Journal of Neuroimmunology 115 Ž2001.

168–175
www.elsevier.comrlocaterjneuroin

Enhanced expression of fractalkine in HIV-1 associated dementia

C.F. Pereira a,) , J. Middel a , G. Jansen b, J. Verhoef a , H.S.L.M. Nottet a
a
Neuroimmunology Section, Eijkman-Winkler Institute, UniÕersity Medical Center, HpG04.614, Heidelberglaan 100, NL-3584 CX Utrecht, Netherlands
b
Neuropathology Section, Department of Pathology, UniÕersity Medical Center, Heidelberglaan 100, NL-3584 CX Utrecht, Netherlands
Received 14 August 2000; received in revised form 16 January 2001; accepted 17 January 2001

Abstract

The CX 3 C chemokine fractalkine was found to be up-regulated in the brain during inflammatory processes. In this study, we tried to
assess the role of fractalkine in HIV-1-associated dementia. Fractalkine expression is up-regulated in the brains of AIDS patients with
HAD. Fractalkine immunoreactivity was mainly detected in astrocytes. In addition, fractalkine expression was found to be up-regulated in
cocultures of astrocytes and HIV-infected macrophages. This up-regulation was dependent on cell–cell contact. We propose that
fractalkine produced during interactions between astrocytes and HIV-infected macrophages plays a role in HAD by regulating the
trafficking of monocytic cells in the brain parenchyma. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Fractalkine; HIV-1 associated dementia; Monocyte-derived macrophages; Primary human astrocytes; Reverse transcriptase-polymerase chain
reaction; Immunohistochemistry

1. Introduction 2000.. Fractalkine receptor, CX 3 CR1, belongs to the G-

Chemokines are chemoattractant cytokines that recruit
phagocytic cells from the blood to local sites of inflamma-
tion. Until 1997, there were three known families of
chemokines: CXC or a chemokines, CC or b chemokines
and C or d chemokines. These families were distinguished
by their cystein motif in the N-terminal loop connected via
cysteine bonds to the more structured core of the molecule
Žb sheets and a helix.. In 1997, two groups described a
new chemokine that was found to belong to a fourth group
of chemokines, CX 3 C or g chemokines ŽBazan et al.,
1997; Pan et al., 1997.. This family is characterized by a
CX 3 C motif in the N-terminal of the chemokine domain
and a mucin-like stalk that links the chemokine domain to
a transmembrane spanning and short intracellular domains.
Fractalkine can be expressed in a soluble or membrane-
bound form ŽBazan et al., 1997; Pan et al., 1997..
Unlike other chemokines, fractalkine is mainly ex-
pressed in the brain. Neurons, astrocytes and endothelial
cells were found to express fractalkine ŽBazan et al., 1997;
Maciejewski-Lenoir et al., 1999; Nishiyori et al., 1998;
Pan et al., 1997; Schwaeble et al., 1998; Tong et al.,
)
Corresponding author. Tel.: q31-30-250-6525; fax: q31-30-254-
1770.
E-mail address: C.F.Pereira@lab.azu.nl ŽC.F. Pereira..
protein-coupled receptor family and is expressed in mi-
croglia, neurons and astrocytes ŽMaciejewski-Lenoir et al.,
1999; Nishiyori et al., 1998; Tong et al., 2000.. Most
likely, fractalkine mediates cellular communication be-
tween neurons and microglia ŽHarrison et al., 1998;
Nishiyori et al., 1998; Zujovic et al., 2000. and between
astrocytes and microglia ŽMaciejewski-Lenoir et al., 1999..
The expression levels of fractalkine were found to be
up-regulated during neuroinflammatory processes, such as
experimental autoimmune encephalomyelitis ŽPan et al.,
1997. and facial motor nerve axotomy ŽHarrison et al.,
1998.. This up-regulation was found to be parallel with
microglial activation. These studies suggest that fractalkine
may be relevant in other neuroinflammatory diseases where
microglial cell activation also plays a role.
Approximately a third of adults and half of children
with AIDS develop human immunodeficiency virus type-1
ŽHIV-1. associated dementia ŽHAD., with consequent pro-
gressive motor and cognitive loss. HAD is characterized
by microglial nodules, monocytic cell infiltration into the
brain, reactive astrocytosis, presence of macrophage-de-
rived multinucleated giant cells in the brain parenchyma
Žthe pathological hallmark of HIV encephalitis., vacuolar
myelopathy, white matter pallor and eventually, neuronal
injury and death ŽKolson et al., 1998; Nottet and Gendel-
man, 1995.. Recently, it was clearly demonstrated that

