absence of the chemokine receptor ccr2 protects...

Absence of the Chemokine Receptor CCR2 Protects AgainstCerebral Ischemia/Reperfusion Injury in Mice

Oliver B. Dimitrijevic, MD; Svetlana M. Stamatovic, MD, PhD; Richard F. Keep, PhD;Anuska V. Andjelkovic, MD, PhD

Background and Purpose—The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor drivingleukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation,including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important rolein regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulatingpostischemic inflammation, BBB breakdown, and vasogenic edema formation.

Methods—CCR2�/� and CCR2�/� mice were subjected to focal transient cerebral ischemia. BBB permeability and brainedema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluoresceinisothiocyanate–albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assessleukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatoryprofiles in CCR2�/� and CCR2�/� mice.

Results—CCR2�/� mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formationin the affected ischemic hemisphere compared with CCR2�/� mice. This reduction in injury was closely associated withreduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition,CCR2�/� mice had reduced expression/production of inflammatory cytokines during reperfusion.

Conclusions—These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brainedema, leukocyte infiltration, and inflammatory mediator expression. (Stroke. 2007;38:1345-1353.)

Key Words: blood-brain barrier permeability � CCL2 � chemokines � inflammation � stroke

Postischemic inflammation is considered a significantcontributor to ischemic brain injury.1–3 The central event

in postischemic inflammation is recruitment of leukocytes,first neutrophils, and then an influx of cells of the monocyte/macrophage lineage.2,3 This is a multifactorial process in-volving chemotactic signals that promote directed migrationof leukocytes, adhesion receptor/ligand interaction at themicrovascular endothelial surface, and matrix metalloprotein-ase production needed for breakdown of the extracellularmatrix and leukocyte extravasation.2,4 The presence of de-structive leukocytes in the brain parenchyma can enhanceischemic injury, but they also further amplify the inflamma-tory response. Therefore, a critical event to control postische-mic injury is regulating leukocyte entry. Crucial mediators ofthese events are the chemokines, multifunctional mediators ofcellular communication in the ischemic brain and key mole-cules in leukocyte recruitment.

Chemokines (chemoattractant cytokines) are a superfamilyof structurally related proinflammatory peptides that mediatecell-specific, directed migration of leukocytes into sites ofinflammation.5 There are 4 different subfamilies (CC, CXC,

CX3C, and C) based on biochemical, structural, and func-tional differences.6 During ischemia/reperfusion, the brainproduces chemokines such as CXCL8 (also known as inter-leukin [IL]-8), CCL2 (MCP-1), CCL3 (MIP-1�), CCL4(MIP-1�), CCL7 (MCP-3), CX3CL1 (fractalkine), andCXCL10.7–14 These contribute to neutrophil and monocyterecruitment into ischemic tissue. Some chemokines, such asIL-8 (or its murine closest equivalent, CXCL5), CCL2, orfractalkine, are critical factors regulating postischemic in-flammation and inducing not only leukocyte recruitment butalso blood-brain barrier (BBB) disruption and leukocyteadhesion to brain endothelial cells.7,8,13 These chemokines arefound in the ischemic penumbra as early as 6 hours ofreperfusion, with peak expression within 24 to 48 hours andlasting up to 5 days.7–14 Chemokine expression is closelycorrelated with infarct size and the progression of ischemiclesions. Manipulation of chemokine expression/activity af-fects ischemic brain injury. Thus, deletion of the CCL2 orCX3CL1 gene in mice or treatment of rabbits with a neutral-izing CXC8 antibody during reperfusion significantly atten-uates infarct size and decreases the inflammatory response

Received August 3, 2006; final revision received October 25, 2006; accepted November 1, 2006.From the Departments of Pathology (O.B.D., S.M.S., A.V.A.), Neurosurgery (R.F.K., A.V.A.), and Integrative and Molecular Physiology (R.F.K.),

University of Michigan, Medical School, Ann Arbor.Correspondence to Anuska V. Andjelkovic, Department of Pathology, University of Michigan, 7520 MSRB I, Ann Arbor, MI 48109-0532. E-mail

[email protected]© 2007 American Heart Association, Inc.

