CXCL12-γ expression is inhibited in neuroinflammation

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nCorrespondence142 Despota Stefan

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Research Report

CXCL12-γ expression is inhibitedin neuroinflammation

Gordana Timotijevica, Filip Petkovicb, Jana Blazevskib, Miljana Momcilovicb,Marija Mostarica Stojkovicc, Djordje Miljkovicb,n

aInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, SerbiabDepartment of Immunology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, SerbiacInstitute for Microbiology and Immunology, School of Medicine, University of Belgrade, Serbia

a r t i c l e i n f o

Article history:

Accepted 30 April 2013

CXCL12 plays a protective role in CNS autoimmunity. Expression of CXCL12-γ, which has

distinct structural and functional properties than the other isoforms of CXCL12, was

Keywords:

CXCL12

Experimental autoimmune

encephalomyelitis

Astrocyte

Micro-blood vessel

Nitric oxide

nt matter & 2013 Publish.1016/j.brainres.2013.04.05

to: Department of Immua Street, 11000 [email protected] (

ticle as: Timotijević, G.,10.1016/j.brainres.2013.

a b s t r a c t

determined in spinal cords of rats immunized to develop experimental autoimmune

encephalomyelitis (EAE). CNS expression of CXCL12-γ was markedly lower in EAE-prone

Dark Agouti rats than in EAE-resistant Albino Oxford rats, both in spinal cord homogenates

and micro-blood vessels isolated from spinal cords. Inhibition of nitric oxide (NO) synthesis

in DA rats upregulated, while donation of NO in AO rats downregulated CNS expression of

CXCL12-γ. NO inhibited CXCL12-γ expression in astrocytes in vitro. A splice variant of

CXCL12-γ which migrates into nucleolus was not detected in spinal cord or astrocytes.

Thus, CXCL12-γ is expressed in the CNS after EAE induction, but its expression is markedly

suppressed in spinal cord affected with full blown inflammation. NO is an important

regulator of CXCL12-γ expression in neuroinflammation.

& 2013 Published by Elsevier B.V.

ed by Elsevier B.V.6

nology, Institute for Biological Research “Siniša Stanković”, University of Belgrade,Serbia. Fax: +381 11 2761433.D. Miljković).

et al., CXCL12-γ expression is inhibited in neuroinflammation. Brain Research (2013),04.056

1. Introduction

Experimental autoimmune encephalomyelitis (EAE) is ananimal model of multiple sclerosis (MS), an inflammatory,demyelinating and neurodegenerative disease of the CNS.Pathogenesis of EAE and MS includes a strong autoimmunecomponent. It has been proposed that CD4+ T cells (helper Tcells—Th) that produce interferon (IFN)-γ or interleukin (IL)-17, i.e. Th1 and Th17 cells have major pathogenic role ininitiation and propagation of CNS autoimmunity (El-behiet al., 2010; Petermann and Korn, 2011). In order to perform

their encephalitogenic actions Th cells have to migrate intothe CNS parenchyma. It has been shown that CXCL12 has aprominent role in inhibition of CNS invasion by immune cells(reviewed in Momcilović et al., 2012). CXCL12, also known asstromal cell derived factor-1 (SDF-1), is a chemokine whichhas an essential role in neural and vascular development,hematopoiesis and in immunity (reviewed in Li andRansohoff, 2008). It acts through a classic G protein–coupledtransmembrane chemokine receptor CXCR4 and throughCXCR7 which primarily functions as a scavenger of CXCL12(reviewed in Timotijevic et al., 2012). Alternative splicing of

dx.doi.org/10.1016/j.brainres.2013.04.056



mailto:[email protected]





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Fig. 1 – CXCL12-γ expression in spinal cord of AO and DA

rats. Rats were immunized with SCH+CFA and sacrificed atthe time of onset, peak or recovery of EAE in DA rats. Healthy(non-immunized) rats were also included in the experiments.Spinal cords were isolated and homogenized. RNA wasisolated from the homogenates at the indicated time points(A,C) or from MBV purified from the homogenates at the peak(B,D) and RT-PCR was performed. CXCL12-γ mRNAexpression (A,B) and ratio of CXCL12-γ mRNA expression toCXCL12 mRNA expression (C,D) are presented as mean+SD ofvalues obtained from 6 rats per group. *po0.05 representsstatistically significant difference between AO and DAsamples.*po0.05 represents statistically significant differencein comparison to healthy and peak samples in AOrats. “po0.05 represents statistically significant differencein comparison to onset and recovery samples within thestrain.

