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AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129;
New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772; and
Cardiac Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, macrophage inflammatory protein-1
, and RANTES,
indicating coupling of CCR5 and CXCR4 to a Gi protein,
as in leukocytes. Cultured macaque neurons demonstrated calcium flux
response to treatment with recombinant SIVmac239 envelope protein,
suggesting a mechanism by which viral envelope could affect neuronal
function in SIV infection. The presence of functional chemokine
receptors on neurons and astrocytes suggests that chemokines could
serve to link inflammatory and neuronal
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although numerous complicated hypothesis have been proposed to explain
how the viral or cellular products of HIV-1-infected cells damage
neurons, the recent discovery that the chemokine receptors CXCR4, CCR3,
and CCR5, which act as coreceptors for HIV entry into lymphocytes and
macrophages (22, 23, 24), are also present on neurons suggests
new and simpler mechanisms. Chemokine receptors are members of the
seven-transmembrane-spanning G protein-coupled receptor superfamily and
thus are thought to exert their biological effects via multiple signal
transduction pathways (25). Most of the chemokine
receptors are expressed by several leukocyte cell types and have been
shown to be coupled to the pertussis toxin-sensitive
Gi proteins (25). CCR3, CCR5, and
CXCR4 have been detected on subpopulations of large hippocampal and
neocortical pyramidal neurons and on glial cells in both normal and
encephalitic human and macaque brains (26, 27, 28, 29, 30, 31), and on
human fetal neurons in vitro (32, 33). CXCR4 and CCR5 mRNA
have also been detected in cultured rat glial and neuronal cells
(34, 35). Studies on cultured murine microglia and
astrocytes, rat hippocampal neurons, and neurons prepared from the
teratoma cell line, NT2, have demonstrated that CCR5 and CXCR4
chemokine ligands induce calcium transients and chemotaxis in these
cell types (36, 37, 38). It is not known whether HIV-1
chemokine coreceptors expressed on cultured human neurons and
astrocytes respond to their chemokine ligands with characteristic
calcium transients.
The presence of chemokine receptors on pyramidal neurons in the
neocortex and hippocampus, areas of HIV/SIV-induced pathology and
clinical impairment (3), is intriguing. Chemokines have
not been considered as a possible neurotoxin, although they are found
in increased amounts within the brains of both patients and macaques
with HIV/SIV encephalitis. The chemokines macrophage inflammatory
protein-1 (MIP-1),3 MIP-1, RANTES, and
IFN-inducible protein of 10 kDa (IP-10) have all been found to be
up-regulated in the brains of macaques with SIV encephalitis in vivo
(16). Increased amounts of MIP-1 and MIP-1 mRNAs
have been detected in microglia and astrocytes in brains of patients
with HIV encephalitis by RT-PCR (15), and monocyte
chemoattractant protein-1 (MCP-1), MIP-1, and RANTES have been
detected within characteristic microglial nodules in postmortem brain
specimens from patients with HIV encephalitis (33).
In addition, stromal-derived factor-1
(SDF-1) has been shown to induce apoptosis in both NT2 cells and
lymphocytes in vitro (39, 40). Finally, in vitro studies
have demonstrated that HIV and SIV envelope proteins, via binding to
CXCR4 and CCR5, induce signal transduction and chemotaxis in
lymphocytes (41, 42), competitively inhibit chemokine
binding to receptors (43), and induce calcium flux and
apoptosis in neurons (39, 44, 45). The expression of
HIV/SIV coreceptors on neurons suggests a possible mechanism by which
chemokines or HIV/SIV proteins can directly interact with these
nonimmune cells, disrupting their normal function.