Stroke is available at http://www.strokeaha.org DOI: 10.1161/01.STR.0000259709.16654.8f

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and brain edema formation.7,8,15 Chemokines may, therefore,be a target in stroke therapy.

The present study assessed the issue of how excluding theactivity of 1 widely expressed chemokine, CCL2, affectsBBB permeability and inflammation development duringcentral nervous system ischemia/reperfusion injury in mice.Owing to the fact that CCL2 exerts its activity via binding toCCR2 receptors, inhibition of CCL2 activity by deletingCCR2 in our experiments offers insight into new prospectivestroke therapies wherein CCR2 antagonists might be used toreduce brain injury.

Materials and MethodsAll procedures were performed in strict accordance with the NationalInstitute of Health’s Guide for the Care and Use of LaboratoryAnimals and were approved by the institutional animal care and usecommittee of the University of Michigan.

Middle Cerebral Artery OcclusionExperiments were performed on male CCR2�/� and CCR2�/� mice(22 to 25 g) on a C57BL/6�129Sv background. CCR2 knockout(KO, �/�) mice were crossed with wild-type (�/�) CCR2 mice toproduce heterozygous (�) CCR2 mice. These were crossbred toproduce additional CCR2 KO mice. This backcrossing was repeatedfor at least 10 generations to ensure that the generated CCR2 KOmice were almost completely on the same C57BL/6�129Sv back-ground as the wild-type mice. All breeding and husbandry of animalswere undertaken under specific pathogen-free conditions at theUniversity of Michigan. Genotypes were determined by polymerasechain reaction on tail biopsy specimens with the use of CCR2 primerpairs (SuperArray Bioscience Co, Inc).

Mice were anesthetized by injection of ketamine (100 mg/kg IP)and xylazine (10 mg/kg IP). Body temperature was maintained at37�0.5°C by means of a heating blanket and a heating lamp duringthe entire experimental procedure. Focal cerebral ischemia wasinduced by left middle cerebral artery occlusion (MCAO) with anintraluminal filament technique. In brief, the right common carotidartery was exposed through a midline incision in the neck. Next, a6–0 silicone suture was introduced into the external carotid arteryand advanced into the internal carotid artery for a distance of 10 to11 mm from the common carotid artery bifurcation, according toanimal weight. MCAO was confirmed by a laser Doppler flow probe(model BPM System, Vasomedics) positioned 3 mm posterior and5 mm lateral to the bregma. After 30 minutes of MCAO, the micewere reperfused by suture withdrawal and then allowed to awakenfrom anesthesia. Sham-operated animals underwent all proceduresexcept MCAO.

Reperfusion periods were 1 to 5 days. During reperfusion,neurologic deficits were evaluated with the following scoringscheme: 0, no visible neurologic deficits; 1, forelimb flexion; 2,contralateral forelimb grips weakly (the operator places the animalon an absorbent pad and gently pulls the tail); 3, circling to theparetic side only when pulled by the tail (the animal was allowed tomove about freely on the absorbent pad); and 4, spontaneouscircling.

Brain Water Content and ElectrolytesBrain water content was measured by the wet/dry weight method.Samples were taken from ischemic and nonischemic hemispheres.After decapitation under deep isoflurane anesthesia, brains wereweighed wet and then oven-dried at 100°C for 48 hours andreweighed. Brain water content (%) was calculated as (wetweight�dry weight)/wet weight�100. After the tissue was dried,electrolytes were extracted with nitric acid, and sodium and potas-sium contents were measured by flame photometry (Corning Corp).

Morphometric Measurement of Infarct VolumeAnimals were humanely killed 1 and 5 days hours after transientMCAO, and the brain was removed and sliced. Slices of brain tissuewere incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC)solution for 1 hour at 37°C. The area of infarction in each slice wasdetermined by a computerized image analysis system, and thevolume of infarction was calculated by multiplying the distancebetween sections. In addition, to account for cerebral edema orresolution of the infarct, an indirect measurement of infarction wasperformed. Infarct volume was calculated as [contralateral hemi-sphere volume�(ipsilateral hemisphere volume�measured injuryvolume)].16 Cresyl violet staining of 200-�m-thick serial sectionswas also used to examine infarct size after 5 days of reperfusion.