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 5 - 5 7 2 8 Ž 0 1 . 0 0 2 6 2 - 4
 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175 169

monocyte infiltration into the brain correlates extremely
well with the occurrence of HAD ŽNottet, 1999.. The
distribution of infected monocytic cells in the brain is
probably related with temporal expression of adhesion
molecules and chemokines ŽNottet et al., 1997.. However,
the mechanisms that regulate the trafficking of these cells
in the brain parenchyma are not yet fully understood.
In this study, we aimed to assess the role of fractalkine
in HAD by studying the expression of this chemokine in
postmortem human brain tissue of AIDS patients with
HAD, and in an in vitro model for HAD consisting of
monocyte-derived macrophages and primary human astro-
cytes.
2. Materials and methods
2.1. Brain tissue
Tissue specimens of the frontal cortex were obtained
from autopsy brain of 10 HIV-1-infected adults and five
control adults Žcause of death was unrelated to HIV-1
infection and did not show any motor or cognitive impair-
ment. ŽBoven et al., 1999a.. All the HIV-1-infected indi-
viduals had developed AIDS at the time of death and
showed decreased levels of CD4 q T cells Ž- 300.. Six of
them developed cognitive and motor impairments. None of
the patients received any anti-retroviral therapy. AIDS or
HIV-associated dementia was a premortem clinical diagno-
sis made by an AIDS specialty physician or neurologist.
The severity of dementia was scored on the Memorial
Sloan–Kettering scale and all demented patients scored at
least 2 or higher. Patients showed a wide variety of
opportunistic infections ŽTable 1. and died because of
various reasons. All patients are well characterized at the
time of death, and the cause of death was never directly
associated with diseases of the central nervous system.
Viral levels were measured by RT-PCR, which showed a
high degree of variation among all AIDS patients Ždata not
shown.. There was no correlation between the presence or
absence of high viral levels and the stage of disease. When
there were no differences between brain tissue of the
studied patients and normal brain tissue, it was scored as
Ano significant changesB.
Total cellular RNA was isolated from frontal cortex
specimens and was stored at y708C. From adjacent re-
gions, 6-mm sections were made, fixated in cold acetone,
and stored at y208C until immunostaining was performed.
Sections from each individual were stained with H & E and
analyzed by a neuropathologist. The neuropathological
findings are given in Table 1.
2.2. Immunohistochemical analysis of brain tissue
Frozen sections of frontal cortex, with underlying white
matter of HIV-1-infected adults with or without HAD,
were stained for fractalkine Žrespectively, patients 7 and 3
from Table 1.. After fixation with acetone, endogenous
peroxidases were blocked by incubation with 1.5% H 2 O 2
for 20 min. Afterwards, the slides were washed with PBS
and incubated with the primary antibody wgoat-anti-frac-
talkine, Santa Cruz Biotechnology, Santa Cruz, CA;
mouse-anti-glial fibrillary acidic protein ŽGFAP., DAKO,
Jena, Denmark; mouse-anti-microtubule associated protein
ŽMAP-2., Boehringer Mannheim, Mannheim, Germanyx
for 1 h at room temperature ŽRT. in 100% humidity. The
slides were washed with PBS and fixated with 4% formal-
dehyde. Subsequently, the slides were washed with PBS
and incubated with the corresponding peroxidase ŽPo.
Žrabbit-anti-goat, DAKO. or alkaline phosphatase ŽAP.
Žgoat-anti-mouse, Southern Biotechnology Associates,
Birminghan, AL.-conjugated secondary antibody for 1 h at