We hypothesize that elevated levels of chemokines and/or virus in the CNS of HIV/SIV-infected subjects could contribute to the pathogenesis of AIDS dementia through direct interactions with chemokine receptors expressed on neurons and astrocytes. Here we show that cultured human and macaque fetal neurons and astrocytes express chemokine receptors immediately ex vivo and after 2 wk in culture. These receptors are present in unique distribution patterns on the surfaces of both neurons and astrocytes, and chemokine ligands produce increases in intracellular calcium in these cell types, which are neutralized by mAbs to each receptor. In addition, neuron and astrocyte responses to chemokines are abolished by pretreatment of cells with pertussis toxin. We also show that gp120 from SIVmac239 produces similar calcium flux responses in fetal macaque neurons, which can be blocked by pretreatment with CCR5 chemokine ligands. These results indicate that neural chemokine receptors signal through pathways similar to those within leukocytes and suggest a direct mechanism by which chemokines and/or viral envelope could contribute to neuronal dysfunction and loss in the neuropathogenesis of AIDS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neuronal cultures.
Fetal brains were obtained from human abortuses between 17 and 20 wk of
gestation from the Brigham and Womens’ Hospital (Boston, MA) with
approval from the human studies committee (IRB protocol 98-08793) and
from second trimester fetal macaques obtained via caesarian section.
Animals were housed in accordance with standards of the American
Association for Accreditation of Laboratory Animal Care. The
investigators adhered to the "Guide for the Care and Use of
Laboratory Animals" prepared by the Committee on Care and Use of
Laboratory Animals of the Institute of Laboratory Resources, National
Research Council. The human and macaque brains were handled
identically. The meninges were removed, and cortical tissue was
mechanically dissociated using sterile scalpel blades and washed in
sterile PBS. Cells were pelleted by centrifugation at 800 rpm for 10
min, and the pellet was subsequently enzymatically dissociated with
0.25% trypsin in the presence of DNase (50 µg/ml) at 37°C for 30
min. Cells were further mechanically dissociated through a 150-µm
pore size nylon mesh filter and washed again in sterile PBS by
centrifugation at 1800 rpm for 10 min. The collected cell pellet was
mechanically dissociated by trituration and further enzymatically
dissociated with DNase (50 µg/ml) for 10 min. After several further
washes with sterile PBS, cells were seeded onto
poly-D-lysine-coated glass coverslips or T75 flasks at a
density of 1 x 106 cells/ml in DMEM/10%
FCS. 5-Fluoro-2'-deoxyuridine (FDU; 10 µM), a uridine analogue that
is toxic to dividing astrocytes, was added to cultures to inhibit
overproliferation of glial cells on days 5, 8, and 12 in vitro. Cells
were used for single- and double-label immunocytochemical techniques
and calcium flux analyses after 
2 wk in culture.
Astrocyte cultures.
Brains were prepared as described above, but seeded instead into T75
flasks at a concentration of 1 x 107
cells/ml. After 1 wk in culture, nonadherent neurons were washed off
with PBS, and adherent cells were treated with 0.25% trypsin for 5 min
at 37°C and reseeded into new T75 flasks at a ratio of 1:2 in
DMEM/10% FCS. After an additional week in culture, cells were again
treated with trypsin and reseeded onto poly-D-lysine-coated
glass coverslips or T75 flasks at a density of 1 x
106 cells/ml. Cells were fed DMEM/10% FCS
biweekly for an additional week and then used for immunocytochemical
and calcium flux analyses. These cultures are 90% pure, as
determined by GFAP immunocytochemical analysis.
