developmental expression patterns of ccr5 and cxcr4 in the rhesus macaque brain

Developmental expression patterns of CCR5 and CXCR4

in the rhesus macaque brain

S.V. Westmoreland a,*, X. Alvarez a, C. deBakker a, P. Aye a, M.L. Wilson a,b,K.C. Williams c, A.A. Lackner a

aDivision of Comparative Pathology, New England Regional Primate Research Center, Harvard Medical School,

One Pine Hill Drive, Southborough, MA 01772-9102, USAbSchool of Veterinary Medicine, University of Illinois Urbana Campus, Jacksonville, IL 62650, USA

cDivision of Viral Pathogenesis, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02115, USA

Received 27 June 2001; received in revised form 10 October 2001; accepted 19 October 2001

Abstract

Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS

development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and

explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5

and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and

immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem,

and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased

significantly ( p < 0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4

expression in resident cells of the CNS. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: CCR5; CXCR4; Chemokine receptors; Brain; Neurons; Glia; Macaque; HIV; SIV

1. Introduction

Patients with AIDS frequently suffer from a debilitating

neurologic syndrome with children exhibiting a higher inci-

dence of dysfunction than do adults (Ammann, 1994; Bel-

man, 1994; Epstein et al., 1986; Wilfert et al., 1994). The

neurologic disease is postulated to result from HIV-induced

neuronal injury and loss (dropout) (Asare et al., 1996; Everall

et al., 1991; Ketzler et al., 1990; Lipton et al., 1991; Masliah

et al., 1992a), particularly apoptotic death (Adle-Biassette et

al., 1995; Gelbard et al., 1995). There is evidence using the

SIV-infected rhesus macaque model that similar neuronal

injury and death occurs in SIV infection (Gonzalez et al.,

2000; Jordan-Sciutto et al., 2000; Li et al., 1999; Marciario et

al., 1999; Tracey et al., 1997). Seven-transmembrane, G-

protein-coupled chemokine receptors have been demonstra-

ted on neurons and glia from both humans and non-human

primates, and may play a role in mediating HIV- and SIV-

induced neuronal injury and death, respectively (Coughlan et

al., 2000; Halks-Miller et al., 1997; Hesselgesser and Horuk,

1999; Horuk et al., 1996, 1997; Lavi et al., 1997; Ohagen et

al., 1999; Qin et al., 1998; Rottman et al., 1997; van der Meer

et al., 2000, 2001; Westmoreland et al., 1998; Zhang et al.,

1998).

We have previously shown that these receptors are func-

tional on rhesus macaque fetal neurons by demonstrating that

the relevant chemokine induces calcium flux (Klein et al.,

1999). Although much has been written about the impor-

tance of chemokine receptors in leukocyte function, chemo-

taxis, and as coreceptors for HIVand SIV cell entry, little has

been published relating to their role in normal physiologic

processes in cells of the central nervous system (CNS).

Emerging data suggest that chemokine receptors are in-

volved in communication between neurons and glia (Dorf

0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0165 -5728 (01 )00457 -X

* Corresponding author. Tel.: +1-508-624-8074; fax: +1-508-624-8181.

E-mail address: [email protected]

(S.V. Westmoreland).

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 122 (2002) 146–158

et al., 2000; Giovannelli et al., 1998; Janabi et al., 1999;

Ohtani et al., 1998), CNS development (Ma et al., 1998; Zou

et al., 1998), and neuronal survival (Araujo and Cotman,

1993; Giovannelli et al., 1998; Meucci et al., 1998). There

are also reports indicating that chemokine receptors mediate

pathologic processes in the cells of the CNS (Adamson et al.,

1996; Adle-Biassette et al., 1995; Gelbard et al., 1995; Kaul

and Lipton, 1999; Krajewski et al., 1997; Meucci et al.,

1998; Ohagen et al., 1999; Zheng et al., 1999). In light of the

growing interest in understanding determinants of neuronal

and glial function and dysfunction, particularly pertinent to

Alzheimer’s, Parkinson’s, and AIDS dementia, it is critically

important to understand the roles that chemokine receptors

have in neuronal and glial communication, survival, and

injury. With this endeavor in mind, we document detailed

information of cellular, regional, and temporal CCR5 and

CXCR4 expression in the CNS of fetal, neonatal, and adult

rhesus macaques.

2. Materials and methods

2.1. Tissue collection and processing

Brain was collected for immunohistochemistry and

immunofluorescence from 14 animals including 1 fetus, 12

neonates, and 1 adult. The fetus, obtained by caesarian

section, was at gestational day 100, which corresponds to

the second trimester of gestation (full term is 165 ± 5 days).

Neonates were sacrificed at 0, 3, 7, 14, 21, and 50 days, as

well as 6 and 9 months of age (roughly equivalent to a 3- to

4-year-old child). Brain tissue from one adult rhesus mac-

aque (greater than 4 years) was also examined. These gesta-

tional and perinatal time points were selected because most

HIV infections in children occur by vertical transmission

during parturition or immediately postpartum (Ammann,

1994; Belman, 1994; Wilfert et al., 1994). The animals used

in this paper were control animals for other studies.

At necropsy, body and organ weights were recorded and

tissues were collected within 1 h of death. A complete set of

brain tissues, including frontal and temporal cortex, hippo-

campus, thalamus, cerebellum, and brain stem, was col-

lected and fixed in 10% neutral-buffered formalin,

embedded in paraffin, sectioned at 6 mm and stained with

hematoxylin and eosin by routine techniques within 2 weeks

of necropsy. Adjacent tissue blocks from the same regions

were collected and snap-frozen in microcentrifuge tubes and

stored at � 80 �C for nucleic acid isolation.

