HIV-1-mediated apoptosis of neuronal cells: Proximal molecular mechanisms of HIV-1-induced encephalopathy

Abstract

The induction of neuronal cell death in vivo has been recognized as a prominent feature of HIV type I (HIV-1) infection leading to HIV-1-induced encephalopathy. Viral and host cell products, released from HIV-1-infected cells, have been implicated as inducers of neuronal cell apoptosis. It is unclear which is more important in this process. Neuronal cells were treated with media bearing HIV-1 virions derived from infected T cells and macrophage or the same set of media depleted of virions. T cell media bearing virus induced high levels of apoptosis, whereas that depleted of virions did not. In contrast, neurons treated with media from infected macrophages induced cell death whether virions were present or depleted by ultracentrifugation. The former initiated a repeatedly and significantly higher degree of apoptosis. These data suggest that exposure of neurons to viral products is critical for the induction of apoptosis, in addition to putative host factors released from virally infected cells. Protein-array analyses identified host cell factors up-regulated from infected macrophages, versus their uninfected counterparts, and these host cell factors may be prime candidates for contributing to neuronal apoptosis. Gene-array analyses also identified mRNAs up-regulated in human neurons after treatment with purified HIV-1 gp120 envelope protein or virus-containing media from HIV-1-infected macrophages. These analyses suggest molecular mechanisms for the induction of apoptosis relating to the exposure of viral and host cell factors and rationally designed approaches toward neuroprotection.

Infection with HIV type I (HIV-1) causes neurodegeneration in patients with AIDS, which is designated HIV-1-associated dementia (HAD) (1). Apoptosis of neuronal cells in HIV-1-infected individuals relates to cognitive and motor dysfunctions (2, 3). The factors initiating HAD seem to be pleotropic and include HIV-1 infection of certain CNS-based cell types that may compromise their natural functions and further lead to the release of viral particles or proteins that trigger apoptosis of neurons (4–6). Alternatively, the infection of macrophages or microglia may enhance the release of proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and IFN-γ that are toxic to neurons (7, 8).

The presence of multinucleated giant cells, along with increased macrophage/microglia activity from the patient's brains infected with HIV-1 (9, 10), suggest that these cells are crucial for initiating neuronal apoptosis in HAD. Indeed, macrophages/microglia are the most productively infected cell types in the brain (11). It has been reported that infected and activated macrophages/microglia release viral proteins (gp120, Vpr, Tat), inflammatory cytokines (TNF-α, IL-1, IL-6), chemokines [stromal-derived factor, regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein, fractalkine], and neurotoxic substances including arachidonic acid metabolites, l-cysteine, and the amine Ntox (12–15), which induce neuronal apoptosis. Although viral proteins may induce apoptosis via interaction with CXC chemokine receptor 4/stromal-derived factor-1α ligand (16–20), mechanistically, neurotoxins also may induce cell death via binding to N-methyl-d-aspartate-type glutamate receptor (21). Finally, immunoinflammatory cytokines may induce apoptosis involving the action of caspases through the TNF/TNF receptor 1 (TNF-R1) receptor or the Fas/Fas ligand receptor (22).

In attempts to understand the potential mechanisms of HIV-1-induced neuronal apoptosis, we examined the effects of purified, recombinant gp120 and virion-depleted supernatant from infected macrophages and T cells as well as virion-containing cell supernatants from these infected cell types on human neurons (from hNT-2 cells) and the murine neuro2A cell line. Both cell lines are well established for the study of neuronal cell apoptosis (5, 6, 23–25). Macrophages, derived from primary human peripheral blood monocytes, were infected with the monocytetropic (CC chemokine receptor 5) HIV-1 isolate, BaL. T cells were infected with the HIV-1 T cell-tropic (CXC chemokine receptor 4) strain, NL4-3. The culture supernatants were harvested from each of the infected cell types, and virus was separated from host cellular factors in these culture media by ultracentrifugation. Neurons then were treated with media containing virus or that depleted of virions from infected macrophages or T cells as well as by direct addition of pure gp120 Envelope protein. The extent of neuronal cell death was determined by the terminal deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) assay, and changes in the transcription of genes, relating to apoptosis after treatment, were assessed by human gene-array analysis.

