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Regulation of CCL2 and CCL3 expression in human brain endothelial cells by cytokines and lipopolysaccharide Chui, Ray; Dorovini-Zis, Katerina Jan 4, 2010

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RESEARCH Open AccessRegulation of CCL2 and CCL3 expression inhuman brain endothelial cells by cytokines andlipopolysaccharideRay Chui, Katerina Dorovini-Zis*AbstractBackground: Chemokines are emerging as important mediators of CNS inflammation capable of activatingleukocyte integrins and directing the migration of leukocyte subsets to sites of antigenic challenge. In this studywe investigated the expression, release and binding of CCL2 (MCP-1) and CCL3 (MIP-1a) in an in vitro model ofthe human blood-brain barrier.Methods: The kinetics of expression and cytokine upregulation and release of the b-chemokines CCL2 and CCL3were studied by immunocytochemistry and enzyme-linked immunosorbent assay in primary cultures of humanbrain microvessel endothelial cells (HBMEC). In addition, the differential binding of these chemokines to the basaland apical endothelial cell surfaces was assessed by immunoelectron microscopy.Results: Untreated HBMEC synthesize and release low levels of CCL2. CCL3 is minimally expressed, but notreleased by resting HBMEC. Treatment with TNF-a, IL-1b, LPS and a combination of TNF-a and IFN-g, but not IFN-galone, significantly upregulated the expression and release of both chemokines in a time-dependent manner. Thereleased CCL2 and CCL3 bound to the apical and basal endothelial surfaces, respectively. This distribution wasreversed in cytokine-activated HBMEC resulting in a predominantly basal localization of CCL2 and apical distributionof CCL3.Conclusions: Since cerebral endothelial cells are the first resident CNS cells to contact circulating leukocytes,expression, release and presentation of CCL2 and CCL3 on cerebral endothelium suggests an important role forthese chemokines in regulating the trafficking of inflammatory cells across the BBB in CNS inflammation.BackgroundThe microenvironment of the brain is tightly regulatedby the blood-brain barrier (BBB) the anatomical basis ofwhich is the cerebral endothelium. The BBB endothe-lium is highly specialized and different morphologically,functionally and immunologically from small and largevessel EC of other organs. Under normal physiologicalconditions, the presence of interendothelial tight junc-tions and absence of a vesicular transport system restrictthe entry of proteins, ions, lipid insoluble non-electro-lytes and circulating haematogenous cells into the brain[1,2]. Yet in response to infectious, inflammatory dis-eases, ischemia, hemorrhage or trauma, there is aninflux of leukocytes to sites of brain damage. Interac-tions between endothelial cells (EC) and circulating leu-kocytes have been increasingly implicated in theinitiation and evolution of inflammatory processes inthe central nervous system (CNS). Thus, molecularchanges induced on the endothelium by cytokines leadto specific interactions with inflammatory cells thatmediate their entry into the brain and accumulation atsites of antigenic challenge [3].Chemokines are a family of chemoattractant cytokinescharacterized by their unique ability to both recruit andactivate a variety of cell types. Currently, there are aboutfifty known chemokine members [4] which are dividedinto four sub-families by virtue of highly conserved N-terminal cysteine motifs (disulfide bonds) and the pre-sence or absence of intervening amino acids. There aretwo major sub-families, which consist of the a- (or* Correspondence: dorovini@interchange.ubc.caDivision of Neuropathology, Department of Pathology and LaboratoryMedicine, Vancouver General Hospital and the University of British Columbia,Vancouver, BC, CanadaChui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1JOURNAL OF NEUROINFLAMMATION© 2010 Chui and Dorovini-Zis; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.CXC) and b- (or CC) chemokines; and two minor sub-families, the g- (or C) and δ-chemokines (or CX3C).Chemokines have been associated with a broad range ofbiological and pathological processes [5-7], whichinclude angiogenesis [8], CNS development [9,10], ather-osclerosis [11], cancer biology [12-14], autoimmune dis-eases [15], nervous system inflammation [16], asthma[17], and haematopoiesis [18].There is considerable evidence that the b-chemokinesCCL2 (MCP-1) and CCL3 (MIP-1a) play an importantrole in CNS inflammation. Several studies have shownthe presence of CCL2 and/or CCL3 in multiple sclerosis(MS) lesions. In acute MS lesions, CCL2, CCL3 andCCL4 are selectively expressed by astrocytes, macro-phages, and microglia in the lesion centre and in thesurrounding white matter, whereas in actively demyeli-nating plaques CCL5 localizes on EC, perivascular cellsand reactive astrocytes [19]. In acute and chronic activeMS lesions, CCL2, along with CCL7 and CCL8, areexpressed primarily by hypertrophic astrocytes and vari-ably by inflammatory cells [20,21]. Significantly lowerCSF levels of CCL2 have been reported in MS patientswith active disease compared to controls and patientswith stable MS [22,23]. In this regard, it has beenshown that CCL2 is “consumed” by T lymphocytes andmonocytes as they migrate across brain EC monolayersin vitro, leading to downregulation of the CCR2 receptorin response to CCL2, which may account for thedecreased CSF CCL2 levels [24]. The expression ofCCL3 has been associated with microglia and macro-phages in the white matter lesions of MS patients [25].In experimental allergic encephalomyelitis (EAE), theanimal model of MS, the onset of the disease coincideswith the mRNA expression of CCL2, CCL3 and otherchemokines [26,27] and the accumulation of CXCL10and CCL2 [28]. In the Lewis rat model, CCL2 mRNAincreased before the onset of clinical signs, peakedwith height of clinical disease, and declined with reso-lution [29]. CCL2 expression was localized on lympho-cytes, macrophages, astrocytes, and EC and correlatedwith disease activity [30]. In chronic relapsing EAE,increased expression of CCL2, CXCL10 and KC wasobserved in astrocytes, whereas infiltrating leukocyteswere the source of CCL3 and CCL5 [31]. In the SJL/Jmouse, the expression of CCL3 correlates with theclinical onset of EAE and administration of anti-CCL3Abs prevents the development of both acute and relap-sing disease and infiltration of mononuclear cells intothe CNS initiated by the transfer of neuroantigen pep-tide-activated T cells [32]. Furthermore, these chemo-kines could drive Th1/Th2 lymphocyte differentiation[33]. Vaccination of