In Vivo Permeability AssayBBB integrity in mice was assessed by measurement of the bloodbrain transfer coefficient (Ki) for fluorescein isothiocyanate (FITC)–albumin with a method based on a previously described proce-dure.17,18 In brief, FITC-albumin was injected as a bolus into afemoral vein 20 minutes before the end of the experiment. Serialarterial blood samples were taken from a femoral artery every 5minutes from 0 to 20 minutes to determine the FITC-albumin plasmaprofile. At the end of the experiment, the mice were killed bydecapitation. Brains were rapidly removed and dissected into rightand left hemispheres. These were weighed and homogenized in50 mmol/L Tris buffer solution (pH 7.4). The homogenates werecentrifuged at 3000 rpm for 30 minutes and the supernatant wascollected. Methanol was added to the collected supernatant (1:1,vol/vol), and the mixture was centrifuged again under the sameconditions. The fluorescence intensity of the supernatant as well asthat of the plasma samples was measured with a fluorescence reader(Bio-Tek Instruments, Inc; emission 485 nm and excitation 540 nm),and concentration was calculated by using a standard curve.19 The Ki

for FITC-albumin was determined according to the following equa-tion developed by Ohno and colleagues17:

Ki ��Cbr�VOCbl�

�Cpl � dt

where Cbr is the concentration of FITC-albumin in brain tissueat the time of decapitation (ng/g), Cbl is the concentration of

Physiologic Variables in the Experimental Mouse Groups

Variables CCR2�/� CCR2�/�

pH

Before 7.34�0.036 7.34�0.023

After 7.26�0.045 7.28�0.13

PCO2

Before 34.4�2.9 34.5�2.5

After 39�4.1 38.4�1.9

PO2

Before 124.05�7 118.45�8.1

After 120.25�3.2 116.05�2.6

Blood glucose, mg/dL

Before 137.5�10.6 134.5�6.36

After 127�14.24 122.5�5.65

rCBF, %

Before 100�0 100�0

During 18�3 17�1

After 91�2 91�3

rCBF indicates regional cerebral blood flow. Other abbreviations are asdefined in text.

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FITC-albumin (ng/mL) in the last blood sample, Vo isregional blood volume (mL/g), and � Cpl � dt is the integral ofthe arterial concentration of FITC-albumin over time t. Vo wasdetermined in a second set of animals in which mice weredecapitated 1 minute after intravenous injection of FITC-al-bumin. If one assumes no extravasation during this shortcirculation time, Vo�Cbr/Cbl. Vo, � Cpl, and Cbl were deter-mined for CCR2�/� and CCR2�/� mice separately, but therewere no significant differences in these values between the 2groups.

ImmunohistochemistryBrain samples were fixed in 4% paraformaldehyde for 18 hours,cryoprotected with sequential immersions in 10% and 20% sucrosesolutions, and then cut into 50-�m-thick coronal sections with afreezing microtome. Afterward, the samples were preincubated inblocking solution (5% bovine serum albumin, 5% normal goatserum, 0.05% Tween, and phosphate-buffered saline) and thenincubated overnight with the primary antibody, rat anti-mouse Ly6G

(BD Bioscience) and anti-myeloperoxidase antibody (HyCult Bio-technology) at 4°C. Reaction was visualized by Texas red–conju-gated anti-rat (Sigma-Aldrich) or anti-rabbit (Vector Laboratories)antibody. All samples were viewed on a confocal microscope (LSM510, Zeiss). For quantification, 50 coronal brain slices (25 slices infront and 25 behind the “middle line” of the visible lesion).Microscope data were acquired with a 10� objective with constantlaser power (45% of laser power), pinhole, zoom, focus, gain, andduration of image capture. A total of 20 images were randomlyselected and captured per slide. The immunolabeled cells werecounted in areas surrounding the ischemic lesion. Five mouse brainsper group were analyzed. In the sham-operated group, brain areaswere analyzed corresponding to those analyzed in ischemic mice.Slides were coded so that the counter was blinded to the identity ofthe slides being counted.

