ORIGINAL COMMUNICATION

The gender-specific association of CXCL16 A181V genepolymorphism with susceptibility to multiple sclerosis, and itseffects on PBMC mRNA and plasma soluble CXCL16 levels:preliminary findings

Ljiljana Stojkovic • Aleksandra Stankovic •

Tamara Djuric • Evica Dincic • Dragan Alavantic •

Maja Zivkovic

Received: 25 February 2014 / Revised: 14 May 2014 / Accepted: 14 May 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract CXC ligand 16 (CXCL16) is a multifunctional

chemokine involved in cell adhesion and chemoattraction

as well as in the scavenging of oxidized lipoproteins.

Experimental data suggest the roles of CXCL16 in patho-

genesis of multiple sclerosis (MS). A181V polymorphism

in the human CXCL16 gene has been associated with the

clinical course of certain chronic inflammatory diseases.

The aim of this study was to analyze the effects of

CXCL16 A181V polymorphism on: (1) susceptibility to

MS and disease course, (2) peripheral blood mononuclear

cells (PBMC) CXCL16 mRNA levels and plasma soluble

CXCL16 levels of patients with MS and healthy controls.

In this study, 459 MS patients and 303 controls were

included. Real-time PCR-based methods were applied for

genotyping of CXCL16 A181V and for CXCL16 gene

expression analysis. Quantitative sandwich enzyme

immunoassay was performed for quantification of plasma

soluble CXCL16. CXCL16 AA genotype had a significant

protective effect on MS susceptibility in women

(OR = 0.53, ±95 % CI = 0.35–0.82, p = 0.004). The V

allele-containing genotypes were associated with signifi-

cantly higher CXCL16 mRNA levels in PBMC of both

female (mean factor = 1.81, S.E. = 1.14–2.77, p \ 0.01)

and male (mean factor = 1.58, S.E. = 1.35–1.73,

p \ 0.01) controls. No significant association of the

CXCL16 polymorphism was established either with

soluble CXCL16 plasma levels or with clinical parameters

and course of MS. The main finding of this study is gender-

specific association of CXCL16 A181V polymorphism

with susceptibility to MS in females. The current results

should be replicated and validated in the larger sample

group.

Keywords CXCL16 � Gene � Polymorphism � A181V �Multiple sclerosis

Introduction

CXC ligand 16 (CXCL16/SR-PSOX-scavenger receptor

that binds phosphatidylserine and oxidized lipoprotein) is a

recently discovered chemokine with unique features:

adhesion of the cells expressing its unique receptor-

CXCR6 [1], scavenging of the oxidized lipoproteins, bac-

teria or apoptotic cells [2, 3] and chemoattraction of the

CXCR6-expressing cells [4]. CXCL16 may exist either as a

transmembrane molecule––acting as an adhesion molecule

and a scavenger receptor, or as a typical soluble chemo-

kine––which acts as a chemoattractant and is created by

ADAM10 metalloproteinase cleavage of the transmem-

brane CXCL16 extracellular chemokine domain [5, 6].

CXCL16 is largely expressed by macrophages/monocytes

[2] and B lymphocytes [7], while its specific receptor,

CXCR6, is present on the surface of various subpopula-

tions of T lymphocytes, including MBP-reactive T cells

[8].

Chemokines and their receptors are expressed in the

central nervous system (CNS) and under pathological

conditions they may be involved in chronic inflammatory

diseases of the CNS, such as multiple sclerosis (MS) [9–

11]. Expression of CXCL16 in the astrocytes was found to

L. Stojkovic � A. Stankovic � T. Djuric � D. Alavantic �M. Zivkovic (&)

Laboratory for Radiobiology and Molecular Genetics, VINCA

Institute of Nuclear Sciences, University of Belgrade,

P.O. Box 522, 11001 Belgrade, Serbia

e-mail: majaz@vinca.rs

E. Dincic

Neurology Clinic, Military Medical Academy, Belgrade, Serbia

123

J Neurol

DOI 10.1007/s00415-014-7379-7

be up-regulated by proinflammatory stimuli, while its

expression in the healthy brain was low and mostly

restricted to brain endothelial cells [12]. Clinical manifes-

tation of the experimental autoimmune encephalomyelitis

correlated well with the expression of CXCL16 in the CNS,

while neutralizing antibodies against CXCL16 attenuated

the development of disease [13]. This was the first indi-

cation of the potential role of CXCL16 in MS pathology.

During inflammatory and autoimmune diseases, such as

MS, the inducible production and release of CXCL16 in the

brain was confirmed by detection of soluble CXCL16 in

the human cerebrospinal fluid [14]. In the postmortem

brains of MS patients, CXCL16 was expressed by foamy

macrophages and microglia around the chronic active MS

lesions [15].