Lewis rats with naked DNAencoding CCL2 and CCL3 prevented EAE [34]. Thesedata suggest that CCL2 and CCL3 have critical andnon-redundant roles in the pathogenesis of autoim-mune CNS inflammation.In this study, we investigated the kinetics of expres-sion and release of CCL2 and CCL3 by resting and cyto-kine or lipopolysaccharide (LPS) treated human brainmicrovessel EC using a well-characterized in vitromodel of the BBB. We show that the constitutiveexpression and release of CCL2 by HBMEC is signifi-cantly upregulated following EC activation in a time-dependent manner. In contrast, CCL3 expression underresting conditions is negligible and its release requiresstimulation by cytokines or LPS. Since leukocyte integ-rin activation by chemokines is required for firm adhe-sion and transendothelial migration, these findingsfurther support the active participation of the BBBendothelium in neuroinflammation.MethodsEndothelial cell culturesHuman brain microvessel endothelial cells (HBMEC)HBMEC were isolated from normal brains at autopsyless than 12 hours post mortem by methods previouslydescribed [35]. The study has complied with all institu-tional policies and was approved by the ethics commit-tees of the University of British Columbia and theVancouver General Hospital. For these studies we usedHBMEC isolated from five individuals ranging in agefrom 30 to 57 years. The cause of death included acutemyocardial infarction (2) and motor vehicle accidents(3) without CNS involvement. Review of the clinicalrecords ensured the absence of any pre-existing neurolo-gical, psychiatric, inflammatory disease or cancer. Thefreshly isolated clumps of HBMEC were seeded ontofibronectin-coated 96 well plates or 100 mm diameterplastic dishes (Corning Costar Corp., Cambridge, MA)and maintained in culture in complete media consistingof M199 supplemented with 10% horse plasma derivedserum (Hyclone Laboratories, Logan, UT), 100 μg/mlheparin, 20 μg/ml endothelial cell growth supplement,300 μg/ml glutamine (all from Sigma Chemical Co., St.Louis, MO) and 1% antibiotic/antimycotic solution (LifeTechnologies Inc.). The cells were maintained at 37°C ina humidified 5% CO2/95% air incubator and culturemedia were changed every other day. Endothelial cellsreached confluence 7 to 10 days after plating. Theendothelial origin and purity of the cells was determinedby their strongly positive perinuclear staining forFVIIIR:Ag and binding of Ulex europeaus I lectin. Pri-mary cultures from a single isolation were used for eachexperiment.Antibodies and cytokinesFor immunocytochemistry, the following primary anti-bodies (Abs) were used: monoclonal mouse anti-humanCCL2 (10 - 40 μg/ml) and CCL3 (10 - 50 μg/ml) wereChui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 2 of 12purchased from Pepro Tech (Rocky Hill, NJ) and Che-micon International Inc. (Temecula, CA); anti-FactorVIII related Ag and Ulex europeaus Ab were both pur-chased from Dako Diagnostics Canada Inc (MississaugaON). Secondary Abs used for these studies included thefollowing: 5 nm gold-conjugated goat anti-mouse IgG(1:40) (Auroprobe LM GAM IgG) and 10 nm gold-con-jugated goat anti-mouse IgG (1:20) (Auroprobe EMGAR IgG, Cedarlane Laboratories Ltd, Hornby, ON).Mouse anti-human follicle stimulating hormone (FSH)(BioGenex Laboratories, San Ramon, CA) was used asirrelevant isotype-matched control Ab. Recombinanthuman tumour necrosis factor a (TNF-a) and lipopoly-saccharide (LPS) were obtained from Sigma ChemicalCo. Recombinant human interferon-g (IFN-g) wasobtained from the NIH AIDS Research and ReferenceProgram. Recombinant human interleukin-1b (IL-1b)was obtained from Inter Medico (Markham, ON). Theconcentrations of cytokines and LPS used were basedon our previous studies and are within the range of con-centrations reported in the blood and CNS in variousinflammatory and infectious diseases.Induction of chemokine expression by cytokines and LPSHBMEC monolayers were grown to confluence in repli-cate wells and incubated with cytokines for 24, 48 and72 hrs. The cytokines used were TNF-a (10 - 100 U/ml), IFN-g (200 - 500 U/ml), IL-1b (10 U/ml), bacterialLPS (5 μg/ml) and combinations of these cytokines.Supernatants of cytokine-treated monolayers were col-lected following the respective time points for detectionof chemokine release by sandwich ELISA.Reverse transcription PCR (RT-PCR)Confluent HBMEC cultures grown on 100 mm diameterfibronectin- coated plates were used untreated or follow-ing 24 hr incubation with TNF-a (100 U/ml) and IFN-g(500 U/ml). The cells of both unstimulated and stimu-lated cultures were removed using a rubber policeman,centrifuged and the cell pellets were frozen at -70°C.Trizol (Life Technologies Inc.) was added to the fro-zen pellet, sonicated and the RNA extracted usingchloroform. The RNA preparation was cleaned up usingan RNeasy kit (Qiagen Inc., Mississauga, ON), accordingto the manufacturer’s instructions. 2 - 5 μg of totalRNA was reverse transcribed for 90 min at 37°C usingMoloney Murine Leukaemia Virus reverse transcriptase(MMLV-RT, USB Corp., Cleveland, OH) and randomhexamer primers (Amersham Pharmacia Biotech Inc.,Piscataway, NJ). PCR was performed using 2.5 U/25 μlreaction AmpliTaq Gold (Applied Biosystems, FosterCity, CA) with primers purchased from Life Technolo-gies Inc. (Table 1) on 1 - 5 μl of cDNA under the fol-lowing conditions: one initial cycle with dissociation at94°C for 8 min, annealing at 55 - 60°C for 30 sec, exten-sion at 72°C for 3 min; then cycling at 94°C for 1 min,55 - 60°C for 30 sec and 72°C for 45 sec on an AppliedBiosystems GeneAmp PCR System 9700 thermalcycler.GAPDH was used as an internal standard. The followingwere used as positive controls: pBluescript-hCCL2 (kindgift from Dr. Teizo Yoshimura, NCI/NIH, MD) andpBR322-hCCL3 (kind gift from Dr. Donald Forsdyke,Queen’s University, Kingston, ON). PCR products wererun on either a 6% polyacrylamide/TBE gel or a 2%agarose/TBE gel containing ethidium bromide (EtBr)and visualized under UV light. The RT-PCR experi-ments were repeated three times for each chemokineusing different primary HBMEC cultures.Light microscopic intracellular localization of CCL2 andCCL3Resting and cytokine treated (TNF-a [10-100 U/ml] andIFN-g [200-500 U/ml]) confluent monolayers cultivatedin triplicate wells of 96 well plates were washed withphosphate-buffered saline (PBS) supplemented with 1%bovine serum albumin (BSA, Sigma Chemical Co.) and1% normal goat serum (NGS), and then fixed- permea-bilized using buffered formaldehyde/acetone containing0.03% Triton X-100 for 10 minutes. The cultures werethen incubated with anti-CCL2 or anti-CCL3 monoclo-nal