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CXCL12 gene gives rise to several isoforms, among whichCXCL12-α and CXCL12-β are considered classical isoforms.CXCL12-γ is an isoform that is special on several grounds.It has extremely high affinity for glycosaminoglycans,heparan sulfate in particular, thanks to a unique highlycationic carboxy-terminal domain that encompasses fouroverlapped BBXB heparan sulfate-binding motifs (Ruedaet al., 2008). This structural specificity makes CXCL12-γ asuperior haptotactic protein with a high potency to promotedirectional migration and selective tissue homing of cells.The other unique property of CXCL12-γ, that stems from thespecificities of its carboxy-terminal domain, is that it can betransported into nucleolus instead of being secreted extra-cellularly (Torres and Ramirez, 2009). Intranucleolar localiza-tion of CXCL12-γ was observed in heart muscle andendothelial cells, and the authors suggested that it mighthave important, although yet undefined, autocrine regulatoryfunction (Torres and Ramirez, 2009).

The interest of the present work was to determine expres-sion of CXCL12-γ in the CNS of rats immunized to developEAE. It was previously shown that high CNS CXCL12 expres-sion was important for resistance of Albino Oxford (AO) ratsto EAE and that its expression in spinal cord homogenates(SCH) and micro-blood vessels (MBV) was heavily impaired inEAE-prone Dark Agouti (DA) rats at the peak of the disease(Miljković et al., 2011). Major source of CXCL12 in the inflamedCNS are astrocytes and blood vessel endothelial cells(reviewed in Momcilović et al., 2012). Thus, the expressionof CXCL12-γ in SCH and MBV of AO and DA rats afterencephalitogenic immunization was analyzed, as well as inastrocytes in vitro. The expression of nucleolar form ofCXCL12-γ was also analyzed in the same samples. HighCXCL12-γ expression in the CNS of AO rats, and low in DArats was demonstrated. Expression of CXCL12-γ in DA rat CNSwas upregulated as a consequence of EAE suppressionimposed by aminoguanidine (AG), but not by methylpredno-silone (MP). A donor of nitric oxide sodium nitroprusside(SNP) inhibited CNS expression of CXCL12-γ in the CNS ofimmunized AO rats and also in astrocytes in vitro. Nucleolarform of CXCL12-γ in the examined samples could not bedetected.

2. Results

2.1. CXCL12-γ expression in SCH and MBV of AO and DArats immunized to develop EAE

CXCL12-γ expression was determined in SCH of rats immu-nized with SCH+CFA and in healthy (non-immunized) rats. Inresponse to the immunization, DA rats developed acutemonophasic disease, passing through typical clinical phases,including onset (9–11 dpi, cs 0.5–1), peak (12–15 dpi, cs 2–3.5)and recovery (16–20 dpi, cs 0–0.5), while AO rats remainedwithout clinically manifested symptoms throughout theobservation period. CXCL12-γ expression was similar inhealthy rats of both strains (Fig. 1A). It was increased in AOrats, but not in DA rats at the time of onset in DA rats.CXCL12-γ expression was downregulated in both strains ondays 12–15 post immunization and then increased on days

Please cite this article as: Timotijević, G., et al., CXCL12-γ expresshttp://dx.doi.org/10.1016/j.brainres.2013.04.056

16–20 post immunization. Thus, pattern of CXCL12-γ expres-sion in spinal cord homogenates was similar in AO and DArats, as it was relatively high at 9–11 dpi and 16–20 dpi andlower at 12–15 dpi (Fig. 1A). Still, at all the time pointsCXCL12-γ expression was higher in AO than in DA rats. Also,at none of the time points CXCL12-γ expression in DA ratswas higher than in samples obtained from healthy rats.Further, CXCL12-γ expression was higher in MBV isolatedfrom spinal cords of AO rats (Fig. 1B). Ratio of CXCL12-γ andtotal CXCL12 mRNA expression was also determined inhealthy and immunized rats. This ratio was similar inhealthy AO and DA rats, as well as at the time of peak inDA rats (Fig. 1C,D). However, significantly higher ratio ofCXCL12-γ to total CXCL12 mRNA expression was observedin AO rats at the time of onset and recovery of DA rats.