The allelic variations of CXCL16 gene have been barely

investigated in human diseases. The missense polymor-

phism A181V (rs2277680), located in exon 4 of the human

CXCL16 gene, has been analyzed in the coronary artery

disease [16, 17] and inflammatory bowel disease [18].

Recent pathway analysis of a genome wide association

(GWA) study in schizophrenia has revealed CXCL16

(rs2277680) as a novel candidate gene [19]. So far, the

association of CXCL16 A181V gene polymorphism with

MS susceptibility has been examined in a single GWA

study [20].

The aim of this study was to analyze the effects of

CXCL16 A181V (rs2277680) missense polymorphism on

susceptibility to MS and disease course, as well as on the

peripheral blood mononuclear cells (PBMC) CXCL16

mRNA expression levels and soluble CXCL16 plasma

levels, in patients with MS and healthy controls.

Materials and methods

Subjects

In this case–control study, the group of patients with MS

consisted of 459 unrelated Caucasian patients from Serbia,

who had been recruited consecutively from Neurology

Clinic of Military Medical Academy (MMA), Belgrade,

Serbia. All patients fulfilled the criteria for clinically def-

inite MS [21] and the disease course was determined

according to clinical data [22]. Expanded disability status

scale (EDSS) [23] and multiple sclerosis severity score

(MSSS) [24] were used for estimation of disease severity.

EDSS enables quantification of disability in patients, while

MSSS relates scores on EDSS to distribution of disability

in patients with comparable periods of disease duration.

Calculation of global MSSS was done according to clinical

data, immediately after the blood specimens for genetic

analysis had been collected. The control group consisted of

303 unrelated healthy volunteers of MMA and ‘‘Vinca’’

Institute of Nuclear Sciences staff, without a history of MS

in their families. Healthy volunteers and MS patients were

of the same ethnical origin (Serbian). The Ethical Com-

mittee of MMA approved this study and each participant

gave their written informed consent to participate in the

study.

Determination of CXCL16 A181V genotypes

Genomic DNA was isolated from peripheral blood cells

using ABI PRISMTM 6100 Nucleic Acid PrepStation DNA

BloodPrepTM kit (Applied Biosystems, Foster City, CA,

USA). TaqMan�SNP genotyping assay C__15885167_10,

designed and tested by Applied Biosystems (Foster City,

CA, USA), was used for detection of A181V (rs2277680)

polymorphism. PCR amplification and determination of

genotypes were performed according to the recommended

protocol, using 7500 Real-Time PCR system (Applied

Biosystems, Foster City, CA, USA) and SDS software

(v1.4.0) (Applied Biosystems, Foster City, CA, USA).

Quantitative real-time reverse transcription-PCR (real-

time RT-PCR) for CXCL16 gene expression analysis

Peripheral blood mononuclear cells from 3 ml of each

peripheral blood sample were separated with lymphocyte

separation medium (PAA, GE Healthcare). PBMC total

RNA was isolated using TRI Reagent (Ambion, Life

Technologies) according to the manufacturer’s instruc-

tions. The quantity of total RNA was assessed on BioSpec-

nano spectrophotometer (Shimadzu Biotech). Quality and

integrity of total RNA were analyzed using RNA 6000

Nano Kit on the 2100 Bioanalyzer (Agilent, USA).

Five hundred nanograms (500 ng) of each sample total

RNA was treated with DNAseI (Fermentas, Lithuania) and

then reverse transcription (RT) was done using First strand

cDNA synthesis kit with oligo-dT18 primers (Fermentas,

Lithuania), in a final reaction volume of 20 ll, according to

manufacturer’s instructions. Real-time PCR analysis was

performed on Applied Biosystems 7500 Real-Time PCR

system, using TaqMan� gene expression assays:

Hs01055223_g1 (AB, Foster City, CA)––for detection and

quantification of CXCL16 gene expression and

Hs99999904_m1 (AB, Foster City, CA)––for detection and

quantification of PPIA (endogenous control) gene expres-

sion, according to the manufacturer’s protocol. In a total

volume of 12.5 ll, each real-time PCR reaction contained

1 ll of the RT product. All samples were run in duplicates.

Differences in CXCL16 mRNA relative expression levels

were determined based on the comparative Ct method,

using Relative Expression Software Tool (REST) 2009

software.