Abs diluted at 10 μg/ml in carrier buffer consistingof PBS containing 5% BSA and 4% NGS. At the end ofthe incubation period, cultures were washed and incu-bated with 5 nm gold-conjugated secondary Ab at 1:40dilution for 1 hr. Following washing in wash buffer, thecultures were incubated with silver enhancement solu-tion (Amersham Life Sciences, Buckinghamshire, Eng-land) and silver deposition was monitored for 22 - 26min. Nuclei were counterstained with Giemsa. Controlsincluded unstimulated HBMEC and cytokine treatedcultures incubated with carrier buffer, isotype-matchedirrelevant Ab (anti-human FSH) or normal mouse IgG(Cedarlane) instead of primary Abs. The cells wereexamined under a Nikon Labophot light microscope.Immunoelectron microscopyConfluent unstimulated HBMEC cultures and culturestreated with TNF-a (10 - 100 U/ml) and IFN-g (200 -500 U/ml) for 24 hrs were washed with PBS containing1% BSA and 20 mM NaN3 and incubated with anti-Table 1 RT-PCR primer sequencesGENE PRIMER SEQUENCECCL2 *F: atg aaa gtc tct gcc gcc ctt ctg t†R: agt ctt cgg agt ttg ggt ttg ctt gCCL3 F: atg cag gtc tcc act gct gcc cttR: gca ctc agc tcc agg tcg ctg aca tGAPDH F: cca tgt tcg tca tgg gtg tga acc aR: gcc agt aga ggc agg gat gat gtt cPCR primer sequences for CCL2 and CCL3 amplify fragments that are 298 and274 base pairs in length, respectively. *F-forward/sense primer. †R-reverse/anti-sense primer.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 3 of 12CCL2 or CCL3 monoclonal Abs at 10 μg/ml in PBScontaining 5% BSA, 4% NGS and 20 mM sodium azide(NaN3) for 30 min at room temperature. Followingwashing in wash buffer, the monolayers were incubatedfor 1 hr with the secondary Ab conjugated to 10 nmgold particles at 1:40 dilution in carrier buffer. Cellswere then washed and fixed with cold 1/2-strength Kar-novsky’s fixative (2.5% glutaraldehyde and 2% parafor-maldehyde in 0.2 M sodium cacodylate buffer) for onehr at 4°C. Following fixation, the cells were washed with0.2 M sodium cacodylate buffer and post-fixed with 1%osmium tetroxide for 1 hr at 4°C. After block stainingwith uranyl magnesium acetate overnight at 4°C, thecultures were washed with sodium acetate buffer, dehy-drated and embedded in Epon-Araldite. Longitudinalstrips cut from the embedded cultures after curing ofthe plastic were re-embedded in Araldite for cross sec-tioning. Thin sections were examined using a Zeiss EM910 electron microscope. One hundred cells from eachresting or treated cultures were photographed under25,000× magnification and the number of gold particlesbound per μm of cell membrane on the apical, basal cellsurface or subendothelial matrix was quantified, takingthe magnification into account.Enzyme-linked immunosorbent assay (ELISA)HBMEC grown to confluence in triplicate wells of 96well plates were used untreated or following incubationwith various cytokines or LPS for 24 - 72 hrs. Thesupernatants were collected and analyzed by sandwichELISA (Quantikine ELISA Kits, R & D Systems, Min-neapolis, MN), as per manufacturer’s instructions.Briefly, culture supernatants and provided standardswere added to microtiter plates coated with the appro-priate capture Ab. The application of detection Ab,along with substrate (TMB), allowed color development,which was stopped with 2N sulfuric acid. Absorbancewas read with an ELISA microtiter plate reader at awavelength of 490 nm. The assay sensitivity was <5 pg/ml for CCL2 and <10 pg/ml for CCL3. The generationof a standard curve using chemokine standards allowedthe calculation of the quantity of chemokines releasedby HBMEC in the culture supernatants.Statistical analysisValues derived from ELISA experiments were analyzedusing analysis of variance (ANOVA) to determine signif-icant differences between treatments. Student’s t testswere used where differences were found. The Tukey testwas also used as a multiple comparison method. Datathat were not normally distributed were subjected toKruskal-Wallis ANOVA on Ranks and Mann-WhitneyU-tests. The chi-square test was used to analyze theimmunoelectron microscopy data. Significant differencesbetween cytokine treated cells and control groups areshown as (*) where p < 0.05.ResultsDetection of CCL2 and CCL3 RNA by RT-PCRRNA extracted from confluent monolayers of HBMECwith and without treatment with 100 U/ml TNF-a and500 U/ml IFN-g for 24 hrs was used to study theexpression patterns of CCL2 and CCL3 in these cul-tures. The CCL2 and CCL3 gene-specific primers (Table1) used for these studies amplified 298 and 274 basepair fragments, respectively. The expression of CCL2RNA from both unstimulated and stimulated HBMECwas similar (Fig. 1A). There appeared to be no detect-able upregulation of RNA expression following cytokinetreatment. CCL2 cDNA cloned into pBluescript wasused as the positive control in these experiments.CCL3 RNA was found in barely discernible levels inunstimulated HBMEC (Fig. 1B). Incubation with 100 U/mlTNF-a and 500 U/ml IFN-g for 24 hrs resulted in increasein RNA expression. The control used for these experi-ments was CCL3 cDNA cloned into a pBluescript vector.ImmunocytochemistryImmunogold silver staining was used to demonstrateintracellular CCL2 and CCL3 protein expression in con-fluent monolayers of unstimulated HBMEC and in cul-tures treated for 24 to 72 hrs with TNF-a, IFN-g, IL-1band LPS, or combinations of TNF-a and IFN-g. Alluntreated cells exhibited positive staining for CCL2 inthe form of fine, black, granular cytoplasmic deposits(Fig. 2A) indicating constitutive protein expression con-sistent with the constitutive RNA expression by RT-PCR. Treatment with cytokines or LPS resulted inincreased diffuse cytoplasmic staining in the majority ofFigure 1 RNA expression of CCL2 (A) and CCL3 (B) by HBMECdetermined by semi-quantitative RT-PCR. Increasing amounts ofRNA (not shown) were used to detect differential expression inunstimulated HBMEC and cells treated with 100 U/ml TNF-a and500 U/ml IFN-g for 24 hrs. After amplification with gene-specificprimers, CCL2 or CCL3 and GAPDH PCR samples were run on a 2%agarose gel after 35 and 25 cycles respectively, under the followingconditions: pre-PCR step (94°C for 8 min, 55 - 60°C for 30 sec and72°C for 3 min) and cycling (94°C for 1 min, 55 - 60°C for 30 secand 72°C for 45 sec). The data shown are from one of threeexperiments for each chemokine with similar results.