HIV infection is characterized by a sustained CD4 helper T-cell loss . An array of complementary direct and indirect mechanisms may contribute to this loss, involving both apoptotic and non-apoptotic modes of CD4 cell death [1-4]. However, increased cell death by apoptosis has been described in both infected and uninfected cells in different experimental models of HIV-1 infection [4-15]. An essential determinant of HIV-1-induced apoptosis is the cell surface-expression of the HIV-1 envelope glycoprotein complex (gp120/gp41)n, which is a potent inducer of apoptosis [9-11]. Although the cellular machinery involved in HIV-1 envelope protein-induced apoptosis has not been fully identified, this process appears to be specific for cell surface expressed HIV-1 envelope protein, because viral particles or soluble forms of envelope glycoprotein do not trigger apoptotic responses in target cells [9,10]. In addition, HIV-1 envelope protein-induced apoptosis appears to be independent of the FAS/CD95 and TNF-R1 apoptotic pathways [11,12]. The membrane-expressed oligomeric gp120/gp41 complex must be functional in terms of CD4 binding and V3 loop structure to elicit the apoptotic cascade in the target cells [9,10]. Moreover, although expression of the CD4-associated kinase p56lck accelerates apoptosis , CD4 mutants unable to bind to p56lck can also deliver the apoptotic signal [14-16]. Therefore, the involvement of additional CD4-independent events and receptor oligomerization in HIV-induced apoptosis has been suggested [15,16].
Chemokine receptors, which interact with gp120 after virion binding to CD4 , are cofactors of HIV entry. CXCR4 allows entry of T-tropic (X4) isolates , whereas other chemokine receptors such as CCR5, are involved in infection by M-tropic (R5) strains . Consequently, the natural ligand of CXCR4, the chemokine SDF-1a, and some small antagonists, such as the bicyclam AMD3100, have been reported to inhibit infection of HIV-1 X4 isolates [20-23]. In the course of the HIV-l envelope interaction with the cell surface, soluble gp120 from R5 isolates appears to behave as an agonist of CCR5 [24-26]. In contrast, signalling associated with the gp120 from X4 isolates has not been uniformly observed in all of the cell lines tested [24-28]. Although productive infection by HIV-1 does not require signalling through chemokine receptors , these transductional events together with CD4-dependent signals, may play a role in HIV-1-induced cell death. Indeed, CXCR4 has been involved in gp120-induced neurone and CD8 apoptosis [26,29], and a very recent report described an unusual CXCR4-dependent CD4 T-cell death-induced by immunocomplexed gp120 ; however, no information is available concerning the role of CXCR4 in cell-surface expressed HIV-1 envelope protein-induced apoptosis in CD4 cells.
In this work a well described cell coculture model [15,31] was used to analyse this role, by determining the effect of CXCR4 ligands and pertussis toxin (PTX) and by studying the signalling capacity of envelope glycoproteins. It was found that HIV-1 envelope induces apoptosis in syncytium-forming and in single uninfected cells. In both cases, apoptosis can be inhibited by blocking CXCR4 with the CXCR4 antagonist AMD3100 or the CXCR4 agonist SDF-1a. Moreover, apoptosis can occur in PTX-treated cells in the absence of G-protein-mediated signalling through the chemokine receptor CXCR4.
Recombinant soluble gp120 and gp160 from the X4 HIV-1 strain IIIB, were obtained from the Medical Research Council AIDS Reagent Project (Potters Bar, UK). SDF-1a was from R&D Systems (Minneapolis, Minnesota, USA).
Cells and culture
CEM, clone 13, and HIV-1-infected H9/IIIB cells were obtained from A. G. Hovanessian, Institut Pasteur, France. SUP-T1 and MT-4 cells were obtained from ATCC (Rockville, Maryland, USA). All cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS) and 2mM glutamine. Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by Ficoll-Hypaque sedimentation and cultured for 2days in RPMI supplemented with 20% FCS, phytohaemagglutinin and interleukin-2 (Sigma; Madrid, Spain). Cocultures of chronically infected H9/IIIB cells with target cells were performed as described  using a 1:10 ratio of infected to uninfected cells. Before addition of infected cells, target cells were incubated with fusion inhibitors (30min, 37ºC), or with 1μ/ml of PTX (1h, 37ºC). Cocultures were incubated at 37ºC and monitored for syncytium formation and ballooning of cells.
