Increased_expression_of_bioactive_chemokines_in_human_cerebromicrovascular_endothelial_cells_and

Journal of Neuroimmunology1011999148–160

https://www.360docs.net/doc/ee5178378.html, r locate r jneuroim

Increased expression of bioactive chemokines in human cerebromicrovascular endothelial cells and astrocytes subjected to

simulated ischemia in vitro

Wandong Zhang a,Catherine Smith a,Anthony Shapiro a,Robert Monette a,

James Hutchison b,Danica Stanimirovic a,)

a Institute for Biological Sciences,National Research Council of Canada,Montreal Road Campus,Building M-54,Ottawa,ON,Canada K1A0R6

b Children’s Hospital of Eastern Ontario,Ottawa,ON,Canada K1H8L1

Received30March1999;received in revised form7July1999;accepted7July1999

Abstract

Leukocyte infiltration into the brain has been implicated in the development of ischemic brain damage.In this study,simulated in vitro

?. ischemia r reperfusion and IL-1b were found to up-regulate both the expression of intercellular adhesion molecule-1ICAM-1in cultured

human cerebromicrovascular endothelial cells HCEC and the adhesion of allogenic neutrophils to HCEC.Both HCEC and human fetal ?.

astrocytes FHAS also responded to IL-1b and to in vitro ischemia r reperfusion by a pronounced up-regulation of IL-8and MCP-1 mRNA and by increased release of IL-8and MCP-1in cell culture media.FHAS were found to release30-times higher levels of MCP-1 than HCEC under both basal and ischemic conditions.However,100u r ml IL-1b induced greater stimulation of both IL-8and MCP-1?.?. secretion in HCEC50and20times above controls,respectively than in FHAS three and two times above controls,respectively.IL-8 was the principal neutrophil chemoattractant released from IL-1b-treated HCEC,since IL-8antibody completely inhibited neutrophil chemotaxis enticed by HCEC media.However,the IL-8antibody neutralized only50%of IL-1b-stimulated neutrophil chemoattractants released from FHAS,and40%–60%of ischemia-stimulated chemotactic activity released by either HCEC or FHAS.These results suggest that simulated in vitro ischemia,in addition to IL-8and MCP-1,stimulates secretion of other bioactive chemokines from HCEC and FHAS.q1999Elsevier Science B.V.All rights reserved.

Keywords:Chemokines;Adhesion molecules;Cerebral endothelial cells;Astrocytes;Human;Hypoxia

1.Introduction

Brain inflammation has been implicated in the pathogenic cascade leading to secondary ischemic brain ?.

damage Kim,1996;Feuerstein et al.,1997.Neutrophils are commonly the first inflammatory cells to infiltrate the ischemic brain,followed by mononuclear phagocytes ?.

Barone et al.,1991;Feuerstein et al.,1994.The recruit-ment of inflammatory cells into the brain is mediated by

leukocyte r endothelial adhesion molecules Kishimoto and

Rothlein,1994;Kim,1996and by the creation of specific

?chemotactic gradients in the ischemic brain tissue Glabin-ski et al.,1995;Feuerstein et al.,1997;Ransohoff and

Corresponding author.Tel.:q1-613-993-3730;fax:q1-613-941-4475;E-mail:danica.stanimirovic@nrc.ca

.?Tani,1998.The reduction of circulating neutrophils i.e., .?.

neutropenia Matsuo et al.,1994,administration of anti-bodies against endothelial or leukocyte-expressed adhesion

?molecules in animal models of cerebral ischemia Chopp .

et al.,1994,and knock-out of the gene encoding the

?.?intercellular adhesion molecule-1ICAM-1Soriano et .

al.,1996,have been shown to limit neutrophil infiltration into the brain and to reduce infarct size and brain swelling.

Chemokines,a family of8–12kDa peptides,have been shown to entice selective leukocyte recruitment at periph-

?. eral inflammation sites Rollins,1997;Baggiolini,1998. Chemokine selectivity to subpopulation of leukocytes is determined by the distribution of four cysteines in a highly

?conserved N-terminal domain,such as that a CXC;proto-

typic member IL-8chemokines primarily attract neu-?.

trophils,b CC;prototypic member MCP-1chemokines

attract both monocytes and lymphocytes,g C;lympho-

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160149

tactin chemokines draw lymphocytes,and d CX C;neu-

rotactin both neutrophils and monocytes Baggiolini et al.,

1997;Bazan et al.,1997.Recent studies have shown that chemokines and their receptors are expressed in a variety

of brain cells,including astrocytes Hayashi et al.,1995;

.?. Gourmala et al.,1997,microglia Ehrlich et al.,1998,

?. macrophages Calvo et al.,1996;Gourmala et al.,1997,

and neurons Horuk et al.,1997.

Chemokines have been suggested to play a role in the pathophysiology of brain injury accompanying autoim-?.?

mune Ransohoff,1997,post-traumatic Fan et al.,1995;

Ransohoff and Tani,1998,and,more recently,post-

ischemic brain inflammation Kim et al.,1995;Matsumoto .?

et al.,1997.Increased levels of MCP-1Kim et al.,1995;

Wang et al.,1995,macrophage inflammatory protein-1

?.?.

MIP-1Kim et al.,1995;Takami et al.,1997,and IL-8

Matsumoto et al.,1997have been detected in the is-chemic rat brain,and systemic administration of anti-IL-8 antibody has been shown to reduce cerebral edema,

blood-brain barrier BBB permeability,and infarct size in

?. experimental models of stroke Matsumoto et al.,1997.

