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(Journal of Leukocyte Biology. 2000;68:429-435.)
© 2000 by Society for Leukocyte Biology

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes

Stefano Aquaro*, Stefania Panti*, Maria Cristina Caroleo{dagger}, Emanuela Balestra*, Alessandra Cenci*, Federica Forbici{ddagger}, Giuseppe Ippolito{ddagger}, Antonio Mastino§, Roberto Testi*, Vincenzo Mollace||, Raffaele Caliò* and Carlo Federico Perno*,{ddagger}

* Department of Experimental Medicine, University of Rome "Tor Vergata", Rome, Italy;
{dagger} Faculty of Pharmacy, University of Calabria, Cosenza, Italy;
{ddagger} IRCCS "L. Spallanzani", Rome, Italy;
§ Institute of Microbiology, University of Messina, Messina, Italy; and
|| Faculty of Pharmacy, University of Catanzaro, Catanzaro, Italy

Correspondence: Stefano Aquaro, M.D., Ph.D., Department of Experimental Medicine, University of Rome "Tor Vergata", via di Tor Vergata 135, 00133 Rome, Italy. E-mail: aquaro{at}uniroma2.it


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ABSTRACT
 
Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces <10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death.

Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.


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INTRODUCTION
 
Macrophages (M/M) infected by human immunodeficiency virus (HIV) are found in all tissues and organs of HIV-infected patients [1 2 3 4 5 ]. These cells represent an important source of HIV during the whole course of the disease and a key cellular reservoir acting as a major impediment to eradication of HIV by highly active antiretroviral therapy (HAART) [6 7 8 ].

Infection of M/M is considered a main pathogenetic event leading to alteration of cognitive dysfunctions typically found during HIV infection [9 10 11 ]. Indeed, because neurons, oligodendrocytes, and brain microvascular endothelial cells are rarely infected in vivo [12 , 13 ], productive viral replication by M/M of the central nervous system (CNS; of microglial origin or derived from monocytes arrived in the CNS through the blood brain barrier) is considered the most relevant cause of neurological impairment found in patients with HIV encephalopathy [14 ]. More than one-third of adults and half of children with AIDS show neurological symptoms [15 16 17 18 19 ]. Although HAART has potently contributed to reduce the prevalence of major HIV-related opportunistic infections, its effect on the incidence of HIV encephalopathy is less pronounced, as demonstrated by a relative increase in recent years of HIV encephalopathy as the AIDS-defining diagnosis [20 ].

The fine mechanisms by which HIV-infected M/M affect cellular homeostasis during HIV encephalopathy are poorly understood still [21 ]. HIV-infected M/M are able to trigger apoptosis of bystander lymphocytes [22 ] through the release of soluble factors (Fas ligand, gp120, etc.) [23 , 24 ]. Apoptosis is a common feature of neurons in HIV-infected patients; the same effect occurs, although at lower prevalence, in astrocytes not infected and not adjacent to HIV-infected M/M [13 ]. Thus, it is conceivable that M/M in the CNS may mediate cellular damage through secretion of soluble factors. Thus, to better understand this latter phenomenon, we have investigated whether HIV-infected M/M can affect the homeostasis of astrocytes and the potential mechanisms underlying this phenomenon.


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MATERIALS AND METHODS
 
Cells
Human primary macrophages
Peripheral blood mononuclear cells (PBMCs) were obtained from the blood of healthy seronegative donors by separation over Ficoll-Hypaque gradient. After separation, PBMCs were seeded at a density of 1.5 x 106 cells/ml in 25 cm2 plastic flasks (Costar, Cambridge, MA) in RPMI 1640 (Gibco, Grand Island, NY) with the addition of 50 units/ml penicillin, 50 µg/ml streptomicin, 2 mM L-glutamine, and 20% heat-inactivated mycoplasma- and endotoxin-free fetal calf serum (FCS; Hyclone, Logan, UT; complete medium). Cells were incubated at 37°C in humidified air containing 5% CO2. After 5 days of culture, nonadherent cells were removed by repeated washings with warm medium. Macrophages obtained with this method resulted in >95% of purity by cytofluorimetric analysis.