cDNA ArrayBrain was sampled from the area around the ischemic lesion with a“pinch-out” method, whereby the visible ischemic lesion was re-moved and the surrounding area (1 mm thick) was collected. Thecorresponding contralateral region was also collected. Total RNA

Figure 1. CCR2�/� mice have significantly reduced brain infarcts, A, Coronal section illustrating typical infarcts (arrows) in CCR2�/� andCCR2�/� mouse brains stained with TTC and cresyl violet (CV) at 5 days of reperfusion. B, Bar graph showing infarct volumes at days 1and 5 after transient MCAO in CCR2�/� (n�9) and CCR2�/� (n�10) mice. Values are means�SD. & indicates P0.001. C, Indirect mea-sure (to correct for edema/infarct resolution) of total infarct volume and (D) cortical and striatal infarct volumes at days 1 and 5 aftertransient MCAO in CCR2�/� and CCR2�/� mice. E, Summary of neurologic scores in CCR2�/� and CCR2�/� mice at days 1 and 5 aftertransient MCAO in CCR2�/� (n�13) and CCR2�/� (n�13) mice. #P0.01 by �2 test.

Dimitrijevic et al CCL2/CCR2 Axis in Brain Ischemia/Reperfusion Injury 1347


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was prepared with Trizol (Invitrogen). Biotinylated cDNA probeswere synthesized with an Ampho-LPR labeling kit (SuperArray).After labeling, the resulting cDNA probe was denaturated andhybridized to the GEArray Q Series mouse inflammatory cytokinegene array (SuperArray). The hybridized membrane was incubatedwith a chemiluminescent substrate and exposed to x-ray film. Theimages were scanned and the relative expression level of each genewas analyzed with software provided by SuperArray Bioscience. Inbrief, the relative value of every gene was obtained by subtractingthe background and comparing with the positive control gene. Brainsamples, with 5 animals per group, were analyzed. In addition,reverse transcription–polymerase chain reaction was performed forselected genes (CXCL5, CCL2, IL-1�, tumor necrosis factor [TNF]-�, and IL-13). All primer sets were purchased from SuperArray.

Cytokine Antibody ArrayA mouse cytokine antibody array (Raybiotech Inc) was used tosimultaneously detect and quantify 22 cytokines in samples collectedfrom the ischemic lesion of the ipsilateral hemisphere and from thecorresponding contralateral side. Tissue samples were homogenizedin 1.8 mL Tris buffer solution (pH 8.5) supplemented with TritionX-100 at a final concentration of 1% and stirred for 12 hours at 4°C.Samples were centrifuged at 100 000g for 60 minutes at 4°C toremove cell debris. The supernatant was collected and protein levelswere evaluated with a bicinchoninic acid protein assay (Pierce).Total protein level was adjusted for every sample to 2 �g/mL. Thecytokine antibody array was performed according to the manufac-turer’s instructions. Brain samples from 5 mice per experimentalgroup (MCAO operated and sham control CCR2�/� and CCR2�/�

mice) were analyzed.

Statistical AnalysisAll values are expressed as mean�SD. One-way ANOVA was used,followed by Bonferroni post hoc analysis as well as a �2 test with theuse of Prism analysis software. P0.05 was regarded as statisticallysignificant.

ResultsPhysiologic parameters (pH, PO2, PCO2, glucose level, andregional cerebral blood flow) before MCAO and after 30minutes of reperfusion were not significantly different betweenwild-type CCR2�/� and CCR2�/� (KO) mice (Table). At 1 and5 days after reperfusion, the infarct volume in CCR2�/� micewas smaller than that in CCR2�/� animals (day 1: CCR2�/�

61�9 vs CCR2�/� 145�3 mm3; day 5: CCR2�/� 64�5 vsCCR2�/� 164�6; P0.001; Figure 1A through 1C). Similarstatistically significant differences were present by indirectmeasurement of infarct volume to correct for brain edema and/orinfarct resolution (P0.001, Figure 1C). There were no signif-icant differences in infarct size measured after TTC and cresylviolet staining at days 1 and 5 of reperfusion. Analyzing theregional distribution of the infarct, we found that in our allexperimental groups, infarct lesion was present in the cortex andstriatum. However, a reduced infarct volume in CCR2�/� micewas associated with parallel reductions in both structures (cortexand striatum) at days 1 and 5 of reperfusion (Figure 1D). The

Figure 2. Absence of CCR2�/� has a protective effect on BBB breakdown and brain edema formation after transient MCAO. Three pa-rameters were examined in CCR2�/� and CCR2�/� mice: (A) influx rate constant (Ki) for FITC-albumin, (B) water content, and (C and D)level of electrolytes (K� and Na�) in ischemic and nonischemic hemispheres at days 1 and 5 after transient MCAO. Values aremean�SD in CCR2�/� (n�7) and CCR2�/� (n�7) mice. *P0.0.05, #P0.01, and &P0.001.



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reduction in infarct size in CCR2�/� mice was associated withless severe neurologic impairment (Figure 1E). There were alsosignificant differences in mortality (P0.001 by �2). Four of 20CCR2�/� mice died before neurologic scoring, whereas all 20CCR2�/� mice survived.

To evaluate brain edema development, brain water, sodiumand potassium contents, and the BBB Ki for FITC-albuminwere measured. Transient MCAO in CCR2�/� mice led to anincrease in BBB permeability for FITC-albumin during reper-fusion (Figure 2A). The BBB disruption was much less inCCR2�/� mice (FITC-albumin Ki at day1 0.041�0.0007 vs0.158�0.017 �L�g�1�min�1 in CCR2�/� mice, P0.01; at day5: 0.034�0.015 vs 0.192�0.051 �L�g�1�min�1 in CCR2�/�

mice, P0.001, Figure 2A). This difference in BBB perme-ability was associated with reduced brain edema (Figure 2B)in the ischemic hemisphere in CCR2�/� mice compared withCCR2�/� mice at days 1 and 5. There were no significantdifferences in brain water content in the contralateral hemi-sphere. The decreased edema formation in CCR2�/� mice wasassociated with less sodium accumulation in the ischemichemisphere (Figure 2C) and reduced potassium loss (Figure2C). The data presented indicate that the absence of CCR2and the activity of its ligand CCL2 have protective effects in

BBB disruption and brain edema development associatedwith ischemic/reperfusion injury.

In CCR2�/� mice, there was a massive infiltration ofneutrophils (myeloperoxidase-positive cells) at days 1 and 5after MCAO. In addition, at day 5, monocytes were present inbrain parenchyma around the ischemic lesion. This pattern ofleukocyte infiltration was significantly changed in CCR2�/�

mice. Monocyte migration (CCL2 is a potent monocytechemoattractant) was markedly reduced, but also there wasmuch less neutrophil infiltration (Figure 3).

To address how blocking the CCL2/CCR2 axis couldhave such broad effects on the inflammatory response, cDNAgene arrays and protein microarrays were used to examine theischemic penumbra at days 1 and 5 of reperfusion. Resultsare shown in Figure 4. In CCR�/� mice, in a comparison of theexpression profile of proinflammatory cytokines and chemo-kines and their receptors at day 5 of reperfusion, there wassignificant mRNA overexpression of a variety of proinflamma-tory cytokines (eg, IL-1�, IL-1�, IL-6, IL-18, IL-17, andTNF-�; �2.5-fold increases) and CC chemokines (eg, CCL2,CCL22, CCL24, CCL4, CCL6, CCL7, CCL8, and CCL9;2-fold increases). On the other hand, at day 1, CCR2�/� micehad significant upregulation of the CXC chemokines (CXCL2,

Figure 3. Mice CCR2�/� mice have a reduced inflammatory response. Infiltration of neutrophils (MPO-positive) and monocytes (Ly-6G–positive) was significantly reduced in CCR2�/� compared with CCR2�/� mice. Representative images of neutrophil (PMN) and monocyte(Mo) infiltration in area around the ischemic lesion at day 5 of reperfusion in CCR2�/� and CCR2�/� mice. Graphs represent percent-ages of neutrophils and monocytes in the cell infiltrate around ischemic lesion. &P0.001. Scale bar�20 �m.