Since neutrophils and monocytes are the inflammatory

?.?

cells commonly found in ischemic brain lesion s Barone

et al.,1991;Feuerstein et al.,1994,two groups of chemokines are likely important in recruiting these cells

into the ischemic brain:a neutrophil chemokines,belong-

ing to a and d families,and b monocyte chemokines, belonging to b family.A prototype chemokine from each

?.?of these families,i.e.,IL-8a family,neurotactin d .?.

family,and MCP-1b family were analyzed in this study

?. in both cultured human cerebral endothelial cells HCEC

and human fetal astrocytes FHAS subjected to simulated in vitro ischemia.The results show that both cell types respond to IL-1b and to in vitro ischemia by up-regulating the expression r secretion of IL-8and MCP-1,and other neutrophil chemoattractants,and are likely important par-ticipants in the process of inflammatory cell recruitment into the ischemic brain.

2.Materials and methods

2.1.Materials

All culture media were obtained from Gibco BRL ?.

Gaithersburg,MD.Fetal bovine serum was purchased

from Hyclone Logan,UT.Endothelial cell growth sup-plement,ITS e Premix,and human fibronectin were pur-

?chased from Collaborative Biomedical Products Bedford, .

MA.Human serum and gelatin were obtained from Sigma ?.

St.Louis,MO.The mouse melanoma cell line,Cloud-?.

man S91clone M-3cells was from the American Type

Culture Collection Rockville,MD.IL-1b,IL-8,antibody

to human ICAM-1clone CD54,and antibody to human

IL-8were obtained from Upstate Biotechnology Lake

Placid,NY.Colloidal gold-conjugated goat anti-mouse IgG and peroxidase-conjugated goat anti-mouse IgG anti-

?bodies were from Accurate Chemical and Scientific West-

.?. bury,NY.Silver-enhancing solution IntenSE e M was

from Amersham Canada Oakville,ON,while the peroxi-

dase substrate,2,2-azinobis3-ethylbenzthiazoline-6-

.?.

sulfonic acid ImmunoPure ABTS was from Pierce

?.?. Rockford,IL.Diethyl pyrocarbonate DEPC,Tri reagent, Trizma,collagenase IV,and the Ficoll gradients, Histopaque-1077and-1119,were obtained from Sigma. Taq DNA polymerase was purchased from Promega ?.

Madison,WI.Superscript RT-II,deoxyribonucleitides,

Oligo dT primer,phenol,and agarose were pur-12–18

chased from Gibco BRL.Chloroform,2-propanol,for-mamide,and formaldehyde were from Fisher Scientific ?.

Nepean,ON.

2.2.Cell cultures

The protocols used in these studies have been approved by the Human Research Ethics Committees of the National Research Council of Canada,Montreal Neurological Insti-?.

tute McGill University,and the Children’s Hospital of Eastern Ontario.

HCEC were isolated using previously described proto-

?. cols Gerhart et al.,1988;Stanimirovic et al.,1996. Briefly,the samples of human temporal cortex removed surgically for the treatment of epilepsy were dissected, minced,homogenized and filtered sequentially through

350and112-m m mesh nylon nets Nitex,Tetko,Elmsford,

NY,USA.The pellets collected from filtrates were cen-

trifuged3,000=g;15min;48C in20%dextran and then filtered through a20-m m mesh nylon net.Microvessels and capillaries retained on the net were dissociated with1 mg r ml type IV collagenase for15min at378C and plated

in growth media containing65%medium M199Earle’s salts,25mM Hepes,4.35g r l sodium bicarbonate and3

mM L-glutamine,10%fetal calf serum,5%human serum,

20%murine melanoma cell mouse melanoma,Cloudman

S91,clone M-3,melanin producing cells-conditioned me-dia,5m g r ml insulin,5m g r ml transferrin,5ng r ml selenium,and10m g r ml endothelial cell growth supple-ment.Endothelial cell colonies emerging from attached microvessels4–5days after seeding were isolated using ?.

cloning rings BELCO Glass,Vineland,NJ and2–3of these cloned colonies were pooled and further passaged. Purity of HCEC cultures generated by these procedures is routinely assessed by the immunocytochemical staining for Factor VIII-related antigen and the lack of staining for smooth muscle a-actin,and is estimated to be)95%. The morphological,phenotypic,biochemical and func-tional characteristics of these HCEC cultures have been

described in detail previously Stanimirovic et al.,1996;

Muruganandam et al.,1997.

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160 150

Cultures of fetal10–18weeks of gestation human

brain astrocytes FHAS were provided by Drs.J.Antel

and W.Yong Montreal Neurological Institute,Montreal, .

QC,Canada.These cultures were prepared using previ-

ously described procedures Yong et al.,1992.Briefly, brain samples were trypsinized,homogenized and filtered through a130-m m mesh.Pellets were then resuspended in a medium containing95%Dulbecco’s modified Eagle’s

?.?.

medium DME4500mg r l glucose and5%fetal bovine serum,and plated onto poly-L-lysine coated tissue culture dishes.Contaminating neurons were completely eliminated after two rounds of trypsinization and passaging.More than95%of cells in these cultures were immunopositive

?.?

for the glial fibrillary acidic protein GFAP Yong et al., .

1992.

2.3.Simulated in?itro ischemia

The term‘simulated in vitro ischemia’is used in this study to describe an in vitro model of combined severe hypoxia and glucose and nutrient deprivation that lacks a blood flow component of in vivo ischemia.The simulated in vitro ischemia was induced as described previously ?.

Stanimirovic et al.,1997a.HCEC and FHAS grown in 35-mm dishes were washed twice in phosphate-buffered ?.

saline PBS,and then subjected to a combination of

w?.

glucose-free Krebs solution containing in mM119NaCl,

x 4.7KCl,1.2KH PO,25NaHCO,2.5CaCl,1MgCl

24322

and severe hypoxia-2%oxygen in an anaerobic cham-?. ber Anaerobic System model1024,Forma Scientific equipped with a humidified,temperature-controlled incu-bator directly accessible within the chamber.The entire system was purged with95%N r5%CO atmosphere.