J Neurol

123

ELISA quantification of soluble CXCL16

Peripheral blood samples were collected with EDTA as an

anticoagulant and centrifuged for 15 min at 1,0009g within

30 min of collection. Plasma samples were then aliquoted

and stored at -20� C until used. For quantification of soluble

CXCL16 in plasma samples, we used Quantikine� Human

CXCL16 Immunoassay (R&D Systems, Minneapolis, MN,

USA) and performed quantitative sandwich enzyme immu-

noassay, according to the manufacturer’s instructions. Sam-

ples and standards were assayed in duplicates. Determination

of the optical density was done using VICTOR3 (PerkinEl-

mer, Waltham, MA, USA) microplate reader set to 450 nm

(wavelength correction at 540 nm). Each sample’s average

optical density value (averaged duplicate readings) was used

for determination of soluble CXCL16 plasma concentration

(ng/ml), from a standard curve generated as a five parameter

logistic (5 PL) curve-fit (http://www.myassays.com/five-

parameter-logistic-curve.assay).

Statistical analysis

Allele frequencies and genotype distributions were estimated

by the gene counting method. Deviation from Hardy–

Weinberg equilibrium was examined using Chi square (v2)

test. Values of continuous variables were compared between

the sample groups by using analysis of variance (ANOVA)

and Kruskal–Wallis ANOVA, or using T test and Mann–

Whitney U test. The logistic regression analysis was

expressed in terms of odds ratio (OR) and its 95 % confi-

dence interval (CI), which represented a measure of strength

of association between the polymorphism and the phenotype

of interest. All statistical tests were performed using Statis-

tica version 5 software package (StatSoft Inc, 1997). The

statistical power and sample size of study for the analyzed

SNP were calculated using PS (v3.0.43) software [25]. REST

2009, relative expression software tool, which incorporates a

pairwise randomization and bootstrapping technique (http://

rest.gene-quantification.info) [26], was used for gene

expression analysis. We performed Bonferroni correction for

multiple testing in the: (a) analysis of one polymorphism

using three Chi square tests, and p \ 0.016 values were

considered statistically significant; (b) analysis of CXCL16

mRNA and soluble CXCL16 in four subgroups, and

p \ 0.0125 values were considered statistically significant.

Results

Controls and MS patients

Characteristics of the study participants are shown in

Table 1. Subgroup of primary-progressive (PP) MS

patients was not included in further analysis, due to the low

number of participants.

CXCL16 A181V polymorphism in controls and MS

patients

Genotype frequencies of CXCL16 A181V polymorphism

were: AA = 34.3 %, AV = 48.2 %, VV = 17.5 % in

controls and AA = 27.5 %, AV = 52.0 %, VV = 20.5 %

in MS (RR ? SP) patients (p = 0.1). There was no devia-

tion from Hardy–Weinberg equilibrium in the control group.

According to the observed genotype frequencies, in further

analysis the A allele recessive model (AV ? VV vs. AA)

was used. The genotype and allele frequencies of A181V

polymorphism in the studied groups overall as well as

divided by gender are shown in Table 2. After Bonferroni

correction for multiple testing, there were no significant

differences in genotype frequencies between the overall MS

patients and controls (OR = 0.72, ±95 % CI = 0.53–0.99,

p = 0.04, the power of the study was 39 % at the signifi-

cance level of p = 0.016) (Table 2). After dividing the

study cohort by gender, there were no significant differences

in genotype frequencies in males (OR = 1.05, ±95 %

CI = 0.66–1.68, p = 0.83) (Table 2). We found a signifi-

cantly lower frequency of the AA genotype only in female

MS patients, compared to female controls, according to the

A allele recessive model (OR = 0.53, ±95 %

CI = 0.35–0.82, p = 0.004) (Table 2). Most importantly,

for this association, we had a study power of 76 %, at the

significance level of p = 0.016 (calculated sample size was

311 female patients to achieve the power of 80 %).

CXCL16 A181V polymorphism and clinical

parameters of MS

There were no significant effects of CXCL16 A181V

polymorphism either on disease onset age, EDSS and

MSSS or on disease course (RR vs. SP) (results not

shown).

Effects of CXCL16 A181V polymorphism on CXCL16

gene expression

We analyzed relative CXCL16 mRNA expression in

PBMC of controls and RR MS patients, according to

A181V genotypes. Significant upregulation of CXCL16

mRNA expression has been revealed for the V allele-

containing genotypes, compared to AA genotype, in both

female controls (mean factor = 1.81, S.E. = 1.14–2.77,

p \ 0.01) and male controls (mean factor = 1.58,

S.E. = 1.35–1.73, p \ 0.01). The relative expression of

CXCL16 mRNA according to A181V genotypes did not

differ significantly either in female or in male patients with

J Neurol

123

MS (Fig. 1). We have detected significantly higher levels

of CXCL16 mRNA in PBMC of the overall RR MS

patients (n = 37) compared to controls (n = 29), regard-

less of A181V genotypes, by a mean factor of 1.49

(S.E. = 0.71–3.11, p = 0.002).