ion is inhibited in neuroinflammation. Brain Research (2013),




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Fig. 2 – CXCL12-γ expression in AG-, or MP-treated DA rats and in SNP-treated AO rats. DA and AO rats were immunized withSCH+CFA. Starting from the day 9 post immunization, DA rats were treated daily with AG (i.p. 400 mg/kg body weight) or MP (i.p. 50 mg/kg body weight) or vehicle (PBS) and AO rats were treated with SNP (i.p. 1 mg/kg body weight) or vehicle (PBS), until thedisease reached its peak in the group of DA rats that has been receiving PBS (day 12 post immunization). At this stage, animalswere sacrificed, SCH and MBV were obtained. RNA was isolated from SCH and MBV and RT-PCR was performed. Clinical score(A,C) is presented as mean +/−SE and CXCL12-γ gene expression (B,D,E) is shown as mean+SD of 8 (A,B), 6 (C,D) or 9 (E) rats pergroup. *po0.05 represents statistically significant difference between the treatment and PBS.

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These results clearly show that the high CNS expression ofCXCL12-γ is specific for the EAE-resistant strain of rats.

2.2. CXCL12-γ expression in the spinal cord of theimmunized DA rats treated with AG, MP or SNP

In order to determine if the expression of CXCL12-γ in DA ratCNS can be correlated to EAE severity, DA rats were immunizedwith SCH+CFA and treated with AG or MP to suppress thedisease. Both AG and MP suppressed clinical symptoms of thedisease in DA rats (Fig. 2A,C). In parallel, CXCL12-γ expressionwas elevated in SCH and MBV of AG-treated rats (Fig. 2B), butnot in SCH of MP-treated rats (Fig. 2D). Thus, it could not beconcluded that the reduction of EAE severity in DA ratscorrelated with upregulation of CXCL12-γ expression in theCNS. As AG is a selective inhibitor of inducible nitric oxidesynthase (iNOS), it was assumed that NO might impair CXCL12-γ expression in the inflamed CNS. To test this assumption,immunized AO rats were treated with a NO donor, SNP.CXCL12-γ expression in SCH of immunized AO rats wassignificantly reduced by this treatment (Fig. 2E). Taken together,the results obtained implied that NO is an important inhibitorof CXCL12-γ expression in neuroinflammation.


2.3. Cytokines stimulate and NO reduces CXCL12-γexpression in astrocytes

The next aim was to investigate if CXCL12-γ was expressed inastrocytes and to test effect of NO on this expression.Astrocytes isolated from newborn rats were stimulated withConASn or IFN-γ+IL-17. Expression of CXCL12-γ was detectedin unstimulated astrocytes and it was upregulated by bothstimuli (Fig. 3A). Thus, CXCL12-γ expression was induced inastrocytes under inflammatory conditions. CXCL12-γ expres-sion was markedly downregulated when ConASn-stimulatedastrocytes were treated with a NO donor, and SNP (Fig. 3B).This indicates that NO profoundly inhibits CXCL12-γ expres-sion in astrocytes.

2.4. Nucleolar fraction of CXCL12-γ is not detected in SCH,MBV and astrocytes

To investigate if nucleolar CXCL12-γ is expressed in inflamedrat CNS, RT–PCR with primers that specifically recognize allCXCL12-γ isoforms was performed and also with primers thatamplify only those cDNAs that have signal peptide code(CXCL12-γSP, as depicted in Fig. 4A). Importantly, onlyCXCL12-γ splice variants without signal peptide code give





-7-6-5-4-3-2-101

AOMBV

DAMBV

AOhom

DAhom

heart

CXCL12- CXCL12- SP

Fig. 4 – Detection of nucleolar CXCL12-γ in SCH and MBV of

AO and DA rats. (A) Schema of full-length CXCL12-γ cDNA(left hand side) and short splice variant CXCL12-γ cDNAwithout signal peptide sequence (right hand side) witharrows indicating primer position. CXCL12 f/r—primers thatamplify all CXCL12 isoforms; CXCL12-γSP f/r–primers thatamplify CXCL12-γ isoform with signal peptide and CXCL12-γf/r that amplify both isoform with signal peptide andalternatively processed CXCL12-γ without signal peptide.Gray rectangle indicates signal peptide sequence and blackrectangle assigns gamma specific C-terminus enclosingnucleolar localization signal. (B) Rats were immunized withSCH+CFA and sacrificed at the time of EAE peak in DA rats.Spinal cords were isolated, SCH prepared and MBV isolated.Also homogenates of adult rat heart were obtained. RNA wasisolated from these samples and RT–PCR was performed.Results are presented as mean+SD obtained from 6 rats pergroup, except for heart, which is obtained from one rat andtherefore presented as mean+SD of two homogenatesamples.

