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The Journal of Immunology, 1999, 163: 1636-1646.
Copyright © 1999 by The American Association of Immunologists

Chemokine Receptor Expression and Signaling in Macaque and Human Fetal Neurons and Astrocytes: Implications for the Neuropathogenesis of AIDS1

Robyn S. Klein2,*, Kenneth C. Williams{dagger}, Xavier Alvarez-Hernandez{dagger}, Susan Westmoreland{dagger}, Thomas Force{ddagger}, Andrew A. Lackner{dagger} and Andrew D. Luster*

* AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129; {dagger} New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772; and {ddagger} Cardiac Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokines are believed to play a role in the neuropathogenesis of AIDS through their recruitment of neurotoxin-secreting, virally infected leukocytes into the CNS. Levels of chemokines are elevated in brains of patients and macaques with HIV/SIV-induced encephalitis. The chemokine receptors CCR3, CCR5, and CXCR4 are found on subpopulations of neurons in the cortex of human and macaque brain. We have developed an in vitro system using both macaque and human fetal neurons and astrocytes to further investigate the roles of these receptors in neuronal response to inflammation. Here we report the presence of functional HIV/SIV coreceptors CCR3, CCR5, and CXCR4 on fetal human and macaque neurons and CCR5 and CXCR4 on astrocytes immediately ex vivo and after several weeks in culture. Confocal imaging of immunostained neurons demonstrated different patterns of distribution for these receptors, which may have functional implications. Chemokine receptors were shown to respond to their appropriate chemokine ligands with increases in intracellular calcium that, in the case of neurons, required predepolarization with KCl. These responses were blocked by neutralizing chemokine receptor in mAbs. Pretreatment of neural cells with pertussis toxin abolished responses to stromal-derived factor-1{alpha}, macrophage inflammatory protein-1{beta}, and RANTES, indicating coupling of CCR5 and CXCR4 to a Gi{alpha} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human immunodeficiency virus type 1-associated neurological disease is a devastating consequence of infection with HIV-1 characterized by behavioral, motor, and memory disturbances (1, 2). It has long been proposed that HIV-1-induced neurologic disease is related to the production of toxins by virally infected macrophages and possibly astrocytes, ultimately resulting in neuronal dysfunction and/or loss (3, 4, 5, 6, 7). Several neurotoxic factors have been proposed based on in vitro studies; these include viral proteins, such as gp120 (8, 9) and tat (10, 11), as well as a variety of immunologically active molecules produced in response to infection (12, 13, 14), including chemokines (15, 16, 17), whose effects on neuronal function are unknown. Many of these factors can be produced by several cell types within the CNS, including macrophages, lymphocytes, and astrocytes (18, 19, 20, 21).

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{alpha} 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{alpha} (MIP-1{alpha}),3 MIP-1{beta}, 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{alpha} and MIP-1{beta} 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{alpha}, 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{alpha} (SDF-1{alpha}) 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fetal macaque and human brain cells express CCR3, CCR5, and CXCR4

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. 1GoA, 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. 1GoB), 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. 1GoC). 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. 1GoD). In addition, ~14% of these cells demonstrated expression of CCR3 (Fig. 1GoD); 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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  		FIGURE 1. Chemokine receptors CCR3, CCR5, and CXCR4 are present on fetal macaque brain cells immediately ex vivo. A, Phase-contrast photograph of dissociated macaque fetal brain cells after 24 h in culture. Clusters of floating neurons (arrow) can be seen overlying adherent glial cells. B, Removal and reculturing of neuronal clusters from mixed cultures produces >95% pure MAP-2-positive neurons. Cells were stained with mAb against MAP-2, which was detected with FITC-conjugated secondary Abs. C, Flow cytometry of nonadherent neural cells, which were >95% neurons and expressed CCR3, CCR5, and CXCR4 in the amounts depicted. D, Flow cytometry of adherent cells after removal via light trypsinization were a mixture of approximately >90% GFAP-positive astrocytes and <10% CD11b-positive macrophages/microglia and expressed CCR5 and CXCR4. Black histograms depict cellular binding of chemokine receptor mAbs and white histograms depict similar experiments using isotype-matched control mAbs. Data are representative of four experiments.

