins (ZO-1, ZO-2 and ZO-3) (Citi and Cordenonsi,1998; Huber et al., 2001), link transmembrane proteins to thecortical actin-based cytoskeleton (Förster, 2008; Michel and Curry,1999; Mitic and Anderson, 1998). Accumulating evidence showsthat MCP1 increases BBB permeability through redistribution ofTJPs and reorganization of actin cytoskeleton in a CCR2-dependentmanner (Dimitrijevic et al., 2006; Stamatovic et al., 2006;Stamatovic et al., 2003; Stamatovic et al., 2005). The molecularmechanisms underlying TJP redistribution, however, are stillelusive.

Here, we show that plasmin-mediated cleavage is crucial forfast MCP1-induced compromise of the BBB. Further mechanisticstudies reveal that MCP1-induced redistribution of ZO-1 ismediated by ezrin-radixin-moesin (ERM) proteins andreorganization of actin cytoskeleton.

SummaryPrevious studies have shown that plasmin cleaves monocyte chemoattractant protein 1 (MCP1; officially known as C-C motifchemokine 2, CCL2) at K104, and this cleavage enhances its chemotactic potency significantly. Accumulating evidence reveals thatMCP1 also disrupts the integrity of the blood–brain barrier (BBB). Here, we show that K104Stop-MCP1, truncated at the K104 whereplasmin would normally cleave, is more efficient than the full-length protein (FL-MCP1) in compromising the integrity of the BBBin in vitro and in vivo models. K104Stop-MCP1 increases the permeability of BBB in both wild-type mice and mice deficient fortissue plasminogen activator (tPA), which converts plasminogen into active plasmin, suggesting that plasmin-mediated truncation ofMCP1 plays an important role in BBB compromise. Furthermore, we show that the mechanisms underlying MCP1-induced BBBdisruption involve redistribution of tight junction proteins (occludin and ZO-1) and reorganization of the actin cytoskeleton. Finally,we show that the redistribution of ZO-1 is mediated by phosphorylation of ezrin-radixin-moesin (ERM) proteins. These findingsidentify plasmin as a key signaling molecule in the regulation of BBB integrity and suggest that plasmin inhibitors might be used tomodulate diseases accompanied by BBB compromise.

Key words: Monocyte chemoattractant protein 1 (CCL2), Blood–brain barrier, Plasmin, Tight junction, Endothelia cells

Accepted 4 January 2011Journal of Cell Science 124, 1486-1495 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.082834

Truncation of monocyte chemoattractant protein 1 byplasmin promotes blood–brain barrier disruptionYao Yao and Stella E. Tsirka*Program in Molecular and Cellular Pharmacology, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA*Author for correspondence (stella@pharm.stonybrook.edu)

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ResultsTruncation by plasmin promotes MCP1-induced BBBcompromiseIt has been shown that mouse MCP1 increases BBB permeabilityboth in vitro and in vivo (Dimitrijevic et al., 2006; Stamatovic etal., 2005). However, it is not known whether plasmin-mediatedcleavage of MCP1 at K104, which enhances its chemotactic activity(Sheehan et al., 2007), affects MCP1-induced BBB compromise.To address this question, we used four recombinant MCP1 proteins,full-length MCP1 (FL), plasmin-noncleavable MCP1 (K104A),constitutively cleaved MCP1 (K104Stop) and MCP1 C-terminalextension (CT), that we previously characterized (Yao and Tsirka,2010). First, we evaluated the cytotoxicity of these MCP1 mutantsin mouse or human BMECs (HBMECs) using lactatedehydrogenase (LDH) cytotoxicity assays. None of these MCP1mutants was cytotoxic to BMECs (supplementary material Fig.S1). Then, we investigated the function of these MCP1 proteins onBBB permeability in a simple in vitro BBB model, which onlyinvolved HBMECs (Alter et al., 2003; Glynn and Yazdanian,1998). Although both FL- and K104Stop-MCP1 were found todecrease transendothelial electrical resistance (TEER) valuessubstantially, the effect of K104Stop-MCP1 was more dramaticthan that of FL-MCP1 (Fig. 1A) at all timepoints. Consistent withthese data, an in vitro permeability assay revealed similar results:both FL- and K104Stop-MCP1 increased the permeabilization of