Recovery i.e.,simulated reperfusion was achieved by

exposing cells to ambient air reoxygenation and replac-ing media with Krebs buffer containing5mM glucose. The culture media and cells were simultaneously harvested at the end of a4-h simulated in vitro ischemia and at4,16, and24h of recovery.Respective controls were maintained in a20%oxygen and Krebs buffer supplemented with 5-mM glucose for matching periods of time.

In parallel experiments,both HCEC and FHAS were also exposed to various concentrations of the pro-in-

?. flammatory cytokine,IL-1b50–100u r ml,and media and cells were harvested at4,8,20,and28h after IL-1b addition.

2.4.RT-PCR

Total RNA was extracted from HCEC and FHAS grown

in35-mm Petri dishes using Tri reagent500m l r dish and subsequently purified using the protocol provided by the manufacturer.The RNA pellets were resuspended in50m l

of DEPC-treated dH O and incubated at558C for10min.

The quality of the RNA was confirmed for each sample

?using formaldehyde-agarose gel electrophoresis.RNA0.5 .?.

m g was then mixed with0.5m g of oligo dT

12–18 primers,heated for10min at708C,and then chilled on ice.

Synthesis of single-stranded cDNA was performed by

reverse transcription428C,1h in a reaction mixture ?.

final volume20m l containing4m l of5=first strand ?.

buffer Gibco BRL,2m l of0.1M DTT,1m l of10mM

y?dNTP,100units of RNase H reverse transcriptase Su-

perScript e II,Gibco BRL,and10m l of DEPC-treated

dH0.The reverse transcriptase was inactivated by heating 2

the reaction mixture at708C for15min.The20-m l of the resulting reaction mixture was diluted with20m l of

dH O,and4m l of the cDNA from this mixture was 2

subsequently used in a50-m l PCR reaction.

Specific primers were designed according to published sequences of the human chemokines IL-8,MCP-1and

neurotactin,and housekeeping genes,b-actin and b-mi-

2 croglobulin,and were synthesized using a PerSeptive ?.

Biosystem Framingham,MA synthesizer.Sequences of the primers used are shown in Table1.

PCR amplifications were carried out in a final volume

of50m l containing1=reaction buffer Promega,1.5

mM MgCl,0.2mM each of four dNTPs,0.4m M each of 2

two pairs of the primers primers for an internal control gene and the primers for either IL-8,MCP-1or neuro-.

tactin,2.5units Taq DNA polymerase,and4m l cDNA. The amplification reactions were performed in a PTC-200

Table1

Sense and anti-sense sequences of primers for the human chemokines,IL-8,MCP-1,and neurotactin,and for the housekeeping genes,b-actin and ?.

b2-microglobulin b2-M used in PCR reactions in this study.The RT-PCR reactions using the paired primers listed in the table generate a289-bp DNA fragment for IL-8,a257-bp fragment for MCP-1,a943-bp fragment for neurotactin,a504-bp fragment for b-actin,and a137-bp fragment for b2-microglobulin,respectively

X X

?.?.

Gene5Primer sense primer3Primer anti-sense primer

X X X X

IL-85-ATGACTTCCAAGCTGGCCGTG-35-CTCCACAACCCTCTGCACCCA-3

X X X X

MCP-15-GCTCGCTCAGCCAGATGCAAT-35-TGGGTTGTGGAGTGAGTGTTC-3

X X X X

Neurotactin5-GATACCTGTAGCTTTGCTCATCC-35-TGGTAGGTGAACATGGCCACC-3

X X X X

b-Actin5-GACTATGACTTAGTTGCGTTA-35-GCCTTCATACATCTCAAGTTG-3

X X X X

b2-M5-CAGCAAGGACTGGTCTTTCTAC-35-GCTACCTGTGGAGCAACCTGC-3

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160151

?. Peltier Thermal Cycler MJ Research,Watertown,MA in ?.

0.2-ml tubes Rose Scientific,Alberta,Canada.All ampli-fications were done using a denaturation step at948C for 30s,annealing step at558C for45s and polymerization step at728C for40s,and were carried out for45cycles.

Aliquots of the PCR10m l and restriction digests were subjected to electrophoresis on a1.5%agarose in a Tris-borate buffer containing0.5m g r ml ethidium bromide,and then photographed.The internal control genes and chemokine genes were linearly amplified during the45 PCR cycle.The relative densities r volumes of the bands on film negatives were measured using a Computing Densito-?.?

meter model300A Molecular Dynamics,Sunnyvale, .

CA and analyzed using ImageQnaNT,version4.1soft-?.

ware Molecular Dynamics.

2.5.Immunocytochemistry

Surface expression of ICAM-1in HCEC was assessed by indirect immunocytochemistry.Briefly,HCEC grown on10m g r ml human fibronectin-coated glass coverslips were washed with PBS and incubated at room temperature with2m g r ml mouse hybridoma monoclonal antibody ?.?.

IgG to human ICAM-1clone CD54for40min in medium M199.Cells were then washed in PBS and incu-bated for1h with a1:50dilution of a4-nm colloidal gold particle-conjugated goat anti-mouse IgG antibody.Cover-slips were then fixed for30s with9.25%formaldehyde and45%acetone in PBS,washed,and incubated in a silver

enhancing solution IntenSE e M for25min.Coverslips were washed and counterstained with Giemsa stain for15 min.Non-specific staining was determined by either the exclusion of the primary antibody,or by replacing primary anti-ICAM-1antibody with the isotype-matched antibody ?.

to GFAP Accurate Chemical and Scientific.