Analysis of chemokine receptor expression by flow cytometry and immunocytochemistry
Brains were prepared as described above and placed in culture in T75 flasks for 1 day, and then enriched neurons and nonneuronal cells were obtained via their differential adherence properties. Whereas nonadherent neurons remained floating in clusters for 1 day, glia, consisting of enriched (>90%) astrocytes and rare (<10%) macrophages/microglia, adhered during the first day. The nonadherent neurons were washed off with PBS, and the adherent cells were lightly trypsinized to remove them from the flasks. Nonadherent neurons and adherent glial cells were then analyzed by flow cytometry at 106/ml for chemokine receptor expression. Briefly, cells were washed twice with PBS and incubated with CXCR4-PE (clone 12G5; 20 µl/test; PharMingen, San Diego, CA), biotinylated CCR5 (clone 5C7; 5 µg/ml; LeukoSite, Cambridge, MA) plus streptavidin 670 (2.5 µg/test; Life Technologies, Grand Island, NY), and CCR3 (clone 7B11; 4–5 µg/ml; LeukoSite, Cambridge, MA) (46) for 20 min at 4°C. Cells incubated with mAbs to CCR3 were washed with PBS and incubated in goat anti-mouse PE (20 µl/test; Becton Dickinson, Franklin Lakes, NJ) for 20 min at 4°C. Cells were then washed in PBS, fixed in 2% paraformaldehyde overnight, and used for analysis. Controls consisted of cells incubated with IgG1-FITC and IgG2a-PE (20 µl/test; Becton Dickinson, Franklin Lakes, NJ), goat anti-mouse PE alone, streptavidin 670 alone, and omission of primary Abs. To determine the percentages of neural cell type populations, aliquots of the same cells were also cytospun onto glass slides and analyzed via immunocytochemistry for expression of neuron-specific microtubule-associated protein-2 (MAP-2; 10 µg/ml; Sigma), the astrocyte marker glial fibrillary acidic protein (GFAP, 5–10 µg/ml; Dako), and CD11b (10 µg/ml; Immunotech, Westbrook, ME), which is a monocyte/myeloid Ag found on all brain macrophages/microglia.
Double-label immunofluorescence and confocal microscopy
In all immunocytochemical experiments, cells were washed twice
with sterile PBS, fixed in 4% paraformaldehyde for 1 h, washed an
additional two times with sterile PBS, and then stored under sterile
PBS until analyzed. Characterization of cell types within our 2- to
3-wk cultures was performed using polyclonal Abs to neuron-specific
enolase (NSE; 1:100; Dako, Carpenteria, CA) and GFAP as above, which
were detected via avidin-biotin-peroxidase complexes (Biogenex, San
Ramon, CA) with diaminobenzadene and Mayer’s hematoxylin counterstain,
as previously described (26). In all experiments, cells
were treated for 1 h with 10% normal horse serum, 10% normal
goat serum, and 5% human AB serum in sterile PBS to block nonspecific
binding before placement of primary Abs. Cultures were found to contain
70–80% neurons and 20–25% astrocytes. The remaining cells
(<5%, NSE-, GFAP-) were
morphologically identifiable as macrophages/microglia. Double-label
immunofluorescence was performed using mAbs to chemokine receptors CCR3
(clone 7B11), CCR5 (biotinylated; clone 5C7), and CXCR4 (clone 12G5) at
5–10 µg/ml and anti-MAP-2 and anti-GFAP as described above,
with or without topro-3 (Molecular Probes, Eugene, OR) staining to
identify cell nuclei. When double immunofluorescence was performed
using chemokine receptor (CCR) and MAP-2 mAbs, cells were treated with
anti-CCR first, and an additional blocking step was performed
before addition of anti-MAP-2. IgG2A isotype controls were used at
a concentration of 10 µg/ml (Dako). Chemokine mAbs and isotype
controls were detected using secondary anti-mouse Abs conjugated
with Texas Red (1/200; Sigma) or streptavidin Texas Red (1/50; Sigma).
MAP-2 and GFAP were detected using FITC-conjugated anti-mouse and
anti-rabbit secondary Abs (1/200; Sigma), respectively. Stained
cells were examined by routine epifluorescence and confocal microscopy.
For epifluorescence, cells were visualized and images captured using a
Vanox S research microscope (Olympus, Lake Success, NY) equipped with
Texas Red and FITC filter sets coupled to an Optronics DEI-750 camera
(Optronics, Goleta, CA). Individual images were then superimposed using
Photoshop (Adobe Systems, Mountain View, CA) to demonstrate the
heterogeneity of chemokine receptor expression in the various cell
types. Confocal microscopy was performed using a Leica TCS SP laser
scanning microscope (Leica, Microsystems, Exton, PA) fitted with a
x100 Leica objective (PL APO, 1.4 NA), and using the Leica image
software. Images were collected at 512 x 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. The step size in the z-axis was
varied from 0.2–0.5 µm to obtain 30–50 slides/imaged field. The
images were transferred to a Macintosh G3 (Apple Computer, Cupertino,
CA) computer and NIH Image v1.61 software was used to render the
images.