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Evans Blue across the monolayer of HBMECs (Fig. 1B), indicatinga decreased BBB integrity. Because HBMECs have endogenousplasmin expression and activity, we explored the possibility thatthe decrease in TEER values observed with FL-MCP1 was due tothe truncation of MCP1 by endogenous plasmin. To block theendogenous plasmin generated by HBMECs, a2-antiplasmin(A2AP), a specific plasmin inhibitor, was used. In the presence ofA2AP, addition of FL-MCP1 did not decrease TEER values (Fig.1A) and was unable to promote infiltration of Evans Blue (Fig.1B), suggesting that the effect of FL-MCP1 observed in the initialexperiments was due to the endogenous HBMEC plasmin activity.Evans Blue can cross the BMEC monolayer by both transcellularand paracellular pathways (Hart et al., 1987; Kawedia et al., 2007).The addition of A2AP alone appeared to modify BBB permeabilityto Evans Blue but not the TEER values. This result suggested thatA2AP might affect the transcellular diffusion of Evans Blue,although it is not at this point clear how A2AP would mediate thiseffect. When we performed the in vitro permeability experimentusing the tracer FITC–Dextran (molecular mass of 4 kDa; Sigma),which only uses the paracellular pathway to go across BMECmonolayer (Hayashi et al., 2004; Neuhaus et al., 2008), onlyK104Stop-MCP1 was able to substantially enhance the leakage ofthe tracer across the co-culture system in the presence of A2AP(supplementary material Fig. S2). This result suggests that there isan important role for plasmin activity in MCP1-induced BBBdisruption. Next, we investigated whether the effect of FL- andK104Stop-MCP1 on BBB permeability was dose-dependent. Asshown in supplementary material Fig. S3, the activity of FL-MCP1increased in a dose-dependent manner, reaching a plateau at 100nM. By contrast, K104Stop-MCP1 was fully active even at lowerconcentrations (50 nM). These data suggest that K104Stop-MCP1is more ‘active’ than FL-MCP1 in this permeability assay.

To better replicate the in vivo BBB conditions, we used a mouseBMEC-astrocyte co-culture system, as astrocytes contribute to theimpermeability of BBB by covering more than 99% of themicrovessels (Kacem et al., 1998; Simard et al., 2003). Consistentwith previous data, in the absence of A2AP, FL- and K104Stop-MCP1 reduced TEER values and increased Evans Blue infiltrationsubstantially (data not shown), whereas, in the presence of A2AP,only K104Stop-MCP1 was effective in compromising the integrityof this in vitro BBB model (Fig. 2A,B). These data further supportthe hypothesis that plasmin-mediated truncation of MCP1 plays avital role in MCP1-induced BBB compromise.

We investigated the integrity of the BBB in wild-type, tPA–/–,MCP1–/– and Ccr2–/– mice, as in vivo the shearing stress effects onBBB are also present. In wild-type or MCP1–/– mice, both FL- andK104Stop-MCP1 enhanced the diffusion of Evans Blue into thebrain parenchyma. In tPA-deficient mice, the FL-MCP1 was not asefficient as the K104Stop-MCP1 in enhancing BBB permeability.As the absence of tPA results in the virtual absence of plasminactivity, the decreased efficiency of FL-MCP1 to compromise theBBB suggests that MCP1-induced disruption of BBB dependsupon the activity of plasmin. Although MCP1–/– mice showedvirtually no compromise of the BBB, adding back recombinantMCP1 to them (Fig. 2C) resulted in significant BBB compromiseby both FL- and K104Stop-MCP1. By contrast, in Ccr2–/– mice,none of these MCP1 proteins was able to increase the diffusion ofEvans Blue from the blood to the brain parenchyma (Fig. 2C),indicating an indispensable role for CCR2 in MCP1-induced BBBleakage.

Fig. 1. Truncated MCP1 compromises the integrity of the HBMECmonolayer. HBMECs seeded in Transwell inserts were treated with theindicated form of MCP1 (100 nM; in the lower chamber, abluminal side) inthe presence or absence of A2AP. Ctr, control (saline). (A)Changes in thetransendothelial electrical resistance (TEER) over time. Results are means±s.d.(n3). *P<0.05 (analyzed using one-way ANOVA followed by Newman–Keuls multiple comparison test) compared with results with FL-MCP1 withoutA2AP. (B)Chances in the permeability of Evan Blue over time. Results aremeans±s.d. (n6). *P<0.05 (comparison with Ctr).

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Plasmin-mediated truncation of MCP1 promotesredistribution of TJPsRedistribution of TJPs has been reported in MCP1-treated BMECs(Stamatovic et al., 2006; Stamatovic et al., 2003; Stamatovic et al.,2005). We further studied whether plasmin-mediated cleavage ofMCP1 is involved in this process. Using HBMECs, we found thatFL- and K104Stop-MCP1 disrupted the integrity of occludindistribution on the cell membrane. K104Stop-MCP1 was morepotent, in that disruption of the BBB was evident from the 30-minute timepoint onwards (Fig. 3A,B showing the 120-minutetimepoint). By contrast, K104A- and CT-MCP1 did not change thestaining pattern of occludin. Similarly, FL- and K104Stop-MCP1treatment led to punctate ZO-1 staining in the cell border, whereasK104A- and CT-MCP1 treatment minimally affected ZO-1 staining(Fig. 3A). We also investigated the redistribution of ZO-1 andoccludin in primary BMECs, and the results were the same asthose from HBMECs (Fig. 3C at the 120-minute timepoint).