Fresh brain tissue from two additional second-trimester

fetuses derived by caesarian section and three adult rhesus

macaques was also harvested for flow cytometry (see

below). Animals were housed in accordance with standards

of the Association for Assessment and Accreditation of

Laboratory Animal Care. Investigators adhered to the Guide

for the Care and Use of Laboratory Animals prepared by the

National Research Council.

2.2. Immunohistochemistry

To evaluate regional distribution and cellular expression

of CCR5 and CXCR4 in normal brain tissue, immunohis-

tochemistry using an avidin–biotin–horseradish-peroxidase

complex (ABC) technique was performed as described with

minor modifications (Westmoreland et al., 1998). Briefly, 6-

mm-thick sections of paraffin-embedded tissue from the

brain regions listed above were baked, deparaffinized,

rehydrated, blocked with a methanolic block (methanol,

H2O2, PBS) for 5 min, and treated with Proteinase K (Dako,

Carpinteria, CA) for 5 min. Slides were incubated with

monoclonal primary antibodies for CCR5 (5C7 used at 10

mg/ml; LeukoSite, Cambridge, MA) or CXCR4 (12G5a used

at 10 mg/ml; PharMingen, San Diego, CA), followed by a

biotinylated secondary antibody (horse–antimouse–biotin),

and then horseradish-peroxidase avidin–biotin complexes

(Vectastain Elite; Vector, Burlingame, CA). Labeling was

detected with the chromogen diaminobenzidine (Sigma, St.

Louis, MO) and slides were lightly counterstained with

Mayer’s hematoxylin. Controls consisted of adjacent sec-

tions stained with isotype- and concentration-matched irrel-

evant antibodies.

2.3. Image analysis and quantification of neuronal chemo-

kine receptors

Immunohistochemical labeling was evaluated by manual

scoring and computerized image analysis as described pre-

viously (Sasseville et al., 1996; Veazey et al., 1998; Wykrzy-

kowska et al., 1998). Briefly, for manual scoring the intensity

(minimal, moderate, or marked) and percentage (0–25%,

26–50%, 51–75%,>75%) of positive cells within each brain

region and within the six neocortical layers were examined

(Adams and Graham, 1994). For image analysis we used an

Olympus Vanox-S research microscope interfaced to a Quan-

timet 500IW image analyzer (Leica, Cambridge, UK) via an

Optronics DEI 750 CCD camera (Goleta, CA) and focused on

the frontal cortex. Eight separate regions of equal area (5000

mm2) from the frontal cortex of each case were analyzed. The

total number of immunopositive cells in each region was

counted automatically according to a contrast threshold that

would detect cells stained with diaminobenzidine.

2.4. Data analysis

Data were compiled for each animal by averaging the

total number of CCR5 or CXCR4 immunopositive cells for

all eight regions analyzed for each case. The data were

expressed as the number of immunopositive cells/5000 mm2.

Standard deviation was assessed for each time point and

added to each graph using Excel 5.0 (Microsoft, Redmond,

WA). One-way Analysis of Variance (ANOVA) was used to

determine the relationship between the age of monkeys and

the expression of CCR5 and CXCR4 (Rosner, 1990). When

the result of one-way ANOVAwas significant the difference

S.V. Westmoreland et al. / Journal of Neuroimmunology 122 (2002) 146–158 147

between individual age groups was determined by the Least

Significant Difference analysis. A p value less than 0.05 was

considered significant for one-way ANOVA.

2.5. Confocal microscopy for CCR5 and CXCR4

To examine the cellular expression pattern of CCR5 and

CXCR4 in neurons and to determine the degree of coex-

pression of the two chemokine receptors in neurons in

animals of varying maturity, we performed double- and

triple-label immunofluorescence and confocal microscopy

using previously described techniques (Klein et al., 1999;

Williams et al., 2001). Briefly, slides from paraffin-embed-

ded tissues were treated as described above for immunohis-

tochemistry and then incubated overnight in the dark at 4 �Cwith monoclonal antibody to CXCR4 (12G5a; 10 mg/ml;

PharMingen). Slides were washed twice in 1X phosphate-

buffered saline with 0.2% fish skin gelatin (PBS-FSG) with

0.1% Triton X-100 (Sigma). Slides were then incubated

with a rabbit anti-mouse ‘‘bridge’’ (1:100; Vector) for 30

min at room temperature, washed, and incubated with a goat

anti-rabbit Alexa 488 fluorochrome (1:1000; Molecular

Probes, Eugene, OR) for 30 min at room temperature. Slides

were washed, treated with an avidin–biotin block (Dako)

for 10 min followed by 10% normal mouse serum for 30

min. The second primary antibody, biotinylated CCR5-5C7

(10 mg/ml), was incubated for 1 h at room temperature.

Slides were washed and incubated with streptavidin Alexa

568 fluorochrome (1:1000; Molecular Probes) for 30 min at

room temperature. Slides were washed and treated with 50

mM Cu3SO4 in ammonium acetate buffer (pH 5.0) for 45

min to quench autofluorescence (Schnell et al., 1999), rinsed

in dH2O, and coverslipped with anti-quenching aqueous

mounting solution (Mowiol 4–88 glycerol DABCO, TRIS

pH 8.5) (Harlow and Lane).