We demonstrated that gp120 alone, virion-containing cell supernatants, and virion-depleted supernatants from infected macrophages induced neuronal cell apoptosis but to differing degrees. The virion-containing supernatant from infected macrophages consistently induced greater levels of apoptosis versus virion-depleted supernatant. Protein-array analyses identified host factors that were up-regulated from infected macrophages that might contribute to the induction of apoptosis. This study indicated that the human CC chemokine I-309, monocyte chemoattractant proteins (MCP-2, MCP-3), ILs (IL-3, IL-5, IL-6, IL-7, IL-8), granulocyte/macrophage colony-stimulating factor, and growth-regulated oncogene were elevated from infected relative to uninfected macrophages, with IL-5 and IL-6 being elevated the most robustly.

The major apoptotic genes, TNF-α, TNF-R1, and TNF-receptor-associated death domain (TRADD) protein, and their downstream products Iκ B kinase (IκK-B) and NF-κB, as well as TNF-related apoptosis-inducing ligand (TRAIL) and caspase 8, were up-regulated in neurons that underwent neuronal apoptosis after treatment with media from macrophages infected with HIV-1. TRADD, TNF-R1, and their downstream products IκK-B and NF-κB, as well as Fas-associated death domain (FADD), caspase 6, caspase 7, and their cleavage substrates, poly(ADP-ribose) polymerase and Apaf1, were up-regulated in neurons treated with gp120. The changes in the expression of such genes, as assessed by mRNA-array analyses, are consistent with the phenomenon of cell death and dissects the synergism that likely exists between cell death induced by viral products as well as host factors.

Materials and Methods

HIV-1 Infection of Primary Human T Lymphocytes and Macrophages. Macrophages were isolated from peripheral blood mononuclear cells of HIV-1-seronegative individuals according to guidelines of the U.S. Department of Health and Human Services. After layering on Histopaque-1077 (Sigma), peripheral blood mononuclear cells were washed and pelleted, and the cell pellet was washed and cultured with RPMI medium 1640 containing 0.05 units of penicillin, 0.05 units of streptomycin, and 10% FBS. Macrophages were isolated by adherence to culture dishes in half RPMI medium 1640 complete and half Dulbecco's minimal essential medium (DMEM). The cultures were maintained for at least 1 week before infection.

The CD4⁺ T lymphocyte SupT1 cell line was infected with HIV-1 strain NL4-3 (X4-tropic) at 10 ng/ml p24 antigen equivalents and maintained in complete RPMI medium 1640. The cells were washed 48 h before culture to remove residual virus and maintained in culture for an additional 5 days. Virus-containing supernatant then was used to infect (10 ng of p24 input) CD8⁺ T lymphocyte- and macrophage-depleted peripheral blood mononuclear cells, which were washed and cultured for 7 days. The cell medium then was harvested and treated to deplete virus particles (see below). Macrophages were washed twice to remove nonadherent cells and then infected with the BaL monocyte-tropic (R5) strain of HIV-1 (input of 100 ng/ml HIV-1 p24 antigen equivalents) for 48 h at 37°C with 5% CO₂. The cells then were washed twice and maintained for another 5 days. The supernatant was centrifuged at 1,800 rpm for 5 min (Beckman Coulter) and passed through a 0.45-μm nylon membrane filter. The supernatant was used for p24 antigen ELISA, and the remainder was ultracentrifuged at 40,000 rpm for 1 h at 4°C (Sorval Ultra 80, Kendro, Asheville, NC). The supernatant after ultracentrifugation was designated as infected macrophage medium without virus (virion-depleted), whereas the one from low-speed centrifugation was designated as infected macrophage medium with virus (virion-containing). HIV-1 p24 antigen ELISA kits (NEN) were used according to the manufacturer's instructions (6).