Next, we quantified the redistribution of TJPs with semi-quantitative western blotting using Triton-X-100 fractions.Consistent with previous reports (Stamatovic et al., 2006;Stamatovic et al., 2003; Stamatovic et al., 2005; Tsukamoto andNigam, 1997; Tsukamoto and Nigam, 1999), FL-MCP1 shiftedoccludin and ZO-1 from the Triton-X-100-soluble fraction to theTriton-X-100-insoluble fraction. K104Stop-MCP1, however, had adramatically amplified change compared with that induced by FL-MCP1. By contrast, K104A- and CT-MCP1 failed to induce theshift of the TJPs (Fig. 4). As the effect of K104Stop-MCP1 wasmore dramatic than that of FL-MCP1, we tested again thepossibility that endogenous plasmin generated by BMECs mighttruncate MCP1, as described for Fig. 1. In the presence of A2APthe shift of the TJPs induced by FL-MCP1 was abrogated, whereasA2AP alone did not affect their distribution (Fig. 4).

Plasmin-mediated truncation of MCP1 changes the actincytoskeletonAs MCP1 treatment leads to the redistribution of TJPs, which areconnected to the actin cytoskeleton directly or indirectly, weexamined the reorganization of F-actin in HBMECs over time. FL-MCP1 increased F-actin over time (Fig. 5A). It peaked at 1 hourafter treatment. K104Stop-MCP1 treatment led to a significantenhancement in the amount of F-actin as early as 15 minutes aftertreatment. This effect was even more dramatic at 2 hours aftertreatment. Although K104A- and CT-MCP1 elevated F-actinslightly at early timepoints, F-actin mainly remained distributed atthe cell border. F-actin levels returned to baseline levels at 2 hoursafter treatment (Fig. 5A). We also treated primary BMECs withvarious MCP1 proteins for 2 hours. Consistently, FL-MCP1increased F-actin expression compared with that in untreated cells.K104Stop-MCP1 treatment resulted in a dramatic enhancement ofthe intensity of F-actin (Fig. 5B). K104A-MCP1 only elevated F-actin staining slightly in the cell border (indicative of the presenceof cortical actin), whereas CT-MCP1 had no effect on F-actinlevels or distribution (Fig. 5B). Quantification of the intensity ofF-actin fluorescence is shown in Fig. 5C. Although K104A-MCP1also significantly increased F-actin intensity, the effect was alsolimited to the cell border (Fig. 5B). Compared with FL-MCP1,K104Stop-MCP1 more efficiently enhanced F-actin (Fig. 5C).These results further support the hypothesis that plasmin-mediatedtruncation is necessary for MCP1-induced BBB compromise.

Fig. 2. Truncated MCP1 compromises BBB integrity. (A)The indicatedform of MCP1 (100 nM) was added to the lower chamber of the BMEC-astrocyte co-culture system. TEER values were determined over time in thepresence of A2AP. Results are means±s.d. (n3). Ctr, control (saline). (B)Cellswere treated as in A. The leakage of Evans Blue across the co-culture systemwas determined spectrophotometrally after treatment with A2AP. Results aremeans±s.d. (n4). *P<0.05 (analyzed using one-way ANOVA followed byNewman–Keuls multiple comparison test) compared with results with FL-MCP1. (C)Infiltration of Evans Blue across the compromised BBB into brainparenchyma was assayed 12 hours after a single injection of MCP1 (1g).Mice injected with saline were used as control. Results [OD (optical density;absorbance) per g of body weight] are means+s.d. (n4). *P<0.05 (analyzedusing one-way ANOVA followed by Newman–Keuls multiple comparisontest) compared with results with FL-MCP1 without A2AP.

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Plasmin-truncated MCP1 induces phosphorylation of ERMproteinsIn an effort to understand how TJPs are redistributed during BBBcompromise, we focused on the ERM proteins, a family of highlyconserved proteins that serve as a linker between the plasmamembrane and the actin cytoskeleton (Louvet-Vallee, 2000). Theseproteins have two states: a dormant state (during which they arelocated in the cytoplasm) and an active state (during which theyare located just beneath the plasma membrane) (Louvet-Vallee,2000). The activation process involves post-translational andconformational changes. It has been shown that phosphorylationon a conserved threonine residue (Thr567 for ezrin, Thr564 forradixin and Thr558 for moesin) suppresses the intramolecularinteraction between the N-terminus and C-terminus of ERM