 
We next determined whether fetal neural cells would continue to express chemokine receptors after longer times in culture. Human and macaque fetal brain cells were treated with FDU to limit astrocyte proliferation, cultured for 2–3 wk, and then analyzed by double immunofluorescence using mAbs to CCR3, CCR5, and CXCR4 and rabbit polyclonal Ab to GFAP. Immunocytochemical characterization of cultured macaque neural cells demonstrated that cultures contained 70–80% small, process-bearing cell types positive for NSE (Fig. 2GoA), 20–25% large cells positive for GFAP (Fig. 2GoB), and <5% small, unstained cells resembling microglia/macrophages (Fig. 2GoA, 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. 2GoC), CCR5 (Fig. 2GoD), and CXCR4 (Fig. 2GoE) but negatively for GFAP; they appear red. The pattern of CXCR4 immunostaining appeared more punctate along the neuronal processes than CCR3 and CCR5 (Fig. 2Go, C–E). Astrocytes that did not stain with chemokine receptor mAbs and were positive for GFAP appear green (Fig. 2Go, C–E). However, there were a small percentage of astrocytes that were doubly positive, appearing yellow (Fig. 2GoD). Fig. 2GoF 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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  		FIGURE 2. CCR3, CCR5, and CXCR4 are present on neural cells in culture. A and B, Replicate 2-wk fetal macaque neural cultures treated with FDU were characterized using polyclonal Abs to NSE (A) and GFAP (B), which were detected via avidin-biotin-peroxidase complexes with diaminobenzadene (brown) and Mayer’s hematoxylin counterstain. A, Fetal macaque neuronal culture demonstrating presence of NSE+ neurons and small NSE- cells (arrowheads), which are presumed to be microglia. B, Fetal macaque neuronal culture demonstrating large GFAP+ contaminating astrocytes. C–F, Replicate 2-wk fetal human neural cultures treated with FDU were double immunofluorescent labeled with mAbs to CCR3 (C), CCR5 (D), CXCR4 (E), and polyclonal Abs to GFAP. Chemokine primary mAbs were detected using Texas Red-conjugated secondary Abs and GFAP primary Abs were detected using FITC-conjugated secondary Abs. Cells were photographed using Texas Red and FITC filter sets, and then images were superimposed. C, A group of red CCR3+GFAP- neurons is seen overlying a green CCR3-GFAP+ astrocyte. D, Red CCR5+GFAP- neurons are seen among mostly green CCR5-GFAP+ astrocytes. Note the yellow CCR5+GFAP+ astrocyte. E, Red CXCR4+GFAP- neurons are seen among green CXCR4-GFAP+ astrocytes. F, An example of a double-labeled CCR5+ (red) and GFAP+ (green) astrocyte viewed under red (left) and green (right) filters. Data presented are representative of four separate staining experiments with both macaque and human fetal neural cultures.

 
Chemokine receptors have unique patterns of distribution on cultured neurons

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. 3Go). CCR3 (red) density was greatest over the neuronal cell body (Fig. 3GoA), while CCR5 (red) was evenly distributed over the neuronal cell body and processes (Fig. 3GoE). 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. 3GoF). 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. 3Go, E and F). Isotype-matched control mAbs revealed that the chemokine receptor staining was specific (Fig. 3GoC). 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. 3Go, B and D). Similar receptor patterns were observed in human and macaque fetal neural cultures.