Effects of CXCL16 A181V polymorphism on soluble

CXCL16 levels

Soluble CXCL16 plasma levels were not significantly

different according to CXCL16 A181V genotypes among

the controls and RR MS patients divided by gender

(Table 3). However, significantly (p = 4 9 10-5) higher

levels of soluble CXCL16 were detected in plasma of the

overall group of RR MS patients (n = 33, 2.98 ± 0.52 ng/

ml), compared to controls (n = 29, 2.45 ± 0.33 ng/ml),

regardless of A181V genotypes.

Discussion

The missense polymorphism A181V in CXCL16 gene has

been associated with fewer chronic inflammatory diseases

[16, 18], but its potential role as a genetic risk factor for

MS susceptibility has been studied recently in a single

GWA study [20]. In our study, the wild type AA genotype

had a significant protective effect on MS susceptibility in

women. The same genotype was associated with signifi-

cantly lower CXCL16 mRNA expression in PBMC of both

female and male control subjects. The association of the

CXCL16 polymorphism was not established either with

soluble CXCL16 plasma levels or with clinical parameters

and course of MS. However, significantly higher levels of

both CXCL16 mRNA and soluble CXCL16 were detected

in MS patients compared to controls, independently on

CXCL16 genotypes.

Multiple sclerosis occurs more frequently in women.

Gender-specific differences regarding the incidence of MS,

its clinical course or response to therapy, may be caused by

gender-specific differences in: the functional characteris-

tics of the immune system, the CNS and the candidate

genes [27]. Therefore, we analyzed CXCL16 A181V

polymorphism, which we selected based on CXCL16

potential function in MS, in the whole group of MS patients

and in both genders separately. The A181V polymorphism

was covered by the Illumina array used in the GWAS

performed by the IMSGC & WTCCC2 and it was not

found among the MS-associated SNPs with genome-wide

Table 1 Characteristics of controls and MS patients

Parameter Controls MS patients (RR ? SP) RR MS SP MS PP MS

(n = 303) (n = 459) (n = 374) (n = 85) (n = 12)

Age (years) 39.6 ± 10.7 38.0 ± 10.2

Gender (female/male) 148/155 284/175 232/142 52/33 7/5

Disease onset age (years) 29.6 ± 8.8 29.5 ± 8.9 29.7 ± 8.7 43.0 ± 10.4

Disease duration (years) 10.2 ± 6.3 7.0 ± 5.5 13.4 ± 7.2 3.5 ± 2.3

EDSS 4.2 ± 1.4 2.6 ± 1.2 5.8 ± 1.6 3.7 ± 1.7

MSSS 5.5 ± 2.2 4.2 ± 2.3 6.9 ± 2.1 7.0 ± 2.0

Values are expressed as mean ± standard deviation (SD)

MS patients: RR MS relapsing-remitting, SP MS secondary progressive, PP MS primary progressive, EDSS expanded disability status scale,

MSSS multiple sclerosis severity score

Table 2 Genotype and allele frequencies of CXCL16 A181V polymorphism in controls and MS patients (RR ? SP), overall and divided by

gender

CXCL16

A181V

Overall p (v2) Males p (v2) Females p (v2)

Controls n (%) Patients n (%) Controls n (%) Patients n (%) Controls n (%) Patients n (%)

(n = 303) (n = 459) (n = 155) (n = 175) (n = 148) (n = 284)

AA 104 (34.3) 126 (27.5) 0.04 47 (30.3) 55 (31.4) 0.83 57 (38.5) 71 (25.0) 0.004*

AV ? VV 199 (65.7) 333 (72.5) 108 (69.7) 120 (68.6) 91 (61.5) 213 (75.0)

Alleles A/V 0.58/0.42 0.53/0.47 0.06 0.57/0.43 0.55/0.45 0.46 0.59/0.41 0.53/0.47 0.06

OR 0.72 0.04 1.05 0.83 0.53 0.004*

±95 % CI 0.53–0.99 0.66–1.68 0.35–0.82

p Pearson’s Chi-square test, OR odds ratio for the AV ? VV vs. AA genotype model, CI confidence interval

* p was corrected for multiple testing and p \ 0.016 values were considered statistically significant