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proteins and promotes a subsequent conformational change(Bretscher et al., 1995; Louvet-Vallee, 2000; Pearson et al., 2000).This then results in the N-terminus interacting with membraneproteins [e.g. CD44 (Tsukita et al., 1994), CD43 (Yonemura et al.,1993) and ICAM-2 (Helander et al., 1996), ICAM-3 (Serrador etal., 1997)] and the C-terminus interacting with the actincytoskeleton (Matsui et al., 1998; Nakamura et al., 1995;Pestonjamasp et al., 1995; Turunen et al., 1994). Here, weinvestigated whether activation of ERM proteins is involved inMCP1-induced BBB disruption. Using a phosphorylation-specificantibody, we found that FL- and K104Stop-MCP1 significantlyenhanced the phosphorylation of ERM proteins over time (Fig. 6),suggesting that phosphorylation of ERM proteins is involved inthe MCP1-induced BBB compromise. In the presence of A2AP,

Fig. 3. Truncated MCP1 induces the redistribution of tight junction proteins. (A)HBMECs were treated with the indicated form of MCP1 (100 nM). Ctr,control (saline); WT, wild-type. At 15, 30, 60 and 120 minutes after treatment, the cells were fixed and immunostained for occludin and ZO-1. (B)Highermagnification of cells incubated with the MCP-1 variants and stained for occludin at the 120-minute timepoint. Scale bar: 15m. (C)Primary mouse BMECs wereexposed to the MCP1 variants (100 nM) for 2 hours. Then, the cells were fixed and immunostained for ZO-1 and occludin. Blue, DAPI staining.

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however, FL-MCP1 failed to induce phosphorylation of ERMproteins, suggesting that plasmin-mediated truncation of MCP1plays a crucial role.

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Plasmin-truncated MCP1 promotes interaction betweenphosphorylated ERM proteins and ZO-1As K104Stop-MCP1 induced phosphorylation of ERM proteins,we next asked whether the N-termini of ERM proteins interactwith TJPs. By performing co-immunoprecipitation experimentswe found that, in the absence of MCP1, phosphorylated ERMproteins did not interact with ZO-1 (Fig. 7). Treatment ofendothelial cells with K104Stop-MCP1 for 1 hour, however,promoted interaction between phosphorylated ERM proteins andZO-1 (Fig. 7). By contrast, occludin failed to interact withphosphorylated ERM proteins, suggesting that the redistribution ofoccludin is not regulated through ERM proteins.

DiscussionThe BBB is a unique structure that separates the CNS from theperipheral nervous system. It mainly comprises BMECs, pericytes

Fig. 4. Truncated MCP1 shifts TJPs from the Triton-X-100-solublefraction to the Triton-X-100-insoluble fraction. HBMECs were exposed tothe indicated form of MCP1 (100 nM) for different periods of time in thepresence or absence of A2AP. Ctr, control (saline). The shift of occludin andZO-1 from the Triton-X-100-soluble fraction (S) to the Triton-X-100-insolublefraction (I) was analyzed by western blotting. Actin was used a loadingcontrol. (A)Representative images of western blots. (B)Quantitative data fromwestern blots. The intensity of target bands was determined using ScionImage. The intensity of occludin and ZO-1 was normalized to that of actin andthe ratios were further normalized to the zero timepoint. Results aremeans±s.d. (n3). **P<0.01 (analyzed using one-way ANOVA followed byNewman–Keuls multiple comparison test) for K104Stop-MCP1 comparedwith FL-MCP1 at each timepoint.

Fig. 5. Truncated MCP1 promotes reorganization of the actincytoskeleton. (A)HBMECs were treated with saline (Ctr) or the indicatedform of MCP1 (100 nM) for different times, and the actin cytoskeleton wasvisualized using Alexa-Fluor-647–phalloidin. (B)Primary mouse BMECstreated with the indicated form of MCP1 (100 nM) for 2 hours wereimmunostained to visualize the actin cytoskeleton. (C)Quantification ofF-actin fluorescence intensity. Values are mean+s.d. (n9). *P<0.05 (analyzedusing Student’s t-tests).

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and astrocytes (Guillemin and Brew, 2004); the BMECs, whichconnect to each other through TJPs, forming the primary barrier,whereas astrocytes, which cover more than 99% of the microvesselswith their endfeet, confer a secondary barrier (Kacem et al., 1998;Simard et al., 2003). Here, we used two different in vitro BBBmodels to assess the effect of plasmin on MCP1-induced BBBcompromise. In both models, we found that the constitutivelyactive K104Stop-MCP1 was functional in increasing BBBpermeability, suggesting that the activation of MCP1 by plasmin isindispensable for MCP1-induced BBB compromise.

