Patent Application: US-62347203-A

Abstract:
the invention relates to the fields of inflammation and immunology and , more specifically , to the field of chemokines and receptors therefor , and their role in neurodegenerative or neuroinflammatory disease . the invention provides a method for identifying a candidate drug compound for the treatment of inflammatory or degenerative brain disease comprising testing the compound for its capacity to modulate or mimic mcp - 1 binding with a chemokine receptor capable of being expressed on brain glial cells , the receptor known in the mouse as l - ccr or in humans as cram - b .

Description:
isoflurane ( forene ™) from abbott ( baar , switzerland ); dulbeccos modified eagle medium from gibcobrl life technologies ( breda , netherlands ); ta vectors pcr2 . 1 and pcrii from invitrogen ( leek , netherlands ); digoxigenin - conjugated utp and alkaline phosphatase conjugated sheep - anti - digoxigenin from boehringer mannheim ( mannheim , germany ); recombinant mouse chemokines from pepro tech ec ltd . ( london , united kingdom ); antibodies for gfap , ed - 1 and mac - 1 from chemicon ( temecula , usa ); fura - 2 am and all other chemicals from sigma - aldrich ( bornhem , belgium ). for treatment with endotoxin , five week old cd - 1 mice were injected intraperitoneally with lps ( 50 μg / 25 g weight ) dissolved in sterile saline solution . control animals received injections with 0 . 9 % nacl alone . at different time points after the injection , animals were decapitated under isoflurane anesthesia ( five animals per time point , three for rna preparation and two for in - situ hybridization ) and brains were removed . brains were lysed in gtc solution for rna preparation and fixed with zamboni &# 39 ; s fixative by perfusion fixation for in - situ hybridization experiments . both raw 264 . 7 and hek 293 cells were maintained in dmem containing 10 % fetal calf serum with 0 . 01 % penicillin and 0 . 01 % streptomycin in a humidified atmosphere ( 5 % co 2 ) at 37 ° c . mixed astrocyte cell cultures were established as described previously ( 28 ). in brief , mouse cortex was dissected from newborn mouse pups (& lt ; 1 d ). brain tissue was gently dissociated by trituration in phosphate - buffered saline and filtered through a cell strainer ( 70 μm ø , falcon ) in dmem . after two washing steps ( 200 × g for 10 minutes ), cells were seeded in culture dishes ( nunc , 10 cm ø ) ( 8 × 10 6 cells / dish ). cultures were maintained 6 weeks in dmem containing 10 % fetal calf serum with 0 . 01 % penicillin and 0 . 01 % streptomycin in a humidified atmosphere ( 5 % co 2 ) at 37 ° c . culture medium was changed the second day after preparation and every six days thereafter . microglia cultures were established as described previously ( 29 ). in brief , floating microglia were harvested from confluently mixed glial cultures and plated on new culture dishes . microglia cultures were pure (& gt ; 95 %) as tested by cell - specific markers ( ed - 1 and mac - 1 ). for calcium measurements , cells were seeded on glass coverslips . for chemotaxis assays , cultured microglia were left in suspension . cells and brain material were lysed in guanidinium isothiocyanate / mercaptoethanol ( gtc ) buffer and total rna was extracted with slight modifications according to chomczynski and sacchi ( 30 ). a ) reverse transcription : 1 μg of total rna was transcribed into cdna as described ( 28 ). potential contaminations by genomic dna were checked for by running the reactions ( 35 cycles ) without reverse transcriptase and using gapdh primers in subsequent pcr amplifications . only rna samples which showed no bands after that procedure were used for further investigation . b ) polymerase chain reaction : 2 μl of the rt - reaction were used in subsequent pcr amplification as described ( 28 ). see table 1 for primer sequences , cycle numbers and annealing temperature . identification of all pcr products were checked by cloning into pcr2 . 