J Neurol

123

significance [20], similar to our results in a whole group of

patients. However, we found a significant protective effect

of the AA genotype on MS susceptibility, exclusively in

women. Although our study involved a limited number of

MS patients and controls, compared to the previous GWA

study, the novel significant result appeared after incorpo-

ration of gender-specific analysis, corrected for multiple

testing and with a study power of 76 %. However, neither

this GWAS nor other GWAS-es in MS had included the

Western Balkan populations. The ethnicity, north to south

gradient of MS prevalence as well as lifestyle could

underlie the heterogeneity of the results. We searched to

find if there were any polymorphisms significantly asso-

ciated with MS in the recent GWAS [20], that could be

A Female controls B Female RR MS patients

1

1.805

0

0.5

1

1.5

2

AA AV + VV

CXCL16 A181V polymorphism

Rel

ativ

e C

XC

L16

mR

NA

ex

pres

sion

1

1.232

0

0.5

1

1.5

AA AV + VV

CXCL16 A181V polymorphism

Rel

ativ

e C

XC

L16

mR

NA

expr

essi

on

C Male controls D Male RR MS patients

1

1.576

0

0.5

1

1.5

2

AA AV + VV

CXCL16 A181V polymorphism

Rel

ativ

e C

XC

L16

mR

NA

ex

pres

sion

1

0.762

0

0.2

0.4

0.6

0.8

1

1.2

AA AV + VV

CXCL16 A181V polymorphism

Rel

ativ

e C

XC

L16

mR

NA

ex

pres

sion

p<0.01*p=0.29

p<0.01*p=0.25

Fig. 1 Effects of CXCL16 A181V polymorphism on CXCL16 gene

expression in PBMC of controls and RR MS patients, according to

gender. The cDNAs from PBMC specimens were used as templates in

real-time qPCR, for relative quantification of CXCL16 mRNA and

housekeeping CYCLA mRNA expression. For each specimen, the

expression level of CXCL16 mRNA was normalized to the house-

keeping gene’s and calculation was done using the REST 2009 soft-

ware. * p was corrected for multiple testing and p \ 0.0125 values

were considered statistically significant. Relative expression of

CXCL16 mRNA in PBMC was: a significantly up-regulated in

female controls who carried the V allele-containing genotypes

(n = 11), compared to those with AA genotype (n = 7), by a mean

factor of 1.805 (S.E. = 1.14–2.77, p \ 0.01); b not significantly

different in female patients with MS who carried the V allele-

containing genotypes (n = 11), compared to those with AA genotype

(n = 8) (mean factor = 1.232, S.E. = 0.713–2.366, p = 0.29); c sig-

nificantly up-regulated in male controls who carried the V allele-

containing genotypes (n = 7), compared to those with AA genotype

(n = 4), by a mean factor of 1.576 (S.E. = 1.353–1.733, p \ 0.01);

d not significantly different in male patients with MS who carried the

V allele-containing genotypes (n = 11), compared to those with AA

genotype (n = 7) (mean factor = 0.762, S.E. = 0.416–1.244,

p = 0.25)

Table 3 Effects of CXCL16 A181 V genotypes on plasma soluble CXCL16 levels in controls and RR MS patients, according to gender

A181V Females Males

AA n AV ? VV n p AA n AV ? VV n p

Controls 2.43 ± 0.37 8 2.41 ± 0.27 9 0.8 2.45 ± 0.41 4 2.57 ± 0.46 8 0.8

MS 3.00 ± 0.55 9 2.99 ± 0.59 16 0.7 2.91 ± 0.55 6 2.99 ± 0.45 12 0.8

Values of plasma soluble CXCL16 levels (ng/ml) are presented as mean ± SD

p Student’s t test

J Neurol

123

somehow linked to CXCL16 gene or its function. We

found that NFKB1 rs228614 and MAPK1 rs2283792 were

associated with susceptibility to MS, although with bor-

derline genome-wide significance. The NFKB1 and

MAPK1 proteins were found to be involved in the acti-

vation of CXCL16 and CXCR6 gene expression, respec-

tively [28, 29]. These potential links could be investigated

in the future.

Recently, it has been shown that in postmortem brain

tissue from MS patients the CXCL16 mRNA has been up-

regulated around the chronic active MS lesions [15].

Herein we have detected significantly higher expression of

CXCL16 mRNA in PBMC of MS patients, compared to

controls. According to our knowledge, the association

between CXCL16 gene polymorphisms and CXCL16

mRNA levels has not been investigated yet. In the current

study, we have found significantly higher CXCL16 mRNA

expression in PBMC of the V allele carriers, compared to

AA genotype, in controls (both females and males). In

patients there has been a lack of association between the

A181V genotypes and CXCL16 mRNA levels. This result

suggests that in inflammatory conditions such as MS

pathogenesis, when CXCL16 gene expression is upregu-

lated, other genetic and epigenetic mechanisms have

impact on CXCL16 gene transcription rather than the

A181V polymorphism. Thus far, we do not have an

explanation for the potential functional effect of the

investigated polymorphism on the gene expression. Still,

CXCL16 gene expression in PBMC as a potential molec-

ular marker of MS should not be ruled out.