Human astrocytoma
The astrocytic cell line was derived from a 51-year-old male patient who presented a large right front-temporal mass (astrocytoma); characteristics of cells derived from this tumor are described elsewhere [25 ]. Cells were expanded and cultured by seeding them in 48-well plastic plates at a density of 100,000 cells/well in complete medium and incubated at 37°C in humidified air containing 5% CO2.

Drug and HIV proteins
3'-Azido-2',3'-dideoxythymidine (AZT; GlaxoWellcome, Middlesex, UK) was dissolved in sterile phosphate-buffered saline (PBS) and stored at -80°C before use. Glycosylated recombinant HIV-1 gp120 was obtained from Medical Research Council Directed Reagent Project (NIBSC, South Mimms, UK). Characteristics of biologically active recombinant HIV-1 Tat protein (gift of Dr. G. Barillari, University of Rome "Tor Vergata", Italy) are described elsewhere [26 , 27 ].

Antibodies anti-Fas
Anti-CD95 hybridoma DX2 [immunoglobulin G1 (IgG1); Ab anti-Fas] was generated by immunizing C3H/He mice with CD95-transfected L cells and fusing immune splenocytes with Sp2/0 myeloma cells [28 ].

Virus, macrophages infection, and supernatants collection
A monocytotropic strain of HIV-1, HIV-1BaL,was used in all experiments; characteristics and genomic sequence of this strain have been described previously [29 , 30 ]. The HIV-1BaL virus was expanded, collected, filtered, and stored in liquid nitrogen. Virus expansion was performed in primary M/M. To do so, 5-day adherent M/M were infected with 300 tissue culture infectious dose (TCID)50 of HIVBaL. After 14 days of infection, the supernatants were collected, titrated, and stored at -80°C before use. Details of this procedure are described elsewhere [31 ]. In some experiments, we utilized supernatants from HIV-infected M/M treated with AZT 0.1 µM at the time of infection. Treatment with AZT was continued thereafter. Uninfected supernatants were obtained from mock-infected M/M of the same donors.

Challenge of astrocytes with supernatants from HIV-infected M/M
After plating and removing culture medium, astrocytes were exposed to supernatants of HIV-infected or mock-infected M/M (with or without AZT, where required) for 4 h; cells were then carefully and repeatedly washed, and cultivated in complete medium. For the assessment of the effect of gp120, tat, and Fas antibody, astrocytic cells were treated with various concentrations of test compounds by using the same culture conditions as HIV-infected supernatants.

HIV detection
HIV-p24 antigen production in supernatants of M/M and astrocytes was assessed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (HIV-p24 gag, Abbott Lab, Pomezia, Italy). Integrated HIV-DNA detection in astrocytes and M/M was performed by polymerase chain reaction (PCR) using primers for pol gene, described elsewhere [30 ].

Trypan blue-exclusion test of cell viability
The dye-exclusion test was used to determine the number of viable cells after exposure of astrocytes to supernatants. At different time points after treatment, astrocytes were trypsinized, exposed to dye, and then examinated visually to determine whether cells take up or exclude dye. The live cells that possess intact cell membranes exclude trypan blue, whereas dead cells do not [32 ].

Evalutation of programmed cell death
Immunocytochemical analysis
The astrocytic cells exposed to HIV-infected supernatants were analyzed for the presence of apoptotic nuclei by in situ TdT-mediated, dUTP-biotin nick-end labeling (TUNEL) [33 ]. Quantitative analysis of apoptotic cells was carried out using a computerized image-analysis system (Axiophot Zeiss microscope equipped with a Vidas Kontron system).