Table 1
Clinical and neuropathological data
Patients Age Žyear.rsex Clinical status MSK a Neuropathologic findings
1 26rm AIDS 0 no significant changes
2 36rm AIDS 0 HIV encephalitis
3 34rm AIDS 0 cerebral toxoplasmosisrcryptococcosis
4 33rm AIDS 0 cryptococcosis, toxoplasmoses, CMV
5 36rm AIDS 3 major atrophy
6 55rm AIDS 3 atrophy
7 39rm AIDS 3 HIV encephalitis
8 62rm AIDS 3 major atrophy
9 37rm AIDS 2 minor atrophy
10 40rm AIDS 3 major atrophy, HIV encephalitis
11 71rf seronegative no significant changes
12 71rm seronegative diffuse gliosis
13 81rf seronegative no significant changes
14 38rm seronegative no significant changes
15 56rf seronegative no significant changes
a
HIV-associated dementia was a premortem clinical diagnosis made by a neurologist. The severity of dementia was scored on the Memorial
Sloan–Kettering ŽMSK. scale. Patients 1–4 and 11–15 did not suffer from motor or cognitive impairments.
170 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175
RT in 100% humidity. Then, slides were washed with PBS
and incubated with a Po-substrate solution containing the
chromogen 3-amino-9-ethyl carbazole, resulting in a red
reaction product, followed by rinsing and counterstaining
with Mayer’s hematoxylin for 1 min, or in AP-substrate
fast blue BB for 10 min. For fractalkinerGFAP double
staining, slides were first incubated with anti-GFAP, fol-
lowed by anti-fractalkine, then with peroxidase-conjugated
secondary antibody, followed by the alkaline phosphatase-
conjugated secondary antibody. For fractalkinerMAP
double staining, slides were first incubated with anti-frac-
talkine, followed by anti-MAP, then with peroxidase-
conjugated secondary antibody, followed by the alkaline
phosphate-conjugated secondary antibody.
No counterstaining was performed in the double stain-
ing procedure, since staining with both fast blue BB and
hematoxylin results in a blue color.
2.3. Isolation and culture of primary human monocytes
and primary human astrocytes
Peripheral blood mononuclear cells were isolated from
heparinized blood from HIV-1-, HIV-2-, and hepatitis
B-seronegative donors and obtained on Ficoll-Hypaque
density gradients. Cells were washed twice and monocytes
were purified by countercurrent centrifugal elutriation.
Cells were ) 98% monocytes by criteria of cell morphol-
¨
ogy on May-Grunwald-Giemsa-stained cytosmears, and
by non-specific esterase staining using a-naphtylacetate
ŽSigma, St. Louis, MO. as substrate. Monocytes were
cultured in suspension at a concentration of 2 = 10 6
cellsrml in Teflon flasks ŽNalgene, Rochester, NY. in
Dulbecco’s modified Eagle’s medium ŽDMEM., with 10%
heat-inactivated human AB serum negative for anti-HIV
antibodies, 10 mgrml gentamycin, and 10 mgrml cip-
rofloxacin ŽSigma.. As previously described, HIV-1 infec-
tion of non-adherent macrophages, especially when using a
low multiplicity of infection, appears much more repro-
ducible than infection of macrophages that were first al-
lowed to adhere ŽBoven et al., 1999a..
Primary human astrocytes were prepared from se-
cond-trimester human fetal tissue, obtained from elective
abortions Žperformed in full compliance with University
Medical Center, Utrecht.. Brain tissue composed of telen-
cephalon with both cortical and ventricular surfaces was
dissected in cold HBSS with 25 mM HEPES and 50
mgrml gentamycin ŽSigma., then transferred to 20 ml