As a potential molecular marker of inflammation, solu-

ble CXCL16 was significantly increased in serum and CSF

of patients with several neuroinflammatory diseases

including MS [14]. We confirmed this result by detecting

significantly higher levels of soluble CXCL16 in plasma of

MS patients, compared to controls. A single study in

Chinese population did not reveal any significant results

regarding the association of A181V polymorphism with

CXCL16 serum levels [30]. Similarly, we found no sig-

nificant association between the soluble CXCL16 plasma

levels and A181V genotypes, either in the overall controls

and patients or divided by gender. Position of A181V

polymorphism in the transmembrane form of CXCL16

protein is close to the cell membrane, indicating this amino

acid substitution may affect the proteolytic cleavage of the

transmembrane CXCL16. The result of our study does not

support this hypothesis, similar to the result of an extensive

functional study by Petit et al. [17]. Petit et al. also revealed

that there were no significant differences in monocyte cell

surface expression levels of the transmembrane CXCL16

protein between donors of the three A181V genotypes and

that the only functional difference between the A181 and

V181 protein variants of CXCL16 was a lack of the

adhesive property, caused by the presence of V at position

181. Considering the findings of our current study—in

which significantly higher frequency of the V allele-con-

taining genotypes of CXCL16 A181V polymorphism was

established in female patients, and the previous findings on

the functional effects of this polymorphism [17], it is likely

that the cell adhesion property of CXCL16 can be of par-

ticular importance in the pathogenesis of MS in women.

Among sex hormones, exclusively female hormones

(estradiol and progesterone) had stimulating effects on

adhesion of mononuclear leukocytes to endothelial cells

activated with TNF-alpha and IFN-gamma [31] and also

having been known for inducible expression of CXCL16

[32, 33]. In light of this fact, the potential significance of

the CXCL16 genotypes’ effects on CXCL16-mediated

adhesion is to be considered in the pathogenesis of MS

particularly in women.

In conclusion, the main novelty of this study is a gender-

specific association of CXCL16 A181V polymorphism

with susceptibility to MS, established in females from

Serbia. We have confirmed the importance of CXCL16 as a

potential molecular marker of MS, but further research is

warranted to elucidate the mechanisms of its transcriptional

and posttranslational regulation. The future extensive

genetic analysis should include investigation of the non-

coding regulatory region of CXCL16 gene as well as

nearby genes within the same haplo-block. The current

results regarding the association of A181V polymorphism

with mRNA and soluble CXCL16 levels should be vali-

dated in the larger sample group. Because this is the first

candidate gene study investigating the association between

CXCL16 A181V polymorphism and MS, in MS patients

and controls from Serbia, a replication of the gender-spe-

cific association found in this preliminary study is essential,

if not in Serbian population then in genetically and envi-

ronmentally similar one.

Acknowledgments This study was funded by Serbian Ministry of

Education and Science Grants No. OI175085 and III41028.

Conflicts of interest The authors declare that they have no conflicts

of interest.

Ethical standards The Ethical Committee of Military Medical

Academy, Belgrade, Serbia, approved this study and each participant

gave their written informed consent to participate in the study.

References

1. Matloubian M, David A, Engel S, Ryan JE, Cyster JG (2000) A

transmembrane CXC chemokine is a ligand for HIV-coreceptor

Bonzo. Nat Immunol 1:298–304

2. Shimaoka T, Kume N, Minami M, Hayashida K, Kataoka H, Kita

T, Yonehara S (2000) Molecular cloning of a novel scavenger

receptor for oxidized low density lipoprotein, SR-PSOX, on

macrophages. J Biol Chem 275(52):40663–40666

J Neurol

123

3. Shimaoka T, Nakayama T, Kume N, Takahashi S, Yamaguchi J,

Minami M, Hayashida K, Kita T, Ohsumi J, Yoshie O, Yonehara

S (2003) Cutting edge: SR-PSOX/CXC chemokine ligand 16

mediates bacterial phagocytosis by APCs through its chemokine

domain. J Immunol 171(4):1647–1651

4. Kim CH, Kunkel EJ, Boisvert J, Johnston B, Campbell JJ,

Genovese MC, Greenberg HB, Butcher EC (2001) Bonzo/

CXCR6 expression defines type 1-polarized T-cell subsets with

extralymphoid tissue homing potential. J Clin Invest

107(5):595–601

5. Gough PJ, Garton KJ, Wille PT, Rychlewski M, Dempsey PJ,

Raines EW (2004) A disintegrin and metalloproteinase 10-med-

iated cleavage and shedding regulates the cell surface expression

of CXC chemokine ligand 16. J Immunol 172(6):3678–3685

6. Abel S, Hundhausen C, Mentlein R, Schulte A, Berkhout TA,

Broadway N, Hartmann D, Sedlacek R, Dietrich S, Muetze B,

Schuster B, Kallen KJ, Saftig P, Rose-John S, Ludwig A (2004)