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 4 of 12cells, as compared to the untreated ones (Figs. 2B-D).Some variation in the intensity of staining between indi-vidual cells in both resting and stimulated cultures wasusually present (Figs. 2A-D), however, no differenceswere detected between HBMEC from different donors.Unstimulated HBMEC monolayers showed uniformlyslight cytoplasmic staining for CCL3 indicating minimalprotein expression at all time points investigated (Fig.3A) consistent with the low RNA expression by RT-PCR. Incubation with TNF-a, IFN-g, IL-1b and LPS, orcombination of TNF-a and IFN-g resulted in increasedintensity of cytoplasmic staining in all cells, with somevariation between EC of the same culture (Figs. 3B-D),but not between different donor endothelial cells.Release of CCL2 and CCL3 by HBMEC in cultureIn order to determine the amount of CCL2 and CCL3released by confluent HBMEC monolayers treated withTNF-a, IFN-g, IL-1b, LPS, or combination of TNF-aand IFN-g, culture supernatants from both resting andstimulated cultures grown in triplicate wells wereremoved at 24, 48 and 72 hrs post stimulation and ana-lyzed by sandwich ELISA. Unstimulated HBMECreleased 10 ~21 ng/ml of CCL2 over 24 - 72 hrs withsignificant increase in release following cytokine treat-ment in a time-dependent fashion (Fig. 4A). Treatmentwith TNF-a at 10 or 100 U/ml increased CCL2 releaseup to ~35 ng/ml at 24 hrs and up to a maximum of~63 ng/ml at 72 hrs. This increase in CCL2 release wastime-, but not concentration-dependent. Incubation withLPS (5 μg/ml) or IL-1b (10 U/ml) resulted in 124% and156% increase in CCL2 release over unstimulated cul-tures, respectively, in a time-, but not concentration-dependent manner. Incubation of HBMEC with IFN-galone resulted in a modest, but not significant upregula-tion of release that was similarly time- but not concen-tration- dependent. Co-incubation with TNF-a (100 U/ml) and IFN-g (200 U/ml) augmented CCL2 release tolevels comparable with TNF-a treatment alone.Figure 2 Intracellular localization of CCL2 in HBMEC by immunogold silver staining. (A) Unstimulated HBMEC constitutively express CCL2as shown by the positive cytoplasmic staining in the form of fine, granular black deposits of silver-enhanced gold particles. Nuclei arecounterstained with Giemsa. (B)-(D) The intensity of staining is markedly increased following incubation with (B) TNF-a (10 U/ml) for 48 hrs, (C)IL-1b (10 U/ml) for 72 hrs and (D) LPS (5 μg/ml) for 48 hrs. (E) Control cultures incubated with secondary antibody only show no staining. Scalebars = 50 μm.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 5 of 12The release of CCL3 by confluent monolayers ofHBMEC was measured by ELISA in culture superna-tants similarly collected at 24, 48 and 72 hr time points.Unstimulated cells did not release any detectable protein(Fig. 4B). Stimulation with 100 U/ml TNF-a resulted insmall amounts of protein release within the first 24 hrsand up to 100 pg/ml over 48 and 72 hrs. Lower concen-trations of TNF-a (10 U/ml), as well as IFN-g at 200and 500 U/ml, failed to induce CCL3 release into themedia. However, co-incubation of HBMEC with TNF-a(100 U/ml) and IFN-g (200 U/ml) significantly augmen-ted CCL3 release to a maximum of 200 pg/ml (p <0.005) in a time-dependent manner. Treatment of themonolayers with IL-1b (10 U/ml) or LPS (5 μg/ml)induced the release of up to 200 - 400 pg/ml of thischemokine. There were some quantitative differences inthe constitutive and stimulated release of both chemo-kines between HBMEC from different donors; however,the pattern of chemokine release after cytokine or LPStreatment was similar in all experiments.Differential binding of CCL2 and CCL3 to the surface ofHBMECChemokines are secreted molecules that exert theiractions via binding to glycosaminoglycans at theendothelial surface and the extracellular matrix. In orderto elucidate the binding patterns of CCL2 to the cellmembrane, an immunoelectron microscopic approachwas employed. An additional advantage of this methodis the ability to visualize both the apical and basal bind-ing of the chemokines. HBMEC cultures were usedFigure 3 Intracellular localization of CCL3 in HBMEC by immunogold silver staining. (A) In untreated cultures, cytoplasmic staining is faintindicating minimal constitutive expression. Nuclei are counterstained with Giemsa. (B)-(D) The density of the granular black cytoplasmic depositsis increased in cultures treated with (B) TNF-a (100 U/ml)+ IFN-g (200 U/ml) for 72 hrs, (C) IL-1b (10 U/ml) for 72 hrs and (D) LPS (5 μg/ml) for 48hrs. (E) Control cultures incubated with secondary antibody only show no staining. Scale bars = 50 μm.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 6 of 12Figure 4 Quantitation of CCL2 and CCL3 release by resting and cytokine stimulated HBMEC by ELISA. (A) Detection of CCL2 protein insupernatants of HBMEC cultures under resting conditions and following incubation with cytokines and LPS. Chemokine release was determinedby sandwich ELISA at the indicated time intervals. Values represent mean release (pg/ml) ± SEM (n = 3). Tukey test p ≤ 0.001; * p < 0.05 ascompared to unstimulated HBMEC. Values shown represent one of two independent, representative experiments. (B) Detection of CCL3 proteinin supernatants of resting HBMEC cultures and monolayers treated with cytokines and LPS. Chemokine release was determined by sandwichELISA at the indicated time intervals. Values represent mean release (pg/ml) ± SEM (n = 3). ANOVA p ≤ 0.001; * p < 0.05 as compared tounstimulated HBMEC. Values shown are the results of one of two independent, representative experiments.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 7 of 12untreated or after stimulation for 24 hrs with cytokines(100 U/ml TNF-a + 500 U/ml IFN-g). A small numberof gold particles indicating the presence of CCL2 wereassociated with the cell surface of untreated HBMEC(Fig. 5A). Most of the gold particles were associatedwith the apical cell surface compared to the basolateralsurface, although this difference did not reach significantdifference (Fig. 5C). In cytokine-treated monolayers,CCL2 was redistributed and bound preferentially to thebasal cell surface and the discontinuous, amorphous,basal lamina-like material underlying the basal surfacecompared to the apical one, as shown by the increasedmean value (Figs. 5B, C). The number of gold particlesbound to the basal cell surface was significantly greaterin cytokine treated versus resting HBMEC (Fig. 5C).Gold particles were not observed along intercellularcontacts. In contrast to CCL2, virtually no gold particleswere identified on the apical cell surface, and only occa-sional gold particles, indicating the presence of CCL3,were bound to the basal cell surface of unstimulatedHBMEC (Figs. 5D, F). Gold particles were not foundalong intercellular contacts. Upon cytokine treatment,Figure 5 Surface