ice-cold Ž48C. DMEMrF12 ŽLife Technologies, Grand
Island, NY. with 10% heat-inactivated FCS. The tissue
was dissociated mechanically by teasing through a Nitex
bag with a glass pestle. Cells were resuspended in medium
and filtered through a 230- and then 140-mm sieve. The
cell suspension was centrifuged, washed twice in media,
then plated in DMEMrF12 containing 10% heat-in-
activated FCS, 50 mgrml penicillin and streptomycin, 100
mgrml neomycin and 2.5 mgrml fungizone ŽLife Tech-
nologies. into 150-cm2 tissue flasks ŽCorning, Corning,
NY. at a cell density of 10 6 cellsrml. Media was ex-
changed every 3 days. Nonadherent microglia and oligo-
dendrocytes were removed by gentle agitation and circular
shaking of cultured cell preparations 10 days after plating.
Cells were then cultured as adherent monolayers in
DMEMrF12 with 10% heat-inactivated FCS, 20 mgrml
gentamycin and 10 mgrml ciprofloxacin.
Cells were identified as astrocytes by immunocytologi-
cal examination. The second passage of the neural cells
Žastrocyte-enriched preparations. was plated in 48-well
plates to calculate purity of the culture. Cells were fixated
with 4% paraformaldehyde for 30 min at RT, washed with
PBS and incubated with 0.1% Triton-X-100 for 30 min.
Afterwards, cells were washed with PBS and incubated
with blockbuffer ŽPBS with 0.5% gelatin, Sigma; 50 mM
glycine, RdH Laborchemikalien, Seelze, Germany; 1%
BSA, Instruchemie Hilversum, Hilversum, The Nether-
lands and 0.1% sodium azide, Merck, Hohenbrunn, Ger-
many. for 20 min. The cells were washed with PBS
and incubated overnight with mouse-anti-GFAP ŽDAKO.
or mouse-anti-MAP-2 ŽBoehringer Mannheim.. Then,
cells were washed with PBS and incubated with a biotiny-
lated horse-anti-mouse antibody ŽVector Laboratories,
Burlingame, CA. for 1 h. Afterwards, cells were washed
with PBS and incubated for 1 h with streptavidine-FITC.
Cells were then washed with PBS and fluorescence was
examined with a Leica DM IRBE microscope. The purity
of the cell preparation was shown to be ) 99% astrocytes
ŽFig. 1B. by positive GFAP staining. The specificity of
each of the Ab staining of CNS cells was demonstrated by
immunostaining sections of human brain tissue Žsee Fig.
3..
2.4. CocultiÕation of HIV-1-infected or uninfected
macrophages and primary human astrocytes
2 = 10 5 primary human astrocytes were propagated as
adherent monolayers in 24-well plates for 24 h before
addition of macrophages. Seven days after isolation of the
monocytes, monocyte-derived macropahges ŽMDM. were
recovered from the Teflon flasks and infected with HIV-
1 Ba-L at a multiplicity of infection of 0.01 for 2 h. HIV-in-
fected and mock-infected MDM were washed twice to
remove unbound virus, and cultured in Teflon flasks for an
additional 5 days to establish a chronic infection. Then,
MDM were washed and added directly or in a chamber
insert Žtranswell. to the 24-well plates containing a mono-
layer of primary human astrocytes in a 1:0.5 or 1:1 ratio.
2.5. RT-PCR detection of fractalkine mRNA
Brain tissue and cocultures of MDM and primary hu-
man astrocytes Žratio of 1:1. were homogenized and lysed
in 1 ml TRIzol ŽLife Technologies, Gaithersburg, MD.
according to the manufacturer’s guidelines. Total RNA
 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175