Calcium flux analysis
Neural cells were prepared as described above, seeded onto 110-µm-thick coverslips, and cultured as above. Cells were washed with PBS and loaded with 5 µM fura-2 (Molecular Probes, Eugene, OR) for 1 h in a dark chamber at 37°C. Cells were then washed with PBS, and DMEM/1% FCS was added to the cultures. Cells were kept at 37°C until analyzed for calcium flux responses (up to 1 h). For calcium flux analysis, coverslips were placed into a 37°C warming chamber and examined under an inverted microscope connected to a spectrofluorometer. Groups of three to eight neurons or one to three astrocytes were analyzed for their responses to stimulation with various chemokines and BSA at a concentration of 100 ng/ml. Additional experiments were performed after pretreatment of human neuronal and astrocyte cultures with 200 ng/ml pertussis toxin or 10 µg/ml mouse mAbs to CCR3 (clone 7B11), CCR5 (clone 2D7), and CXCR4 (clone 12G5). Fetal macaque neurons were also tested for response to 10 nM recombinant SIVmac239 gp120 (gift from J. Sodroski, purified as previously described (47)). This particular envelope protein is monomeric and thus does not require CD4 to bind CCR5 (47, 48). For experiments with neurons, cells were predepolarized with 20 mM KCl before exposure to chemokines. Calcium flux tracings were analyzed for the maximum increase in intracellular calcium according to the formula [Ca]i = kd [(R - Rmin/(Rmax - R)], assuming a Kd of 224 nM, and R is the ratio of fluorescence at 340 and 380 nM, as previously described (49). Rmax reflects the amount of calcium increase after treatment with the nonspecific calcium ionophore ionomycin (5 µg/ml), and Rmin reflects the level of calcium following calcium chelation with Tris-EDTA. Calcium concentrations are expressed as the mean ± SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fetal macaque brain cells were examined immediately ex vivo to
determine the percentage of neuronal and glial cells expressing
chemokine receptors before culture. Populations were separated via
their differential adherence properties, as described in
Materials and Methods, with neurons being nonadherent and
astrocytes and microglia being adherent, and analyzed by flow cytometry
at 106 cells/ml for chemokine receptor
expression. In Fig. 1
A, a
phase-contrast photograph depicts fetal neural cells containing
floating clusters of neurons (arrow) overlying adherent glial cells.
When the nonadherent cells were removed and cultured separately, >95%
of these cells were positive for expression of the neuronal marker
MAP-2 (Fig. 1B), with contaminating cells positive for the
astrocyte marker GFAP or the microglia marker CD11b (data not shown).
Flow cytometric analysis of nonadherent macaque brain cells
demonstrated that 13% of the neurons stained positive for CCR3,
34% were positive for CCR5, and 28% were positive for CXCR4 (Fig. 1C). These percentages are consistent with the heterogeneity
in neuronal expression observed in adult macaque brain in vivo
(26). Similar analysis of chemokine receptor expression
was performed on the adherent cell population, which contained >90%
astrocytes and <10% macrophages/microglia. Approximately 90% of
cells stained positive for CCR5, and 86% were positive for CXCR4 (Fig. 1D). In addition, 14% of these cells demonstrated
expression of CCR3 (Fig. 1D); however, most of the
CCR3+ cells are likely to be
macrophages/microglia based on immunocytochemical characterization of
cytospun samples (see Materials and Methods; data not
shown). The data suggest that astrocytes express CCR5 and CXCR4.