2.6.ELISA

An enzyme-linked immunosorbent assay ELISA was used to quantify the levels of ICAM-1expressed in HCEC and the levels of IL-8and MCP-1in culture media of HCEC and FHAS.For ICAM-1measurements,HCEC

?4 cultures grown in96-well microtiter plates2=10

cells r100m l per well at378C in5%CO were sequen-

tially incubated with a primary monoclonal anti-human

ICAM-1antibody2m g r ml for1h at378C,followed by 1:500diluted peroxidase-conjugated goat anti-mouse IgG in PBS for45min at378C.Non-specific binding sites were

blocked with2%bovine serum albumin BSA in PBS for 30min at378C.After each incubation,the plates were washed three times with PBS.Color was developed by the addition of100-m l of a1mg r ml solution of the horse-

radish peroxidase substrate,2,2-azinobis3-ethylbenz-

thiazoline-6-sulfonic acid diammonium salt ImmunoPure

ABTS.The reaction was stopped after5min by the addition of an equal volume of1%SDS to each well.The

optical density O.D.of the developed color was read at

405nm using a SpectraMAX Molecular Devices,Menlo .

Park,CA microplate reader.

The levels of IL-8and MCP-1released from variously treated HCEC and FHAS were quantified by a‘sandwich’ELISA using commercially obtained ELISA kits,Quan-

?. tikine human IL-8kit R&D System,Minneapolis,MN

and ID Elisa e MCP-1kit ID Labs Biotechnology,Lon-.?5. don,ON.Either HCEC or FHAS2=10cells r dish grown in35-mm dishes were used in these experiments. Aliquots of culture media were collected,centrifuged at 14,000rpm for5min at48C,and the ELISA assays were carried out as instructed by the manufacturers.

2.7.Neutrophil adhesion and chemotaxis

Human neutrophils were isolated from fresh,EDTA-treated venous blood obtained from adult healthy volun-?

teers medical staff at Children’s Hospital of Eastern On-.

tario.The neutrophil-containing band was separated on

?discontinuous polysucrose–sodium diatrizoate Histo-

paque-1077and-1119gradients as described by English

and Andersen1974.Contaminating erythrocytes were removed by brief hypotonic lysis in ice-cold0.15%NaCl. Microscopic examination and acetic acid–Cresyl violet staining indicated that more than98%of the remaining cells were neutrophils.The neutrophils were then labeled

?. with10m M calcein-AM Molecular Probe,OR,USA in PBS for20min at378C.The cells were centrifuged at room temperature for5min at1400rpm,washed twice with PBS,and re-suspended in PBS at a final density of 2.5=106cells r ml.

Confluent monolayers of control or treated HCEC grown in96-well microtiter plates were washed twice with PBS

?4.

and calcein-labeled neutrophils5=10cells r100ul were then added to each well for1h at378C.Non-adherent neutrophils were removed by two rapid washes with100 m l PBS and the intensity of the fluorescence remaining in each well was determined using a CytoFluor2350fluores-

cence microplate reader Millipore,Bedford,MA equipped with485r22nm excitation and530r25nm emission filters.The numbers of neutrophils adhering to the HCEC monolayers were calculated using a standard curve gener-

ated from the fluorescence values of increasing1000–.

100,000numbers of labeled neutrophils in suspension. Alternatively,variously treated HCEC monolayers were pre-labeled with the fluorescent dye,3,3X-dipropylthiadi-

??..?. carbocyanine iodide DiSC51m M,20min prior to

the addition of calcein-labeled neutrophils and fluorescent

images were generated using a Zeiss LSM410Carl Zeiss,

Thornwood,NY inverted laser scanning microscope ?.

LSM equipped with an Argon r Krypton ion laser and a Zeiss LD achroplan20=,0.4NA objective.The images

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160 152

were generated using the488-nm and the568-nm excita-tion lines with a FT655dichroic mirror and a FT560 beam splitter positioned in the light path.Emitted fluores-

cence of both calcein and DiSC5was collected simulta-

neously after being filtered through a510–525nm band-pass filter and a610-nm long-pass filter,respectively. Confocal apertures for each recorded wavelength were adjusted to a full width half maximum of20m m.Standard image processing was performed to enhance brightness

and contrast using ImageSpace software Molecular Dy-.

namics.

Chemotaxis of calcein-AM labeled neutrophils induced by media collected from variously treated HCEC and FHAS,or known chemoattractants,in the presence or absence of the anti-IL-8antibody,was assessed by a quantitative in vitro method using a ChemoTx a101-5?.

Neuro Probe,Gaithersburg,MD assembly consisting of a 96-well plate and a polycarbonate filter membrane,as

described by Junger et al.1993.Briefly,the wells of the plates were loaded with media collected from variously treated cells or solutions containing known concentrations of chemoattractants,framed filter was positioned on top, 50,000neutrophils suspended in20m l of matching non-conditioned media were applied on the top of each mem-brane r well,and the assembly was incubated for90min at 378C.The numbers of neutrophils transmigrated into wells of the96-well plate were quantified by measuring intensity

of fluorescence excitation r emission:485r530nm in a

CytoFluor2350reader Millipore,against the standard curve constructed with increasing numbers of labeled neu-trophils.

3.Results

3.1.Effects of simulated in?itro ischemia on the expres-sion of ICAM-1in HCEC and the adhesion of allogenic neutrophils to HCEC

Simulated in vitro ischemia4h followed by a24-h recovery resulted in an up-regulation of ICAM-1in HCEC

?. as determined by immunocytochemistry Fig.1A and B ?.

and ELISA Fig.1G.The same duration of ischemia r re-covery effected a2–3-fold increase in a number of allo-genic neutrophils firmly adhering to HCEC monolayers as

?observed r quantified by confocal microscopy Fig.1D and .?.