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Fig. 3 – CXCL12-γ expression in astrocytes. Rat astrocytes(1.5�105/ml) were cultivated in the absence (Ctrl) or presenceof ConASn (20%) or IFN-γ (10 ng/ml)+IL-17 (50 ng/ml) for 24 h(A). Also, they were treated with ConASn in the absence orpresence of SNP (200 μM) for 24 h (B). Subsequently, RNA wasisolated and RT–PCR was performed. Results from threeindependent experiments are presented as mean+SD.*po0.05 represents statistically significant difference tovalues obtained in Ctrl.

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rise to nucleolar CXCL12-γ. Thus, the difference betweenexpression of mRNA amplified with CXCL12-γ primers andCXCL12-γSP primers indicates that there is nucleolar CXCL12-γ present in a sample. However, such difference was notdetected in SCH and MBV of AO and DA rats at the peak ofEAE (Fig. 4B). It was also not observed in SCH at the onset orthe recovery, or in unstimulated or ConASn or IFN-γ+IL-17-stimulated astrocytes (data not shown). In order to verify thatthe methodology for detection of nucleolar CXCL12-γ wasoperative, it was applied on the heart homogenate samples.As a result, much higher expression of CXCL12-γ in compar-ison to CXCL12-γSP was detected (Fig. 4B), thus indicating thatthere was high nucleolar CXCL12-γ expression in this tissueand that our methodology was appropriate for its detection.This is simple and highly efficient methodology that can beused easily for screening of nucleolar CXCL12-γ in othertissues and cell types.

3. Discussion

CXCL12-γ is a fascinating chemokine for at least two func-tional properties. Both of these properties are dependent onits structural specificity contained in C-terminal region. First,in comparison to other chemokines it has superior bindingaffinity to glycosaminoglycans and therefore predominantlyacts as glycosaminoglycane-bound molecule and not as asoluble molecule. This property makes it a perfect candidatefor generation of haptotactic gradients (Rueda et al., 2008).Second, it is the only known chemokine that can be localizedwithin the nucleus, or even more specifically in the nucleolus(Torres and Ramirez, 2009). These two characteristics indicatethat CXCL12-γ might perform functions distinct to CXCL12-αand CXCL12-β. In line with such an assumption, the


difference in the expression of total CXCL12 and CXCL12-γin inflamed rat CNS were observed. While total CXCL12expression in AO rats had the highest value at the time ofEAE peak in DA rats (Miljković et al., 2011), CXCL12-γ expres-sion was higher at the onset and at the recovery than at thepeak. This implies that CXCL12-γ might have distinct role inneuroinflammation in comparison to other CXCL12 isoforms.Still, all CXCL12 isoforms act through two common receptors,CXCR4 and CXCR7 (Momcilović et al., 2012), although theiraffinity for receptor binding differs. It is known that CXCL12-γbinds to CXCR4 with a higher affinity than CXCL12-α. Inter-estingly, the increase in binding affinity does not have to betranslated in stronger activity of an isoform. CXCL12-γ wasreported to have the greatest anti-HIV activity, but theweakest chemotactic activity through CXCR4 (Altenburget al., 2007; Altenburg et al., 2010). Furthermore, it has beensuggested that depending on CXCL12 isoform to which it





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binds, heparan sulfate differentially orchestrates the chemo-kine mediated directional cell kinesis (Laguri et al., 2007).Thus, CXCL12 isoforms act through same receptors and bindto same extracellular structures but the outcome they pro-duce in this way may diverge. Hence, different isoforms ofCXCL12 may have the same role in EAE, but changes in theirexpression relative to each other might modulate the inten-sity of their effect. This implies that different isoforms ofCXCL12 might have the same role in EAE and that theirdifferential expression serves for tuning of the intensity ofthe effect. This could be one of plausible explanations for thedivergent expression of CXCL12-γ and total CXCL12 in immu-nized AO rats, i.e. for the decrease of CXCL12-γ expression inthe CNS of AO rats at the time of the peak of EAE in DA rats. Itis important to emphasize that there was no difference inCXCR4 expression by cells infiltrating spinal cord of AO andDA rats immunized with encephalitogenic emulsion(Miljković et al., 2011). Hence, modulation of CXCL12 andnot of CXCR4 expression seems to be important for protectiveeffects of CXCL12 in EAE.