Isotype-matched control mAbs did not produce staining beyond background
autofluorescence. These experiments, performed on fetal brain cells
immediately ex vivo, suggest that a significant fraction of fetal brain
cells express chemokine receptors in vivo.
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A), 20–25% large cells
positive for GFAP (Fig. 2B), and <5% small, unstained
cells resembling microglia/macrophages (Fig. 2A,
arrowheads). Cultured human neural cells contained the same cell types
in similar percentages (data not shown). Immunofluorescence analysis of
chemokine receptor expression in human neurons and astrocytes
demonstrated that subpopulations of neurons and astrocytes express
chemokine receptors in vitro, consistent with our published in vivo
data (26) and the ex vivo data described above. These
cultures contained neurons that stained positively for CCR3 (Fig. 2C), CCR5 (Fig. 2D), and CXCR4 (Fig. 2E) but negatively for GFAP; they appear red. The pattern of
CXCR4 immunostaining appeared more punctate along the neuronal
processes than CCR3 and CCR5 (Fig. 2, C–E). Astrocytes that
did not stain with chemokine receptor mAbs and were positive for GFAP
appear green (Fig. 2, C–E). However, there were a small
percentage of astrocytes that were doubly positive, appearing yellow
(Fig. 2D). Fig. 2F demonstrates the expression of
CCR5 in an astrocyte (red staining, left panel); the
cellular identity was verified by the coexpression of GFAP (green
staining, right panel). Isotype-matched mouse mAb controls
did not show staining beyond background autofluorescence. Experiments
using cultured macaque brain cells gave similar results (data not
shown). In general, we observed that the numbers of chemokine
receptor-positive neurons and astrocytes decreased after several weeks
in culture compared with cells tested immediately ex vivo. Thus,
subpopulations of neurons and astrocytes can continue to express
chemokine receptors for at least 2–3 wk.
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Given the subtle differences in immunocytochemical staining
patterns of the three chemokine receptors on neurons, we next examined
their expression on neurons and astrocytes via confocal microscopy.
Cells were immunostained as described above and additionally stained
with topro-3 to distinguish the nuclei of cells. The subtle differences
in immunocytochemical staining patterns described above were much more
evident with the increased resolution of confocal microscopy (Fig. 3). CCR3 (red) density was greatest over
the neuronal cell body (Fig. 3A), while CCR5 (red) was
evenly distributed over the neuronal cell body and processes (Fig. 3E). CXCR4 (red), however, was present along the axonal
membrane but was more concentrated in the axon hillock, a cone-shaped
portion of the cell body from which the axon arises, which is known to
be important for summating presynaptic potentials (Fig. 3F).
All neurons appeared to contain a pool of intracellular receptors
within the cell body, as evident by their presence in all optical
sections through the cell. CCR5 and CXCR4 (both red) were also detected
in GFAP-positive (green) astrocytes, with CCR5 more evenly distributed
over and inside the cell and CXCR4 found predominantly along the edges
of the cell membrane (Fig. 3, E and F).
Isotype-matched control mAbs revealed that the chemokine receptor
staining was specific (Fig. 3C). Differential interference
contrast (DIC) images of cells whose staining patterns did not
delineate their entire axonal processes are provided to demonstrate
complete cellular structures (Fig. 3, B and D).
Similar receptor patterns were observed in human and macaque fetal
neural cultures.
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). CCR3 and MAP-2 showed the highest
degree of colocalization (Fig. 4A), while CCR5 and CXCR4 had
fewer colocalized sites with MAP-2, and multiple surface CXCR4 (red)
receptor sites could be detected along the processes. Isotype-matched
control mAbs detected with Texas Red (red)-conjugated secondary Abs
demonstrated no staining of the cell body or processes and only
intracellular MAP-2 staining (green; Fig. 4D). The
differences in the distribution patterns of these receptors is likely
to impact their physiological responses to ligand binding and warrants
further study.