The transmembrane CXC-chemokine ligand 16 is induced by

IFN-gamma and TNF-alpha and shed by the activity of the dis-

integrin-like metalloproteinase ADAM10. J Immunol

172(10):6362–6372

7. Wilbanks A, Zondlo SC, Murphy K, Mak S, Soler D, Langdon P,

Andrew DP, Wu L, Briskin M (2001) Expression cloning of the

STRL33/BONZO/TYMSTRligand reveals elements of CC, CXC,

and CX3C chemokines. J Immunol 166(8):5145–5154

8. Calabresi PA, Yun SH, Allie R, Whartenby KA (2002) Chemo-

kine receptor expression on MBP-reactive T cells: CXCR6 is a

marker of IFNgamma-producing effector cells. J Neuroimmunol

127(1–2):96–105

9. D’Aversa TG, Weidenheim KM, Berman JW (2002) CD40-

CD40L interactions induce chemokine expression by human

microglia: implications for human immunodeficiency virus

encephalitis and multiple sclerosis. Am J Pathol 160(2):559–567

10. Mahad DJ, Howell SJ, Woodroofe MN (2002) Expression of

chemokines in the CSF and correlation with clinical disease

activity in patients with multiple sclerosis. J Neurol Neurosurg

Psychiatry 72(4):498–502

11. Satoh J, Nanri Y, Tabunoki H, Yamamura T (2006) Microarray

analysis identifies a set of CXCR3 and CCR2 ligand chemokines

as early IFNbeta-responsive genes in peripheral blood lympho-

cytes in vitro: an implication for IFNbeta-related adverse effects

in multiple sclerosis. BMC Neurol 6:18

12. Ludwig A, Schulte A, Schnack C, Hundhausen C, Reiss K,

Brodway N, Held-Feindt J, Mentlein R (2005) Enhanced

expression and shedding of the transmembrane chemokine

CXCL16 by reactive astrocytes and glioma cells. J Neurochem

93(5):1293–1303

13. Fukumoto N, Shimaoka T, Fujimura H, Sakoda S, Tanaka M,

Kita T, Yonehara S (2004) Critical roles of CXC chemokine

ligand 16/scavenger receptor that binds phosphatidylserine and

oxidized lipoprotein in the pathogenesis of both acute and

adoptive transfer experimental autoimmune encephalomyelitis.

J Immunol 173(3):1620–1627

14. le Blanc LM, van Lieshout AW, Adema GJ, van Riel PL, Ver-

beek MM, Radstake TR (2006) CXCL16 is elevated in the

cerebrospinal fluid versus serum and in inflammatory conditions

with suspected and proved central nervous system involvement.

Neurosci Lett 397(1–2):145–148

15. Hendrickx DA, Koning N, Schuurman KG, van Strien ME, van

Eden CG, Hamann J, Huitinga I (2013) Selective upregulation of

scavenger receptors in and around demyelinating areas in mul-

tiple sclerosis. J Neuropathol Exp Neurol 72(2):106–118

16. Lundberg GA, Kellin A, Samnegard A, Lundman P, Tornvall P,

Dimmeler S, Zeiher AM, Hamsten A, Hansson GK, Eriksson P

(2005) Severity of coronary artery stenosis is associated with a

polymorphism in the CXCL16/SR-PSOX gene. J Intern Med

257(5):415–422

17. Petit SJ, Wise EL, Chambers JC, Sehmi J, Chayen NE, Kooner

JS, Pease JE (2011) The CXCL16 A181V mutation selectively

inhibits monocyte adhesion to CXCR6 but is not associated with

human coronary heart disease. Arterioscler Thromb Vasc Biol

31(4):914–920

18. Seiderer J, Dambacher J, Leistner D, Tillack C, Glas J, Niess JH,

Pfennig S, Jurgens M, Muller-Myhsok B, Goke B, Ochsenkuhn

T, Lohse P, Reinecker HC, Brand S (2008) Genotype–phenotype

analysis of the CXCL16 p.Ala181Val polymorphism in inflam-

matory bowel disease. Clin Immunol 127(1):49–55

19. Lee YH, Kim JH, Song GG (2013) Pathway analysis of a gen-

ome-wide association study in schizophrenia. Gene

525(1):107–115

20. International Multiple Sclerosis Genetics Consortium, Wellcome

Trust Case Control Consortium 2 (2011) Genetic risk and a pri-

mary role for cell-mediated immune mechanisms in multiple

sclerosis. Nature 476:214–219

21. Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Fi-

lippi M, Fujihara K, Havrdova E, Hutchinson M, Kappos L,

Lublin FD, Montalban X, O’Connor P, Sandberg-Wollheim M,

Thompson AJ, Waubant E, Weinshenker B, Wolinsky JS (2011)