1 ( invitrogen ) and subsequent sequencing . primers to amplify the full - length sequence for mouse ccr12 have been chosen according to the sequence for l - ccr ( accession number : ab009384 ). the full - length mouse ccr12 coding sequence was amplified from cdna derived from lps - stimulated microglia with the following primers : forward , 5 ′- tatcaagcaacctgcctcaa ( seq id . no : ______ ); backward 5 ′- tggcataaaacaatgtgaagaga ( seq id no : ______ ). sequence similarity searches using the mouse ccr12 sequence and human databases gave high homology of mouse ccr12 with the human orphan chemokine receptor cram - b ( accession number : af015525 ). the following primers were designed to get the full - length sequence for the human ccr12 . forward , 5 ′- cccagtgggcagtctgaa ( seq id no : ______ ; backward , 5 ′- cttgcatttggtggatgcta ( seq id no : ______ . the resulting pcr products were cloned in pcr2 . 1 ( invitrogen ) for sequencing and subcloned into the bamhi - not i sites of pcdna 3 . 1 ( invitrogen ) for transfection . 1 μg of the plasmid was transfected with 6 μl fugene ( roche molecular biochemicals ) in hek 293 cells according to the manufacturer &# 39 ; s instructions . stably transfected cells were selected with g418 500 μg / ml for approximately two weeks and the resulting cell clones were checked by rt - pcr for ccr12 mrna expression . paired alignment of the mouse ccr12 with other ccrs was performed using the alignment tool clustalw at european bioinformatics institute ( ebi ), homepage http :// www . ebi . ac . uk . for calcium measurements , cells were cultured on poly - l - lysine coated glass coverslips . in order to load the cells with fura - 2 am , the cells were incubated for 30 minutes at 37 ° c . in loading buffer containing : ( in mm ) nacl 120 , hepes 5 , kcl 6 , cacl 2 2 , mgcl 1 , glucose 5 , nahco 3 22 , fura - 2 am 0 . 005 ; ph 7 . 4 . the cover - slips were fixed in a perfusion chamber ( 37 ° c .) and mounted on an inverted microscope . fluorometric measurements were done using a sensicam ccd camera supported by axolab ® 2 . 1 imaging software . digital images of the cells were obtained at an emission wavelength of 510 nm using paired exposures to 340 and 380 nm excitation wavelength sampled at a frequency of 1 hz . fluorescence values representing spatial averages from a defined pixel area were recorded on - line . increases in intracellular calcium concentrations were expressed as the 340 / 380 ratio of the emission wavelengths . compounds were administered using a pipette positioned at a distance of 100 - 300 μm from the cells . cell migration in response to chemokines was assessed using a 48 - well chemotaxis microchamber ( neuroprobe ). chemokine stock solutions were prepared in pbs and further diluted in medium for use in the assay . culture medium without chemokines served as a control in the assay . 27 μl of the chemoattractant solution or control medium were added to the lower wells ; lower and upper wells were separated by a polyvinylpyrrolidone - free polycarbonate filter ( 8 μm pore size ) and 50 , 000 cells per 50 μl were used in the assay . determinations were done in hexaplicate . the chamber was incubated at 37 ° c ., 5 % co 2 in a humidified atmosphere for 120 minutes . at the end of incubation , the filter was washed , fixed in methanol and stained with toluidine blue . migrated cells were counted with a scored eyepiece ( 3 fields ( 1 mm 2 ) per well ) and migrated cells per chamber were calculated . the data are presented as mean values ± s . d . and were analyzed by students t - test . p values ≦ 0 . 01 were considered significant . immunohistochemistry and in situ hybridization were carried out as described ( 31 ). in brief , prior to immunohistochemical processing and between the incubation steps , the sections were washed in 0 . 9 % saline dissolved in 0 . 05 m tris , ph 7 . 4 ( tbs ). all antisera were diluted in tbs containing 0 . 3 % triton x - 100 , 1 % bovine serum albumin ( bsa ) and heparin ( 5 mg / ml ). sections were preincubated in 5 % bsa in tbs for 30 minutes and incubated overnight with gfap and ed - 1 . antibody - antigen reactions were detected using the biotin - streptavidin method and the complex was visualized with diaminobenzidine ( dab )/ h 2 o 2 . in the case of fluorescence detection , fitc - conjugated streptavidin was used to visualize the antibody - antigen complex . for in situ hybridization , ccr12 pcr product was cloned into the dual promoter pcr ii vector and linearized . ccr12 sense and antisense probes were synthesized by run - off transcription and the use of digoxigenin - conjugated utp according to the manufacturer &# 39 ; s protocol ( boehringer mannheim ). slides were rinsed in pbs and digested with 10 μg / ml proteinase k for 0 . 