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Although the normal physiological functions of chemokine receptors
in the CNS are unknown, cultured astrocytes, like lymphocytes, have
been shown to respond to SDF-1 and MIP-1 in chemotaxis and
calcium flux assays (38). Thus, we sought to determine
whether activation of neuronal chemokine receptors would produce
increases in intracellular calcium. Using a microscope-based
fluorometer, enriched populations of fura-2-loaded neurons and
astrocytes were analyzed in groups of three to eight neurons or one to
three astrocytes for their relative fluorescence upon treatment with
100 ng/ml of chemokine. Chemokines were chosen for their known
abilities to bind to chemokine receptors CCR3 (eotaxin, RANTES), CCR5
(MIP-1, RANTES), and CXCR4 (SDF-1). All these chemokines have
been shown to induce calcium flux in various leukocyte cell types
(25, 49). As shown in Figs. 5-9, cultured neurons and astrocytes from
both fetal macaque and human brain demonstrated increases in
intracellular calcium concentrations upon treatment with chemokines.
Although the tracings within each panel have been adjusted to the same
scale, the individual tracings were generated using groups of neurons
or astrocytes composed of varying numbers of cells and thus cannot be
quantitatively compared. In neuronal cultures, this response was
significantly enhanced by predepolarization with 20 mM KCl (Fig. 5).
Fig. 5A demonstrates this augmentation in the response of
cultured macaque neurons to RANTES, and Fig. 5B demonstrates
the responses of cultured human neurons to various chemokines after
predepolarization with KCl. BSA, used as a protein control, did not
elicit calcium flux responses (Fig. 5A), nor did treatment
with the chemokine GRO, which is a known ligand for chemokine
receptor CXCR2 (Fig. 5C). Neuronal responses to chemokine
ligands demonstrated homologous, but not heterologous, receptor
desensitization, thus remaining refractory to additional stimulation
with the same chemokine shortly (<50 s) after calcium flux responses
(Fig. 5C). In cases where one chemokine binds multiple
receptors, such as RANTES (ligand for CCR1, CCR3, and CCR5), additional
calcium responses to chemokine ligands for only one of these receptors
were blocked (Fig. 5C). In contrast to neurons, cultured
human astrocytes did not require predepolarization with KCl to respond
to chemokines (Fig. 6). Astrocyte
responses to SDF-1, RANTES, and MIP-1 were similar to those of
neurons; however, astrocytes did not respond to eotaxin. This was not
surprising given that CCR3 was not detected on this cell type in
appreciable amounts (Fig. 1
D). The addition of mAbs to CCR5
(clone 2D7), CCR3 (clone 7B11), and CXCR4 (clone 12G5) before treatment
with MIP-1, eotaxin, and SDF-1, respectively, blocked neuronal
responses to these ligands, but not to ligands that signal through
other, nonneutralized receptors, demonstrating the specificity of these
responses (Fig. 7). Pretreatment with 200
ng/ml pertussis toxin abolished both neuronal and astrocyte responses
to RANTES, MIP-1, and SDF-1 (Fig. 8). In pertussis toxin-treated neurons,
treatment with eotaxin gave inconsistent results, eliciting calcium
flux in approximately half the experiments (data not shown). These
results indicate that CNS chemokine receptor responses are specific and
appear to be coupled to a pertussis toxin-sensitive G protein(s), as
they are on leukocytes.
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B).
Chelation treatment with Tris-EDTA allowed determination of minimum
intracellular calcium concentrations (Fig. 8B).
Intracellular neuronal calcium concentrations were calculated to range
from 40.5 ± 16.2 nM and increased to 146 ± 51.4 nM
(4-fold) after predepolarization with KCl. After predepolarization,
the calcium concentrations declined to a new baseline of 83.7 ±
29.6 nM and were increased to 202 ± 95.2 (5-fold from the
original baseline, but only 2- to 3-fold from the new baseline) after
treatment with chemokines. Astrocyte responses were calculated to be
3-fold from baseline after treatment with chemokines. SIVmac239 gp120 induces calcium flux in culture fetal macaque neurons
Fig. 9 depicts representative
tracings of fetal macaque neuronal responses to 10 nM recombinant
monomeric SIVmac239 gp120, a concentration equal to the
Kd for CD4-independent binding of
SIVmac239 gp120 to CCR5 cloned from rhesus macaques (47).