To confirm the neuronal phenotype of analyzed cells,

selected tissues were also incubated with a monoclonal anti-

body to microtubule associated-protein-2 (MAP-2), a neu-

ron-specific marker. In these studies the sections were first

labeled with anti-MAP-2 (1:500; Sigma) and goat anti-mouse

Alexa 488 (Molecular Probes), blocked with 1% normal

mouse serum (Sigma), incubated with a biotinylated-anti-

CCR5 (as above) followed by streptavidin Alexa 568, and

lastly with CXCR4 conjugated to Cychrome (PharMingen).

Slides were examined using a Leica TCS SP laser scan-

ning confocal microscope equipped with three lasers (Leica

Microsystems, Exton, PA) using Leica software. Images

were collected at 512� 512 pixel resolution. The stained

cells were optically sectioned in the z-axis, and the images in

the different channels (photomultiplier tubes) were collected

simultaneously after compensating for bleed-through in

different channels. The step size in the z-axis varied from

0.2 to 0.5 mm to obtain 30–50 optical sections per field. The

images were transferred to a Macintosh G4 computer, and

NIH image 1.6 and Photoshop 6.0 (Adobe Systems, Moun-

tain View, CA) were used to assemble the images.

2.6. Analysis of chemokine receptor expression by flow

cytometry

In addition to examining chemokine receptor expression

in tissues, we examined chemokine receptor expression in

neurons from three fetal brains and immediately ex vivo glia

isolated from three adult brains. The protocol for isolation of

neurons from fetal rhesus brain has been previously pub-

lished (Klein et al., 1999). Briefly, fetal brain was harvested

immediately after caesarian section. The cortex was disso-

ciated mechanically with sterile scalpel blades, washed in

sterile PBS, and centrifuged at 800 rpm for 10 min. The

pellet was then enzymatically dissociated with trypsin

(0.05%) in the presence of DNase (50 mg/ml) for 20 min at

37 �C. Dissociated cells were washed with PBS, passed

through a 150-mm nylon mesh, washed, pelleted again, and

resuspended in tissue culture medium consisting of DMEM

supplemented with gentamycin (20 mg/ml), 0.01% dextrose,

and 5% FBS at a concentration of 3� 106 cells per ml. Cells

were seeded in T75 flasks for 1 day. Non-adherent neurons

were gently washed of the adherent glial cells and analyzed

by flow cytometry at 106 cells/ml for chemokine receptor

expression. As previously described (Klein et al., 1999),

neurons were incubated with monoclonal antibodies to either

biotinylated CCR5 (clone 5C7; LeukoSite) or CXCR4-PE

(clone 12G5a; PharMingen). All samples were incubated in

the dark at 4 �C for 30 min and then washed in PBS. Cells

were then resuspended and fixed in 2.5 ml RPMI 5% FCS

and 2% paraformaldehyde. Samples were analyzed on a

Becton Dickinson FACS Calibur using Cell Quest software.

Glia were isolated from adult rhesus brain obtained

within 30 min of necropsy using a previously described

procedure (Yong et al., 1992). Meninges were removed and

tissues were mechanically dissociated using sterile scalpel

blades. Dissociated tissues were washed three times in PBS

by centrifugation at 800 rpm for 10 min and enzymatically

dissociated with 0.25% trypsin in the presence of DNase (50

mg/ml) at 37 �C for 45 min. Dissociated cells were washed

twice with PBS and subjected to a 30% isotonic percol

linear gradient at 15,000 rpm for 30 min to separate myelin

from glial cell bodies. Recovered cells were washed twice

with PBS and adjusted to a concentration of 1�106 cells

per ml per data point. Cells were incubated for 30 min on ice

in PBS blocking buffer containing 10% goat serum, 5%

fetal calf serum, and 1% human serum, washed, and

incubated with biotinylated anti-CCR5 or anti-CXCR4 for

30 min on ice. Cells were washed with PBS and incubated

with streptavidin PE (for CCR5) or goat-anti-mouse PE (for

CXCR4) for 30 min on ice. Cells were then washed and

fixed for 15 min on ice with 2% paraformaldehyde. The

cells were made permeable by incubating for 15 min with

0.1% saponin. Cells were spun down and incubated with

CD11b� FITC (10 mg/ml; Immunotech, Westbrook, ME)

for 30 min on ice. Cells were washed with PBS, and

resuspended in 2% paraformaldehyde for analysis by flow

cytometry using a FACS Calibur (Becton Dickinson, Moun-

S.V. Westmoreland et al. / Journal of Neuroimmunology 122 (2002) 146–158148

tain View, CA). At least 10,000 events were collected per

sample. Cells were analyzed according to CD11b positivity.

Chemokine receptor expression on CD11b+ macrophage/

microglia and CD11b� astrocytes and oligodendrocytes

was quantitated.