E and fluorometry Fig.1H.The exposure of HCEC to

IL-1b100u r ml for24h caused a dramatic up-regu-

lation of ICAM-1Fig.1C and G,and an increase in the

?. number of neutrophils3–4-fold above control adhering

to IL-1b-pretreated HCEC Fig.1F and H.We have previously reported that both cytokines and in vitro-ischemia stimulate the transcription of ICAM-1in HCEC ?.

Stanimirovic et al.,1997a.In both cases,increased neu-trophil adhesion to HCEC was blocked by a combination

of anti-ICAM-1and anti-CD18antibodies Stanimirovic et .

al.,1997a.

3.2.Effects of simulated in?itro ischemia and IL-1b on the expression r secretion of chemokines in HCEC and FHAS

3.2.1.The expression of neurotactin in HCEC and FHAS

The low levels of expression of the recently discovered,

brain-enriched neutrophil chemoattractant with a CX C

3?.

motif Pan et al.,1997,neurotactin,was detected by RT-PCR in the whole human brain homogenate,as well as in HCEC,FHAS,and human umbilical vein endothelial ?.?.

cells HUVEC in culture data not shown.Neurotactin mRNA expression in HCEC and FHAS was not affected by either of the following stimuli:in vitro ischemia r re-

covery,IL-1b50–100u r ml;4–24h,and phorbol ester ?.?.

20–40nM;6h data not shown,whereas a transient induction of neurotactin mRNA was detected in HUVEC

?. stimulated with100u r ml IL-1b for8h data not shown. Increased expression of fractalkine r neurotactin mRNA by IL-1b and TNF a has previously been demonstrated in

?. peripheral human endothelial cells Bazan et al.,1997, and brain microglia in mice with experimental autoim-

mune encephalomyelitis Pan et al.,1997.These experi-ments suggested that neurotactin is likely not critically involved in ischemia-induced HCEC r neutrophil interac-?.

tion s in vitro.

3.2.2.Effects of simulated in?itro ischemia on the expres-sion and secretion of IL-8in HCEC and FHAS In vitro ischemia resulted in a transient up-regulation of

IL-8mRNA in HCEC at4h of recovery Fig.2A.IL-8 mRNA returned back to control levels at16and24h of ?.

recovery Fig.2A.The levels of immunoreactive IL-8 released into HCEC media increased as early as4h after ?.

ischemia Fig.2B and continued to accumulate in culture

media up to16h of recovery Fig.2B.

In FHAS,IL-8mRNA was already up-regulated at the end of a4-h ischemia and throughout the period of post-

ischemic recovery up to24h Fig.3A.The maximal

up-regulation was seen at4-h post-ischemia Fig.3A.The increased mRNA expression was accompanied by up to 3-fold increases in the levels of immunoreactive IL-8in ?.

FHAS media Fig.3B.

The expression of two housekeeping genes,b-actin and

b-microglobulin Table1,used alternatively in various 2

experiments,was not affected by ischemia r recovery pro-

tocols in either HCEC or FHAS Fig.2A and,Figs.

3–5A.

3.2.3.Effects of simulated in?itro ischemia on the expres-sion and secretion of MCP-1in HCEC and FHAS The MCP-1mRNA was up-regulated at4,16and24h

?. after transient in vitro ischemia in both HCEC Fig.4A

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160153

Fig.1.Effects of simulated in vitro ischemia r reperfusion or IL-1b on the expression of ICAM-1in HCEC A–C,G and the adhesion of homologous ?.?.?. neutrophils to HCEC monolayers D–F,H.ICAM-1expression was determined by immunocytochemistry panels A–C and by ELISA G,and

?.?.?.

neutrophil adhesion was assessed by fluorescence confocal microscopy panels D–F and fluorometry H as described in Section2.A basal expression of ICAM-1in HCEC.Insert micrograph shows a non-specific staining in which primary anti-ICAM-1antibody was replaced with the isotype-matched ?.?.

anti-GFAP antibody;D basal level of neutrophil adhesion to control HCEC monolayer;B and E HCEC were exposed to4h ischemia r24h recovery;?.

C and F HCEC were exposed to100u r ml IL-1b for24h.The images shown are representative of at least five independent experiments.Each bar in G and H represents the mean"S.D.of six replicate wells in one of at least three experiments yielding similar results.Asterisks indicate significant w?.x

differences p-0.01;ANOVA followed by the Fisher’s protected least square difference PLSD multiple comparisons compared to control levels.a ?.?.

Indicates significant difference p-0.01;ANOVA compared to in vitro ischemia I4r R24.

()W.Zhang et al.r Journal of Neuroimmunology 1011999148–160

154Fig.2.Effects of simulated in vitro ischemia r reperfusion on IL-8mRNA ?.expression in HCEC A and the secretion of immunoreactive IL-8in ?.?5.HCEC media B .Confluent HCEC ;2=10cells r dish were exposed to 4-h simulated in vitro ischemia and the indicated periods of recovery as described in Section 2.IL-8mRNA expression was determined by a ?.?.semi-quantitative RT-PCR A .Volumes of IL-8bands closed bars were ?.?.expressed as a percentage of control gene b -actin bands open bars amplified in same PCR reactions.IL-8secretion in cell media of control ?.?.?.open bars and ischemic cells closed bars was quantified by ELISA B in same experiments.Each bar represents the mean "S.D.of four sepa-rate gels and ELISA determinations,each performed in duplicate.Aster-w isks indicate a significant difference p -0.01;ANOVA followed by x Fisher’s PLSD multiple comparisons from corresponding control levels.