localization of CCL2 and CCL3 by immunoelectron microscopy. (D, E) Surface localization of CCL2 on HBMEC byimmunoelectron microscopy. (A) In resting monolayers, gold particles indicating the presence of CCL2 are bound mostly to the apical cell surface(arrow). (B) In cultures treated with TNF-a (100 U/ml) + IFN-g (500 U/ml) for 24 hrs gold particles are preferentially bound to the subendothelialbasal lamina-like material (arrows). Arrowheads in (A) and (B) indicate the basal cell surface. Scale bars = 0.25 μm. (C) Quantification of the numberof gold particles bound to the apical and basal cell surface of unstimulated HBMEC shows no significant difference between apical and basalbinding (p ≥ 0.1). In cytokine-treated HBMEC cultures, there is a significant increase in the number of gold particles bound to the basal cell surfaceand the basal lamina-like material versus the apical surface (p ≤ 0.01). The number of gold particles at the basal cell surface is significantly greater inactivated versus resting HBMEC (p ≤ 0.01). (D, E) Surface localization of CCL3 on HBMEC by immunoelectron microscopy. (A) In resting monolayersoccasional gold particles are bound to the basal cell surface only (arrow). (B) Following treatment with TNF-a (100 U/ml) + IFNg (500 U/ml) for 24hrs gold particles are preferentially bound to the apical surface (arrow). Arrowheads in (A) and (B) indicate the basal cell surface. Scale bars = 0.25μm. (F) Quantification of the number of gold particles bound to the apical and basal cell surfaces of unstimulated and cytokine-treated HBMECcultures shows no statistically significant differences between the different groups (p ≥ 0.15).Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 8 of 12CCL3 localized predominantly along the apical cell sur-face, whereas the number of gold particles associatedwith the basal cell surface remained the same. Thus,although binding to the basal surface was similarbetween untreated and treated HBMEC, the mean num-ber of gold particles associated with the apical surfacewas greater in cytokine activated cells (Figs 5E, F).DiscussionThe entry of inflammatory cells into the brain is a criti-cal event in the pathogenesis of inflammatory and infec-tious diseases, as well as non-inflammatory conditionsof the CNS, such as stroke and trauma. As an anatomi-cal and immunological barrier, the BBB plays a centralrole in the recruitment of leukocytes in acute andchronic CNS inflammation. It is now well establishedthat the transendothelial movement of leukocytes is amulti-step process, each step being mediated by specificinteractions between EC adhesion molecules and theirligands on leukocytes [36]. Some of the molecularmechanisms involved in the trafficking of leukocytesacross the BBB have been recently elucidated andinvolve membrane interactions between adhesion mole-cules induced on cerebral EC by inflammatory media-tors and integrins or glycosylated ligands on leukocytes.Recent studies from this laboratory have shown that theadhesion and transendothelial migration of resting andactivated T lymphocytes across the BBB depend uponthe activation status of the endothelium and the T cellsand are mediated by receptor-ligand interactions thatare specific for each step and for each class of leuko-cytes [37,38].In vivo and in vitro studies have established a criticalrole for chemokines in the leukocyte adhesion cascade.Binding of chemokines to their G-protein coupledreceptors on leukocytes triggers inside-out signalingleading to rapid integrin activation and firm adhesion tothe endothelium [39]. Although the expression of che-mokines by glial cells in the CNS has been relativelywell documented [21,40], human cerebrovascular che-mokine expression has not been fully characterized. Inthe present study we show that CCL2 is constitutivelyexpressed by HBMEC with marked increase of the intra-cellular protein and a 3-fold increase in the release ofCCL2 into the media following treatment with TNF-a,IL-1b and LPS. Although IFN-g alone had no effect onCCL2 release by HBMEC, combination of the highestconcentrations of TNF-a and IFN-g resulted in a 3-foldincrease of the CCL2 levels in the supernatants.Constitutive expression of CCL2 mRNA has been pre-viously reported in porcine brain EC with upregulationupon stimulation with TNF-a [41]. In vitro studiesusing rat brain and retinal EC lines showed constitutiveexpression of CCL2 and increased release into themedia following activation with TNF-a, IL-1b and IFN-g[42]. Both the constitutive release at 2,000 - 2,700 pg/mland the stimulated release at 5,500 pg/ml were muchlower compared to HBMEC (up to 20,000 pg/ml and60,000 pg/ml, respectively), which may reflect speciesdifferences in CCL2 expression and release. In extracer-ebral endothelium models, CCL2 RNA transcripts havebeen demonstrated in human aortic, pulmonary arteryand umbilical vein endothelial cell (HUVEC) cultures, aswell as in freshly removed human arteries and veins[43]. Expression of CCL2 mRNA has been detected inresting and cytokine activated HUVEC and human brainEC after IFN-g stimulation [44]. Exposure of human cer-ebrovascular EC to hypoxic astrocyte-conditioned mediafor 4 - 8 hrs increased the release of CCL2 from a lowconstitutive level of 100 pg/ml to a modest 600 - 700pg/ml, both significantly lower than the levels obtainedin the present study [45]. This may be related to theshort time of exposure to hypoxic media. Similarly,expression of CCL2 was induced in human brain-derived EC by endothelin-1 and ischemia [46]. A modestincrease in CCL2 release, up to 1,800 pg/ml, has beenreported in a human brain EC line after incubation withheat-killed Streptococcus suis [47]. The same strain hadno effect on CCL2 expression by HUVEC. In additionto cytokines, injection of the HIV Tat1-72 protein intothe mouse hippocampus was shown to increase theexpression of CCL2 on brain vascular endothelium [48].Substantial evidence indicates the importance of CCL2in the induction and propagation of the inflammatorycascade. Thus, CCL2 was shown to stimulate T cellmigration across microvascular endothelium [49] and tomediate the firm adhesion of monocytes under flowconditions [50]. In the CNS, knockout models for CCL2and CCR2 provide strong evidence for the importanceof the CCL2-CCR2 interaction [51,52]. Recent observa-tions indicate that glia-derived CCL2 regulates thedevelopment of EAE by attracting TNF-a and iNOS-producing dendritic cells and macrophages to the CNS[53]. Furthermore, a recent study investigating potentialmechanisms for HIV entry into the CNS indicates thatCCL2 enhances the transmigration of HIV-infected leu-kocytes