5 hour at 37 ° c . subsequently , sections were rinsed in 2 × ssc ( 1 × ssc : 150 mm nacl , 15 mm na citrate ), dehydrated in an ethanol series and dried . sections were hybridized overnight at 60 ° c . in a solution containing 50 % formamide , 0 . 3 m nacl , 10 mm tris ( ph 8 . 0 ), 1 mm edta , 0 . 05 % trna , 1 × denhardt &# 39 ; s solution and 10 % dextran sulfate . final probe concentrations in the hybridization buffer were 1 - 5 ng / μl . after hybridization , the sections were treated with 10 μg / ml of ribonuclease a for 0 . 5 hour at 37 ° c . and washed in 0 . 1 × ssc at 65 ° c . the immunological detection of the digoxigenin - labeled rna - rna complex was preceded by a 0 . 5 hour pre - incubation at room temperature in 0 . 1 m tris , 0 . 15 m nacl , ph 7 . 5 ( buffer 1 ), containing 5 % bsa . slides were incubated for two hours at room temperature with the alkaline phosphatase conjugated sheep - anti - digoxigenin , diluted 1 : 500 in buffer 1 , containing 2 % bsa . after thorough rinsing in buffer 1 and a ten minute pre - incubation in an alkaline buffer solution ( abs : 0 . 1 m tris , 0 . 1 m nacl , 0 . 05 m mgcl 2 , 6h 2 o , ph 9 . 5 ), the alkaline phosphatase was revealed with a freshly prepared solution of 0 . 34 mg / ml nitroblue tetrazoleum and 0 . 17 mg / ml 5 - bromo - 4 - chloro - 3 indolyl phosphate in abs . endogenous non - intestinal phosphatase activity was inhibited by the addition of levamisole ( 0 . 24 mg / ml ) to the staining solution . the color development was done overnight and terminated by placing the slides in a buffer solution , consisting of 0 . 01 m tris , 1 mm edta , ph 8 . 5 . the dark purple precipitate indicating the presence of hybridized mrna was revealed with bright - field microscopy . control experiments included hybridization with digoxigenin - labeled sense probes and hybridization after treatment of the sections with rnase . the expression of an orphan lps - inducible chemokine receptor ( l - ccr ) in the mouse macrophage cell line raw 264 . 7 was previously described by shimada et al . ( 27 ). later , we came to designate l - ccr as ccr12 , and we use this term hereinafter for the sake of convenience . in order to investigate possible mrna expression of this receptor in other cell types , we designed primers for rt - pcr experiments and validated the primers using cdna derived from raw 264 . 7 cells . results similar to those described by shimada et al . ( 1998 ) were found ; mrna for ccr12 was strongly up - regulated in raw cells by stimulation with lps ( 100 ng / 2 hours ) ( fig1 ). using rt - pcr analysis ( 35 cycles ), no other mrna encoding mouse ccrs ( ccr1 - 8 and d6 ) was detected in cdna derived from control or lps - stimulated raw 264 . 7 cells . this indicates that ccr12 is the only β - chemokine receptor in these cells . genomic mouse dna served as a positive control for the primers ( ccr1 - 8 and d6 ) used . in cultured mouse microglia , mrna for ccr1 , 3 and 5 was detected ( fig2 a ). no mrna for ccrs 2 , 4 , 6 , 7 , 8 and d6 was found in these cells ( 35 cycles rt - pcr ) ( data not shown ). untreated microglia did show basal expression levels for ccr12 mrna and this expression was up - regulated by two hours stimulation with 100 ng / ml lps ( fig2 b ). similar but less pronounced effects were found after two hours stimulation with 10 and 1 ng / ml lps ( data not shown ). lps induction of ccr12 mrna in cultured microglia peaked at two hours and declined to baseline expression after eight hours ( data not shown ). using rt - pcr , mrna expression for ccr1 and 5 was detected in cultured astrocytes ( fig2 c ). no other ccr mrna ( 2 , 3 , 4 , 6 , 7 , 8 and d6 ) was found in these cells ( 35 cycles rt - pcr ) ( data not shown ). similar to microglia , untreated cultured astrocytes showed basal mrna expression