Both CXCL12-γ and total CXCL12 are highly expressed inEAE-resistant AO rats that have limited infiltration ofimmune cells into the CNS (Miljković et al., 2011). On thecontrary, they are weakly expressed in EAE-prone DA ratsafter immunization, especially at the peak of the disease.This is obvious both in SCH and MBV and indicates thatCXCL12-γ might contribute to the observed anti-inflammatory role of CXCL12 in EAE (Miljković et al., 2011).The assumption is further substantiated with upregulation ofCXCL12-γ in the CNS of AG-treated DA rats that were partiallyprotected from EAE. However, MP-treated DA rats were alsoprotected from EAE, but this treatment did not induceCXCL12-γ expression in the CNS of DA rats. A major differ-ence between AG and MP is that the former is a specificinhibitor of iNOS activity (Misko et al., 1993), while the latteris a general immunosuppressive that has been shown ineffi-cient in reducing iNOS expression in MS patients (Lindquistet al., 2011). Also, MP, but not AG inhibits IFN-γ and IL-17expression in SCH of immunized DA rats (Miljković et al., 2009and our unpublished observation). This is important as ourresults on astrocytes imply that proinflammatory cytokinesstimulate and NO inhibits CXCL12-γ expression in the CNS.Thus, MP ameliorates EAE in a way that prevents upregula-tion of CXCL12-γ in the CNS. On the other hand, AG treatmentallows production of CXCL12-γ stimulators and at the sametime inhibits NO. This implies that NO is an importantregulator of CXCL12-γ generation in the inflamed CNS. Suchan assumption is substantiated in experiments where SNPinhibited CNS CXCL12-γ expression in immunized AO rats.The effect of NO on CXCL12-γ could be one of the ways inwhich this reactive nitrogen species contributes to EAE andMS pathogenesis.

It was previously reported that CXCL12-γ was expressed inthe developing, neonatal and adult CNS of mice (Franco et al.,2009). More specific data can be added to this that CXCL12-γ isexpressed in spinal cord endothelial cells and in brainastrocytes under inflammatory conditions. Strong expressionof CXCL12-γ protein was recently shown on synovialendothelial cells of rheumatoid arthritis patients (Santiagoet al. 2012). However, it was proposed that CXCL12-γ protein


expressed on synovial endothelial cells was of exogenousorigin, i.e. produced by other cell types. Indeed, it waspreviously reported that synovial endothelial cells did notexpress CXCL12 mRNA, but still had abundant proteinexpression of this chemokine (Pablos et al., 2003). Actually,CXCL12 produced by fibroblast-like synoviocytes was boundto endothelial cells through heparan sulfate. A similarmechanism may be operative with CXCL12-γ, especially ifits high affinity for binding of heparan sulfate is considered.However, CXCL12-γ mRNA was detected in MBV of rat CNS,which is concordant with our previous report that CXCL12gene was expressed in MBV (Miljković et al., 2011). Thedifference in the origin of CXCL12 expressed on synovialand spinal cord endothelial cells could be explained by aspecificity of CNS endothelial cells that are constituents ofblood brain barrier and thus have distinct structural andfunctional properties in comparison to the most of bloodendothelial cells present outside of the CNS. Indeed, endothe-lial cells are heterogenic and their functions, including geneexpression profile, are intensively influenced by the sur-rounding cells and tissues (Ribatti et al., 2002). One also hasto consider a possibility that CXCL12 and CXCL12-γ mRNA areat least partly, produced by pericytes, smooth muscle cellsand astrocytes that might also be present in MBV isolates.

Astrocytes expressed CXCL12-γ in vitro, under the influ-ence of ConASn, as a mimic of an inflammatory surrounding.They also responded with elevated CXCL12-γ expression toIFN-γ and IL-17, marker cytokines of Th1 and Th17 cells,respectively. This is of particular interest for neuroinflamma-tion as Th1 and Th17 cells are considered to be major culpritsin EAE and MS (El-behi et al., 2010; Petermann and Korn,2011). Upregulation of CXCL12-γ in response to the inflam-matory cytokines might be regulatory feedback that is opera-tive in AO rats and which prevents invasion of the CNS byimmune cells. Still some other molecules released inneuroinflammation obviously counteract induction ofCXCL12-γ expression in DA rat CNS and in that way pre-sumably promote CNS infiltration. One of the candidates forsuch function is NO, as already elaborated in this section.