2.7. Reverse-transcriptase-polymerase chain reaction (RT-

PCR) for detection of CCR5 and CXCR4 mRNA stand-

ardized to rhesus beta-globin

Total RNA was isolated from blocks of frozen frontal

cortex gray matter, adjacent to those used for immunohis-

tochemistry, using the RNEasy Mini Kit (Qiagen, Valencia,

CA) from all animals except the fetuses. Reverse transcrip-

tion (RT) was performed on 3 mg of RNA using the

GeneAmp RNA PCR Kit (Perkin-Elmer, Foster City, CT)

according to manufacturer’s instructions. Briefly, conditions

for RT reactions consisted of 10 min at 15 �C, 1 h at 42 �C,and 5 min at 99 �C. PCR was performed on 15 mg of newly

transcribed cDNA using 20 pmol of CXCR4 primers (s-50-

ATA-TAC-ACT-TCA-GAT-AAC-TAC-ACC-30 and as-50-

CAT-AGG-AAG-TTC-CCA-AAG-TAC-C-30) to produce a

311 bp product, CCR5 primers (s-50-GCT-CTA-CTC-ACT-

GGT-GTT-CAT-C-30 and as-50-TCA-TGA-TGG-TGA-

AGA-TAA-GCC-3) to produce a 620-bp product or Beta-

globin primers 50-CTT GGG AGT GAA GAA ACT GC and

30-TAG CCT CAG ACT CTG TTT GG. cDNA was dena-

tured for 2 min at 92 �C, then amplified within a linear range

for 35 cycles at 92 �C for 30 s, 61 �C for 30 s, and 72 �C for

30 s, followed by an extension time of 10 min at 72 �C.Amplification was performed in a Perkin-Elmer 9700 ther-

mocycler with 100 mM of deoxynucleoside triphosphates,

1.5 mM MgCl2, 2.5 IU of taq DNA polymerase and buffer

(50 mM KCl, 10 mM Tris, pH 9.0) in a 50-ml reaction. Eightmicroliters of amplified PCR reaction products with 2 ml ofgel loading buffer were electrophoresed through 2% agarose

gel, stained with ethidium bromide, and visualized under UV

light. Appropriate controls for contamination during PCR

were negative. Images were captured with densitometry

assayed using the Gel-Doc 2000 system with Quantity One

software (Bio Rad Laboratories, Hercules, CA). The density

of the CCR5 and CXCR4 bands were standardized to the

density of amplified rhesus beta-globin from each of the

samples. Values for CCR5 and CXCR4 mRNA were ex-

pressed as the ratio of signal obtained for chemokine recep-

tors relative to rhesus beta-globin signals and graphed using

Excel 98 (Microsoft).

3. Results

3.1. Expression pattern of CCR5 in the brain of rhesus

macaques

We used immunohistochemistry to examine CCR5

expression in resident cells of the cortex, hippocampus,

thalamus, cerebellum, and brain stem from rhesus macaques

ranging from the third trimester of gestation to adult. We

evaluated the results by individual manual scoring based on

frequency and intensity of immunopositive cells. Manual

scoring of the frontal cortex included evaluation of each of

the six neocortical neuronal layers.

Expression of CCR5 was present in all animals and

increased with age. In a third-trimester fetus, scattered

cortical neurons were weakly immunopositive for CCR5

while numerous neurons of the subcortical nuclei expressed

moderate-to-marked amounts of CCR5 in the cytoplasm of

the perikaryon and processes (Fig. 1A). In addition, there

was strong vessel-associated immunopositivity for CCR5 in

fetal brain (Fig. 1B), which was still detectable after 50 days

of age (Fig. 1E). At birth, there was minimal CCR5

expression in most of the pyramidal neurons of the cortex,

hippocampus (CA1–4), and the granular neurons of the

dentate gyrus. Purkinje cells expressed abundant cytoplas-

mic CCR5 in a granular or vesicular pattern (Fig. 1C) while

neurons within the granular layer had modest CCR5. Epen-

dymal and choroid plexus cells were uniformly CCR5

immunopositive. Occasional CCR5 cells within periventric-

ular and perivascular collections of germinal matrix cells

were also CCR5 positive.

Between 3 and 14 days of age, expression of CCR5

increased in neocortical neurons in all layers. In addition,

moderate or marked expression was present in greater than

75% of CA1–4 pyramidal hippocampal and dentate gyrus

neurons. CCR5 expression was also marked in brain stem

neurons by 3 days of age (Fig. 1D). CCR5 expression in glia

within the cortical white matter was not detectable until 21

days of age, but remained detectable thereafter (Fig. 1E,

inset). CCR5 was expressed within the cytoplasm of the

glial cell bodies as well as in the processes.

By 50 days of age, CCR5 expression in cortical neurons

was comparable to adult levels (Figs. 1E and 2). Also, CCR5

expression in cortical neurons emerged as a bimodal or

differential expression pattern where large pyramidal neo-

cortical neurons expressed much higher levels of CCR5 than

did neighboring neurons (Figs. 1E, arrows; 2). This pattern

was first evident in neocortical layer V where a subpopula-

tion of large pyramidal neurons (25–50% of neurons in that

layer) exhibited marked cytoplasmic expression of CCR5

while a population of smaller neurons (50–75% of layer V

neurons) expressed only minimal CCR5. This bimodal

expression pattern was more prominent in the 6-month-old

animal and included neocortical layers III and V, reached

adult proportions by 9 months of age in layers III, V, and VI

(Fig. 2), and persisted in adults.