Fluorescein-activated cell sorter (FACS) analysis
Astrocytic cells were gently detached from plastic 6 days after exposure to HIV-infected supernatants. Aliquots of 5 x 105 cells were centrifuged at 300 g for 5 min; pellets were washed with PBS, placed on ice, and overlaid with 0.5 ml of a hypotonic fluorochrome solution containing 50 µg/ml propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. After gentle resuspension in this solution, cells were left at 4°C for 30 min, in absence of light, before analysis. Propidium iodide-stained cells were analyzed with a FACScan Flow Cytometer (Becton Dickinson, Rutherford, NJ); fluorescence was measured between 565 and 605 nm. Data were acquired and analyzed by the Lysis II program (Becton Dickinson).


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RESULTS
 
HIV-infected M/M can affect the homeostasis of astrocytes without causing their infection
In a first set of experiments, we investigated the effects of HIV-infected M/M on viability of astrocytes. Alteration of cellular viability could be found starting at day 3 after virus challenge; at day 6, >52% of cells exposed for 4 h to cell-free supernatants of HIV-infected M/M were dead. Ten days after exposure, a dramatic disruption of cell monolayer could be detected (Fig. 1 ), with >83% of astrocytes dead; by contrast, no alteration of cell viability could be found in astrocytes treated with supernatants of mock-infected M/M (Fig. 2 ). To verify the possible relation of this phenomenon with the presence of HIV infection, we assessed the virus production in the supernatants of astrocytic cell cultures. Although repeatedly checked, all supernatants tested showed no detectable p24 gag, suggesting the absence of productive infection of astrocytes (unpublished results). To exclude a limited and/or nonproductive infection, the presence of viral DNA was assessed in such astrocytes. Indeed, no viral DNA was present 3, 6, and 9 days post-exposure to infected supernatants derived from M/M (unpublished results). It is worth noting that the expression of CD4 on the surface of astrocytes assessed by cytofluorimetric analysis was repeatedly negative (unpublished results). Thus, the astrocytes disruption that occurred upon exposure to HIV-infected supernatants of M/M was not related to HIV infection.



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  		Figure 1. Disruption of astrocytic monolayer induced by exposure to supernatants from HIV-infected macrophages. Representative photomicrographs (optical microscopy x40) of astrocyte cell monolayers: after 6 days of exposure to supernatants of mock-infected M/M (A), after 6 days (B), and after 10 days (C) of exposure to supernatants of HIV-infected M/M. Experiments were repeated four times with reproducible results.



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  		Figure 2. Human HIV-infected macrophages affect homeostasis of astrocytes. Viabilty of astrocytes was checked by Trypan blue-dye exclusion at days 3, 6, and 10 after exposure to supernatants of mock-infected M/M (shaded), or HIV-infected M/M (solid), and in astrocytes not exposed to M/M supernatants (dots). Values are the mean out of three independent experiments. Error bars represent the standard deviations.

HIV-infected M/M, not mock-infected M/M, trigger apoptosis of astrocytes
The huge difference of viability between astrocytes exposed or not exposed to HIV-infected M/M supernatants prompted us to evaluate the role of apoptosis in this phenomenon. As described in Figure 3 , 32% of astrocytes exposed to supernatants of HIV-infected M/M showed hypodiploid DNA by cytofluorimetric analysis 6 days after exposure; at the same time, only 2% and 9% of cells were found positive in the controls not exposed, or exposed to, supernatants of mock-infected M/M, respectively (Fig. 3) . Later analysis of apoptosis was made impossible by the complete disruption of monolayer of astrocytic cells exposed to HIV-infected M/M supernatants (control astrocytes remained healthy; see Fig. 1 ).




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  		Figure 3. Apoptosis in astrocytes exposed to supernatants of HIV-infected macrophage supernatants. A strong increase of hypodiploid nuclei was found at day 6 in astrocytes exposed to HIV-infected M/M supernatants (C), compared with cells exposed to mock-infected M/M supernatants (B) or cells not exposed (A) to M/M supernatants. The figure represents a typical experiment out of three.