As observed with chemokine treatment, neuronal responses to gp120 were
enhanced by predepolarization with 20 mM KCl, and increases in
intracellular calcium were 2- to 3-fold above the predepolarized
baselines (Fig. 9a). This response desensitizes MIP-1
signaling, which binds CCR5, and is desensitized by pretreatment with
RANTES, which binds CCR3 and CCR5 (Fig. 9). These results suggest that
binding of SIVmac239 gp120, which can bind CCR5 in a CD4-independent
manner, produces neuronal calcium flux responses similar to those
observed with chemokine ligands and are likely mediated by
CCR5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution patterns of chemokine receptors CCR3, CCR5, and CXCR4 on neurons were quite different. CCR3 was most concentrated within the cell body of neurons, while CCR5 and CXCR4 were found on the axonal processes and axon hillock regions. Similar differences in the distribution patterns of these receptors on cultured human neurons have been reported by others (33). Astrocyte expression of CCR5 and CXCR4 also differed, with a high density of CCR5 found within intracellular pools and CXCR4 detected along the cell membranes. The numbers of astrocytes expressing CCR5 and CXCR4 in vitro appeared to decline after prolonged time in vitro compared with the amount detected on cells immediately ex vivo, which may explain why these receptors have not been reported in some other studies (28, 31). This decline in receptor expression in culture may be due to exposure to serum factors or loss of neuronal factors in these purified cultures. Coculturing experiments with neurons and astrocytes may address these observations.
The differential distribution of CCR3, CCR5, and CXCR4 on fetal macaque
and human neurons may have relevance for synaptic activation and signal
processing. Chemokine receptors concentrated near the neuronal cell
body or axon hillock may regulate amplification of synaptic signals
resulting from activation of other receptor types, such as NMDA or
-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors,
on distant dendritic sites (50). The requirement of
neuronal predepolarization for chemokine calcium flux responses also
supports a neuromodulatory role. Thus, the presence of chemokines in
the CNS during inflammatory states may lead to alterations in neuronal
and astrocyte signaling that may contribute to an impairment of
neuronal function.
During infection with HIV-1 or SIV, increased levels of MIP-1,
MIP-1, and RANTES are detected in the brains of macaques and humans
with AIDS encephalitis (15, 26). In addition, others have
demonstrated the in vivo and in vitro expression of monocyte
chemoattractant protein-1, SDF-1, and IP-10 by activated astrocytes
(20, 21, 34, 51, 52). Thus, there are ample sources of
chemokines during CNS infection with HIV/SIV. Here we examined the
effects of these immunologically active molecules on neurons
themselves. We focused on chemokines whose receptors are both known to
be present on neurons and act as HIV/SIV coreceptors. We show that
treatment of neurons and astrocytes with chemokine ligands for CCR3,
CCR5, and CXCR4 produces a 2- to 3-fold increase in intracellular
calcium. The specificity of these responses was demonstrated in
experiments using neutralizing chemokine receptor mAbs. Although CXCR2
has been detected on neurons in adult brain tissue specimens
(37), our fetal neuronal cultures did not respond to one
of its known ligands, GRO, which is secreted by spinal cord
astrocytes and acts on oligodendrocytes (53). The
expression of CXCR2 by fetal brain cells has not been investigated.
Responses to RANTES, SDF-1, and MIP-1 were abolished by
pretreatment of neurons and astrocytes with pertussis toxin,
demonstrating that these receptors are coupled to G proteins in these
cells that are similar to those in leukocytes (Fig. 7).