Although the in vitro systems replicate some of the anatomicalstructures of BBB, they lack the shear stress, which plays animportant role in maintaining the dynamic properties and integrityof BBB. Hence, we also performed the Evans Blue permeabilityassay in vivo (in wild-type and MCP1–/– mice) and obtained similarresults to those described above for the in vitro model. However,the effect was more potent in MCP1–/– mice than in wild-typemice. It has been shown that MCP1 binds to and reduces membrane

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CCR2 levels (probably through endocytosis) (Jung et al., 2009;Yao and Tsirka, 2010). The dramatic change observed in MCP1–/–

mice could be due to higher membrane CCR2 density, as in theabsence of MCP1 there would be no recycling of CCR2 off theplasma membrane. To examine whether plasmin activity isindispensable, we also conducted experiments in mice deficient fortPA, which converts plasminogen into active plasmin. As expected,only K104Stop-MCP1 was functional in tPA–/– mice. Ccr2–/– micewere used as negative controls. Consistent with previous reportsthat Ccr2–/– mice were resistant to MCP1-induced BBBcompromise, none of the recombinant MCP1 proteins disruptedBBB integrity in these mice, suggesting that MCP1 exerts itsBBB-compromising effect solely through CCR2.

Stamatovic et al. showed that loss of CCR2 on astrocytes didnot affect MCP1-induced BBB disruption in the BMEC-astrocyteco-culture system, whereas loss of CCR2 on BMECs significantlydecreased BBB permeability (Stamatovic et al., 2005), suggestingthat MCP1 exerts its function by binding to CCR2 on BMECs. To

Fig. 6. Truncated MCP1 induces phosphorylation ofERM proteins. Mouse BMECs were treated with saline(Ctr, control) or the indicated form of recombinant MCP1proteins (100 nM) with or without A2AP over time. Thephosphorylated ERM proteins (p-ERM) were determinedusing a phosphorylation-specific antibody for ERMproteins. The level of total ERM (t-ERM) proteins is alsoshown. Quantitative data of western blots are shown asmean+s.d. (n3). *P<0.05 (compared with the zerotimepoint control).

Fig. 7. Truncated MCP1 promotes interactionbetween phosphorylated ERM proteins and ZO-1.Mouse BMECs were treated with K104Stop-MCP1 orsaline (0 h) for 1 hour. Cell lysates were collected andused for co-immunoprecipitation experiments.(A)Immunoprecipitation (IP) with an anti-(phosphorylated ERM) antibody (p-ERM) andimmunoblotting (IB) with anti-ZO-1 and anti-occludinantibodies. (B)Immunoprecipitation with an anti-ZO-1antibody and immunoblotting with anti-(phosphorylatedERM) and anti-occludin antibodies.(C)Immunoprecipitation with an anti-occludin antibodyand immunoblotting with anti-(phosphorylated ERM)and anti-ZO-1 antibodies. Anti-(mouse IgG) antibodywas used as control (Ctr).

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maintain the stability of the microenvironment in the brain, BMECsconnect to each other through tight junctions, where a lot ofadhesion molecules, such as the TJPs, bind to each other, sealingthe intercellular gaps. This structure makes the permeability of thebarrier highly dependent on the expression and localization ofTJPs. Our immunofluorescence data showed that plasmin-truncatedMCP1 disrupted occludin and ZO-1 staining in the borders ofHBMECs, suggesting a disruption of tight junction structure. Astight junction disassembly involves formation of large proteincomplexes and increased association of TJPs with the actincytoskeleton, which is Triton-X-100 insoluble, we utilized detergentextractability to analyze the integrity of BBB biochemically.Semi-quantitative western blotting revealed a shift of occludin andZO-1 from the Triton-X-100-soluble fraction to the Triton-X-100-insoluble fraction. Although TJPs are connected to the actincytoskeleton directly or indirectly, they are not normally affixed toit, which allows them to be extracted mostly in the Triton-X-100-soluble fraction. The translocation from the Triton-X-100-solublefraction to the Triton-X-100-insoluble fraction supports thecompromise of tight junction structures, suggesting that truncatedMCP1 induces BBB disruption through redistribution of TJPs.Consistently, we found that K104Stop-MCP1 significantlyenhanced stress fiber formation in endothelial cells, suggestingreorganization of actin skeleton. These data suggest that uponMCP-1 binding to endothelial CCR2, a property that all MCP-1mutants that retain the N-terminal portion of the protein exhibit, anincrease in cortical actin is observed. However, the next step of theprocess, the internalization of CCR2 and re-arrangements of actincytoskeleton to allow the cells to retract/reorganize the TJPs, istaking place only when the active FL- and K104Stop-MCP-1proteins are present (Yao and Tsirka, 2010).