AZT treatment of M/M infected by HIV prevents apoptosis and necrosis of astrocytes
To assess the ability of AZT to prevent astrocytic cell death, we treated HIV-infected M/M with AZT at a concentration of 0.1 µM (sufficient to inhibit >90% virus infectivity). Supernatants from HIV-exposed, AZT-treated M/M were collected 14 days after infection: consistent with previous data [34 ], production of p24 was very low (>95% inhibition compared with untreated controls; unpublished results). Immunocytochemical studies performed by TUNEL showed that necrosis and apoptosis were absent in astrocytic cells exposed to HIV-infected M/M treated with AZT at day 6 (Fig. 4 ) or day 10 (unpublished results). Treatment with AZT of astrocytes before the exposure of HIV-infected M/M supernatants did not reverse apoptosis in these cells (unpublished results). Thus, productive HIV infection of M/M is mandatory to induce death of astrocytes, and later treatment with AZT, once HIV particles have been produced, does not reverse such effect.



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  		Figure 4. AZT treatment of HIV-infected M/M prevents apoptosis in astrocytes. For this analysis, astrocytes were seeded in slides at a density of 20,000 cells/slide and cultivated at 37°C, 5% CO2 in humidified incubator for 24 h before exposure to HIV-infected (B), mock-infected (A), or AZT-treated HIV-infected (C) M/M supernatants. Astrocytes were also exposed to gp120 at a concentration of 1 nM (D). TUNEL analysis assessed at day 6 shows an increase of positive cells in astrocytes exposed to supernatants of HIV-infected M/M (arrowheads in B), and no significant presence of apoptotic cells was shown in the other groups (A, C, and D). These are representative photomicrographs (optical microscopy x40) out of three independent experiments.

Role of Tat and gp120 in mediating apoptosis of astrocytes
Because gp120 and tat, previously described as viral proteins involved in apoptosis, are produced by HIV-infected M/M, we treated the astrocytes with different concentrations of gp120 or tat to evaluate the possible involvement of these viral proteins in apoptotic events occurring in our cultures. TUNEL analysis shows that neither necrosis nor apoptosis is induced even by the highest concentration used (1 nM) of gp120 (Fig. 4) ; shown to be effective in other systems, Tat 1 ng/ml induced modest levels of apoptosis (8% of cells) compared with untreated controls at day 6 after treatment (2%; unpublished results).

Fas/Fas-ligand interactions are involved in the apoptosis of astrocytes
Fas ligand is upregulated by HIV infection in M/M [35 ] and was described previously as responsible for the HIV-M/M-triggered apoptosis of T lymphocytes in vivo [23 ] and in uninfected T lymphocytes in vitro [36 ]. For this reason, the role of Fas ligand was analyzed by adding blocking anti-Fas antibodies to cultures of astrocytes incubated with HIV-infected M/M supernatants. As shown in Figure 5 , Fas-blocking antibodies completely reversed in a dose-dependent manner the death of astrocytes found 6 days after exposure to HIV-infected M/M in the absence of such antibody.



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  		Figure 5. Anti-Fas antibody reverse-programmed cell death in astrocytes exposed to supernatants of HIV-infected macrophages. Expression of apoptosis was detected by TUNEL at day 6. Quantitative analysis of positive cells was carried out with a computerized image analysis system. Dose-dependent decrease of apoptotic astrocytes after treatment with Ab anti-Fas (shaded). Composed to HIV-infected M/M supernatants not exposed to anti-Fas antibody (solid). Data from three independent experiments are shown. Error bars represent the standard deviation.


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DISCUSSION
 
This study shows that M/M productively infected by HIV affect homeostasis of astrocytes. This phenomenon occurs in the absence of infection of target cells and is mediated mainly by Fas ligand, a factor known to regulate astrocytic survival and functions [37 ]. Soluble proteins of HIV, commonly present in the supernatants of HIV-infected cells, do not seem to play a major role in this contest.

Virus production is necessary for the production and release by infected M/M of Fas ligand and the other factors able to trigger astrocytic damage. This suggests that the continuous and abundant virus production by M/M in the CNS is essential for the induction of cellular damage typically found in HIV-infected patients, and stresses the crucial role played by these cells in the pathogenesis of HIV infection.