Detection of apoptosis
At 24h, apoptosis in the cocultures was determined by different methods. Total apoptosis (associated with syncytia plus single-cell killing) was determined by a radioactive TUNEL (RANEL) assay coupled with b-IMAGER quantification. Briefly, cells were washed twice with phosphate-buffered saline (PBS) and fixed in 3% paraformaldehyde for 30min at room temperature (5¥106 cells/ml), 20μl of the cell suspensions were allowed to dry in Lab-Tek II chamber slides. Once dried, the cells were treated with 70% ethanol for 20min at -20ºC, dried and stored at -20ºC until analysis. For the radioactive TUNEL (RANEL) assay, slides were rehydrated, treated with proteinase K (3μg/ml for 10min in PBS), washed three times with PBS, and incubated for 40min at 37ºC in 75μl (per slot) of labelling mixture containing 5U of terminal deoxynucleotidyl transferase (tdt), 50mM cacodylate buffer (pH 6.8), 0.5mM CoCl2, 50μM dithiothreitol and 0.25μCi [a-33P]dCTP. After labelling, slides were incubated for 15min in saline buffer (30mM trisodium citrate, 300mM NaCl), washed six times with PBS and dried. Labelling was performed in triplicate, and a blank slot was treated in parallel without addition of tdt. Emission from labelled cells and controls was visualized and counted in a b-IMAGER (Biospace; Paris, France) [32,33].
The death of single uninfected cells was determined by fluorescence-based TUNEL assays or cell cycle analysis after propidium iodide staining. For fluorescence-based TUNEL assays (In-Situ Cell Death Detection Kit, Boeringher Mannheim Biochemicals, Barcelona, Spain) cells were fixed in 3% paraformaldehyde, washed twice with PBS and permeabilized in 70% ethanol for 30min at -20ºC. Labelling was performed according to the manufacturer‚s instructions. For propidium iodide staining, washed cells were incubated for 90min in labelling solution (0.1mg/ml propidium iodide, 0.1 % Triton X100 in PBS) and analysed. In both cases fluorescent antibody cell sorting (FACS) analysis (FACSCalibur; Becton Dickinson, Mountain View, California, USA) was performed gating single cells as measured by forward and side light-scatter pattern using the CellQuest software (Becton Dickinson). As a positive control for apoptosis, the cells (106/ml) were incubated with 100ng/ml of the agonist anti-CD95/FAS monoclonal antibody (Mab) CH11 (IgM; Immunotech, Marseille, France).
Analysis of expression of cell surface antigens.
Leu-3a antibody [IgG1, phycoerythrin-labelled or fluorescein isothiocyanate (FITC)-labelled; Becton Dickinson] and the 12G5 MAb (IgG2a, R&D Systems) were used to determine CD4 and CXCR4 expression, respectively. FITC-labelled goat anti-mouse antibody (Southern Biotechnology Associates Inc., Birmingham, Alabama, USA) was used for indirect staining. Before incubation with each antibody (4ºC, 30min), cells were washed in ice-cold PBS; cells were fixed in PBS containing 1% formaldehyde. In CXCR4 downregulation assays, CEM cells were incubated with SDF-1a or H9/IIIB cells at 37ºC; before incubation with antibodies; sequential washes with PBS, glycine buffer pH 2.0, and PBS were performed in order to remove unbound and bound ligand. Acid treatment did modify 12G5 labelling. CEM cells were identified by staining cocultures with Leu3a MAb, due to the lack of CD4 expression by H9/IIIB cells.
Measurement of cytosolic free calcium
Intracellular calcium levels were measured in a Fluoroscan (Labsystems; Helsinki, Finland) using Fluo-3 loaded cells. Briefly, cells were washed twice with buffer A containing 140mM NaCl, 5mM KCl, 5mM glucose, 1.2mM CaCl2, 1mM MgCl2, 2mM Na2HPO4, 0.3mM KH2PO4, 10mM HEPES, pH 7.0, and loaded with 10μM Fluo-3-AM (Sigma) at 107 cells/ml in the same buffer for 30min at 37ºC. At this time one volume of buffer B (buffer A supplemented with 5% inactivated FCS, pH 7.4), was added to the cell suspension and incubated for a further 30min at 37ºC. Cells were then washed twice with buffer B, resuspended at 107 cells/ml and stored at room temperature during the experiment. For calcium measurements aliquots of this cell suspension were preincubated for 1min at 37ºC in a total volume of 200μl of buffer B in a 96-well flat bottom plate. Agonists and antagonists were added at the indicated times and calcium levels were determined by monitoring fluorescence (483nm excitation, 530nm emission) every 2s in duplicate wells. Maximum and minimum fluorescence values were determined after addition of Triton X-100 and EGTA, respectively.