3.2. Expression pattern of CXCR4 in the brain of rhesus

macaques

Expression of CXCR4 in the CNS was similar to that of

CCR5 with some exceptions. Fetal expression of CXCR4

was slightly greater than that seen for CCR5 with moderate


expression in neocortical neurons, but less intense expres-

sion in the thalamic neurons and neurons of subcortical

nuclei. Also, CXCR4 was generally less intense than CCR5

in the neocortical neurons after birth (Fig. 2). Cortical

neuronal expression was low until 14 days of age when it

increased (Fig. 2). Similar to CCR5, CXCR4 expression in

neocortical neurons increased with age and also evolved

into a subtle bimodal expression pattern, but the degree of

differential expression of CXCR4 was much less prominent

than that seen with CCR5 (Fig. 2). CXCR4 was detectable

in 50–75% of hippocampal pyramidal neurons in regions

CA1–4 and in over 75% of granular neurons of the dentate

gyrus by 3 days of age, similar to CCR5 (Fig. 1F). CXCR4

expression in glial cells in the cortical white matter was

minimal, but detectable as early as 3 days of age. Glial ex-

pression increased slightly after 21 days of age and re-

mained detectable into adulthood. Unlike CCR5, CXCR4

glial expression was limited to the cytoplasm of the cell

body and did not include glial processes.

Minimal vessel-associated CXCR4 expression was ob-

served compared to the intense expression of CCR5. Al-

though vessel-associated CXCR4 was detected in the fetus,

by 14 days of age vessel-associated expression of CXCR4

was undetectable by immunohistochemistry, whereas ves-

sel-associated CCR5 expression persisted (Fig. 1E, arrow-

heads).

Fig. 1. A–F: Immunohistochemistry for CCR5 (A–E) and CXCR4 (F) expression in rhesus fetal and neonatal brain. There is abundant CCR5 expression in

subcortical nuclei (A) and associated with cortical vessel in fetal brain (B), the perikaryon of Purkinje cells in day 1 neonate (C), brain stem neurons in 3-

day-old neonate (D), and in the perikaryon and processes of neurons in frontal cortex layer III in a 50-day-old rhesus (E). Arrows highlight two

neighboring neurons with differential expression of CCR5. Vessel-associated CCR5 expression indicated by arrowheads. CCR5 glial expression in cortical

white matter demonstrated in inset. CXCR4 is expressed in hippocampus and dentate gyrus of 3-day-old rhesus macaque (F). Avidin–biotin horseradish

peroxidase technique with diaminobenzidine chromogen and Mayers hematoxylin counterstain. Original magnification, � 60 (A, B, E inset), � 40 (C, E,

F), � 20 (F).


Fig. 2. A–H: Comparison of CCR5 and CXCR4 expression by immunohistochemistry in frontal cortical neurons in rhesus neonates over time: CCR5 expression in neonates at birth (A), 14 days (B), 50 days (C),

and 9 months of age (D); CXCR4 expression in neonates at birth (E), 14 days (F), 50 days (G), and 9 months of age (H). Note the significant increase in staining intensity over time. This change is more

pronounced for CCR5 than CXCR4. Avidin–biotin–horseradish peroxidase complex technique with diaminobenzidine chromogen and Mayer’s hematoxylin counterstain. Original magnification, � 20.

S.V.Westm

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al./JournalofNeuroimmunology122(2002)146–158

151

3.3. CCR5 and CXCR4 neuronal expression increases with

age

Because there appeared to be an increase in the neuronal

expression of CCR5 and CXCR4 with age, we used manual

scoring (Fig. 2; data incorporated in text) and digital image

analysis (Fig. 3) to quantify the change. Based on these data,

the number of CCR5 and CXCR4 immunopositive neurons

in the frontal cortex increased four- to five-fold between birth

and 9 months of age (Figs. 2 and 3). This age-dependent

increase in CCR5 and CXCR4 expression by cortical neu-

rons was statistically significant ( p < 0.00001) from birth to

9 months of age. The most dramatic increase in expression

occurred in cortical layers III, V, and VI in pyramidal

neurons.

At birth, CCR5 was expressed at minimal levels in

neurons in all six neocortical layers with slightly higher

levels of expression in the pyramidal neurons of layer III

(Fig. 2A). By 14 days of age, CCR5 immunoreactivity in

the frontal cortex increased significantly compared to day 0

based on Least Significant Difference (LSD) analysis (Figs.

2B and 3A). Around 50% to 75% of neurons in neocortical

layers III, V, and VI were strongly immunopositive for

CCR5 (Fig. 2). In the 50-day-old animal, CCR5 expression

in cortical neurons again increased significantly by LSD

analysis in layers III, V, and VI compared to the 14-day-old

animal (Figs. 2C and 3A). Expression of CCR5 increased

again in cortical neurons in the 9-month-old animal (Fig.

2D), but the difference from the 50-day-old animal was not

statistically significant (Fig. 3A).

At birth, CXCR4 expression was minimal in only 10–

20% of neocortical neurons limited to layer III (Fig. 2E). By

14 days of age, moderate-to-marked CXCR4 expression

was detectable in 50% to 75% of the neurons in neocortical

layers III and V (Fig. 2F). The increase in cortical neuronal

expression between days 0 and 14 was statistically signifi-

cant ( p < 0.00001) (Fig. 3B). By 50 days of age, CXCR4

immunoreactivity in neurons increased, particularly in pyr-

amidal neurons of neocortical layers III, V, and VI (Fig. 2G).

CXCR4 neuronal immunoreactivity increased, but not sig-

nificantly (Fig. 3B), between 21 days and 9 months of age.

The expression of CXCR4 increased to moderate or marked

expression in up to 75% of neurons in all neocortical layers

in a 9-month-old juvenile (Fig. 2H). Although expression of

CCR5 was significantly increased through 50 days of age

(Fig. 3A), the expression of CXCR4 did not change sig-

nificantly after 21 days of age (Fig. 3B).