for ccr12 , which also was up - regulated after two hours stimulation with lps ( 100 ng / ml ) ( fig2 d ). treatment with 1 and 10 ng / ml lps had a similar but less pronounced effect ( data not shown ). in cultured astrocytes , a comparable time dependency for the lps effects was detected as it was found for cultured microglia ( data not shown ). no ccr12 mrna expression was detected in cdna derived from cultured cortical neurons ( data not shown ). in order to verify the results obtained with rt - pcr , in situ hybridization was combined with immune histochemistry . mixed glial cultures were stimulated for two hours with lps ( 100 ng / ml ) and stained with ed - 1 and gfap to detect microglia and astrocytes respectively . ed - 1 - positive microglia ( brown reaction product ) showed a positive signal for ccr12 in situ hybridization ( purple reaction product ) ( fig4 a ). note that an in situ signal is also visible in ed - 1 - negative cells , which might be , in this case , an astrocyte ( arrowhead ) ( fig4 a ). ccr12 - positive astrocytes ( purple reaction product ) ( arrows ) became visible by staining with gfap ( brown reaction product ) ( fig4 b ). note that also gfap - negative cells are in situ positive , which in this case is most likely a microglial cell ( arrowhead ) ( fig4 b ). mice were injected intraperitonally with lps ( 50 μg / 25 g weight ) or with 0 . 9 % nacl and brains were removed after 2 , 4 , 8 , 12 and 24 hours for rt - pcr analysis or in situ hybridization . injection with a control nacl solution did not affect expression of ccr12 mrna in brain tissue ( data not shown ). in contrast , injection of lps induced the expression of ccr11 mrna 2 , 4 and 8 hours after the injection . twelve hours after the injection of lps , ccr12 mrna expression returned to baseline levels ( fig3 ). these results were verified by in situ hybridization experiments . no ccr12 mrna - positive cells were found in control brains ( fig5 a ). two hours after injection of lps , many ccr12 - positive cells were observed in the cortex of the lps - treated mice ( fig5 b ). twenty - four hours after the injection , the ccr12 in situ hybridization signal returned to control levels ( fig5 c ). combinations of in situ hybridization ( purple reaction product ) ( fig5 d ) and immuno - histochemistry ( gfap fluorescence ) ( fig5 e ) revealed that gfap - positive astrocytes express ccr12 mrna in mouse cortex ( see fig5 f for overlay of sd and e ). for technical reasons , it was not possible to colocalize ccr12 mrna with microglial markers in vivo . since ccr12 mrna - positive and gfap - negative cells were found in the brain , it is suggested that there are cell types different from astrocytes expressing ccr12 mrna , possibly microglia as observed in cell culture studies . effect of mcp - 1 and rantes ( ccl 5 ) on chemotaxis and calcium signaling of raw 264 . 7 cells in order to find possible chemokine ligands for ccr12 , chemotactic activity and mobilization of intracellular calcium were determined in lps - treated raw cells . mcp - 1 induced concentration - dependent chemotaxis of raw cells with an ec50 value of approximately 0 . 1 nm ( fig6 a ). similar results were obtained using rantes , which was less potent with an ec50 value of approximately 1 nm ( fig6 a ). the cc chemokine mip - 1α ( ccl3 ) did not induce chemotaxis in raw cells ( data not shown ). both chemokines rantes and mcp - 1 were also found to induce intracellular calcium transients in raw cells ( fig6 b ). in order to further investigate its agonist responsivity , we cloned mouse ccr12 from lps - treated microglia and subsequently the receptor was expressed in hek 293 cells . sequencing of the glial ccr12 revealed 99 % identity with the sequence previously published for the orphan receptor ( 27 ). mok - transfected hek cells did not migrate towards a chemotactic gradient of mcp - 1 , whereas ccr12 - transfected hek cells &# 39 ; concentration dependently migrated in response to mcp - 1 ( fig7 a ). moreover , mcp - 1 induced intracellular calcium transients in ccr12 - transfected hek cells ( fig7 b ). among several other chemokines found in the brain ( rantes , fractalkine ( cx3cl1 ), mip - 1α , mip - 1β ( ccl4 ), mip - 3α ( ccl20 ), ip - 10 ( cxcl10 ), mcp - 2 ( ccl8 ), mcp - 3 ( ccl7 ) and slc ( ccl21 )), only rantes , mcp - 2 and mcp - 3 were found to induce chemotaxis of ccr12 - transfected hek cells ( table 3 ). in a set of preliminary experiments , we performed chemotaxis assays with hek cells expressing the human ccr12 and verified that mcp - 1 is also a chemokine ligand for human ccr12 ( data not shown ). stimulation of chemotaxis of cultured mouse astrocytes by mcp - 1 has already been shown by heesen et al . ( 1996 ). effects of mcp - 1 on cultured microglia were only shown so far for rat microglia ( 20 , 22 ) and fetal human microglia ( 23 ) but not for mouse microglia . we , therefore , determined the effects of mcp - 1 on intracellular calcium transients and chemotaxis of cultured mouse microglia . similar to microglia from other species , 10 nm mcp - 1 - induced chemotaxis of cultured mouse microglia : migration of untreated cells , 29 ± 13 ( cells / mm 2 ), migration of cells stimulated with 10 nm mcp - 1 170 ± 42 ( cells / mm 2 ) ( n = 4 ). chemotaxis was determined as described in materials and methods . moreover , intracellular calcium transients in cultured microglia were observed upon stimulation with mcp - 1 ( data not shown ). [ 0078 ] table 2 comparison of ccr12 with all other cloned mouse ccrs by nucleic acid sequence alignment beta chemokine percentage id after alignment receptor ( mouse ) accession number with glial ccr12 ccr1 u28404 52 . 8 ccr2 u51717 49 . 6 ccr3 u29677 54 . 3 ccr4 x90862 51 . 5 ccr5 u83327 53 . 1 ccr6 ab009369 51 . 6 ccr7 l31580 50 . 2 ccr8 z98206 56 . 6 ccr9 aj132336 49 . 1 ccr10 af215982 / af215983 48 . 0 d6 y12879 50 . 0 [ 0079 ] table 3 effect of various chemokines on chemotaxis of ccr12 - transfected hek 293 cells chemotactic effect on ccr12 - transfected hek chemokine ( 100 nm ) cells rantes + mcp - 1 + mcp - 2 + mcp - 3 + mip - 1 α − mip - 1β − mip - 3 α − fractalkine − ip - 10 − slc − [ 0080 ] table 4 expression profile of ccr mrna in cultured glial cells from human ( h ), mouse ( m ) and rat ( r ). astrocytes microglia h m r h m r publication ccr1 + 19 + 20 − + 32 ccr2 − 24 + 20 − 18 − − + 32 ccr3 − − 18 − 20 + 33 − + 32 ccr4 − 19 − 20 − − 32 ccr5 − + 18 + 20 + 33 − + 32 c57bl6 / j mice were obtained from jackson laboratories and housed in groups of four , with free access to food and water . young adult mice ( 8 - 12 weeks ) were used . recombinant myelin oligodendrocyte glycoprotein ( mog ) ( residues 35 - 55 ), obtained from s . amor from the bprc at tno in rijswijk , was used as antigen . immunization of the animals was done with 200 μg mog peptide , added to pbs , emulsified by sonication for ten minutes at room temperature , in incomplete freund &# 39 ; s adjuvant ( ifa ) supplemented with 60 μg of mycobacterium . the mice were injected subcutaneously on days 0 and 7 at two sites on the back . in addition , the mice were also given i . p . 200 ng of pertussis toxin dissolved in phosphate - buffered saline ( pbs ). the pertussis toxin was given immediately and 24 hours later after immunization with the antigen in complete freund &# 39 ; s adjuvant ( cfa ). the scoring and weighing of the animals started on day eight . grade zero meant there were no symptoms at all . at grade one the tail was paralyzed . grade two was reached when the righting reflex was impaired . grade three was reached when one of the hind limbs was paralyzed . grade four was given when both hind limbs were paralyzed . grade five is a moribund state in which all limbs are completely paralyzed . intermediate scores of 0 . 5 were given . we terminated the mice at grade 3 . 0 . after the termination of the mice , using isoflurane as anesthetic , the spinal cord and the brain were taken out and frozen in liquid nitrogen for rna analysis . for in situ hybridization , the mice were perfused with 4 % paraformaldehyde ( pfa ) in pbs and consequently the spinal cord and brain were taken out and put in a fixative . the tissue was embedded in tissuetek , frozen and cut with a reichert - jung frigocut 2800 microtome . sections were caught on double - coated glasses . using in situ hybridization assays , a strong increase of the ccr11 chemokine receptor was observed during eae . this shows that the receptor is involved in ms pathology . accordingly , the antagonists of ccr11 are of use in ms therapy . testing chronic obstructive pulmonary disease ( copd ) in mice mice and in vivo procedures two weeks prior to the experiment , 8 - 10 week old mice c57bi / 6 - j were purchased from harlan . for sensitization ( day 0 ), 24 mice were injected intraperitoneally with ovalbumine ( ova ) 0 . 