Coculture of HIV-1 infected and uninfected cells induces apoptosis in single cells
Chronically infected H9/IIIB cells can be used as apoptosis inducer in cocultures with different CD4 cell lines and PBMC . In this model, the cytopathic effect of HIV-1 is manifested by the rapid syncytium formation, cell-to-cell virus spread, and increased levels of cell death by apoptosis . Cell death of uninfected cells is the result of death of cells fused into syncytia and single-cell killing. In order to quantify these two components total, single-cell- plus syncytium-associated apoptosis was studied by a radioactive tdt-dCTP nick-end-labelling assay (RANEL) , and single-cell killing was assayed by FACS analysis of propidium iodide- or TUNEL-labelled cells. Results, summarized in Fig. 1, show the occurrence of increased levels of apoptosis after 24h of cocultivation of H9/IIIB cells with CEM cells. Total apoptosis reached similar levels in the absence or the presence of zidovudine, thus indicating that events involved in membrane fusion but not in productive viral spread are responsible for the apoptosis observed. Increased single-cell apoptosis was observed in CEM cells cocultured with H9/IIIB cells. Similar results were obtained by using TUNEL or propidium iodide assays. Apoptosis was observed in the presence of 0.5μM zidovudine indicating that HIV-1 envelope protein-induced single-cell killing by apoptosis can occur in the absence of productive infection of target cells.
Single-cell killing occurs in CD4 cells irrespective of FAS sensitivity
In order to identify apoptotic single cells as uninfected cells, double staining of cells was performed by using the fluorescein-labelled anti-CD4 MAb Leu3a and propidium iodide. Fig. 2 shows dot plots of double labelled CEM, SUP-T1 cells and PBMC after treatment for 24h with H9/IIIB cells or with the anti-FAS MAb CH11. All target cells used showed increased levels of apoptosis when cultured in the presence of infected cells; moreover, double labelling clearly showed that cells undergoing apoptosis still maintain CD4 cell surface expression. In cocultures of PBMC with H9/IIIB cells, the double staining revealed that apoptosis occurs mainly in the CD4 T-cell population, which showed increased levels of hypodiploid cells after 24h of coculture. The percentage of apoptotic CD4 T cells was 16% and 2.5% in the presence or the absence of H9/IIIB cells, respectively. In contrast, only a slight increase from 2.1% to 3.6% was observed in the apoptosis occurring in CD4-negative cells.
Parallel experiments were performed using the anti-FAS MAb CH11 as an apoptosis inducer. This treatment triggered apoptosis in both the CD4 and CD4-negative cell subsets of PBMC and in CEM cells, but failed to trigger apoptosis in SUP-T1 cells, although all of these cells express cell-surface CD95 . The lack of correlation between sensitivity to HIV-1 envelope- and FAS-induced apoptosis suggests that the mechanism of HIV-1 envelope protein-induced apoptosis bypasses the FAS receptor, as has been reported by others [11,12].
HIV-1 envelope glycoproteins do not mimic SDF-1a signalling
It is well known that gp120 is able induce intracellular signals by binding to CD4, and that these signals may play an important role in the induction of apoptosis . It has also been shown that gp120 can signal through chemokine receptors, although discordant results have been reported [24-27]. For this reason the signalling capacity of envelope glycoproteins was evaluated in this cell system. SDF-a elicited intracellular calcium flux (Fig. 3) and CXCR4 downregulation in CEM cells and PBMC at low concentration (data not shown). However, the addition of gp120 and gp160 (final concentration 10μg/ml) failed to induce calcium flux in these cells, and to modify SDF-a-induced calcium flux (Fig. 3). Thus, the HIV-1 recombinant envelope glycoproteins do not seem to act as either antagonist or agonist of CXCR4 in PBMC and CEM cells. The possibility of signalling through CXCR4 by HIV-1 envelope glycoprotein complex expressed on the cell surface was also investigated. Because of the experimental difficulties hypothetical signalling was examined by taking advantage of the downregulation of the CXCR4 receptor-induced by SDF-1a . CEM cells were incubated with an excess of H9/IIIB cells (a ratio of 1 infected cell per uninfected cell was used) and assayed for CXCR4 expression at different times. Under these experimental conditions, SDF-1a but not H9/IIIB treatment-induced CXCR4 downregulation in CEM cells, thus suggesting that functional envelope glycoprotein complex does not mimic SDF-1a signalling (data not shown).