Human Protein Cytokine Array. A human protein cytokine-array kit was purchased from RayBiotech (Norcross, GA). Briefly, the membranes were blocked with a blocking buffer, and then 1 ml of medium from either infected or noninfected macrophage or T lymphocyte cultures was added and incubated at room temperature for 2 h. The membranes were washed, and 1 ml of primary biotin-conjugated antibody was added and incubated at room temperature for 2 h. The membranes were incubated with 2 ml of horseradish peroxidase-conjugated streptavidin at room temperature for 30 min. The membranes were developed by using enhanced chemiluminescence-type solution, exposed to film, and processed by autoradiography.

hNT-2 Cell Cultures. NT-2 neuronal precursor cells were purchased from Stratagene (Stratagene cloning system), cultured, and differentiated with retinoic acid into mature human neurons as described (5, 6).

Viral Envelope Protein gp120 Treatment. Viral Envelope protein gp120 from HIV-1 BaL (gp120 1 mg/ml: endotoxin levels <10 endotoxin units/ml) was purchased from Intracel (Rockville, MD) and added at concentrations from 1 to 100 ng/ml in the cell-culture medium. After 48 h the medium was removed, and cells were washed twice with complete DMEM/F12 medium. The cells were cultured for another 3 days before DNA fragmentation and TUNEL assay.

gp120-Capture ELISA. A gp120-capture ELISA kit was purchased from ImmunoDiagnostics (Woburn, MA). According to the instructions, 100 μl of macrophage medium with virus and depleted of virus was diluted 1:10 and 1:2 in diluent buffer and tested along with positive references of gp120 from 250 ng/ml to 130 pg/ml. The ELISA plate was read at 450 nm.

TUNEL Assays. In situ cell-death detection kits, TMR red and AP, were purchased from Roche (Indianapolis). Human neurons and neuro2A cells in chamber slides were washed with PBS (pH 7.4) twice, fixed with 4% paraformaldehyde solution in PBS at room temperature for 1 h, and assayed for apoptosis according to the manufacturer's instructions (5, 6). The cells were analyzed with fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA) and semiquantitated by using a charge-coupled device array camera for fluorescence brightness red/green/blue values (0–255) yielding relative intensities as described (26).

Human Apoptosis Gene Microarray. The hNT-2-derived neurons were grown and matured in T25 flasks. The total RNA then was isolated from untreated or treated neurons by using the RNAgents kit from Promega. Apoptosis gene-microarray filters were obtained from Sigma–Genosys (Woodlands, TX) and used according to the instructions. Gene expression-level differences were quantitated by Sigma–Genosys.

Results

HIV-1-Related Apoptosis of Human Neurons. The objectives of our in vitro studies were directed toward discerning the primary factors involved in the induction of neuronal cell apoptosis that may relate to HIV-1 infection in vivo. First, as shown in Fig. 1, the levels of apoptosis, as determined by TUNEL staining, in human neurons increased as the concentration of the recombinant gp120 protein rose from 1 to 100 ng/ml (relative intensity values for: no treatment, 1, 10, and 100 ng/ml of 12, 75, 157, and 189, respectively; P < 0.0001 for all treatments compared with control by double-tailed Student's t tests). These findings also were observed across species for gp120 treatment of murine neuro2A cells (data not shown). As such, a dose-dependent increase in neuronal cell apoptosis is demonstrated by these studies using a macrophage-tropic (R5) HIV-1 envelope surface glycoprotein.

Fig. 1.

HIV-1 gp120-mediated apoptosis of human neurons. Pure, recombinant HIV-1 gp120 from an R5 (macrophage)-tropic strain was added to mature human neuron (from hNT-2 cells) cultures at increasing doses. The levels of apoptosis were determined by TUNEL after 5 days. Images are shown at ×20 magnification.

Our next objective was to determine whether virus itself, derived from HIV-1-infected cells, induced neuronal cell apoptosis to a degree lesser or greater than putative host factors that have been induced to be expressed and subsequently released from cells infected by the virus. Both human primary CD4⁺ T cells and macrophages were infected in vitro by HIV-1 using the X4 and R5 viral strains, NL4-3 and BaL, respectively. Culture medium was harvested from the HIV-1-infected cell cultures, and a portion of the medium was ultracentrifuged to deplete viral particles. Uninfected cell medium, virus-depleted medium, and virus-containing medium from primary T cells and macrophages then were added to human neuron cultures and assayed for apoptosis.