Although the redistribution of TJPs, which leads to theformation of gaps between endothelial cells, and the reorganizationof actin cytoskeleton, which promotes the formation of contractileforces, have been shown to cause the compromise of BBBintegrity, the mechanisms underlying the shift of TJPs are notclear. It is thought that a phosphorylation event contributes to thetranslocation of TJPs (Clarke et al., 2000; Farshori and Kachar,1999; Hirase et al., 2001; Stamatovic et al., 2006; Stamatovic etal., 2003; Ward et al., 2002). The kinases responsible for thephosphorylation involve members of PKC family and Rho-associated kinase (ROCK) (Stamatovic et al., 2006; Stamatovic etal., 2003), although it is not clear how phosphorylation of TJPsleads to their subcellular redistribution. MCP1 has been shown toactivate ROCK through RhoA in endothelial cells (Stamatovic etal., 2006). One of the substrates for ROCK is myosin light chainphosphatase (MLCP), which functions in opposition to myosinlight chain kinase (MLCK) (Hicks et al., 2010) [MLCKphosphorylates myosin light chain (MLC) and promotes actin–myosin contraction, resulting in contractile forces (Hicks et al.,2010; Stamatovic et al., 2003; Stephan and Brock, 1996; vanNieuw Amerongen et al., 2000)]. Our data clearly show that thepresence of MCP1 leads to increased phosphorylation of ERMproteins, which subsequently bind to ZO-1 and the actincytoskeleton. Our model (Fig. 8) is that by binding to CCR2,MCP1 activates intracellular kinases, which inducephosphorylation of ERM proteins, leading to their conformationchange. The phosphorylated ERM proteins then bind to ZO-1 andthe actin cytoskeleton. In addition, MCP1 also activates RhoA,which further activates ROCK (Stamatovic et al., 2006). ROCKis then expected to phosphorylate the regulatory subunits of MLCP,

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leading to inhibition of phosphatase activity. The effect of MLCKwould outweigh that of MLCP, resulting in increasedphosphorylation of MLC, which would cause enhanced andprolonged actin–myosin contraction. The contraction generatescontractile forces and pulls ZO-1 away from TJ, leading todisruption of BBB integrity. It should be noted, however, that thephosphorylated ERM proteins do not interact with occludin uponMCP1 treatment, suggesting the translocation of occludin is notmediated by ERM proteins. Whether there is another ERM-likeprotein that mediates shift of occludin or if the redistribution ofoccludin is mediated by other mechanisms is unclear.

Besides MCP1, many other cytokines, including interleukin(IL)-1, IL-4, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)-aand interferon (IFN)- (Ahdieh et al., 2001; Blamire et al., 2000;Coyne et al., 2002; Paul et al., 2003; Ross and Joyner, 1997;Wojciak-Stothard et al., 1998; Yang et al., 1999; Youakim andAhdieh, 1999), have been shown to change the structure of tightjunctions and promote the formation of stress fibers in epithelialand endothelial cells. Whether these molecules induce BBBcompromise using the same signaling pathways is not clear;however, on the basis of the conserved function and high expressionlevel of ERM proteins in epithelial and endothelial cells, we wouldassume the same signaling pathways are activated upon the cytokinetreatment.

Our work suggests that plasmin-mediated truncation of MCP1is indispensable for MCP1-induced BBB compromise. Moreover,we have shown that BBB compromise involves reorganization ofthe actin cytoskeleton and redistribution of TJPs, with theredistribution of ZO-1 mediated by ERM proteins. However, whichkinase(s) phosphorylates ERM proteins and what mediates theredistribution of occludin need further investigation.

Materials and MethodsAnimal experimentationAll the mice used were bred in the Division of Laboratory Animal Resources(DLAR) at Stony Brook University. They had free access to food and water, andwere maintained in a 12-hour light–12-hour dark cycle. All the procedures wereapproved by the Institutional Animal Care and Use Committee (IACUC).

Fig. 8. Proposed model for MCP1-induced BBB compromise. Upon MCP1treatment, ERM proteins are phosphorylated by unknown kinases. Thephosphorylated ERM proteins then bind to ZO-1 and the actin cytoskeleton.By binding to CCR2, MCP1 also activates RhoA, and Rho-associated kinases,which phosphorylate and inactivate MLCP, leading to enhancedphosphorylation of MLC and thus prolonged actin–myosin contraction. Thecontractile forces then pull ZO-1 away from the cell-cell border, resulting in adisrupted BBB.

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1493Truncated MCP1 disrupts the blood–brain barrier

Generation of His6-tagged MCP1 proteinsFL-, K104A-, and K104Stop-MCP1 were subcloned without the signal peptideinto pET vectors with an N-terminal His6 tag (Sheehan et al., 2007). CT-MCP1was subcloned into a pTYB1 vector with an N-terminal His6 tag (Yao and Tsirka,2010). BL21 cells were transformed with these constructs and expression of thetarget proteins was induced for 5 hours using isopropyl-b-D-thiogalactopyranoside.Recombinant proteins were purified using a cobalt affinity resin (Clontech)according to the manufacturer’s instructions. The column fractions were analyzedby Tris-Tricine SDS-PAGE (16% gels) by Coomassie Blue staining. The purifiedproteins were confirmed by immunoblotting using 1:1000 anti-MCP1 antibodies(Serotec and Cell Sciences) or 1:1000 anti-His6 antibody (Santa CruzBiotechnology).