3.4. Most cortical neurons that express CCR5 are also

CXCR4 positive

To determine if CCR5 and CXCR4 were expressed in the

same cells, double-label confocal microscopy was per-

formed. At birth we found that 60–70% of neurons coex-

pressed CCR5 and CXCR4 (Fig. 4A). The remaining

neurons were strongly positive only for CCR5 (Fig. 4A,

arrowhead). No neurons were positive for CXCR4 alone.

Most labeling for CCR5 and CXCR4 in neurons was

localized within the perikaryon in a vesicular or granular

pattern and extended into the dendritic processes and axon.

There was also strong vessel-associated CCR5 immunopo-

Fig. 3. A, B: Box plots of immunopositive cells for CCR5 (A) and CXCR4

(B) per 5000 mm2 area in frontal cortex. Standard deviation and p values

were calculated and data were graphed as box plots using Excel 98 software

(Microsoft). Horizontal lines in box plots, generated with Statview software

(Abacus, Berkeley, CA), represent (bottom to top) lowest, first quartile,

median, third quartile, and highest number of immunopositive cells/5000

mm2 area. NS = not significant.


Fig. 4. A–E: Confocal microscopy images in panels A–D are double-label immunofluorescence of CCR5 and CXCR4 in the frontal cortex in a newborn (A),

6-month-old juvenile (B), and 5-year-old adult rhesus macaque (C, D). In panels A–D, images for the individual channels are shown on the left side of the

larger composite panel as insets of CXCR4 (green), CCR5 (red), and either differential interference contrast (DIC) or the nuclear stain ToPro3 (blue). The

composite panels represent superimposition of individual channels with colocalization of CCR5 and CXCR4 appearing yellow. Panel A illustrates a subclass of

CCR5-single positive neurons (arrowhead) and vessel-associated CCR5 (arrow) in a newborn rhesus macaque. Panel B shows a subpopulation of neurons that

have more intense expression of CCR5 than CXCR4 and thus appear red-orange in a 6-month-old animal. Panel C represents coexpression of CCR5 and

CXCR4 by the majority of cortical neurons. Panel D is a similar neuron at higher magnification demonstrating colocalization of CCR5 and CXCR4 at and

below the cellular membrane and within the cytoplasm. Panel E is a triple-label immunofluorescence image of MAP-2 (neuron-specific microtubule associated

protein–2; green) positive neurons from an adult rhesus macaque colabeled with CCR5 (red) and CXCR4 (blue) to verify cells as neurons. White bar at the

bottom of each image represents 10 mm.


sitivity (Fig. 4A, arrow). In animals 3 days of age or older,

most neurons were double positive for CCR5 and CXCR4,

although 15–30% expressed more intense CCR5 than

CXCR4. In animals 6 months of age or older, the population

of CCR5 single-positive cortical neurons was no longer

evident (Fig. 4B,C). However, some pyramidal cortical

neurons did express a relatively higher level of CCR5 (Fig.

4B, arrows; C). In these CCR5, CXCR4 double-positive

neurons, the two chemokine receptors were often present on

or just below the plasma membrane and within the cytoplasm

in a vesicular pattern (Fig. 4D).

To confirm that the CCR5 and CXCR4 positive cells

were neurons, triple-label immunofluorescence with the

neuron marker microtubule-associated protein-2 (MAP-2)

was performed on adult frontal cortex (Fig. 4E).

3.5. CCR5 and CXCR4 are present on the cell surface of

neurons and glia

To determine the percentage of neurons that express

CCR5 and CXCR4 on their surface, we used flow cytometry

to analyze neurons isolated from three second-trimester

fetuses using techniques described previously (Klein et al.,

1999). These data demonstrate CCR5 expression on the

plasma membrane of 34–47% of rhesus fetal neurons (Fig.

5A) and CXCR4 on 28–51% of the cells. Since viable

neurons cannot be isolated from juvenile or adult brain, we

were unable to directly compare neuronal surface chemo-

kine receptor expression between fetuses and adults. How-

ever, the analysis confirms that CCR5 and CXCR4 are

expressed on the surface of subsets of fetal rhesus macaque

neurons in addition to being expressed in the cytoplasm and

suggests that there may be considerable variation of

between individuals.

In addition to characterizing the expression of chemokine

receptors on neurons, we have further characterized glial

cell expression of CCR5 and CXCR4 (Klein et al., 1999;

Westmoreland et al., 1998). CXCR4 expression in glial cells

in the cortical white matter was minimal, but was detectable

as early as 3 days of age, and increased slightly after 21 days

of age and remained detectable into adulthood. CCR5

expression in glia within the cortical white matter was not

detectable until 21 days of age, and remained detectable

thereafter (Fig. 1E, inset). CCR5 was expressed within the

cytoplasm of the glial cell bodies and the processes.