As shown in Fig. 2, only a very modest level of apoptosis was observed in human neurons treated with medium from uninfected macrophages. The cells treated with infected macrophage medium, depleted of virus by ultracentrifugation (≈75% depletion as assessed by detecting the presence of HIV-1 p24 antigen in the medium by using ELISA), yielded an intermediate level of cell death. It is noteworthy, however, that gp120 was demonstrated to be depleted in the medium from 13.76 to <0.176 ng/ml, as measured by an ELISA capture technique. Neurons treated with infected macrophage medium containing virus, however, underwent the greatest degree of cell death (Fig. 2; relative intensities of 27, 197, and 114 for additions of medium alone, infected macrophage medium, and infected, virus-depleted macrophage medium, respectively; P < 0.0001 for infected macrophage medium with depletion of virus compared with control and also for infected macrophage medium containing virus versus that depleted of virus). The aggregating growth pattern of mature human neurons makes it somewhat difficult to quantify precisely the actual levels of cell apoptosis with the various treatments. However, neuron aggregates, chosen for viewing and observed in multiple independent experiments, were approximately the same size; therefore, the intensity of TUNEL staining and semiquantitative relative intensity analyses should reasonably reflect the overall qualitative level of apoptosis for each treatment.

Fig. 2.

HIV-1 virion-mediated apoptosis of human neurons. Cell cultures were treated with medium appropriate for optimal neuronal cell growth (control) (Upper Left), medium cultured from macrophages not infected with HIV-1 (Upper Right), culture medium from HIV-1-infected macrophages depleted of virus particles by ultracentrifugation (Lower Left), and culture medium from infected macrophages containing HIV-1 particles (Lower Right). Images are shown at ×20 magnification.

The results of treating human neurons with media from T cells, containing or depleted of virus particles, were different. Media from uninfected T cells or infected T cells depleted of virus had little or no indication of apoptosis (Fig. 3). However, T cell medium containing virus particles and added to neurons initiated vigorous apoptosis (relative intensities of 11, 118, and 16 for addition of medium alone, infected T lymphocyte medium, and T cell medium depleted of virus, respectively). It is of interest that the virus-depleted medium from infected macrophages induced apoptosis, albeit significantly lower than virus-containing macrophage supernatant, whereas that from T cells did not.

Fig. 3.

HIV-1 virion-mediated apoptosis of human neurons. Mature human neuronal cell cultures were treated with culture medium from primary T lymphocytes not infected with HIV-1 (Left), culture medium from HIV-1-infected T lymphocytes depleted of virus particles by ultracentrifugation (Center), and culture medium from infected T lymphocytes containing intact HIV-1 particles (Right). Images are shown at ×20 magnification.

Human Cytokine Protein Array from Infected Macrophages and T Cells. It has been proposed that host products from HIV-1-infected macrophages, which become activated as a consequence of infection, may alter neuronal physiology and induce apoptosis (27). A human cytokine-array approach, to quantitatively detect the presence of proteins in cell medium, was performed to identify specific host factors induced to express and be released from HIV-1-infected macrophages or T cells.

The data from a representative array are shown in Fig. 4. No factors seemed to be down-regulated in the HIV-1-infected macrophages relative to uninfected cells. These array analyses indicated that IL-5, I-309, IL-6, granulocyte/macrophage colony-stimulating factor, and MCP-3 were the factors most significantly up-regulated for expression and released from the infected macrophages, although MCP-2, IL-3, IL-7, IL-8, growth-regulated oncogene, and RANTES also were upregulated modestly. Human CC chemokine I-309, MCP, RAN-TES, and growth-regulated oncogene as well as cytokines IL-3, IL-5, IL-6, and IL-8 have been shown to be produced by macrophages and astrocytes (28, 29). In three series of arrays, IL-5 and IL-6 were up-regulated dramatically in all three, whereas I-309 was up-regulated robustly in two of three and increased modestly in one. MCP was elevated in two of three sets of arrays (data not shown).