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  		FIGURE 3. Fetal macaque brain cells demonstrate differential distribution of chemokine receptors CCR3, CCR5, and CXCR4. Replicate 2-wk fetal macaque neural cultures treated with FDU were double immunolabeled with mAbs to CCR3, CCR5, and CXCR4 and polyclonal Abs to GFAP. Chemokine primary mAbs were detected using Texas Red (red)-conjugated secondary Abs, and GFAP primary Abs were detected using FITC (green)-conjugated secondary Abs. Nuclei were counterstained with topro-3 (blue). Cells were imaged using a Leica TCS SP confocal microscope. A, Confocal imaging of CCR3+GFAP- neuron. Note that most CCR3 is in the neuronal cell body. B, DIC image of the same cell as that in A. C, Isotype-matched control detected using Texas Red (red)-conjugated secondary Abs demonstrates no red staining of cell body or processes; only nuclei staining with topro-3 is detected. D, DIC image of same cells as those in C. E, Confocal imaging of a CCR5+GFAP- neuron and a CCR5+GFAP+ astrocyte (inset). Both cells contain a pool of CCR5 receptor. F, Confocal imaging of a CXCR4+GFAP- neuron and a CXCR4+GFAP+ astrocyte (inset). Note that most receptors are located along the axon in the neuron and along the edge of the cell membrane in the astrocyte. Data presented are representative of two separate staining experiments with both macaque and human (not shown) fetal neural cultures. Bars represent 100 µm.

 
To confirm the intracellular chemokine receptor expression pattern, additional imaging studies were performed on neurons double labeled with mAbs to chemokine receptors and MAP-2, an intracellular neuronal marker that is also highly expressed within neuronal cell processes. CCR3, CCR5, and CXCR4 (all red) and MAP-2 (green) were colocalized intracellularly (yellow) to varying degrees (Fig. 4Go). CCR3 and MAP-2 showed the highest degree of colocalization (Fig. 4GoA), 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. 4GoD). 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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  		FIGURE 4. Fetal human brain cells demonstrate colocalization of chemokine receptors CCR3, CCR5, and CXCR4 with MAP-2, an intracellular neuronal cell marker. Replicate 2-wk fetal human neural cultures treated with FDU were double immunolabeled first with mAbs to CCR3, CCR5, and CXCR4 and then with mAbs to MAP-2. Chemokine primary mAbs were detected using Texas Red (red)-conjugated secondary Abs, and MAP-2 primary Abs were detected using FITC (green)-conjugated secondary Abs. Nuclei were counterstained with topro-3 (blue). Cells were imaged using a Leica TCS SP confocal microscope. A, Confocal imaging of CCR3+MAP-2+ neuron. Note that most of the label for CCR3 is in the neuronal cell body, colocalizing with MAP-2 and appearing yellow. B, Confocal imaging of a CCR5+MAP-2+ neuron. There is a small pool of intracellular CCR5 that colocalized with MAP-2 (yellow). C, Confocal imaging of a CXCR4+MAP-2+ neuron. There is less intracellular CXCR4, with most of the receptors located along the MAP-2- cell membrane of the axon. D, Isotype-matched control detected with Texas Red (red)-conjugated secondary Abs demonstrates no staining; however, MAP-2 staining is evident throughout cell body and processes (green). Data presented are representative of two separate staining experiments with both macaque (not shown) and human fetal neural cultures.