Fig. 1. Immunocytochemical identification of neural cells in human primary astrocyte-enriched brain cell cultures to calculate purity of the culture.
Antibodies were directed against microtubule-associated protein 2, MAP-2 Žneurons, A. and glial fibrillary acidic protein, GFAP Žastrocytes, B..
Magnification of 200 = .
was dissolved in diethylpyrocarbonate ŽDEPC.-treated wa-
ter; 1 mg of RNA was used for the synthesis of comple-
mentary DNA, and PCR reactions were performed as
described previously ŽBoven et al., 1999a.. Fractalkine
primers were 5X-TGACGAAATGCAACATCACG-3X
Žsense. and 5X-TCTGTGCTTCCTGGGAAGAA-3X Žanti-
sense.. GAPDH primers were 5X-CCATGGAGAAG-
GCTGGGG-3X Žsense. and 5X-CAAAGTTGTCATG-
GATGACC-3X Žantisense.. For semi-quantification, every
primer pair was tested at different cycle numbers to deter-
mine the linear range. GAPDH mRNA levels were mea-
sured at 25 cycles, whereas cDNA had to be subjected to
35 cycles to detect fractalkine mRNA. Aliquots of 5 ml of
the biotinylated PCR product were semi-quantitatively ana-
lyzed using a fluorescent digoxigenin detection ELISA kit
ŽBoehringer Mannheim., according to manufacturer’s pro-
tocol as described previously ŽBoven et al., 1999a.. The
digoxigenin labeled probe for fractalkine was 5X-GGAC-
CACCCCTGCCGCCGGG-3X and for GAPDH was 5X-
CTGCACCACCAACTGCTTAGC-3X . All data were nor-
malized against GAPDH mRNA levels, which was used as
an internal standard.
2.6. Statistical analysis
Data were compared, and a two-tailed Student’s t-test
was used to determine P values.
2.7. Immunocytochemical analysis of fractalkine protein
expression in primary human astrocytes cultiÕated alone
or with HIV-1-infected and uninfected macrophages
Twenty-four hours after cocultivation of macrophages
and primary human astrocytes, monocultures of primary
171
human astrocytes and cocultures, allowing cell–cell con-
tact between primary human astrocytes and HIV-infected
or uninfected MDM, Žratio of 1:0.5 or 1:1. were fixated
with 4% paraformaldehyde for 30 min at RT, washed with
PBS and incubated with 0.1% Triton-X-100 for 30 min.
Afterwards, cells were washed with PBS and incubated
with blockbuffer ŽPBS with 0.5% gelatin, Sigma; 50 mM
glycine, RdH Laborchemikalien; 1% BSA, Instruchemie
Hilversum and 0.1% sodium azide, Merck. for 20 min.
The cells were washed with PBS and incubated overnight
with goat-anti-fractalkine ŽSanta Cruz Biotechnology..
Then, cells were washed with PBS and incubated with a
biotinylated donkey-anti-goat antibody ŽSanta Cruz
Biotechnology. for 1 h. Afterwards, cells were washed
with PBS and incubated for 1 h with streptavidine-FITC.
Cells were then washed with PBS and fluorescence exam-
ined with a Leica DM IRBE microscope.
3. Results
3.1. Fractalkine mRNA leÕels are eleÕated in the brains of
AIDS patients with HAD
A semi-quantitative fluorescence assay was used to
quantify the expression levels of fractalkine in brain tissue
of the frontal cortex of the individual patients described in
Table 1. Fractalkine mRNA expression levels were ex-
pressed as relative fluorescence units ŽRFU.. Patients were
divided in three groups: AIDS patients with or without
HAD and HIV-1-seronegative patients. mRNA levels of
fractalkine, expressed in RFU, are shown in Fig. 2.
Fractalkine levels were found to be significantly elevated
in brains of AIDS patients with HAD when compared with
172 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175