1 mg / mouse in phosphor - buffered saline ( pbs ). for induction of allergic response ( day 8 ), 18 mice were challenged for five minutes with a 2 % ova aerosol in pbs . for control experiments , six mice were challenged for five minutes with pbs . for allergen provocation ( day 15 ), 18 mice were challenged for 20 minutes with a 1 % ova in pbs aerosol , six mice were challenged for 20 minutes with pbs ( control ). respectively 1 , 3 and 6 hours after ova challenge and three hours after pbs challenge ( control ), three mice were terminated . both lungs of all challenged mice were isolated and immediately placed in liquid nitrogen . on days 16 , 17 and 18 , mice were repetitively challenged for 20 minutes with 1 % ova ( n = 9 ) and pbs ( n = 3 , control ). on day 18 , respectively 1 , 3 and 6 hours after ova challenge and three hours after pbs challenge ( control ), three mice were terminated . lungs were isolated and immediately placed in liquid nitrogen . all challenge protocols were performed in a specially designed perspex cage with an internal volume of 9 l . a summary of the challenge protocol is given in fig9 . rna was isolated from mouse lung according to chomczynski and sacchi ( 1987 ) and reverse transcribed into cdna in a final volume of 20 μl containing 1 μg rna , 10 μl of h 2 o , 1 μl of an oligo -( dt ) adapter antisense primer ( ap ), 2 μl of 10 × pcr - buffer , 2 μl of 25 mm mgcl 2 , 1 μl of 10 mm dntps , 2 μl of 0 . 1 m dtt and 1 μl of superscript ii rt . after 50 minutes incubation at 42 ° c ., the reaction was terminated by heating at 70 ° c . for 15 minutes . finally , 1 μl of rnase h was added and incubated at 37 ° c . for another 20 minutes . subsequently , 1 μl of the rt reaction was amplified by , respectively , 28 thermal cycles using specific primers for mouse gapdh with a primer annealing temperature of 60 ° c ., 30 thermal cycles with specific primers for mouse ccr2 ( primer annealing at 56 ° c . ), 30 thermal cycles with specific primers for mouse ccr11 ( primer annealing at 56 ° c .) and 32 thermal cycles with specific primers for mouse mcp - 1 ( primer annealing at 56 ° c .) ( fig2 a , 2b ). each reaction mixture contained 1 μl of the rt reaction , 5 μl of 10 × pcr - buffer ( invitek ), 2 . 5 μl of 50 mm mgcl 2 , 0 . 5 μl of 10 mm dntps ( invitek ), 1 μl of each primer , 39 μl of h 2 o and 0 . 1 μl taq - polymerase ( invitek ). the thermal cycle consisted of one minute denaturation at 94 ° c . ; 1 . 5 minutes primer annealing and one minute amplification at 72 ° c . pcr was terminated by another seven minutes of extension at 72 ° c . pcr products were size fractioned on a 1 . 5 % agarose gel . compared to the control , mcp - 1 mrna expression is increased at respectively 1 ( n — 3 ), 3 ( n = 3 ) and 6 ( n = 3 ) hours after allergen provocation . ccr11 mrna expression seems to be slightly increased at one hour ( n = 3 ) after allergen provocation . ccr2 mrna expression seems to be stable . gapdh mrna expression was comparable in all mouse lung tissues ( fig1 ). day 18 , after 4 days of repetitive allergen provocation with 1 % ova compared to control , mcp - 1 , ccr11 and ccr2 mrna expression is increased at respectively 1 ( n = 3 ), 3 ( n = 3 ) and 6 ( n = 3 ) hours after allergen challenge . gapdh mrna expression was comparable in all mouse lung tissues ( fig1 ). these data show a clear involvement of ccr11 in the copd mouse model and shows a role of ccr11 antagonists in the treatment of obstructive airway diseases such as asthma . 3 . murphy , p . m ., m . baggiolini , i . f . charo , c . a . hebert , r . horuk , k . matsushima , l . h . miller , j . j . oppenheim and c . a . power . 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