Cell cultureHuman BMECs (HBMECs) were kindly provided by Monique Stins (Johns HopkinsUniversity, Baltimore, MD). The cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM), supplemented with 10% fetal bovine serum (FBS), 10% NuSerum,1� sodium pyruvate, 1� nonessential amino acid, 1� vitamin mixtures, 15 units/mlheparin, 30 g/ml endothelial cell growth supplement (ECGS), 100 units/ml penicillinand 100 g/ml streptomycin, at 37°C under a 5% CO2 atmosphere. Mouse BMEC(CRL2299) and mouse astrocyte (CRL2541) cell lines were purchased from theATCC and cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillinand 100 g/ml streptomycin at 37°C under a 5% CO2 atmosphere.

Primary mouse BMECs were isolated and cultured as previously described withminor modifications (Calabria et al., 2006; Deli et al., 2003). Briefly, brains wereremoved and cut into two hemispheres. The meninges were removed by rolling thehemispheres on sterile Whatman chromatography paper. The cortices were mincedand triturated, followed by collagenase (0.7 mg/ml) and DNase I (39 units/ml)digestion at 37°C for 1 hour. Then the solution was diluted with DMEM andcentrifuged at 1000 g for 8 minutes at 4°C. The pellet was resuspended in 20% BSAand centrifuged at 1000 g for 20 minutes at 4°C. The pellet was further digested withcollagenase and dispase (1 mg/ml), and DNase I (39 units/ml) at 37°C for 1 hour.Then the solution was diluted with DMEM and centrifuged at 700 g at 4°C for 7minutes. The pellet was re-suspended and layered over a 33% continuous Percollgradient and centrifuged in a JA-20 rotor at 3000 rev. per minute for 10 minutes at4°C. The microvessel layer was collected using an 18-gauge needle and diluted intoDMEM. After centrifuging at 700 g for 10 minutes at 4°C, the pellet was re-suspended in complete culture medium [DMEM supplemented with 20% bovineplatelet-poor plasma-derived serum (Biomedical Technologies), 100 g/ml heparin,1 ng/ml basic fibroblast growth factor, 4 g/ml puromycin, 100 units/ml penicillinand 100 g/ml streptomycin] and plated on type IV collagen (400 g/ml) andfibronectin (100 g/ml) pre-coated plates. The culture medium was changed every24 hours after initial plating. From day three after the initial plating, complete culturemedium without puromycin was used.

Cytotoxicity assayCytotoxicity was performed using the LDH Detection Kit (Roche) according to themanufacturer’s instructions. Briefly, mouse BMECs or HBMECs were seeded into96-well plates at a density of 1�104 cells per well and maintained in DMEM with1% FBS. The next day, the medium was replaced with fresh DMEM plus 1% FBSand the cells were treated with 100 nM mouse FL-, K104A-, K104Stop-, CT-, orhuman MCP1 overnight. The medium was collected and used to determine LDHlevels. Cells treated with 2% Triton X-100 were used as control to determine themaximal LDH release. The data were converted into a percentage of the maximalLDH release.

Transendothelial electrical resistance assayFor the simple BBB model, HBMECs were plated in the upper chamber of 0.4 mTranswell inserts. For the co-culture system, mouse astrocytes were plated onto thelower side of the 0.4 m Transwell inserts. After 4 hours, the inserts were invertedand mouse BMECs were seeded in the upper chamber, as described previously(Dimitrijevic et al., 2006; Stamatovic et al., 2005). When the cells reached confluence,100 nM FL-, K104A-, K104Stop- or CT-MCP1 was added to the lower chambers.After different periods of time, TEER values were measured using the EVOMEpithelial Volt/Ohm meter (World Precision Instruments). The resistance of emptyinserts were also measured and subtracted for calculation of final TEER values(/cm2).

In vitro permeability assayThe permeability of BBB was assessed in vitro, as described previously (Eugenin,2006). Briefly, HBMECs or the co-culture system was prepared as described above.Sterile Evans Blue or FITC–Dextran was added into the upper chamber and 100 nMFL-, K104A-, K104Stop- and CT-MCP1 were added to the lower chambers. Atdifferent timepoints, 100-l samples from the lower chamber were collected and 100l of fresh medium with 100 nM MCP1 was added back to the lower chamber. Thecollected samples were read at 620 nm (for Evans Blue dye) or 488 nm (for FITC-Dextran) to quantify the concentrations of infiltrated tracers. Empty inserts wereused to measure the maximal absorbance reading, which signifies the complete

disruption of the BBB. The absorbance values were converted to percentage ofpermeability (%) with respect to the maximal absorbance reading.