across the BBB via the upregulated expressionof CCR2 [54].In contrast to CCL2, the constitutive RNA and proteinexpression of CCL3 by HBMEC is negligible. Further-more, resting HBMEC do not constitutively releaseCCL3 into the media. Treatment with TNF-a and IFN-gincreased RNA expression and incubation with indivi-dual cytokines or LPS upregulated protein expressionand induced CCL3 release. However, under identicalexperimental conditions, the stimulated CCL2 releasewas typically two orders of magnitude greater thanCCL3 release. IFN-g alone has no effect on cerebralChui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 9 of 12endothelial CCL3 expression, however, when combinedwith TNF-a, a synergistic effect resulted in higher pro-tein levels compared to TNF-a alone. Expression ofCCL3 by EC has been addressed in a limited number ofstudies. In animals, CCL3 has been reported in murinebone marrow EC [55] and in the endothelium of epi-neurial and endoneurial vessels following transection ofthe rat sciatic nerve [56]. Furthermore, expression ofthis chemokine was induced in a murine endothelial cellline by alloantigen-primed T cells [57]. In humans,endothelial expression of CCL3 has been documented inHUVEC incubated with activated platelets [58] orexposed to diamide [59] and LPS [60]. In addition,CCL3 has been localized to EC of blood vessels andsplenic sinusoids in the hemophagocytic syndrome [61].According to a recent report, transmigration of bonemarrow-derived dendritic cells across mouse brain ECmonolayers was increased in the presence of CCL3 con-centration gradients [62]. The expression of CCL3 bycerebrovascular endothelium under resting and inflam-matory conditions has not been previously addressed.It is now well established that immobilization of che-mokines by binding to glycosaminoglycans on the lumi-nal EC surface enhances leukocyte adhesion, whilebinding to the abluminal surface and subendothelialmatrix promotes their directional migration to sites ofinflammation [63]. The present study shows that, follow-ing their release into the culture media, CCL2 and CCL3bind to the surface of HBMEC and to the discontinuousbasal lamina-like material under the basal cell surface ina polarized manner, which is distinct for each chemokine.Thus, under resting conditions, CCL2 binds preferentiallyto the apical (luminal) surface of HBMEC, whereas CCL3shows minimal binding only to the basal (abluminal) ECsurface, which is consistent with our protein synthesisand release results. In cytokine activated HBMEC, bind-ing of CCL2 is redistributed towards the basal cell surfaceand that of CCL3 preferentially on the apical surface.These findings suggest that CCL3 may be primarilyresponsible for the initial recruitment and activation ofCCR1 and/or CCR5 expressing cells, whereas CCL2 playsa greater role in establishing the chemotactic gradientsnecessary for the directional cell migration into the brainparenchyma. In accordance with these observations, pre-vious studies have demonstrated the presence of specificand separate binding sites for CCL2 and CCL3 along theabluminal surface of human brain microvessels [64]. Thedifferential binding of CCL2 and CCL3 to cerebral EC inan inflammatory milieu lends support to previous studieswhich, taking into account the CCL2 and CCL3 expres-sion patterns and Ab therapy studies, suggest that CCL3controls mononuclear cell accumulation during acuteEAE, whereas CCL2 controls cellular infiltration duringrelapsing disease indicating that acute and relapsing EAEare regulated by the differential expression of CCL2 andCCL3 [65].Previous in vitro studies from this laboratory havedocumented the expression and cytokine upregulation ofthe b-chemokines CCL4 and CCL5 by HBMEC [66] andtheir role in enhancing adhesion of memory and acti-vated CD4+ T lymphocytes to cytokine treated HBMEC[67]. The present study points towards substantial differ-ences in cytokine regulation of protein release among thefour b-chemokines. The release of CCL2 and CCL3 isupregulated by TNF-a, IL-1b, LPS and TNF-a + IFN-g,but not IFN-g alone, in contrast to CCL5 which respondsto TNF-a, IFN-g and LPS, but not IL1-b. The release ofCCL4 is augmented by LPS and combinations of TNF-awith IFN-g or IL-1b, but not by single cytokine treat-ments. Overall, the release of CCL2 under resting and sti-mulated conditions is much greater than that of CCL3,CCL4 and CCL5. Additional differences exist in the dis-tribution of bound chemokines to HBMEC. In restingmonolayers, CCL2 and CCL5 are bound preferentially tothe apical EC surface, CCL4 to both apical and basal sur-faces and CCL3 only to the basal surface. Upon cytokinestimulation, this polarized expression is reversed for allfour chemokines, with CCL2, CCL4 and CCL5 now pre-sent preferentially along the basal surface and suben-dothelial matrix, and CCL3 distributed mostly along theapical surface. These differences strongly suggest differ-ential and possibly temporal roles of these chemokines inthe regulation of leukocyte transendothelial migration inCNS inflammation.ConclusionsThe studies reported herein demonstrate that brainmicrovessel EC synthesize CCL2 and CCL3 with signifi-cant upregulation after cytokine and LPS activation. Theconstitutive and stimulated production and release ofCCL2 is quantitatively greater compared to CCL3. Thepolarized distribution of these chemokines on HBMECunder resting and simulated inflammatory conditionspoints towards possibly distinct functions, with CCL3on the apical surface promoting leukocyte adhesionthrough integrin activation and CCL2 on the basal sur-face directing their migration across the BBB. Thesefindings further emphasize the important role played bythe cerebral microvascular endothelium in regulatinginflammatory responses at the BBB.AcknowledgementsThe authors thank Mrs. Rukmini Prameya for skillful technical assistance withprimary endothelial cell cultures. This work was supported by MultipleSclerosis Society of Canada Grant 20R51514 (to KDZ).Authors’ contributionsRC carried out the experiments and statistical analysis and contributed tothe preparation of the manuscript. KD-Z conceived and designed the studyChui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 10 of 12and prepared the manuscript. Both authors have read and approved thefinal version of the manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 4 September 2009Accepted: 4 January 2010 Published: 4 January 2010References1. Rapoport SI: Sites and functions of the blood-brain barrier. Blood-brainbarrier in physiology and medicine New York: Raven PressRapoport SI 1976,43-86.2. Pardridge WM: Introduction to