Fig. 4.

Human cytokines up-regulated and released from primary macrophages infected with HIV-1. Human protein-array analysis was used to determine the differences in the release of human cytokines from uninfected primary macrophages versus HIV-1-infected macrophages. (A) Data from the array analyses with filter 1, treated with medium from uninfected cells (Left), and filter 2, treated with medium from HIV-1-infected macrophages (Right). The short-stemmed arrows point to the position of the IL-5-array positions, and the long-stemmed arrows point to the I-309-array positions. Autoradiographs of the arrays were scanned to determine the density of the proteinarray positions. (B) The values from scans were adjusted based on the intensity of control spots on the filter corners, and the level increases for specific cytokines are shown. This is one representative series of arrays from three experimental repeats.

I-309 is a monocyte chemoattractant (28), and MCP likely regulates migration of peripheral blood mononuclear cells through the blood–brain barrier (21), whereas RANTES and other cytokines are involved in cellular communication, survival, and differentiation (30). Select cytokines were up-regulated from infected macrophages (Fig. 4); however, a similar array analysis from uninfected versus HIV-1-infected CD4⁺ T lymphocytes demonstrated no differences in the levels of secreted cytokines from this cell type (data not shown). TNF-α, IL-1β, and IL-6 had been demonstrated to have prominent roles in the host–virus interaction, including HIV-1 infection (11, 31).

Human Apoptosis Gene Expression. In defining the host apoptosis genes involved in cell death induced by gp120 alone or HIV-1 products produced from infected macrophages or T cells, it is quite important to assess molecular mechanisms involved in the induction of apoptosis. Table 1 shows apoptosis-related genes up-regulated in response to gp120 viral protein-induced neuronal apoptosis as well as HIV-1-infected macrophage medium-induced neuronal apoptosis. As Table 1 indicates, α-tubulin and growth arrest-specific gene (GAS1) showed similar increases in both sets of treated neural cells. TNF-R1, TRADD, IFN-γ R2, mitogen-activated death domain (MADD), NF-κB subunit, IκK-B, and 14-3-3 protein are also up-regulated in both gp120-induced and infected-macrophage medium-induced apoptotic neurons. TRAIL was up-regulated 1.7-fold as well as caspase 8 and TNF-α in infected macrophage medium-induced apoptosis, but this was not observed in gp120-induced apoptosis. Instead, FADD was up-regulated 1.7-fold along with caspase substract cleavages, caspase 6, caspase 7, poly(ADP-ribose) polymerase, and Apaf1 in gp120-induced apoptotic neurons.

Table 1. Human apoptosis gene array of human neurons.

Virus-containing media	Fold increase	HIV-1 BaL gp120 treatment	Fold increase
HLA-A 0201 heavy chain	3.2	α-Tubulin	3.44
α-Tubulin	3.09	TRADD	3.17
TNF-RI	2.7	IFN-γ R2	2.8
GAS1	2.37	GAS1	2.54
β-Actin	2.34	MADD	2.42
Cyclophilin A	2.34	NF-κ B subunit	2.26
IFN-γ R2	2.04	I κ B	2.05
Cox-1	2	14-3-3 protein	2.03
NF-κ B subunit	2	Apaf1	1.97
14-3-3 protein	1.98	PARP	1.94
I κ B	1.94	RbAp48	1.92
MADD	1.88	IGF-1 R	1.92
TRADD	1.83	RB1	1.88
p27	1.8	Rb2/p130	1.86
TRAIL	1.79	ARC	1.85
GITRL	1.77	Caspase 6	1.84
IGF-1R	1.77	GALECTIN-3	1.81
DAXX	1.76	DFF40	1.79
RBQ-3	1.76	Bcl-W	1.78
RbAp48	1.76	TNF-R1	1.76
p21	1.73	TDAG8	1.75
L19	1.73	FADD	1.74
TNF-α	1.71	AIF	1.74
Caspase 8	1.68	TFAR15	1.73
TRANK	1.67	NAIP	1.73
DR6	1.67	c-Myc	1.72
IL-4 Ra	1.64	Survivin	1.72
IL-10 Ra	1.64	BimEL	1.71
DAD-1	1.6	Mcl-1	1.7
PAK1B	1.6	RBP2	1.7
TRAF6	1.6	Caspase 7	1.7
XIAP	1.56

The mRNA expression levels for apoptosis-related genes increased in human neuronal cells after treatment with virus-containing media from primary macrophage infected with HIV-1 or treatment with HIV-1 gp120.