CXCL12-γ is abundantly expressed in the brain of man, ratand mice, yet its nucleolar splice variant has been detected inthe heart, but not in the brain of mice (Franco et al., 2009;Torres and Ramirez, 2009). It can be added to this thatnucleolar CXCL12-γ is expressed in rat hearts, but not in theCNS samples. As there are no data about functional proper-ties of nucleolar CXCL12-γ we were interested in investigatingif this molecule might be important for pathogenesis of EAE.Still, the lack of its expression in the CNS tissue of both EAE-resistant AO rats and EAE-prone DA rats after encephalito-genic immunization shows that nucleolar CXCL12-γ is notrelevant for EAE in rats. The inability of astrocytes stimulatedwith proinflammatory cytokines to express nucleolarCXCL12-γ contributes to the view that this molecule has norole in neuroinflammation. Still, there is a possibility thatstudies of its expression in murine EAE and on humansamples will give different results.

In conclusion, it can be reported that CXCL12-γ isexpressed in the inflamed CNS and that its expression ismodulated by cytokines and NO. Its high expression in theCNS of EAE-resistant rats in comparison to EAE-prone rats





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suggests that CXCL12-γ has a protective role in neuroinflam-mation. However, functional studies with CXCL12-γ knock-outs and/or with specific inhibitors of CXCL12-γ are neededfor testing this assumption. Also, further exploration of theinflammation-related regulation of CXCL12-γ expression inthe CNS is warranted.

4. Experimental procedure

4.1. Experimental animals, EAE induction, evaluation andtreatment with aminoguanidine, methylprednisolone andsodium nitroprusside

Inbred Dark Agouti (DA) and Albino Oxford (AO) rats wereobtained from the animal breeding facility of the Institute forBiological Research “Siniša Stanković“ (Belgrade, Serbia). Theanimals 2–3 months of age, sex matched in each experiment,were maintained in the same animal facility. Animal experi-ments were approved by the local Ethics Committee (Institutefor Biological Research ”Siniša Stanković“ No 2-22/10). Thedisease was induced with injection of 100 μl of rat spinal cordhomogenate (SCH) in PBS (50% w/v) mixed with equal volumeof Complete Freund's adjuvant (CFA, Difco, Detroit, MI) (SCH+CFA). Rats were monitored daily for clinical signs of EAE,and scored according to the following scale: 0, no clinicalsigns; 1, flaccid tail; 2, hind limb paresis; 3, hind limbparalysis; and 4, moribund state or death. Intermediatescores were assigned if neurologic signs were of lowerseverity than typically observed. Different phases of EAEwere defined as follows: onset, 9–11 dpi, clinical score (cs)0.5–1; peak, 12–15 dpi, cs 2–3.5; recovery, 16–20 dpi, cs 0–0.5.

For the treatment with aminoguanidine (AG), methylpred-nisolone (MP) or sodium nitroprusside (SNP) DA and AO ratswere immunized with SCH+CFA. Starting from the day 9 postimmunization (one day before the first neurologic deficitsusually appear in DA rats), rats were treated daily withaminoguanidine hydrochloride (Sigma-Aldrich, i.p. 400 mg/kgbody weight) or MP (Hemofarm, Vršac, Serbia, i.p. 50 mg/kgbody weight) or SNP (Sigma-Aldrich, i.p. 1 mg/kg body weight)or vehicle (PBS), until the disease reached its peak in the groupof DA rats that has been receiving PBS. At this stage, animalswere sacrificed, SC was isolated, homogenized and MBV wereobtained.

4.2. Isolation of spinal cord micro Blood vessels

Micro-blood vessels (MBV) were isolated from the SC of ratsfollowing the procedure of Ge and Pachter (2006). In brief,meninges and large vessels were cleared from the SC and theSC minced and homogenized in ice-cold PBS. Resultinghomogenates were centrifuged and re-suspended in 6 ml ofan 18% (w/v) Dextran solution (FP70 molecular weight 65,000–73,000, Serva, Heidelberg, Germany) in RPMI 1640 mediumand centrifuged at 2000g at +4 1C for 20 min. The foamymyelin layer and dextran supernatant were removed andthe pellet containing the crude vascular fraction was re-suspended in PBS. Vascular suspensions were next passedthrough a 40 mm cell strainer (BD Biosciences, San Jose, CA) toremove blood cell contamination. After rinsing the cell


strainer with PBS, the trapped micro-vessels were collectedand pelleted. MBV were used for RNA isolation immediately.