the Blood-Brain Barrier: Methodology, Biologyand Pathology Cambridge University Press 1998.3. Engelhardt B: Molecular mechanisms involved in T cell migration acrossthe blood-brain barrier. J Neural Transm 2006, 113:477-485.4. Goncharova LB, Tarakanov AO: Why chemokines are cytokines while theirreceptors are not cytokine ones?. Curr Med Chem 2008, 15:1297-1304.5. Mackay CR: Chemokines: immunology’s high impact factors. Nat Immunol2001, 2:95-101.6. Fernandez EJ, Lolis E: Structure, function, and inhibition of chemokines.Annu Rev Pharmacol Toxicol 2002, 42:469-499.7. Rossi D, Zlotnik A: The biology of chemokines and their receptors. AnnuRev Immunol 2000, 18:217-242.8. Keane MP, Strieter RM: The role of CXC chemokines in the regulation ofangiogenesis. Chem Immunol 1999, 72:86-101.9. Adler MW, Rogers TJ: Are chemokines the third major system in thebrain?. J Leukoc Biol 2005, 78:1204-1209.10. Adler MW, Geller EB, Chen X, Rogers TJ: Viewing chemokines as a thirdmajor system of communication in the brain. Aaps J 2005, 7:E865-870.11. Sasayama S, Okada M, Matsumori A: Chemokines and cardiovasculardiseases. Cardiovasc Res 2000, 45:267-269.12. Nomura T, Hasegawa H: Chemokines and anti-cancer immunotherapy:anti-tumor effect of EBI1-ligand chemokine (ELC) and secondarylymphoid tissue chemokine (SLC). Anticancer Res 2000, 20:4073-4080.13. Murphy PM: Chemokines and the molecular basis of cancer metastasis. NEngl J Med 2001, 345:833-835.14. O’Hayre M, Salanga CL, Handel TM, Allen SJ: Chemokines and cancer:migration, intracellular signalling and intercellular communication in themicroenvironment. Biochem J 2008, 409:635-649.15. Arimilli S, Ferlin W, Solvason N, Deshpande S, Howard M, Mocci S:Chemokines in autoimmune diseases. Immunol Rev 2000, 177:43-51.16. Huang D, Han Y, Rani MR, Glabinski A, Trebst C, Sorensen T, Tani M,Wang J, Chien P, O’Bryan S, Bielecki B, Zhou ZL, Majumder S, Ransohoff RM:Chemokines and chemokine receptors in inflammation of the nervoussystem: manifold roles and exquisite regulation. Immunol Rev 2000,177:52-67.17. Lukacs NW: Role of chemokines in the pathogenesis of asthma. Nat RevImmunol 2001, 1:108-116.18. Lapidot T, Dar A, Kollet O: How do stem cells find their way home?. Blood2005, 106:1901-1910.19. Simpson JE, Newcombe J, Cuzner ML, Woodroofe MN: Expression ofmonocyte chemoattractant protein-1 and other beta-chemokines byresident glia and inflammatory cells in multiple sclerosis lesions. JNeuroimmunol 1998, 84:238-249.20. McManus C, Berman JW, Brett FM, Staunton H, Farrell M, Brosnan CF: MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: animmunohistochemical and in situ hybridization study. J Neuroimmunol1998, 86:20-29.21. Voorn Van Der P, Tekstra J, Beelen RH, Tensen CP, Valk Van Der P, DeGroot CJ: Expression of MCP-1 by reactive astrocytes in demyelinatingmultiple sclerosis lesions. Am J Pathol 1999, 154:45-51.22. Franciotta D, Martino G, Zardini E, Furlan R, Bergamaschi R, Andreoni L,Cosi V: Serum and CSF levels of MCP-1 and IP-10 in multiple sclerosispatients with acute and stable disease and undergoingimmunomodulatory therapies. J Neuroimmunol 2001, 115:192-198.23. Sorensen TL, Ransohoff RM, Strieter RM, Sellebjerg F: Chemokine CCL2 andchemokine receptor CCR2 in early active multiple sclerosis. Eur J Neurol2004, 11:445-449.24. Mahad D, Callahan MK, Williams KA, Ubogu EE, Kivisakk P, Tucky B, Kidd G,Kingsbury GA, Chang A, Fox RJ, Mack M, Sniderman MB, Ravid R,Staugitis SM, Stins MF, Ransohoff RM: Modulating CCR2 and CCL2 at theblood-brain barrier: relevance for multiple sclerosis pathogenesis. Brain2006, 129:212-223.25. Balashov KE, Rottman JB, Weiner HL, Hancock WW: CCR5(+) and CXCR3(+)T cells are increased in multiple sclerosis and their ligands MIP-1alphaand IP-10 are expressed in demyelinating brain lesions. Proc Natl AcadSci USA 1999, 96:6873-6878.26. Glabinski AR, Tani M, Tuohy VK, Tuthill RJ, Ransohoff RM: Central nervoussystem chemokine mRNA accumulation follows initial leukocyte entry atthe onset of acute murine experimental autoimmune encephalomyelitis.Brain Behav Immun 1995, 9:315-330.27. Godiska R, Chantry D, Dietsch GN, Gray PW: Chemokine expression inmurine experimental allergic encephalomyelitis. J Neuroimmunol 1995,58:167-176.28. Ransohoff RM, Hamilton TA, Tani M, Stoler MH, Shick HE, Major JA,Estes ML, Thomas DM, Tuohy VK: Astrocyte expression of mRNA encodingcytokines IP-10 and JE/MCP-1 in experimental autoimmuneencephalomyelitis. Faseb J 1993, 7:592-600.29. Hulkower K, Brosnan CF, Aquino DA, Cammer W, Kulshrestha S, Guida MP,Rapoport DA, Berman JW: Expression of CSF-1, c-fms, and MCP-1 in thecentral nervous system of rats with experimental allergicencephalomyelitis. J Immunol 1993, 150:2525-2533.30. Berman JW, Guida MP, Warren J, Amat J, Brosnan CF: Localization ofmonocyte chemoattractant peptide-1 expression in the central nervoussystem in experimental autoimmune encephalomyelitis and trauma inthe rat. J Immunol 1996, 156:3017-3023.31. Glabinski AR, Tani M, Strieter RM, Tuohy VK, Ransohoff RM: Synchronoussynthesis of alpha- and beta-chemokines by cells of diverse lineage inthe central nervous system of mice with relapses of chronic experimentalautoimmune encephalomyelitis. Am J Pathol 1997, 150:617-630.32. Karpus WJ, Lukacs NW, McRae BL, Strieter RM, Kunkel SL, Miller SD: Animportant role for the chemokine macrophage inflammatory protein-1alpha in the pathogenesis of the T cell-mediated autoimmune disease,experimental autoimmune encephalomyelitis. J Immunol 1995, 155:5003-5010.33. Karpus WJ, Kennedy KJ: MIP-1alpha and MCP-1 differentially regulateacute and relapsing autoimmune encephalomyelitis as well as Th1/Th2lymphocyte differentiation. J Leukoc Biol 1997, 62:681-687.34. Youssef S, Wildbaum G, Maor G, Lanir N, Gour-Lavie A, Grabie N, Karin N:Long-lasting protective immunity to experimental autoimmuneencephalomyelitis following vaccination with naked DNA encoding C-Cchemokines. J Immunol 1998, 161:3870-3879.35. Dorovini-Zis K, Prameya R, Bowman PD: Culture and characterization ofmicrovascular endothelial cells derived from human brain. Lab Invest1991, 64:425-436.36. Ley K, Laudanna C, Cybulsky MI, Nourshargh S: Getting to the site ofinflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol2007, 7:678-689.37. Wong D, Prameya R, Dorovini-Zis K: In vitro adhesion and migration of Tlymphocytes across monolayers of human brain microvessel endothelialcells: regulation by ICAM-1, VCAM-1, E-selectin and PECAM-1. JNeuropathol Exp Neurol 1999, 58:138-152.38. Wong D, Prameya R, Dorovini-Zis K: Adhesion and migration ofpolymorphonuclear leukocytes across human brain microvesselendothelial cells are