?.and FHAS Fig.5A .MCP-1levels in HCEC media increased initially at 4h of recovery and reached a 3-fold increase above the corresponding controls at 16and 24h ?.after ischemia Fig.4B .In FHAS,immunoreactive MCP-1levels were already elevated at the end of ischemia and were 2–2.5-fold higher than corresponding control levels ?.at all times of recovery Fig.5B .

Both basal and ischemia-stimulated levels of immunore-active MCP-1released into FHAS media were 20–30

?times higher than those released by HCEC i.e.,ng r ml vs..?.pg r ml Fig.4B and,Fig.5B .

3.2.

4.Effects of IL-1b on the expression and secretion of IL-8and MCP-1in HCEC and FHAS

IL-1b strongly up-regulated the expression of IL-8mRNA and MCP-1mRNA in both HCEC and FHAS at

Fig.3.Effects of simulated in vitro ischemia r reperfusion on the expres-?.sion of IL-8mRNA in FHAS A and the secretion of immunoreactive ?.?5.IL-8in FHAS media B .Confluent FHAS ;2=10cells r dish were exposed to 4-h simulated in vitro ischemia and the indicated periods of recovery as described in Section 2.IL-8mRNA expression was deter-?.?mined by semi-quantitative RT-PCR A .Volumes of IL-8bands closed .?.bars were expressed as a percentage of control gene b -actin bands ?.open bars amplified in same PCR reactions.IL-8secretion in cell media ?.?.of control open bars and ischemic cells closed bars was quantified by ?.ELISA B in same experiments.Each bar represents the mean "S.D.of four separate gels r experiments.Asterisks indicate a significant difference ?p -0.01;ANOVA followed by the Fisher’s PLSD multiple compar-.isons from corresponding control levels.

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W.Zhang et al.r Journal of Neuroimmunology1011999148–160

155

Fig.4.Effects of simulated in vitro ischemia r reperfusion on the expres-

sion of MCP-1mRNA in HCEC A and the secretion of immunoreactive

?.?5. MCP-1in HCEC media B.Confluent HCEC;2=10cells r dish were exposed to4-h simulated in vitro ischemia and the indicated periods of recovery as described in Section2.MCP-1mRNA expression was

determined by a semi-quantitative RT-PCR A.Volumes of MCP-1?.

bands closed bars were expressed as a percentage of control gene ?.?.

b2-microglobulin bands open bars amplified in same PCR reactions.

MCP-1secretion in cell media of control open bars and ischemic cells ?.?.

closed bars was quantified by ELISA B in same experiments.Each bar represents the mean"S.D.of four separate gels r experiments.Asterisks

indicate a significant difference p-0.01;ANOVA followed by the

Fisher’s PLSD multiple comparisons from corresponding control levels.

8,20,and28h after addition not shown.However,a striking difference was observed in the levels of im-munoreactive chemokines released in media of IL-1b-treated HCEC and FHAS.Whereas IL-1b increased IL-8 levels in FHAS media2–3times above the corresponding time controls,HCEC exposed to IL-1b released abundant amounts of IL-8superseding the control levels by as much ?.

as40times Fig.6.Similarly,IL-1b induced more pro-

nounced release of MCP-1in HCEC10times above .?.?. control than in FHAS2–4times above control Fig.6.

3.3.Effects of simulated in?itro ischemia and IL-1b on the neutrophil chemoattractant acti?ity released in the media of HCEC and FHAS

Media collected from control,ischemic,or IL-1b-stimulated cells were tested for their ability to

entice

Fig.5.Effects of simulated in vitro ischemia r reperfusion on the expres-

sion of MCP-1mRNA in FHAS A and the secretion of immunoreactive

?.?5. MCP-1in FHAS media B.Confluent FHAS;2=10cells r dish were exposed to4-h simulated in vitro ischemia and the indicated periods of recovery as described in Section2.MCP-1mRNA expression was

determined by a semi-quantitative RT-PCR A.Volumes of MCP-1?.

bands closed bars were expressed as a percentage of control gene ?.?.

b-actin bands open bars amplified in same PCR reactions.MCP-1

?.?secretion in cell media of control open bars and ischemic cells closed .?.

bars was quantified by ELISA B in same experiments.Each bar represents the mean"S.D.of four separate gels r experiments.Asterisks

indicate a significant difference p-0.01;ANOVA followed by the

Fisher’s PLSD multiple comparisons from corresponding control levels.

()W.Zhang et al.r Journal of Neuroimmunology 1011999148–160

156Fig.6.Effects of IL-1b on IL-8and MCP-1secretion by HCEC and ?.FHAS.100u r ml of IL-1b closed bars or the same volume of solvent ?.open bars were added to cells for 24h,and IL-8and MCP-1levels secreted in cell media were subsequently determined by ELISA as described in Section 2.Each bar represents the mean "S.D.of four ?.separate experiments each performed in triplicate n s 12.Asterisks ?.indicate a significant difference p -0.01;Student’s t -test from corre-sponding control levels.

chemotaxis of freshly isolated human neutrophils.In vitro ischemia significantly increased neutrophil chemoattractant ?.capacity of cell media from both HCEC Fig.7A and ?.FHAS Fig.7B .FHAS media attracted 1.4–2times more neutrophils than HCEC media collected from the same ?.number r density of cells Fig.7,suggesting that FHAS released either higher levels or more potent neutrophil chemoattractants than HCEC.Neutrophil chemotactic ac-tivity was also markedly elevated in media of IL-1b -?.stimulated HCEC and FHAS Fig.7A and B .