Gi1 and Gi2 have been
purified from adult bovine brain (54) and levels of
Gi2 mRNA have been shown to increase
significantly from embryonic day 16 to adulthood in the rat cerebral
cortex (55). In contrast to RANTES, SDF-1, and
MIP-1, application of eotaxin yielded inconsistent calcium flux in
the presence of pertussis toxin, suggesting coupling of CCR3 to an
alternative nonpertussis toxin-sensitive G protein in neurons. More
complete comparisons between the signal transduction pathways of
chemokine receptors in neurons and leukocytes will be of interest.
Neuronal responses to both chemokines and gp120 differed from those observed in astrocyte cultures, in that they were affected by membrane potential. Predepolarization of neurons with 20 mM KCl was required for meaningful calcium flux responses to all ligands tested. Other studies have shown similar requirements for calcium transients induced by gp120 in rat neuronal preparations (44). This finding suggests neuronal chemokine signaling may be modulated by signals produced by other membrane activity. Alternatively, the abnormal presence of chemokines in the brain may alter neuronal responses to more classical neurotransmitters present. Qiu et al. have recently reported that chronic IL-6 treatment of developing granule neurons increases the membrane and current response to NMDA, producing an enhanced calcium signal to IL-6 and enhanced neurotoxicity in vitro (56). Although IL-6 is a member of a different receptor family, these results demonstrate the modulation of a normal neurotransmitter receptor by a cytokine receptor on neurons. In addition, calcium elevation in astrocytes has been shown to increase their glutamate release, producing NMDA receptor activation in hippocampal neurons. Thus, the responses of both neurons and astrocytes to chemokines may be responsible for neuronal dysfunction through a variety of mechanisms.
Gp120 has been known to induce neurotoxicity via mechanisms that affect both calcium concentrations and NMDA receptor activity (57). Our finding that gp120 produces increases in intracellular calcium in neurons that appear to be chemokine receptor mediated suggests an additional mechanism for neuronal dysfunction during HIV/SIV CNS infection. Others have shown that HIV/SIV viral envelope proteins bind to chemokine receptors, produce calcium flux responses in a variety of nonprimate neuronal cell preparations, and produce apoptosis in neurons in vitro and in vivo (5, 34, 39, 43, 44). We have also recently found that treatment of our neuronal preparations with various chemokines produced an increase in the level of apoptosis (unpublished observation). In addition, quantification of the neuronal marker N-acetylaspartate using proton magnetic resonance spectroscopy in brain extracts of control and SIV-infected macaques demonstrated reduced N-acetylaspartate levels in the SIV-infected animals compared with controls (58), suggesting SIV-induced neuronal loss. Interestingly, imaging studies using proton magnetic resonance spectroscopy also suggest damage to neurons outside of demyelinated plaques in patients with multiple sclerosis (59), a disease in which chemokine levels are also known to be elevated (21). Thus the presence of chemokines and viral proteins may produce persistent calcium influx leading to either aberrant neuronal signaling and/or loss through apoptosis, suggesting mechanisms for both the subtle and lethal effects of HIV/SIV infection on neurons.
The normal function of chemokine receptors on neurons is unknown. However, the recent finding that cerebellar development was abnormal in mice lacking CXCR4 (60) suggests that this receptor is important for CNS development. Similar developmental abnormalities have not been seen in mice deficient in CCR5 (61). However, when CNS chemokine levels are elevated during CNS inflammatory states, the presence of neural chemokine receptors may have consequences for local neuronal function and survival and may also allow neurons to relay information that regulates the global CNS response to inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robyn S. Klein, AIDS Research Center, Massachusetts General Hospital, 149 13th Street, Room 5212D, Charlestown, MA 02129. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; IP-10, IFN-inducible protein of 10 kDa; SDF-1, stromal-derived factor-1; MAP-2, microtubule-associated protein-2; GFAP, glial fibrillary acidic protein; FDU, 5-fluoro-2'-deoxyuridine; GFAP, glial fibrillary acidic protein; NSE, neuron-specific enolase; DIC, differential interference contrast; NMDA, N-methyl-D-aspartate.
Received for publication February 12, 1999. Accepted for publication May 14, 1999.
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