To better define and quantitate the surface expression of

CCR5 and CXCR4 on glial cells, we used flow cytometry to

examine chemokine receptor expression in different types of

glia (CD11b+ microglia/macrophages and CD11b� astro-

cytes and oligodendrocytes) isolated immediately ex vivo

from brain of three adult rhesus macaques. Flow cytometric

analysis of dissociated glial cells double-stained with CD11b

Fig. 5. A–C: Flow cytometric analysis of surface expression of CCR5 and CXCR4 on fetal neurons and adult glial cells. Percentage of CCR5 and CXCR4

surface expression on neurons cultured from fetal rhesus macaque demonstrated by flow cytometry (A); percentage of analyzed cells expressing CCR5 and

CXCR4 on sorted CD11b+ microglia and CD11b� astrocytes/oligodendrocytes harvested immediately ex vivo from normal adult macaque brain (B);

Histograms of original flow data summarized in table 5B (C).


and CCR5 or CXCR4 (Fig. 5B,C) revealed that the number

of CD11b+ microglia that express CCR5 or CXCR4 is

roughly comparable to the number of astrocytes and oligo-

dendrocytes combined indicating that microglia are the

predominant glial cell population expressing CCR5 and

CXCR4. In addition, the number of CD11b+ microglia and

CD11b� astrocytes/oligodendrocytes expressing CXCR4

on their surface is higher than those expressing CCR5

(summarized in Fig. 5B). On average, close to 50% of

CD11b+ microglia express CXCR4 on their surface, while

only an average of 12–13% express CCR5. CXCR4 also has

higher surface expression on CD11b� astrocytes and oli-

godendrocytes averaging 46% of the sorted cells compared

to 4–5% expressing CCR5 in all three animals analyzed. In

general, CD11b+ and CD11b� glial cells together express

more CXCR4 than CCR5.

3.6. CCR5 and CXCR4 mRNA expression in frontal cortex

evaluated by RT-PCR

To confirm the specificity of the CCR5 and CXCR4

immunohistochemistry, we analyzed mRNA from frozen

tissue adjacent to the section of frontal cortex for the

presence of CCR5 and CXCR4 specific mRNA. RT-PCR

was performed on total RNA and we compared mRNA

levels by age of the animal relative to the expression of

rhesus beta-globin mRNA. Although numerous cell types

are present in the gray matter analyzed, most of the cells

expressing chemokine receptors based on immunohisto-

chemistry and immunofluorescence in the cortical gray

matter are neurons. CXCR4 mRNA detected by RT-PCR

had the highest signal in newborns and decreased with age

(Fig. 6A,B). Likewise, the strongest RT-PCR signal of

CCR5 mRNA was from newborns (Fig. 6A,C). Since RT-

PCR has limited usefulness in quantitative analysis, we are

developing in situ hybridization to further explore the

relative changes in CCR5 and CXCR4 protein and RNA

levels.

4. Discussion

We have demonstrated previously that the chemokine

receptors CCR5 and CXCR4, which also function as cor-

eceptors for HIV/SIV, are present and functional on resident

CNS cells, including neurons and glia in normal and SIV-

infected adult rhesus macaques (Klein et al., 1999; West-

moreland et al., 1998). In this study, we have further

characterized the expression of CCR5 and CXCR4 individ-

ually and together in rhesus on neurons and glia relative to

age of the animal. The earliest sites of expression of these

chemokine receptors were the hippocampal CA1–4 neu-

rons, Purkinje cells, subcortical nuclei, and the pyramidal

neurons of neocortical layer III. The observation that neuro-

nal expression of CCR5 and CXCR4 in the cortex increased

significantly with age is of considerable interest. In addition,

these two chemokine receptors were commonly coexpressed

in neurons at the cell membrane and in the cytoplasm of the

perikaryon and processes with the intensity of CCR5

expression often greater than that of CXCR4. In contrast,

CCR5 expression in glia was often less intense and was

expressed on the surface of fewer cells than CXCR4. In

addition, microglia are the predominant glial cell type that

expressed CCR5 and CXCR4. Overall, the cellular expres-

sion of the important HIV/SIV coreceptors, CCR5 and

CXCR4 is complex and varies by cell type, brain region,

and age.

CCR5 and CXCR4 expression increased significantly

( p < 0.05) with age from birth to 9 months. Expression of

both CCR5 and CXCR4 was widespread in neurons of all

neocortical layers, but was most prominent in the pyrami-

dal neurons of layers III, V, and VI. By 50 days of age,

CCR5 was also notably bimodal in neurons of those layers

Fig. 6. A–C: CCR5 and CXCR4 mRNA detected by RT-PCR (A) of 3 mgtotal RNA isolated from adult rhesus macaque frontal gray matter.

Amplified products were visualized on a 2% ethidium-bromide impreg-

nated agarose gel: CXCR4 (top), CCR5 (middle), and beta-globin (bottom).

Graphs of CXCR4 (B) and CCR5 (C) RT-PCR products representative of

mRNA levels normalized to rhesus beta-globin mRNA determined by

densitometry and Excel 98 (Microsoft).


where large pyramidal neurons expressed higher levels of

CCR5 than did neighboring neurons. CCR5 expression in-

creased significantly up to 50 days of age, whereas CXCR4

had a slower rate of increase after 21 days. Although there

was a distinguishable subpopulation of neurons that was

strongly CCR5-single positive early in development, most

neurons were double-positive for CCR5 and CXCR4. The

differential expression pattern of CCR5 and CXCR4 docu-

mented here in rhesus neurons suggests a possible deter-

minant of selective vulnerability of subpopulations of neu-

rons reported in humans infected with HIV (Petito et al.,

1999).