3.3.1.Effects of anti-IL-8antibody on neutrophil chemo -taxis enticed by media of ischemic HCEC and FHAS

In order to examine to what extent IL-8contributed to the neutrophil chemotactic activity of media collected from ischemic and IL-1b -treated HCEC and FHAS,the media ?.were pre-treated with an anti-IL-8antibody 5–10m g r ml .The ability of anti-IL-8antibody to neutralize IL-8activity was confirmed in a neutrophil chemotaxis assay against ?.recombinant human IL-8Fig.8B .Both HCEC and FHAS media collected from ischemic cells and pre-treated with ?the blocking concentrations of anti-IL-8antibody 10

.m g r ml exhibited 40%–60%less chemotactic activity as ?.compared to the same untreated media Fig.8A .Anti-IL-8antibody inhibited up to 90%of chemotactic activity in media of IL-1b -stimulated HCEC and up to 50%in media ?.of IL-1b -stimulated FHAS Fig.7A .Anti-IL-8antibody pretreatment of control cell media inhibited 10%–20%of ?.media chemotactic activity data not shown .Anti MCP-1antibody did not affect neutrophil chemotactic activity in media of either ischemia-or IL-1b -treated HCEC or FHAS ?.data not shown .

The data suggest that,in addition to IL-8,in vitro ischemia stimulate secretion of various other neutrophil chemoattractants r chemokines from both HCEC and FHAS.However,IL-8appears to be a principal

neutrophil

Fig.7.Effects of simulated in vitro ischemia r reperfusion and IL-1b on chemotaxis of allogenic neutrophils enticed by media conditioned by ?.?.?.?.?5HCEC A and FHAS B .HCEC A and FHAS B ;2=10cells of .each cell type r dish were exposed to 4h of simulated in vitro ischemia and the indicated periods of recovery,or to 100u r ml of IL-1b for the indicated periods of time.Cell media were then collected and used to entice migration of fluorescently labeled allogenic neutrophils as de-scribed in Section 2.Each bar represents the mean "S.D.of six replicate wells in one of at least three experiments yielding similar results.?Asterisks indicate a significant difference p -0.01;ANOVA followed .by the Fisher’s PLSD multiple comparisons from corresponding control levels.

()

W.Zhang et al.r Journal of Neuroimmunology1011999148–160

157

?.?.

Fig.8.Effects of anti-IL-8antibody IL-8Ab on neutrophil chemotaxis induced by media conditioned by ischemic HCEC or FHAS A,or by ?.?.

recombinant human IL-8B.HCEC and FHAS were exposed to4-h ischemia and24-h recovery I4r R24,and cell media conditioned by control cells for ?.?.

28h open bars and ischemic cells closed bars were collected and divided into two aliquots;one of the aliquots was pre-treated with the indicated ?.?.

concentration of anti-IL-8Ab for1h black bars.Non-treated and anti-IL-8Ab-pretreated cell media aliquots A,as well as aliquots of Kreb’s solution

containing indicated concentration of IL-8or IL-8pre-incubated for30min in the presence of anti-IL-8Ab B were used to entice migration of allogenic neutrophils as described in Section2.Each bar represents the mean"S.D.of six replicate wells in one of at least three experiments yielding similar

?.?results.Asterisks indicate a significant difference p-0.01;ANOVA from corresponding control levels.a Indicates a significant difference p-0.01;

ANOVA followed by the Fisher’s PLSD multiple comparisons from either ischemia r recovery or IL-8alone.

chemoattractant released in media of ischemic or IL-1b-exposed HCEC.

4.Discussion

This study provides the evidence that two human brain cell types closely associated with the BBB,cerebral en-

?.?.

dothelial cells HCEC and astrocytes FHAS,are poten-tially important sources of neutrophil chemoattractants in brain ischemia.The study shows that in vitro ischemia ?.

stimulates a the expression of ICAM-1in cultured HCEC,?.?.

b neutrophil adhesion to HCEC,

c the expression r secretion of chemokines IL-8an

d MCP-1in both HCEC

and FHAS,and d the release of possibly other bioactive neutrophil chemoattractants from both HCEC and FHAS. Whereas FHAS were the principal source of MCP-1,HCEC were shown to respond more vigorously to IL-1b than FHAS.

Cytokine-induced release of IL-8Brown et al.,1994;

.?. Kaplanski et al.,1997and MCP-1Parry et al.,1998 from human peripheral endothelial cells,as well as from ?.?. rat Gourmala et al.,1997and human Aloisi et al.,1995

?. astrocytes and microglia Ehrlich et al.,1998have previ-ously been described.The induction of IL-8expression has also been shown in HUVEC subjected to hypoxia ?.

Karakurum et al.,1994.This study shows for the first time that simulated in vitro ischemia r recovery induces the expression r secretion of IL-8and MCP-1in both HCEC and FHAS.Ischemic induction of IL-8and MCP-1mRNA expression in HCEC and FHAS may be due to either increased transcription and r or mRNA stabilization.Acti-

vation of nuclear factor k B NF-k B by ischemia may play a role in ischemic induction of pro-inflammatory chemokines,since NF-kB has been shown to mediate the transcription of both cytokines and adhesion molecules in

other cell types Collins et al.,1995and to be activated by

?. hypoxia and oxidative stress Collins et al.,1995.Alterna-tively and r or concomitantly,ischemia can induce secre-tion r release of factors that cause activation of cells through autocrine mechanisms.For example,hypoxia has been shown to induce IL-1expression r release from peripheral

?. human endothelial cells Schreenewas et al.,1992,as well as the IL-1-dependent release of nitric oxide and prosta-

?. glandins from human astrocytes Mollace et al.,1997.