The death receptors Fas/Apol1, TNF-R1, and TRAIL seem to be involved in neuronal apoptosis, as indicated by our gene-array studies. Although the full spectrum of host proteins likely involved in cell-death pathways has not been characterized thoroughly, it is known that FADD likely is a universal adaptor used by death receptors to initiate the action of caspase 8 (32). In this study, the death genes seemed to largely involve TNF-α/TNF-R1 receptor and FasL/Fas receptor pathways in both gp120 directly induced neuronal apoptosis and infected macrophage medium-induced neuronal apoptosis. Moreover, the TRAIL receptor was up-regulated after induction of neuronal apoptosis after treatment with virus-containing macrophage medium. Two pathways were likely engaged in the initiation of apoptosis from the treatment of neurons with HIV-1-infected macrophage cell medium. The relatively modest increases in the transcription of apoptosis-related genes by mRNA-array analyses would be expected, because apoptosis occurs mostly with proteins that exist in the cell and do not require de novo synthesis.

Discussion

The molecular mechanisms that underlie the neuronal cell apoptosis relating to HAD may vary. Currently there are two major hypotheses regarding neuronal cell injury or death within HIV-1-infected individuals: induction of neuronal apoptosis directly by viral proteins or indirectly via soluble factors released from HIV-1-infected cells. These mechanisms may not be mutually exclusive (21). CXC chemokine receptor 4 and CC chemokine receptor 5 can be expressed on neurons, and these coreceptors might facilitate gp120 binding, thereby inducing cell death. In some studies, blocking access of virus to the chemokine receptors seems to occlude the initiation of apoptosis (19, 33). However, neurons do not display the CD4 protein, the primary receptor for efficient HIV-1 entry into cells. Nonetheless, extensive literature indicates that gp120 induces neuronal death (16, 20, 34). High concentrations of gp120, added to pure neuronal or neuroblastoma cell lines in the nanomolar range, are toxic to such cells in vitro (21). The number of HIV-1-infected cells in the brain does not always correlate well with viral load or even necessarily the extent of HAD in HIV-1-seropositive patients (10). Thus, soluble factors released from infected macrophages or perhaps other cell types could also contribute to neuronal cell death.

This study examined the role that viral and cellular products play in apoptosis of human neurons as a model cellular system for HAD. Recombinant gp120, as well as supernatant from HIV-1-infected T lymphocytes and macrophages, enriched or depleted of viral components was added to neurons.

It was clear that the cell medium from infected macrophages or containing gp120 induced the highest levels of neuronal cell death. Of interest and in contrast to findings by Allan and Rothwell (35), proteomics-array analyses indicated that TNF-α and IL-1β were not up-regulated after infection of macrophages in vitro, at least in the time frame of our analyses. Instead, other genes were up-regulated, including IL-5, which was increased 4-fold. An up-regulation and release of granulocyte/macrophage colony-stimulating factor was observed, and this protein could be a reasonable candidate for the induction of neuronal cell apoptosis. IL-5 is secreted by T cells and mast cells and plays a role in the activation and proliferation of both eosinophils and B cells (36–38). IL-5, produced by a variety of CNS cells, was demonstrated to be involved in the interaction between brain and immune cells (39). IL-5 in the CNS has been known as a macrophage/microglial mitogen, and IL-3 and granulocyte/macrophage colony-stimulating factor are involved in the cytokine-immune cascades in areas affected by multiple diseases (40). The detection of I-309 up-regulated from HIV-1-infected macrophages has potential importance. The enhanced release of this chemokine as well as MCP-3 likely serves to recruit additional phagocytes across the blood–brain barrier to HIV-1-infected monocytes/microglia in the brain, inducing additional dysfunction of bystander neurons.