pared with AIDS patients without HAD ŽFig. 3A.. To

determine which cells produced fractalkine in the brains of
AIDS patients with HAD, we performed double-stainings
of fractalkine and MAP-2 Žcellular marker for neuronal
cells. or GFAP Žcellular marker for astrocytes.. Fractalkine
protein immunoreactivity was found to be associated with
astrocytes ŽFig. 3D. but not with neurons ŽFig. 3C..

3.3. Fractalkine mRNA expression was found to be up-reg-

Fig. 2. mRNA levels of fractalkine in the frontal cortex of postmortem
brain tissue of AIDS patients with ŽHD. or without ŽH. HAD and
HIV-1-seronegative patients ŽC., expressed as relative fluorescence units
ŽRFU.. Results are representative of at least three independent PCR
experiments. ) P - 0.05 Žaccording to Student’s t-test..
the two other groups Ž P - 0.05 according to Student’s
t-test.. The mean RFU for AIDS patients with HAD is
139 " 36, while for AIDS patients without HAD and
HIV-1-seronegative patients is 68 " 27 and 78 " 40, re-
spectively.
3.2. Fractalkine immunoreactiÕity was mainly detected in
astrocytes
Fractalkine immunoreactivity and cellular localization
was determined by immunohistochemistry on frozen sec-
tions of frontal lobes of AIDS patients with and without
dementia. In Fig. 3, the immunohistochemical analysis of
patients representatives of the two groups is shown.
Fractalkine protein was found to be overexpressed in
brains of AIDS patients with HAD ŽFig. 3B. when com-
ulated in cocultures of primary human astrocytes and
HIV-infected macrophages and this up-regulation requires
cell–cell contact between HIV-1-infected macrophages and
primary human astrocytes
Since fractalkine expression was up-regulated in astro-
cytes in brain tissue of AIDS patients with HAD, and since
massive infiltration of HIV-1-infected monocytic cells into
the brain occurs in HAD, the effect of the interactions
between HIV-1-infected MDM and primary human astro-
cytes on fractalkine production was investigated in vitro.
Astrocytes, HIV-infected and uninfected MDM were cul-
tured separately, and cocultures of astrocytes and HIV-in-
fected or uninfected MDM were performed. Results are
depicted in Fig. 4. Fractalkine mRNA expression levels
were expressed as relative fluorescence units ŽRFU..
Fractalkine mRNA expression levels increased in cocul-
tures of uninfected or HIV-infected MDM and astrocytes
as compared to all other culture conditions Ž P - 0.05
according to Student’s t-test.. Furthermore, fractalkine
mRNA expression by cocultures of HIV-infected MDM
and astrocytes is significantly elevated when compared
with cocultures of uninfected MDM and astrocytes condi-

Fig. 3. Immunohistochemical staining of brain tissue sections obtained from subcortical white matter of patients representative of the group of AIDS
patients without HAD ŽA. and with HAD ŽB–D.. ŽA, B. Fractalkine immunostaining. Fractalkine is stained in red, and all cell nucleus present in the
section were counterstained with Mayer’s hematoxylin Žin blue.. ŽC. Double-staining for fractalkine and MAP-2. ŽD. Double-staining for fractalkine and
GFAP. Fractalkine is stained in red. MAP-2 and GFAP are stained in blue. ŽA and B. =200 and ŽC and D. =400.
 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175 173

Fig. 4. Fractalkine mRNA expression by uninfected macrophages Žm., HIV-infected macrophages ŽHm., primary human astrocytes ŽA., cocultures
allowing cell–cell contact between primary human astrocytes and macrophages ŽA q m. or HIV-infected macrophages ŽA q Hm., primary human
astrocytes cocultured with a chamber insert Žtranswell. containing HIV-infected macrophages ŽA ŽHm.., and HIV-infected macrophages cultured in a
chamber insert with primary human astrocytes in the lower compartment ŽHm ŽA... Fractalkine mRNA detected is expressed as relative fluorescence units
ŽRFU.. Results are representative of at least three independent experiments, and individual samples were analyzed three times by semi-quantitative PCR.
)
P - 0.05 compared with the monocultures Žaccording to Student’s t-test.. ) ) P - 0.05 compared with A q m Žaccording to Student’s t-test..