Both direct and indirect autocrine and paracrine mecha-nisms of CEC activation are likely important in cerebral ischemia in vivo.For example,elevated messages r levels

of pro-inflammatory cytokines IL-1b Feuerstein et al.,

1994;Arvin et al.,1996and TNF a Fan et al.,1995;Betz .

et al.,1996have been shown in the brain after both focal and global ischemia in animals,as well as in the cere-

?. brospinal fluid of stroke patients Tarkowski et al.,1997. The most likely source of these cytokines are astrocytes and microglia,since both cell types readily produce and

?. respond to cytokines in vitro Aloisi et al.,1995and show morphologic and immunocytochemical evidence of activa-

tion in ischemia in vivo Ridet et al.,1997.Therefore,in

()

W.Zhang et al.r Journal of Neuroimmunology1011999148–160 158

addition to ischemia itself,the glial-derived cytokines are probable triggers of CEC activation,since rat CEC have

been shown to express receptors for cytokines Van Dam .

et al.,1996,and HCEC have been demonstrated to re-spond to cytokines IL-1b and TNF a by a pronounced up-regulation of adhesion molecules ICAM-1,VCAM-1

and E-selectin McCarron et al.,1995;Stanimirovic et al., .

1997a,b.Cytokines have also been shown to facilitate neutrophil adhesion to HCEC monolayers in vitro ?.

Stanimirovic et al.,1997a,and to stimulate secretion of various vasoactive and pro-inflammatory mediators from

?. HCEC,including prostaglandins Stanimirovic et al.,1993

and endothelins Skopal et al.,1998.In agreement with these observations,IL-1b proved to be a potent stimulator of ICAM-1,IL-8and MCP-1expression in HCEC in this study.Whereas IL-1b and ischemia induced quantitatively similar responses in FHAS,the effects of IL-1b on IL-8 and MCP-1release from HCEC highly surpassed those induced by ischemia,suggesting that HCEC are highly responsive cellular target for cytokines.

Temporal profiles of IL-8and MCP-1stimulation by transient simulated in vitro ischemia in HCEC and FHAS detected in this study were remarkably similar to those seen in animal models of cerebral ischemia and corre-sponded well to the time-course of cellular infiltration into the ischemic brain in vivo.For example,IL-8mRNA in FHAS was transiently increased4–16h after ischemia, whereas IL-8increases in vivo have been detected at6h

of reperfusion in the ischemic rabbit brain Matsumoto et .

al.,1997.Similarly,elevated MCP-1mRNA was seen throughout the recovery period up to24h in both HCEC and FHAS,and was also detected at6,24and48h in the rat brain after the onset of the middle cerebral artery ?.?.

occlusion MCAO Kim et al.,1995.In another study, MCP-1mRNA expression in rat ischemic cortex peaked at

24h after either permanent or transient MCAO Wang et .

al.,1995.Parenthetically,polymorphonuclear leukocytes ?.

PMN were shown to first appear in the brain4h after MCAO,peak at48–72h and decrease thereafter,whereas macrophages,some of which originate from the circulating blood,infiltrate somewhat later than PMNs and tend to

persist longer in the ischemic brain Barone et al.,1991;

Feuerstein et al.,1994.

Biological activity of neutrophil chemoattractants re-leased in media of HCEC and FHAS subjected to either ischemia or IL-1b and measured by their ability to elicit chemotaxis of allogenic neutrophils were not entirely at-tributable to the presence of IL-8in these media.For example,whereas IL-8antibody inhibited a major portion of the IL-1b-stimulated neutrophil chemoattractants in HCEC,it neutralized only50%of chemotactic activity in FHAS media.Furthermore,only40%–60%of the is-chemia-stimulated chemotactic activity in media of both HCEC and FHAS was eliminated by IL-8antibody,sug-gesting that,in addition to IL-8,ischemic HCEC and FHAS release other bioactive neutrophil chemoattractants.The nature of these chemoattractants is presently unknown, but may include other members of the a-family,such as MIP-2and IP-10,both shown to increase in the brain

following injury Hausmann et al.,1998,or human‘gro’family,murine equivalents of which were shown to be

up-regulated in the rat brain after MCAO Liu et al., .

1993.

The exact mechanisms by which neutrophils transmi-grate across the BBB in ischemia and inflammation are poorly understood.The firm,ICAM-1-upheld adhesion of neutrophils to CEC has been considered a seminal step in ?.

this process Arvin et al.,1996;Ransohoff,1997.Other studies have demonstrated that endothelial cell surface

presentation of chemokines Glabinski et al.,1995;Baggi-.

olini,1998and the proximity of presented chemokines to ?.

target cells Baggiolini,1998play crucial roles in trigger-ing molecular changes in target cells necessary for chemo-taxis to occur,such as the expression of integrins and

?. cytoskeletal rearrangements DelPozo et al.,1996.More-over,elaboration of chemokines by sentinel cells may be responsible for inducing r sustaining strong adhesive inter-

?actions between rolling leukocytes and endothelium Bag-.

giolini,1998.Therefore,it is plausible to suggest that the expression r presentation of chemokines including IL-8and MCP-1by HCEC is a decisive event in initiating leukocyte transmigration across the BBB.This study further suggests that HCEC and FHAS likely cooperate in attracting, marginalizing and mobilizing neutrophils and monocytes into the brain parenchyma during cerebral ischemia.There-fore,chemokines released r presented at the BBB may represent attractive and accessible therapeutic targets to attenuate post-ischemic brain inflammation.

Acknowledgements

This study is supported by the Astra Canadian Neuro-

?.?. protection Network ACNN and by a grant a ST2718 from the Heart and Stroke Foundation of Ontario.We thank Dr.Edith Hamel,and Dr.Voon Wee Yong and Dr. Jack Antel of Montreal Neurological Institute for provid-ing human temporal lobe biopsies and primary cultures of fetal human astrocytes,respectively.We gratefully ac-knowledge Mrs.Rita Ball’s expert technical assistance with human cerebral endothelial cell cultures.

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