 
Chemokines induce calcium flux in cultured fetal neurons and astrocytes

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{alpha} and MIP-1{beta} 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{beta}, RANTES), and CXCR4 (SDF-1{alpha}). All these chemokines have been shown to induce calcium flux in various leukocyte cell types (25, 49). As shown in Figs. 5Go-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. 5Go). Fig. 5GoA demonstrates this augmentation in the response of cultured macaque neurons to RANTES, and Fig. 5GoB 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. 5GoA), nor did treatment with the chemokine GRO{alpha}, which is a known ligand for chemokine receptor CXCR2 (Fig. 5GoC). 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. 5GoC). 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. 5GoC). In contrast to neurons, cultured human astrocytes did not require predepolarization with KCl to respond to chemokines (Fig. 6Go). Astrocyte responses to SDF-1{alpha}, RANTES, and MIP-1{beta} 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. 1GoD). The addition of mAbs to CCR5 (clone 2D7), CCR3 (clone 7B11), and CXCR4 (clone 12G5) before treatment with MIP-1{beta}, eotaxin, and SDF-1{alpha}, respectively, blocked neuronal responses to these ligands, but not to ligands that signal through other, nonneutralized receptors, demonstrating the specificity of these responses (Fig. 7Go). Pretreatment with 200 ng/ml pertussis toxin abolished both neuronal and astrocyte responses to RANTES, MIP-1{beta}, and SDF-1{alpha} (Fig. 8Go). 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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  		FIGURE 5. Fetal macaque and human neurons respond to chemokine with increases in intracellular calcium. Replicate 2-wk fetal macaque and human neural cultures treated with FDU were loaded with 5 µM fura-2, and groups of three to eight neurons were visualized and analyzed using a microscope based single-cell calcium flux apparatus for their responses to 100 ng/ml of chemokine. A, Fetal macaque neurons exposed to RANTES require predepolarization with 20 mM KCl for maximum response. BSA (10 nM), used as a control, does not induce a response. B, Fetal human neurons similarly require predepolarization with 20 mM KCl to respond to SDF-1{alpha}, MIP-1{beta}, eotaxin, and RANTES. C, Top and middle tracings, Fetal human neurons demonstrate homologous, but not heterologous, receptor desensitization. Note in the middle tracing that RANTES, which binds both CCR3 and CCR5, desensitized the eotaxin response. Bottom tracing, GRO{alpha}, which binds CXCR2, did not induce calcium flux responses in chemokine-responsive neurons. Data are presented as the relative ratio of fluorescence at emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of two to five separate experiments with different neuronal preparations.

 


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  		FIGURE 6. Fetal human astrocytes respond to chemokines with increases in intracellular calcium. Replicate cultures of 90% pure astrocytes were generated via repeated replating, loaded with 5 µM fura-2, and groups of one to three astrocytes were visualized and analyzed using a microscope-based, single-cell, calcium flux apparatus for their response to 100 ng/ml of chemokine. Astrocytes respond to SDF-1{alpha}, MIP-1{beta}, and RANTES, but not to eotaxin. Data are presented as the relative ratio of fluorescence at an emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of three separate experiments with different astrocyte preparations.

 


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  		FIGURE 7. Human fetal neurons do not respond to chemokines after pretreatment with neutralizing chemokine receptor mAbs. Replicate 2-wk fetal human neural cultures treated with FDU were loaded with 5 µM fura-2, and groups of three to eight neurons were visualized and analyzed using a microscope-based, single-cell, calcium flux apparatus for their response to 100 ng/ml of chemokines after pretreatment with neutralizing chemokine receptor mAbs. Human fetal neuronal responses to MIP-1{beta}, eotaxin, and SDF-1{alpha} were abolished by pretreatment with neutralizing mAbs to CCR5 (a; clone 2D7), to CCR3 (b; clone 7B11), and to CXCR4 (c; clone 12G5), respectively. Responses to chemokines that bind nonneutralized receptors, however, were preserved. Data are presented as the relative ratio of fluorescence at an emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of four separate experiments per mAb with two different neuronal preparations.

 


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  		FIGURE 8. Human fetal neurons and astrocytes do not respond to chemokines after pretreatment with pertussis toxin. Replicate 2-wk fetal human neural cultures treated with FDU were loaded with 5 µM fura-2, and groups of three to eight neurons were visualized and analyzed using a microscope-based, single-cell, calcium flux apparatus for response to 100 ng/ml of chemokine after treatment with 200 ng/ml pertussis toxin for 1 h. A, Human fetal neuronal responses to MIP-1{beta}, RANTES, and SDF-1{alpha} were abolished by pretreatment with pertussis toxin. B, Human fetal astrocyte responses to MIP-1{beta} and RANTES were abolished by pretreatment with pertussis toxin, but demonstrated nonspecific calcium flux response to the calcium ionophore ionomycin. Tris-EDTA was used to chelate calcium and generate the Rmin for calcium concentration calculations. Data are presented as the relative ratio of fluorescence at an emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of six separate experiments with three different neuronal preparations.