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CXCL5, CXCL9, and CXCL12 as well as CX3CL1). A similarpattern was found in protein expression. As shown in Figure 5,there was significant upregulation of proinflammatory cytokineslike IL6, IL-12, TNF-�, and the chemokine CC at day 5compared with day 1, whereas the CXC chemokine familyshowed increased activity at day 1 only.

This inflammatory response during reperfusion was signif-icantly reduced in CCR2�/� mice. Most proinflammatorycytokines and chemokines (and their receptors) were under-expressed in the CCR2�/� group at the level of mRNA andprotein. Exceptions were a group anti-inflammatory cyto-kines (IL-4, IL-5, and IL-13) whose transcript and protein

Figure 4. A, Inflammatory profile of CCR2�/� and CCR2�/� mice after MCAO. A, cDNA gene array analysis of inflammatory cytokinesand receptors expressed in area around ischemic lesion at days 1 and 5 after transient MCAO in CCR2�/� and CCR2�/� mice. Valuesin the table represent differences between CCR2�/� and CCR2�/� mice at days 1 and 5 of reperfusion. The data were analyzed by scat-terplot. nd� represents absent genes or genes with 1.5-fold differences between CCR2�/� and CCR2�/� mice. B, Reverse transcrip-tion–polymerase chain reaction for CCL2, CXCL5, IL-1�, TNF-�, and IL-13 at days 1 and 5 after transient MCAO in CCR2�/� andCCR2�/� mice. These results confirmed the pattern of gene expression found with the GeneArray.



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expressions were upregulated in CCR2�/� mice (2- to 3-foldincrease, P0.001). Thus, the absence of CCR2 has aprofound effect on the inflammatory response during reper-fusion as well as affecting BBB permeability and brain edemaformation.

DiscussionThe present study investigated how blocking the CCL2/CCR2 axis with a CCR2 KO affects ischemia/reperfusioninjury. Our results indicate that a lack of CCR2 greatlyreduces brain edema formation and BBB disruption, as wellas decreasing leukocyte infiltration. This study also showed

that CCR2�/� mice had decreased expression of a wide rangeof proinflammatory cytokines during reperfusion. The impli-cations of these findings are discussed next.

Brain edema is a leading complication in ischemia andischemia/reperfusion-induced brain injury.20 The mecha-nisms of brain edema formation after focal ischemia arecomplex. The classic, simplistic pathobiology of ischemia-evoked cerebral edema includes a cytotoxic component (sec-ondary to postischemic energy failure) and a vasogeniccomponent (secondary to breakdown of the BBB and extrav-asation of protein-rich plasma filtrate), although some othermechanisms, such as intrahemispheric diaschisis or hypome-

Figure 5. Protein levels for inflammatory media-tors (cytokines and their receptors, chemokines,and matrix metalloproteinases) analyzed inbrain tissue taken from around the ischemiclesion. The data were obtained by normalizingobtained values against positive controls pres-ent on the membrane and against those ofsham-operated mice.



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tabolism, inflammatory mediators, neurohumoral responses,vascular endothelial growth factor, and upregulation of waterchannels, are under intensive investigation.21–23 In general,cytotoxic edema occurs soon after cessation of blood flow,whereas vasogenic edema occurs later.

Proinflammatory mediators are considered potential causesof vasogenic brain edema. Their effect could be (1) direct, bydisrupting junctional complexes between endothelial cells ofthe BBB and/or (2) indirect, by inducing leukocyte migrationand production of a secondary wave of proinflammatorymediators.24,25 Thus, proinflammatory cytokines (eg, IL-1�,IL-1�, TNF-�, IL-6, and granulocyte macrophage–colonystimulating factor), mediators of oxidative injury (peroxini-trite, superoxide, or H2O2), and adhesion molecules (intercel-lular adhesion molecule-1, vascular cell adhesion molecule-1,and selectins) regulate the magnitude and persistence ofinflammation but may also directly reduce tight junctionintegrity and enhance edema formation.3,4,19,21,22,26,27 Chemo-kines may have similar effects. Neutralizing antibodiesagainst IL-8 and CINC1 prevent BBB breakdown and reducebrain edema formation.8,28,29 The current study has shownthat the CCL2/CCR2 axis also has an effect on BBBdisruption and edema formation during ischemia/reperfusionas well in regulating leukocyte migration.