Diagnostic criteria for multiple sclerosis: 2010 revisions to the

McDonald criteria. Ann Neurol 69(2):292–302

22. Lublin FD, Reingold SC (1996) Defining the clinical course of

multiple sclerosis: results of an international survey. National

Multiple Sclerosis Society (USA) Advisory Committee on clini-

cal trials of New Agents in Multiple Sclerosis. Neurol

46:907–911

23. Kurtzke JF (1983) Rating neurologic impairment in multiple

sclerosis: an expanded disability status scale (EDSS). Neurol

33:1444–1452

24. Roxburgh RH, Seaman SR, Masterman T, Hensiek AE, Sawcer

SJ, Vukusic S, Achiti I, Confavreux C, Coustans M, le Page E,

Edan G, McDonnell GV, Hawkins S, Trojano M, Liguori M,

Cocco E, Marrosu MG, Tesser F, Leone MA, Weber A, Zipp F,

Miterski B, Epplen JT, Oturai A, Sørensen PS, Celius EG, Lara

NT, Montalban X, Villoslada P, Silva AM, Marta M, Leite I,

Dubois B, Rubio J, Butzkueven H, Kilpatrick T, Mycko MP,

Selmaj KW, Rio ME, Sa M, Salemi G, Savettieri G, Hillert J,

Compston DA (2005) Multiple sclerosis severity score: using

disability and disease duration to rate disease severity. Neurol

64(7):1144–1151

25. Dupont WD, Plummer WD (1990) Power and sample size cal-

culations: a review and computer program. Control Clin Trials

11:116–128

26. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression

software tool (REST) for group-wise comparison and statistical

analysis of relative expression results in real-time PCR. Nucl Ac

Res 30(9):e36

27. Greer JM, McCombe PA (2011) Role of gender in multiple

sclerosis: clinical effects and potential molecular mechanisms.

J Neuroimmunol 234:7–18

28. Izquierdo MC, Sanz AB, Mezzano S, Blanco J, Carrasco S,

Sanchez-Nino MD, Benito-Martın A, Ruiz-Ortega M, Egido J,

Ortiz A (2012) TWEAK (tumor necrosis factor-like weak inducer

of apoptosis) activates CXCL16 expression during renal tubulo-

interstitial inflammation. Kidney Int 81(11):1098–1107

29. Patel DN, Bailey SR, Gresham JK, Schuchman DB, Shelhamer

JH, Goldstein BJ, Foxwell BM, Stemerman MB, Maranchie JK,

Valente AJ, Mummidi S, Chandrasekar B (2006) TLR4-NOX4-

AP-1 signaling mediates lipopolysaccharide-induced CXCR6

expression in human aortic smooth muscle cells. Biochem Bio-

phys Res Commun 347(4):1113–1120

J Neurol

123

30. Wang KD, Liu ZZ, Wang RM, Wang YJ, Zhang GJ, Su JR, Kang

XX (2010) Chemokine CXC Ligand 16 serum concentration but

not A181V genotype is associated with atherosclerotic stroke.

Clin Chim Acta 411(19–20):1447–1451

31. Cid MC, Kleinman HK, Grant DS, Schnaper HW, Fauci AS,

Hoffman GS (1994) Estradiol enhances leukocyte binding to

tumor necrosis factor (TNF)-stimulated endothelial cells via an

increase in TNF-induced adhesion molecules E-selectin, inter-

cellular adhesion molecule type 1, and vascular cell adhesion

molecule type 1. J Clin Invest 93(1):17–25

32. Wuttge DM, Zhou X, Sheikine Y, Wagsater D, Stemme V, Hedin

U, Stemme S, Hansson GK, Sirsjo A (2004) CXCL16/SR-PSOX

is an interferon-gamma-regulated chemokine and scavenger

receptor expressed in atherosclerotic lesions. Arterioscler Thromb

Vasc Biol 24(4):750–755

33. Hofnagel O, Luechtenborg B, Plenz G, Robenek H (2002)

Expression of the novel scavenger receptor SR-PSOX in cultured

aortic smooth muscle cells and umbilical endothelial cells. Ar-

terioscler Thromb Vasc Biol 22(4):710–711

J Neurol

123