tions Ž P - 0.05 according to Student’s t-test.. The RFU
for cocultures of uninfected or HIV-infected MDM and
astrocytes is 155 " 11 and 237 " 6, respectively. The RFU
for astrocytes, uninfected MDM and HIV-infected MDM
are 32 " 9, 2 " 1 and 7 " 6, respectively.
To determine whether soluble factors produced by HIV-
1-infected MDM had any effect on fractalkine expression
by astrocytes and vice versa, cocultures of HIV-infected
MDM and astrocytes were also performed in a transwell
system. HIV-infected MDM were cultured in a chamber
insert with astrocytes in the lower compartment. This
coculture system did not allow any cell–cell contact.
mRNA could be isolated separately from astrocytes and
MDM, and RT-PCR was performed. Fractalkine mRNA
expression levels were expressed as relative fluorescence
units ŽRFU.. When astrocytes were cocultured with HIV-
infected MDM in a transwell system, there was no signifi-
cant up-regulation of fractalkine expression in any of these
cell types when compared to astrocytes or HIV-infected
MDM cultivated alone Ž P ) 0.05 according to Student’s

Fig. 5. Fractalkine protein immunoreactivity in in vitro cocultures or monocultures. ŽA. Fractalkine protein expression by primary human astrocytes. ŽB.
Fractalkine protein expression by cocultures of primary human astrocytes and HIV-infected macrophages Žcell cocultured in a ratio of 1:0.5.. ŽC.
Fractalkine protein expression by cocultures of primary human astrocytes and HIV-infected macrophages Žcell cocultured in a ratio of 1:1.. ŽD. Same
experimental conditions as in ŽC., but at a higher magnification. Immunocytochemical staining was performed 24 h after establishment of the cocultures.
ŽA–C. =100 and ŽD. =200.
174 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175
t-test.. Thus, fractalkine mRNA up-regulation during astro-
cyte-HIV-infected MDM interactions requires cell–cell
contact.
3.4. Fractalkine protein expression is up-regulated in co-
cultures of primary human astrocytes and HIV-infected
macrophages when compared with primary human astro-
cytes cultiÕated alone or with uninfected macrophages
To corroborate our observations from the RT-PCR as-
say, fractalkine protein expression patterns in primary
human astrocytes, cultivated alone or with HIV-infected or
uninfected macrophages, were determined by immunocyto-
chemical analysis. The cells were cocultivated for 24 h
before the staining was performed. The results are depic-
ted in Fig. 5. Fractalkine protein was found to be over-
expressed in cocultures of astrocytes and HIV-infected
macrophages ŽFig. 5B–D. when compared to astrocytes
cultivated alone ŽFig. 5A.. Moreover, increased amount of
HIV-infected macrophages in the cocultures correlates with
increased amount of fractalkine protein expression ŽFig.
5B–C.. Cocultures of astrocytes and uninfected MDM
showed no increase in fractalkine protein expression, as
compared with astrocytes cultivated alone Ždata not shown..
4. Discussion
A large percentage of HIV-1-positive individuals de-
velop neurological problems. The pathogenic mechanisms
of this phenomenon are still not fully understood. Since the
discovery that chemokine receptors can act as coreceptors
for HIV infection ŽCombadiere et al., 1998; Deng et al.,
1996; Dragic et al., 1996; Feng et al., 1996., there has
been several attempts to correlate chemokine and
chemokine receptor expression patterns with HIV-1-associ-
ated dementia ŽBoven et al., 1999b; Conant et al., 1998;
He et al., 1997; Meucci et al., 1998; Schmidtmayerova et
al., 1996; Shieh et al., 1998; Tong et al., 2000.. With this
study, we aimed to establish a correlation between the
CX 3 C chemokine fractalkine and HAD. Several conclu-
sions can be drawn from this study.
First, fractalkine is overexpressed in the brains of AIDS
patients with HAD. This finding is supported by a previous
study, where it was shown that fractalkine was up-regu-
lated in the brain tissue of pediatric patients with HAD
ŽTong et al., 2000.. Although it is assumed that fractalkine
is associated with neuroinflammation ŽHarrison et al., 1998;
Pan et al., 1997., the role of fractalkine in neuroinflamma-
tory processes is not yet clear. Some studies indicate