The observation that TNF-α and IL-1β were not elevated after infection of macrophage is supported by other studies. Khanna et al. (41) reported that most HIV-1 variants were incapable of eliciting TNF-α secretion. Importantly, Foli et al. (42) demonstrated that high levels of TNF-α release were elicited by pathogens or processes other than HIV-1 as inductive agents in infected individuals. Bacterial lipopolysaccharide readily stimulates monocyte-derived macrophages to produce TNF-α or IL-1β. Some studies of apoptosis may therefore relate to the effects of the potential presence of endotoxin. Li et al. (43) found that TNF-α mRNA levels on tonsillar tissue from uninfected individuals were not different from those from HIV-1-infected individuals. There was no correlation between the level of HIV-1 gene expression in these tissues and the level of TNF-α expression. It is possible that elevated IL-1β or TNF-α in HAD patients may be from infected astrocytes, because astrocytes are also natural regulators of CNS immune functions (44).

Although all models have limitations, we have clearly demonstrated that the HIV-1 gp120 envelope protein induces neuronal cell apoptosis, and this induction of apoptosis seems to be conserved across species. gp120-mediated apoptosis is fully consistent with previous studies (16, 18, 20, 38, 45). We have demonstrated further that supernatant from infected macrophage cultures, depleted of virus particles, induced apoptosis although to a significantly lesser degree than the same cell medium containing viral particles. Only virus-containing medium from infected T cells was capable of initiating neuronal cell death. These data suggest that soluble factors released from infected macrophages may indeed be candidates capable of inducing neurotoxicity or death but are likely somewhat less critical than intact virions or individual free viral proteins such as gp120, Vpr, and Tat (5, 26, 46, 47).

The levels of apoptosis induced by virion-containing macrophage medium were clearly higher than that induced by virion-depleted macrophage medium and seemed comparable with that initiated by the direct addition of nanomolar concentrations of pure gp120. These observations suggest that viral products likely play a significant role in neuronal cell death and perhaps for HAD induction in vivo rather than sole release of host cell proinflammatory cytokines or other neurotoxic moieties. Although levels of gp120 were depleted significantly in macrophage medium after separation, free Tat and Vpr are also likely to still be present in the medium. Thus, the percent of apoptosis induced solely by macrophage cellular factors is likely to be somewhat lower than demonstrated in these studies.

We detected and characterized the up-regulation of genes known to participate in critical transduction pathways related to cell death that involve or relate to both hypotheses stated above. Previous studies demonstrated that neurotoxic factors from infected macrophages cocultured with astrocytes induced neuronal apoptosis via the N-methyl-d-aspartate subtype (NMDAR)/bcl-2 pathway (23). The NMDAR antagonist memantine rescued neuronal injury induced by gp120 in cultured neurons as well as in gp120 transgenic mouse (48, 49).

Our study demonstrated that the TNF/TNF-R1 and Fas/Fas Ligand pathways might both be engaged in neuronal cell apoptosis, because genes in these pathways were up-regulated within the treated neuronal cells that underwent apoptosis. This result is consistent with other reports (22, 50) that indicate NF-κB activation may be involved in TNF-α-induced apoptosis in the HAD. In addition, TRAIL was detected from infected macrophage-induced neuronal cell apoptosis in our study. Related observations indicated apoptotic neurons colocalized with HIV-1-infected macrophages that expressed TRAIL, and neutralizing antibody against TRAIL, but not human TNF-α or Fas L, blocked neuronal apoptosis in an HIV-1-infected murine brain model (51). Other critical viral and environmental cofactors also may alter HIV-1-induced neuronal death (26, 52).

The present studies identify two major mechanisms involved in inducing apoptosis of neurons. Although not mutually exclusive, the interpretation of the data strongly favors a direct and very significant role for the presence of viral products in the initiation of neuronal cell apoptosis and as such may represent those critical microenvironmental factors most necessary to induce neuronal cell deficits and death in vivo.