 
To test fura-2 loading and to determine maximum intracellular calcium concentrations, ionomycin, a calcium ionophore, was used to nonspecifically activate all calcium channels (Fig. 8GoB). Chelation treatment with Tris-EDTA allowed determination of minimum intracellular calcium concentrations (Fig. 8GoB). 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. 9Go 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. 9Goa). This response desensitizes MIP-1{beta} signaling, which binds CCR5, and is desensitized by pretreatment with RANTES, which binds CCR3 and CCR5 (Fig. 9Go). 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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  		FIGURE 9. Fetal macaque neurons respond to recombinant SIVmac239 gp120 with increases in intracellular calcium. Replicate 2-wk fetal human neural cultures treated with FDU were loaded with 5 µM fura-2, and groups of three to eight neurons were visualized and analyzed using a microscope-based, single-cell, calcium flux apparatus for response to 10 nM SIVmac239 gp120. a, Fetal macaque neurons exposed to SIVmac239 gp120 require predepolarization with 20 mM KCl for maximum response. The gp120-induced response desensitizes a response to 100 ng/ml MIP-1{beta}. b, Response to 10 nM SIVmac239 gp120 is desensitized by pretreatment with 100 ng/ml RANTES. Data are presented as the relative ratio of fluorescence at an emission frequency of 510 nm and excitation frequencies of 340 and 380 nm and are representative of four separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that the chemokine receptors CCR3, CCR5, and CXCR4 are present in a subpopulation of macaque and human neurons, and CCR5 and CXCR4 are present on astrocytes immediately ex vivo and continue to be expressed on neurons and astrocytes for at least 2–3 wk in culture. Thus, chemokine receptor expression is not a cell culture artifact, providing an in vitro model in which to further examine its role in neuronal physiology. We also demonstrated that chemokine receptors on primate neurons respond to their chemokine ligands with transient increases in intracellular calcium. These data support the in vivo observations that chemokine receptors are present on subpopulations of neurons and astrocytes and suggest that these receptors are functional. The presence of functional chemokine receptors on neurons and astrocytes provides a mechanism by which viral envelope or the chemokines known to be produced during CNS HIV/SIV infection could disrupt normal neurophysiology, contributing to the clinical manifestations of AIDS dementia complex.

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 {alpha}-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{alpha}, MIP-1{beta}, 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{alpha}, 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{alpha}, and MIP-1{beta} 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. 7Go). Gi1{alpha} and Gi2{alpha} have been purified from adult bovine brain (54) and levels of Gi2{alpha} 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{alpha}, and MIP-1{beta}, 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.


   Acknowledgments
 
We thank LeukoSite (Cambridge, MA) for providing mAbs to CCR3 and CCR5; Dr. Fred Beber and Ms. Kathleen Sirois from the Department of Pathology, Brigham and Women’s Hospital (Boston, MA) for assistance in the completion of this study.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants AI01519 (to R.S.K), CA69212 and AI40618 (to A.D.L.), RR00168 (to New England Regional Primate Research Center), NS37654 (to K.C.W.), and NS30769 (to A.A.L) and a grant from the Defense Advance Research Planning Agency. A.A.L. is a recipient of an Elizabeth Glazer Scientist Award, and A.D.L. is a Culpepper Scholar in Medicine. Back

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: Back

3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; IP-10, IFN-inducible protein of 10 kDa; SDF-1{alpha}, stromal-derived factor-1{alpha}; 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. Back

Received for publication February 12, 1999. Accepted for publication May 14, 1999.


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