fractalkine as a neurotoxic molecule ŽPan et al., 1997.,
while others show it as neuroprotective ŽTong et al., 2000;
Chapman et al., 2000; Harrison et al., 1998; Meucci et al.,
1998; Schwaeble et al., 1998.. Another study indicates that
at low concentrations, fractalkine is involved in cell regula-
tion, while at high concentrations, it has a proinflammatory
role ŽMaciejewski-Lenoir et al., 1999.. A recent study
shows that individuals homozygous for a structural variant
of CX 3 CR1 progress more rapidly to AIDS ŽFaure et al.,
2000..
Second, astrocytes are probably the main source of
fractalkine production in the brains of AIDS patients with
HAD. This finding is supported by a previous study, where
it was shown that fractalkine expression is up-regulated in
astrocytes following stimulation with pro-inflammatory cy-
tokines ŽMaciejewski-Lenoir et al., 1999.. On the other
hand, it has been reported that fractalkine is mainly ex-
pressed by neurons, but this expression is down-regulated
ŽHarrison et al., 1998. or is not up-regulated ŽSchwaeble et
al., 1998. following neuroinflammatory processes. Interest-
ingly, a recent study showed that fractalkine expression is
up-regulated in neurons during HAD ŽTong et al., 2000..
This difference with our results can be explained by the
different experimental conditions. Tong and colleagues
performed immunohistochemistry in paraffin-embedded
tissue, while we used frozen tissue. Also, they used sam-
ples from pediatric patients, while we used samples from
adult patients. Differences in neuropathologies are seen in
children as compared to adults, and also, the neurological
symptoms are more frequent, and sometimes occur more
rapidly in pediatric cases compared with adult cases. Since
the CNS is developing in young infants and myelinization
is incomplete for 3 years, it might be indeed expected that
HIV-induced macrophage-mediated neuronal damage is
more prevalent. Although very speculative, fractalkine
might be differentially expressed during the development
of the CNS, and when expressed on neurons in the brains
of pediatric patients, they might be more vulnerable to the
macrophage-mediated damage, since it is well known that
neuronal fractalkine mediates communication with the
CX 3 CR1-expressing cells of the mononuclear system
ŽHarrison et al., 1998; Nishiyori et al., 1998.. Astrocytic
expression of fractalkine might therefore be more neuro-
protective than neuronal expression, and this might help to
explain the more chronic nature and lower incidence of
HAD in adult AIDS patients.
Fractalkine has also been indicated to attract and pro-
mote adhesion of leukocytes ŽBazan et al., 1997; Imai et
al., 1997., and also to mediate leukocyte migration across
an in vitro transmigration system ŽImai et al., 1997; Tong
et al., 2000.. Although it was shown that cell surface-bound
fractalkine can be induced on activated primary endothelial
cells ŽBazan et al., 1997., the expression of fractalkine on
blood–brain barrier endothelium has not yet been demon-
strated. A immunohistochemical study showed fractalkine
expression on perivascular areas of LPS-treated murine
brain tissue, but this expression was found to be related
with microglia staining ŽPan et al., 1997.. Since astrocytes
are part of the blood–brain barrier, it is reasonable to think
that fractalkine expression by these astrocytes could play a
role in the recruitment of monocytic cells into the brain.
 C.F. Pereira et al.r Journal of Neuroimmunology 115 (2001) 168–175
We propose that fractalkine produced during interac-
tions between astrocytes and HIV-infected monocytic cells
plays a role in HAD by regulating the trafficking of these
cells in the brain parenchyma, and possibly by recruiting
monocytic cells to the brain.
Acknowledgements
This work was supported by grants from the Training
and Mobility of Researchers of the European Commission
ŽERBFMBICT983314. and the Royal Netherlands
Academy of Sciences and Arts. Dr. Hans S.L.M. Nottet is
a fellow of the Royal Netherlands Academy of Sciences
ˆ
and Arts and Candida da Fonseca Pereira is a Marie Curie
fellow.
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