The interaction of M/M with HIV is quite complex. In vitro virus production by M/M is quite abundant ( particulary if calculated on a per-cell basis) and may last several weeks after virus challenge. Recent data from our group have demonstrated that M/M infected by HIV (but not uninfected M/M) produce and secrete a neurokine, nerve growth factor (NGF), known for its neurotrophic and immunomodulatory effect [38 39 40 41 42 43 44 45 ]. The production of NGF is essential for the survival of HIV-infected M/M and thus for the long-term production of virus particles: Indeed, deprivation of autocrine NGF activates the expression of low-affinity receptors (p75) for NGF on M/M surface and triggers their apoptosis [38 ]. It is conceivable that the mechanisms of production and release of NGF represent the key event leading to the long-term survival of HIV-infected M/M also in the CNS.

In our model, we demonstrate that disruption of astrocyte viability is induced by HIV-infected M/M in the absence of target-cell infection. This result suggests that M/M may alter the homeostasis of bystander cells also in the CNS by acting through secreted soluble factors that affect viability of cells not infected and not necessarily adjacent to infected M/M [13 ]. Nevertheless, we cannot exclude that astrocytes may get in vivo-infected by HIV. Indeed, the cells used in our experiments belong to an astrocytoma cell line that may not fully represent the overall characteristics of normal astrocytes [25 ]. Recent papers have shown that cells other than M/M, and in particular endothelial cells and astrocytes themselves, can be a target of HIV infection in the CNS [46 47 48 ]. Whether their infection is productive, however, is still a matter of debate. In the case of astrocytes, the relative frequency of proviral DNA is not accompanied by production and release of abundant virus particles [49 ]. This phenomenon seems to be related to a cellular block (typical of astrocytes) of the function of rev, a key virus protein able to regulate expression of transcripts encoding for viral structural proteins, and thus essential for an abundant virus production [50 51 52 ].

On the basis of these findings, it is conceivable that astrocytes may play a dual role in the pathogenesis of HIV-related encephalopathy. From one side, some of them can be a non- (or poorly) productive cellular reservoir of HIV in the brain. At the same time, the disruption of their function, dependent on the productive infection of M/M, may in turn affect homeostasis of neurons (cells, whose direct infection by HIV is still a matter of investigation) and alter the functionality of blood-brain barrier (constituted by various cell types including astrocytes), typically disrupted in advanced stages of the diseases [53 ].

It is worth noting that CD95/Fas plays a pivotal role in the induction of astrocytic death also in conditions not related to HIV infection [37 ]. This suggests that the brain inflammation and degeneration found in HIV-infected subjects can be related (at least in part) to a single mechanism common to other diseases and helps in explaining the reversibility of neuronal symptoms described by many authors in patients treated with antivirals (see below).

The results shown in this paper may have relevance also from the therapeutical point of view. M/M are quite sensitive to the antiviral effect of inhibitors of reverse transcriptase [54 ]. At the same time, however, only few of them (AZT and d4T above the others) cross efficiently the blood-brain barrier and reach concentrations in the cerebrospinal fluid above those required for HIV inhibition [55 ]. AZT and d4T are able to reverse cognitive dysfunctions and neurological damage in patients with HIV encephalopathy [56 , 57 ]. Indinavir, a protease inhibitor (the only class of drugs able to inhibit virus release from macrophages already infected by HIV and thus carrying integrated proviral genome at the time of treatment), is also able to reach sustained concentrations in the cerebrospinal fluid [58 ]. Thus, the overall characteristics of the pathogenesis of HIV encephalopathy strongly support the importance of maintaining, in a multidrug approach, at least one anti-HIV drug able to cross the blood-brain barrier and reach concentrations in the CNS sufficient to inhibit virus replication.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the AIDS Project of the Istituto Superiore di Sanità (ISS), Italy; Biomed Project of European Community; and Ricerca Corrente of IRCCS L. Spallanzani. S. A. was supported by a grant from ISS. We thank Mrs. Tania Guenci, Fabbio Marcuccilli, Franca Serra, and Patrizia Saccomandi for technical help. We also thank Dr. G. Barillari (University of Rome "Tor Vergata") for supplying HIV tat protein.


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