differentially regulated by endothelial cell adhesionmolecules and modulate monolayer permeability. J Neuroimmunol 2007,184:136-148.39. Constantin G: Chemokine signaling and integrin activation in lymphocytemigration into the inflamed brain. J Neuroimmunol 2008.40. Weiss JM, Downie SA, Lyman WD, Berman JW: Astrocyte-derivedmonocyte-chemoattractant protein-1 directs the transmigration ofleukocytes across a model of the human blood-brain barrier. J Immunol1998, 161:6896-6903.41. Zach O, Bauer HC, Richter K, Webersinke G, Tontsch S, Bauer H: Expressionof a chemotactic cytokine (MCP-1) in cerebral capillary endothelial cellsin vitro. Endothelium 1997, 5:143-153.42. Harkness KA, Sussman JD, Davies-Jones GA, Greenwood J, Woodroofe MN:Cytokine regulation of MCP-1 expression in brain and retinalmicrovascular endothelial cells. J Neuroimmunol 2003, 142:1-9.Chui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 11 of 1243. Li YS, Shyy YJ, Wright JG, Valente AJ, Cornhill JF, Kolattukudy PE: Theexpression of monocyte chemotactic protein (MCP-1) in human vascularendothelium in vitro and in vivo. Mol Cell Biochem 1993, 126:61-68.44. Frigerio S, Gelati M, Ciusani E, Corsini E, Dufour A, Massa G, Salmaggi A:Immunocompetence of human microvascular brain endothelial cells:cytokine regulation of IL-1beta, MCP-1, IL-10, sICAM-1 and sVCAM-1. JNeurol 1998, 245:727-730.45. Stanimirovic D, Satoh K: Inflammatory mediators of cerebral endothelium:a role in ischemic brain inflammation. Brain Pathol 2000, 10:113-126.46. Chen P, Shibata M, Zidovetzki R, Fisher M, Zlokovic BV, Hofman FM:Endothelin-1 and monocyte chemoattractant protein-1 modulation inischemia and human brain-derived endothelial cell cultures. JNeuroimmunol 2001, 116:62-73.47. Vadeboncoeur N, Segura M, Al-Numani D, Vanier G, Gottschalk M: Pro-inflammatory cytokine and chemokine release by human brainmicrovascular endothelial cells stimulated by Streptococcus suisserotype 2. FEMS Immunol Med Microbiol 2003, 35:49-58.48. Toborek M, Lee YW, Pu H, Malecki A, Flora G, Garrido R, Hennig B,Bauer HC, Nath A: HIV-Tat protein induces oxidative and inflammatorypathways in brain endothelium. J Neurochem 2003, 84:169-179.49. Cai JP, Hudson S, Ye MW, Chin YH: The intracellular signaling pathwaysinvolved in MCP-1-stimulated T cell migration across microvascularendothelium. Cell Immunol 1996, 167:269-275.50. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MAJr, Luster AD, Luscinskas FW, Rosenzweig A: MCP-1 and IL-8 trigger firmadhesion of monocytes to vascular endothelium under flow conditions.Nature 1999, 398:718-723.51. Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD: Resistance toexperimental autoimmune encephalomyelitis in mice lacking the CCchemokine receptor (CCR)2. J Exp Med 2000, 192:1075-1080.52. Huang DR, Wang J, Kivisakk P, Rollins BJ, Ransohoff RM: Absence ofmonocyte chemoattractant protein 1 in mice leads to decreased localmacrophage recruitment and antigen-specific T helper cell type 1immune response in experimental autoimmune encephalomyelitis. J ExpMed 2001, 193:713-726.53. Dogan RN, Elhofy A, Karpus WJ: Production of CCL2 by central nervoussystem cells regulates development of murine experimentalautoimmune encephalomyelitis through the recruitment of TNF- andiNOS-expressing macrophages and myeloid dendritic cells. J Immunol2008, 180:7376-7384.54. Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, Berman JW:CCL2/monocyte chemoattractant protein-1 mediates enhancedtransmigration of human immunodeficiency virus (HIV)-infectedleukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. J Neurosci 2006, 26:1098-1106.55. Cheng LM, Wang QR: [Hematopoietic inhibitors elaborated by bonemarrow endothelial cells]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2002,10:485-491.56. Taskinen HS, Roytta M: Increased expression of chemokines (MCP-1, MIP-1alpha, RANTES) after peripheral nerve transection. J Peripher Nerv Syst2000, 5:75-81.57. Kobayashi H, Koga S, Novick AC, Toma H, Fairchild RL: T-cell mediatedinduction of allogeneic endothelial cell chemokine expression.Transplantation 2003, 75:529-536.58. Cha JK, Jeong MH, Bae HR, Han JY, Jeong SJ, Jin HJ, Lim YJ, Kim SH,Kim JW: Activated platelets induce secretion of interleukin-1beta,monocyte chemotactic protein-1, and macrophage inflammatoryprotein-1alpha and surface expression of intercellular adhesionmolecule-1 on cultured endothelial cells. J Korean Med Sci 2000, 15:273-278.59. Yang L, Zhu X, Zhao X, Deng Z: Expression of macrophage inflammatoryprotein 1 alpha in the endothelial cells exposed to diamide. J HuazhongUniv Sci Technolog Med Sci 2003, 23:219-222.60. Deng ZD, Qu ZL, Yang LM: [Lipopolysaccharide induces expression ofmacrophage inflammatory protein-1alpha in human umbilical veinendothelial cells]. Zhonghua Bing Li Xue Za Zhi 2003, 32:449-452.61. Teruya-Feldstein J, Setsuda J, Yao X, Kingma DW, Straus S, Tosato G,Jaffe ES: MIP-1alpha expression in tissues from patients withhemophagocytic syndrome. Lab Invest 1999, 79:1583-1590.62. Zozulya AL, Reinke E, Baiu DC, Karman J, Sandor M, Fabry Z: Dendritic celltransmigration through brain microvessel endothelium is regulated byMIP-1alpha chemokine and matrix metalloproteinases. J Immunol 2007,178:520-529.63. Tanaka Y, Adams DH, Shaw S: Proteoglycans on endothelial cells presentadhesion-inducing cytokines to leukocytes. Immunol Today 1993, 14:111-115.64. Andjelkovic AV, Spencer DD, Pachter JS: Visualization of chemokinebinding sites on human brain microvessels. J Cell Biol 1999, 145:403-412.65. Kennedy KJ, Strieter RM, Kunkel SL, Lukacs NW, Karpus WJ: Acute andrelapsing experimental autoimmune encephalomyelitis are regulated bydifferential expression of the CC chemokines macrophage inflammatoryprotein-1alpha and monocyte chemotactic protein-1. J Neuroimmunol1998, 92:98-108.66. Shukaliak JA, Dorovini-Zis K: Expression of the beta-chemokines RANTESand MIP-1 beta by human brain microvessel endothelial cells in primaryculture. J Neuropathol Exp Neurol 2000, 59:339-352.67. Quandt J, Dorovini-Zis K: The beta chemokines CCL4 and CCL5 enhanceadhesion of specific CD4+ T cell subsets to human brain endothelialcells. J Neuropathol Exp Neurol 2004, 63:350-362.doi:10.1186/1742-2094-7-1Cite this article as: Chui and Dorovini-Zis: Regulation of CCL2 and CCL3expression in human brain endothelial cells by cytokines andlipopolysaccharide. Journal of Neuroinflammation 2010 7:1.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralChui and Dorovini-Zis Journal of Neuroinflammation 2010, 7:1http://www.jneuroinflammation.com/content/7/1/1Page 12 of 12


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