In vivo permeability assayIn vivo permeability assays were performed as described previously with minormodifications (Ohno et al., 1980; Parathath et al., 2006; Wu and Tsirka, 2009; Yepeset al., 2003). Briefly, wild-type (C57BL6, wt), MCP1–/–, Ccr2–/–, and tPA–/– micewere anesthetized by injecting atropine (0.6 g per g of body weight) and avertin(0.02 ml per g of body weight) intraperitoneally. A burr hole was then drilled forhippocampal injection at stereotaxic coordinates of –2.5 mm posterior to bregma, 1.7mm lateral from midline and 1.6 mm in depth. Then, 1 g of FL-, K104A-,K104Stop- or CT-MCP1 was delivered in 2.5 l of saline over 2 minutes. The needlewas kept in place for 2 minutes to prevent reflux. A total of 2.5 l of saline wasinjected as a control. Evans Blue (2%) was injected into the systematic circulationvia the eye. At 12 hours after the MCP1 injection, the mice were anesthetized again,followed by transcardiac perfusion with PBS to remove the blood from the vessels.Brains were removed, divided into ipsilateral (ipsi) and contralateral (cont)hemispheres and weighed (W). Each hemisphere was homogenized in PBSsupplemented with 0.5% Triton X-100, and centrifuged at 21,000 g. Evans Blue inthe supernatant was calculated as follows: Evans BlueA620 ipsi/Wipsi–A620 cont/Wcont.

Immunofluorescent stainingHBMECs or primary BMECs were plated onto coverslips and treated with MCP1as above. After fixation, in –20°C methanol for 10 minutes or in 4% paraformaldehydefor 20 minute, the cells were incubated overnight with mouse anti-occludin antibody(1:300; Zymed) (Sugimoto et al., 2008) and rabbit anti-ZO-1 antibody (1:300;Zymed) followed by fluorescent anti-(mouse Ig) or anti-(rabbit Ig) antibodies (1:1000;Invitrogen). Next, the cells were washed and mounted on slides using Fluoromountwith DAPI. For phalloidin staining, the cells were fixed with 4% paraformaldehydeand stained with 1:30 Alexa-Fluor-647–phalloidin overnight at 4°C. The stainedsamples were examined using confocal microscopy (Zeiss LSM510).

Triton X-100 fractionationTriton X-100 fractionation was performed as described previously with minormodifications (Fey et al., 1984; Stamatovic et al., 2006; Stamatovic et al., 2003;Stamatovic et al., 2005). Briefly, extracting buffer (10 mM Tris-HCl pH 7.4, 100mM NaCl, 300 mM sucrose, 0.5% Triton X-100 and protease inhibitor cocktail) wasadded on top of a confluent monolayer of HBMECs. Extraction was performed bygently rocking at 4°C for 20 minutes. Collected supernatants were defined as theTriton-X-100-soluble fraction. The residue of cells, which still adheres to the culturedishes, was washed twice with PBS supplemented with protease inhibitor cocktailand lysed with radioimmunoprecipitation assay buffer (10 mM Tris-HCl pH 7.4, 140mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100 and proteaseinhibitor cocktail). Collected supernatants were defined as the Triton-X-100-insolublefraction. Both Triton-X-100-soluble and -insoluble fractions were prepared by adding4� SDS sample loading buffer and heating at 95°C for 10 minutes.

Western blottingThe samples were resolved using SDS-PAGE and transferred onto PVDF membranes.Target proteins were visualized using mouse anti-occludin, rabbit anti-ZO-1 andrabbit anti-actin antibodies (all at 1:1000), and 1:500 rabbit anti-(phosphorylatedERM) antibody (Santa Cruz Biotechnology), and the densities of the bands weremeasured using the NIH Image 1.63 software or the LI-COR Odyssey program.

Co-immunoprecipitationCells treated with or without K104Stop-MCP1 were lysed with RIPA buffer. Thelysates were incubated with anti-(phosphorylated ERM) antibody, anti-ZO-1 antibodyor anti-occludin antibody for 2 hours at 4°C. Then, 25 l of protein A- and G-conjugated agarose beads (Santa Cruz Biotechnology) was added to pull down theimmunocomplexes. After incubating at 4°C for 2 hours, the beads were collected bycentrifugation and washed four times with PBS. After last wash, 20 l of 2� sampleloading buffer was added and the resulting protein complexes were immunoblottedwith the antibodies and visualized using the LI-COR Odyssey scanner. Total Racwas determined in a similar manner, using cell lysates.

StatisticsResults are shown as means±s.d. One-way ANOVA followed by the Newman–Keulsmultiple comparison test was used to analyze differences between two groups.

We thank the members of the Tsirka laboratory for helpfuldiscussions and members of the Frohman laboratory for reagents. Thiswork was partially supported by a SigmaXi grant-in-aid (to Y.Y.) andNIH R0142168 (to S.E.T.). Deposited in PMC for release after 12months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/9/1486/DC1

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