How does the CCL2/CCR2 axis modulate BBB permeabil-ity and edema? In our opinion, CCL2 also has direct andindirect effects on the BBB. Our recent in vitro studies onischemia/reperfusion injury to brain endothelial cells showedthat CCL2 via endothelial CCR2 directly altered brain endo-thelial tight junction proteins.30 In addition, prolonged expo-sure to exogenous CCL2 in vivo also disrupts the BBB andcauses brain edema formation. The depletion of monocytesonly partially ameliorated this effect, suggesting a directeffect of CCL2 on BBB integrity.19 In the current experi-ments, we cannot exclude the possibility that the reduction inCCL2-induced infiltration of leukocytes in CCR2�/� micemight also contribute to reduced BBB disruption and brainedema formation, but it is likely that direct effects on barriertight junction proteins also contribute.

An intriguing finding in this study is that the absence ofCCR2 reduced neutrophil as well as monocyte infiltration.Two possible reasons for this are as follows: (1) CCL2diminishes expression of adhesion molecules on endothelialcells and, in this case, prevents leukocyte–endothelial cellinteraction; and (2) CCL2 may directly modulate cytokinesand CXC chemokines that may alter the inflammatory re-sponse. For example, in hindlimb ischemia and renal ische-mia/reperfusion, the absence of CCR2 is closely associatedwith decreased leukocyte adhesion, expression of adhesionmolecules (intercellular adhesion molecule), and selectinexpression.31 Analysis of chemokine expression also indi-cates that CCL2 can reduce the expression of chemoattrac-tants for neutrophils, such as MIP-2 (CXCL2).11 CCR2 is alsodetected on neutrophils under in vivo conditions.32

CCL2 is also a very important factor for modulation of theTh1/Th2 response.33 Specifically, CCL2 regulates the Th1type of response and expression of associated cytokines. Anabsence of CCR2 and diminished CCL2/CCR2 axis activitychange the balance to the Th2 type of response and the

associated Th2 cytokine profile (IL-4, IL-5, and IL-13). Forexample, CCR2�/� mice have increased levels of IL-4, IL-5,and IL-13 in renal reperfusion injury or in allergic responsesto Aspergillus and a significantly reduced inflammatoryresponse and lesion size.32,33 As such, it may not be surprisingthat diminishing CCL2/CCR2 axis activity by abolishingCCR2 receptors in brain ischemia/reperfusion injury affectsthe cytokine expression profile, switching it in the directionof an anti-inflammatory phenotype.

What is the significance of our findings? This studyindicates that the CCL2/CCR2 axis plays a pivotal role inischemia/reperfusion injury. This axis not only regulatesleukocyte entry but also has a major role in regulating theexpression of other proinflammatory mediators. Furthermore,CCL2 via CCR2 could also be involved directly or indirectlyin inducing vasogenic edema formation. Taken together, itappears that CCL2 could be a good marker of secondary brainischemia/reperfusion injury, owing to the fact that the CCL2level can be measured in cerebrospinal fluid or plasma inpatients. These results in particular indicate that CCR2 mightbe a good therapeutic target for inhibiting the effects of CCL2during cerebral ischemia/reperfusion and ameliorating braininjury in stroke.

Sources of FundingThis work was supported by grant (AVA) NS 044907 from theNational Institutes of Health.

DisclosuresNone.

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Oliver B. Dimitrijevic, Svetlana M. Stamatovic, Richard F. Keep and Anuska V. AndjelkovicIschemia/Reperfusion Injury in Mice

Absence of the Chemokine Receptor CCR2 Protects Against Cerebral

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