4.3. Reagents, cells and cell cultures

RPMI-1640 medium and fetal calf serum (FCS) were from PAALaboratories (Pasching, Austria). IFN-γ and SNP were fromSigma-Aldrich (St. Louis, MO) and IL-17 was from R&DSystems (Minneapolis, MN). Culture medium was HEPES-buffered RPMI-1640 medium with 5% FCS, 2 mM L-glutamine,antibiotics, and sodium pyruvate. Spleens were isolated fromuntreated DA rats and mechanically disrupted, passedthrough 40-μm nylon mesh filter and the resulting suspen-sion was collected by centrifugation. Spleen cells (5�106/ml)were stimulated with concanavalin A (ConA, Sigma-Aldrich,5 mg/ml) for 48 h and subsequently cell-free culture super-natants (ConASn) were collected. Astrocytes were isolatedfrom mixed glial cell cultures prepared from brains of new-born DA rats as previously described (McCarthy and de Vellis,1980). Astrocytes were grown in the culture medium supple-mented with 4 g/l glucose and they were purified by repeti-tion of trypsinization (0.25% trypsin and 0.02% EDTA, bothfrom Sigma-Aldrich) and re-plating. The cells used in theseexperiments were obtained after third passage and were495% positive for glial fibrilar acidic protein (GFAP) ando3% positive for CD11b, as deduced by cytofluorimetricanalysis. Astrocytes were seeded at 1.5�105/ml/well of 24-well plates (Sarstedt, Nümbrecht, Germany) and stimulatedwith ConASn (20%) or IFN-γ(10ng/ml)+IL-17 (50 ng/ml). Theywere also treated with SNP (200 μM).

4.4. Real time reverse transcription–polymerase chainreaction

In order to isolate total RNA a mi-Total RNA Isolation Kit(Metabion, Martinsried, Germany) was used. Reverse tran-scription (RT) was performed by using random hexamerprimers and MMLV (Moloney Murine Leukemia Virus),according to the manufacturer's instructions (Fermentas,Vilnius, Lithuania). Prepared cDNAs were amplified by usingPower SYBRs Green PCR Master Mix (Applied Biosystems,Foster City, CA) according to the recommendations of themanufacturer in a total volume of 20 ml in an ABI PRISM 7000Sequence Detection System (Applied Biosystems). Thermo-cycler conditions comprised an initial step at 50 1C for 5 min,followed by a step at 95 1C for 10 min and a subsequent 2-stepPCR program at 95 1C for 15 s and 60 1C for 60 s for 40 cycles.The PCR primers (Metabion) were as follows: β-actin forwardprimer 5'-GCT TCT TTG CAG CTC CTT CGT-3'; β-actin reverseprimer 5'-CCA GCG CAG CGA TAT CG-3'; CXCL12 forwardprimer 5'-GAT TCT TTG AGA GCC ATG TC-3'; CXCL12 reverseprimer 5'-GTC TGT TGT TGC TTT TCA GC-3'. CXCL12-γforward primer 5'-AGC CAG TCA GCC TGA GCT AC-3';CXCL12-γ reverse primer 5'-CCC CAC TTT TTC TTC TCTGC-3'. CXCL12-γSP forward primer 5'-AGC CAG TCA GCCTGA GCT AC-3'; and CXCL12-γSP reverse primer 5'-CCC CACTTT TTC TTC TCT GC-3'. Accumulation of PCR products wasdetected in real time and the results were analyzed with 7500System Software (Applied Biosystems) and presented as 2−dCt,





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dCt being the difference between Ct values of specific geneand the endogenous control (β-actin).

4.5. Statistical analysis

The results are presented as mean+SD or mean +/−SE ofvalues obtained in repeated experiments. A Student's t-test(two-tailed) was performed for statistical analysis. A p valueless than 0.05 was considered statistically significant.

Acknowledgments

This work was supported by the Ministry of Education,Science and Technological Development of the Republic ofSerbia (173035, 175038, and 173013).

r e f e r e n c e s

Altenburg, J.D., Broxmeyer, H.E., Jin, Q., Cooper, S., Basu, S.,Alkhatib, G., 2007. A naturally occurring splice variant ofCXCL12/stromal cell-derived factor 1 is a potent humanimmunodeficiency virus type 1 inhibitor with weakchemotaxis and cell survival activities. J. Virol. 81, 8140–8148.

Altenburg, J.D., Jin, Q., Alkhatib, B., Alkhatib, G., 2010. The potentanti-HIV activity of CXCL12gamma correlates with efficientCXCR4 binding and internalization. J. Virol. 84, 2563–2572.

El-behi, M., Rostami, A., Ciric, B., 2010. Current views on the rolesof Th1 and Th17 cells in experimental autoimmuneencephalomyelitis. J. Neuroimmune Pharmacol. 5, 189–197.