Although CCR5 and CXCR4 protein increased in the

neocortex with age, mRNA decreased or was variable over

time and did not parallel protein levels. A similar lack of

correlation between mRNA and protein levels has been

observed for a variety of proteins in other experimental

systems and the reasons are not always clear (Fields, 2001;

Gygi SP, 1999). In this instance, the low CXCR4 mRNA

and high protein seen in older neonates and adults may

represent increasing protein stability with age. In addition,

chemokine receptors may recycle to the neuronal surface, as

has been demonstrated in leukocytes (Feniger-Barish et al.,

1999) and endothelium (Andjelkovic and Pachter, 2000)

allowing for high protein expression without significant de

novo protein synthesis. The higher CXCR4 and CCR5

mRNA expression in newborns and young neonates coin-

cident with lower protein expression may also reflect higher

mRNA stability.

A recent publication described limited-to-no expression

of CXCR4 in human children younger than 3.5 and 4.5

years of age, roughly equivalent to 3 to 4 months of age in a

rhesus. Our study suggests that expression in the rhesus

begins earlier and increases more rapidly than that seen in

human infants and children. Although there is some dis-

crepancy between the two studies as to the age of onset of

expression of CCR5 and CXCR4, both studies show that

expression of these two important chemokine receptors

increases with age in infancy and in young juveniles. In

addition to possible species differences between humans and

rhesus in the onset of expression of neuronal chemokine

receptors, particularly CXCR4, explanation of the discrep-

ancy in results may relate to rapid isolation and embedding

of rhesus tissue in experimental settings.

The dynamic expression pattern of these two G-protein-

coupled chemokine receptors suggests that these receptors

potentially have multiple complex functions, many of which

are still unknown. Growing evidence suggests that these

receptors and their chemokines are involved in multiple

functions in neural cells, including cellular communication,

CNS development, and neuronal survival, injury, and death.

One of the first proposed functions is to facilitate commu-

nication between glial cells and neurons in the CNS.

Astrocytes have been shown to produce chemokine ligands,

such as SDF-1a, GROa2, and fractalkine, that bind chemo-

kine receptors found on neurons (Giovannelli et al., 1998;

Janabi et al., 1999; Ohtani et al., 1998). Another role of

chemokines and their receptors in the brain is to promote

normal CNS development. CXCR4 has been shown to be

important in cerebellar development in mice (Ma et al.,

1998; Zou et al., 1998). A third function of chemokine

receptors is to promote neuronal survival, demonstrated by

the effects of GROa2 (Giovannelli et al., 1998), fractalkine

(Giovannelli et al., 1998; Meucci et al., 1998), and IL-8

(Araujo and Cotman, 1993) on different neuronal popula-

tions. Conversely, chemokines and chemokine receptors on

neurons can also mediate neuronal injury and death. Apop-

tosis of cultured neurons can be induced by SDF-1amediated through CXCR4 (Kaul and Lipton, 1999; Zheng

et al., 1999). Also, human fetal neurons treated with super-

natant from HIV-1-infected monocyte-derived macrophages

demonstrate signs of stress, injury, and apoptosis partially

mediated through CXCR4 (Zheng et al., 1999). In addition,

recombinant HIV-1IIIB gp120 and SIVmac251 gp120 induce

apoptosis in rat fetal neurons mediated through CXCR4,

which is blocked with pretreatment of neurons with SDF-1a(Meucci et al., 1998).

In conclusion, data presented here demonstrate early

onset and rapid increase in neuronal CCR5 and CXCR4 in

the rhesus fetus, neonate, and juvenile which reaches adult

levels by 9 months of age, roughly comparable to 9 years of

age in a human child. In addition, there are complex differ-

ences in neuronal CCR5 and CXCR4 expression with

regard to brain region and subpopulation of neurons in the

rhesus that have been similarly suggested in human brain

(Horuk et al., 1997). This complex and differential expres-

sion pattern of chemokine receptors may contribute to the

selective vulnerability of subpopulations of neurons ob-

served in HIV neuropathogenesis (Masliah et al., 1992b).

Lastly, although CCR5 and CXCR4 proteins increase with

age in the rhesus, this does not occur solely due to a parallel

increase in mRNA. This suggests that there are additional

control mechanisms of chemokine receptor expression, such

as post-translational modifications and/or recycling of

receptors which have been previously documented (Vila-

Coro et al., 1999).

Given the emerging significance of chemokine receptors

in neuronal and glial communication, function, and survival,

this class of receptors warrants further study. In addition,

CCR5 and CXCR4 have been implicated as potential

mediators of direct neuronal injury by HIV and SIV viral

proteins, like gp120, or of indirect neuronal injury through

chemokines. Theoretically, aberrant or excessive activation

of these abundant neuronal receptors by either of these

‘‘ligands’’ may disrupt normal physiologic signals or initiate

inappropriate signals that could contribute to neuronal

dysfunction and loss (death) in AIDS. Since neuronal

CCR5 and CXCR4 expression increases with maturation

of the rhesus infant, and similarly the human child, it is

likely that the potential for damage to neurons from SIV- or

HIV-induced chemokines, cytokines, or viral proteins medi-

ated through chemokine receptors could increase with age.


Acknowledgements

We thank Drs. Prabhat Sehgal and Angela Carville and the

rest of the clinical staff for excellent care of neonatal animals

used in this study. We also thank Liz Curran, and the

pathology technicians for assistance with tissue harvesting

and record keeping, Bill Kennedy and Dan Shvetz for

technical support, Kristen Toohey for exceptional photo-

graphic and graphics assistance, and Doug Pauley for edito-

rial review.

This work was supported by Public Health Service grants

NS30769, NS37654, NS40237, MH61192, RR00168, and

RR00150. A. Lackner is a recipient of an Elizabeth Glaser

Scientist Award.

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