Franco, D., Rueda, P., Lendínez, E., Arenzana-Seisdedos, F., Caruz,A., 2009. Developmental expression profile of theCXCL12gamma isoform: insights into its tissue-specific role.Anat. Rec. (Hoboken) 292, 891–901.

Ge, S., Pachter, J.S., 2006. Isolation and culture of microvascularendothelial cells from murine spinal cord. J. Neuroimmunol.177, 209–214.

Laguri, C., Sadir, R., Rueda, P., Baleux, F., Gans, P., Arenzana-Seisdedos, F., Lortat-Jacob, H., 2007. The novel CXCL12gammaisoform encodes an unstructured cationic domain whichregulates bioactivity and interaction with bothglycosaminoglycans and CXCR4. PLoS One 2, e1110.

Li, M., Ransohoff, R.M., 2008. Multiple roles of chemokine CXCL12in the central nervous system: a migration from immunologyto neurobiology. Prog. Neurobiol. 84, 116–131.

Lindquist, S., Hassinger, S., Lindquist, J.A., Sailer, M., 2011. Thebalance of pro-inflammatory and trophic factors in multiple


sclerosis patients: effects of acute relapse andimmunomodulatory treatment. Multiple Scler. 17, 851–866.

McCarthy, K.D., de Vellis, J., 1980. Preparation of separateastroglial and oligodendroglial cell cultures from rat cerebraltissue. J. Cell Biol. 85, 890–902.

Miljković, Z., Momcilović, M., Miljković, D., Mostarica-Stojković, M.,2009. Methylprednisolone inhibits IFN-gamma and IL-17expression and production by cells infiltrating central nervoussystem in experimental autoimmune encephalomyelitis. J.Neuroinflammation 6, 37.

Miljković, D., Stanojević, Z., Momcilović, M., Odoardi, F., Flügel, A.,Mostarica-Stojković, M., 2011. CXCL12 expression within theCNS contributes to the resistance against experimentalautoimmune encephalomyelitis in Albino Oxford rats.Immunobiol. 216, 979–987.

Misko, T.P., Moore, W.M., Kasten, T.P., Nickols, G.A., Corbett, J.A.,Tilton, R.G., McDaniel, M.L., Williamson, J.R., Currie, M.G.,1993. Selective inhibition of the inducible nitric oxidesynthase by aminoguanidine. Eur. J. Pharmacol. 233, 119–125.

Momcilović, M., Mostarica-Stojković, M., Miljkovic, D., 2012.CXCL12 in control of neuroinflammation. Immunol. Res. 52,53–63.

Pablos, J.L., Santiago, B., Galindo, M., Torres, C., Brehmer, M.T.,Blanco, F.J., García-Lázaro, F.J., 2003. Synoviocyte-derivedCXCL12 is displayed on endothelium and inducesangiogenesis in rheumatoid arthritis. J. Immunol. 170,2147–2152.

Petermann, F., Korn, T., 2011. Cytokines and effector T cellsubsets causing autoimmune CNS disease. FEBS Lett. 585,3747–3757.

Ribatti, D., Nico, B., Vacca, A., Roncali, L., Dammacco, F., 2002.Endothelial cell heterogeneity and organ specificity.J. Hematother. Stem Cell Res. 11, 81–90.

Rueda, P., Balabanian, K., Lagane, B., Staropoli, I., Chow, K.,Levoye, A., Laguri, C., Sadir, R., Delaunay, T., Izquierdo, E.,Pablos, J.L., Lendinez, E., Caruz, A., Franco, D., Baleux, F.,Lortat-Jacob, H., Arenzana-Seisdedos, F., 2008. TheCXCL12gamma chemokine displays unprecedented structuraland functional properties that make it a paradigm ofchemoattractant proteins. PLoS One 3, e2543.

Santiago, B., Izquierdo, E., Rueda, P., Del Rey, M.J., Criado, G.,Usategui, A., Arenzana-Seisdedos, F., Pablos, J.L., 2012.CXCL12γ isoform is expressed on endothelial and dendriticcells in rheumatoid arthritis synovium and regulates T cellactivation. Arthritis Rheum. 64, 409–417.

Timotijević, G., Mostarica Stojković, M., Miljkovic, D., 2012.CXCL12: role in neuroinflammation. Int. J. Biochem. Cell. Biol.44, 838–841.

Torres, R., Ramirez, J.C., 2009. A chemokine targets the nucleus:CXCL12-gamma isoform localizes to the nucleolus in adultmouse heart. PLoS One 4, e7570.


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CXCL12-γ expression is inhibited in neuroinflammation

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