CXCR4 ligands inhibit apoptosis in a cell type-dependent manner
In spite of the lack of measurable gp120-induced CXCR4 signalling, the role of this chemokine receptor in HIV-1 envelope-driven membrane fusion led us to evaluate its role in HIV-1 envelope protein-induced apoptosis. For this reason, cocultures of H9/IIIB with different target cells in the presence of the CXCR4 agonist SDF-1a were performed . In contrast with the strong effect of SDF-1a on intracellular calcium flux, and the CXCR4 downregulation induced at low concentrations, SDF-1a had little effect on HIV-1 envelope protein-induced apoptosis in SUP-T1 and CEM cells. Both cell lines, which express high levels of CXCR4 (Fig. 4a), showed marked resistance to SDF-1a inhibition with 50% inhibitory concentration (IC50) values higher than 0.1 and 1μM, respectively (Fig. 4b). Conversely, cell lines expressing low levels of CXCR4, such as PBMC or MT-4 cells (Fig. 4a) were sensitive to the inhibitory effect of SDF-1a on HIV-1 envelope protein-induced apoptosis, with IC50 values of 10-25nM (Fig. 4b). The inhibitory effect on apoptosis induction was correlated with the effect of SDF-1a on the formation of syncytia. Indeed, this chemokine efficiently inhibited syncytium formation in MT4 cells and PBMC, but failed to completely inhibit fusion between H9/IIIB and CEM or SUP-T1 cells.
HIV-1-induced apoptosis does not require G-protein-mediated CXCR4 signalling
The inhibition of HIV-1 envelope protein-induced apoptosis by SDF-1a suggested the active role of CXCR4 in apoptosis induction. To address definitively the role of G-protein-mediated signalling through CXCR4 in HIV-1 envelope protein-induced apoptosis cocultures of H9/IIIB cells were performed with CEM cells pretreated with 1μg/ml of the G-protein inhibitor PTX, or the CXCR4 antagonist AMD3100 . It has been reported that PTX blocks both calcium responses induced by SDF-1a and phosphorylation of Pyk2 induced by gp120 [24,34] by inactivating the Gi-protein heterotrimer. Treatment of CEM cells with PTX did not show any protective effect on HIV-1-induced apoptosis, in spite of a total inhibition of SDF-1a-induced calcium flux (data not shown). Neither the total nor the single-cell apoptosis levels in the presence of H9/IIIB cells were modified by pretreatment of the cells with PTX, confirming that gp120-induced G-protein-mediated CXCR4 signalling is not required for apoptosis induction. Consistent with the lack of involvement of G-protein, both the CXCR4 agonist and the antagonist AMD3100 showed inhibitory activity on total apoptosis induced by H9/IIIB cells in CEM cells. The agonist SDF-1a induced a 33% inhibition, whereas the antagonist AMD3100 inhibited apoptosis induction completely, and did not induce apoptosis in cultures of CEM cells in the absence of H9/IIIB cells (Fig. 5). The extent of apoptosis inhibition was correlated with the inhibition of cell-cell fusion as monitored by syncytium formation after 24h of coculture (Fig. 5). Control coculture showed more than 50% of cells fused into syncytia, whereas AMD3100 completely inhibited syncytium formation and SDF-1a showed only a slight inhibitory effect. Pretreatment of CEM cells with PTX did not modify syncytium formation (Fig. 5).
Multiple levels of virus-cell interactions can contribute to CD4 T-cell death ; however, a number of in vitro models have shown that the most rapid and CD4 T cell-specific killer action is associated with the HIV-1 envelope glycoprotein complex expressed on the cell surface [9-11]. Experimental approaches designed to analyse specifically cell-to-cell, envelope-mediated killing showed the classical apoptotic nature of the death mechanism . We have used a coculture model to determine the role of CXCR4 in this cell-to-cell HIV-1 envelope protein-induced apoptosis. The complete dependence on gp120 contact with target cells makes this system highly suitable for addressing this question. In addition, single-cell and total apoptosis were quantified by a RANEL assay coupled with b-Imager analysis .
The CXCR4 ligands SDF-1a and AMD3100 were able to block apoptosis induction by H9/IIIB cells (Figs 3 and 5). However, the inhibitory activity of SDF-1a was surprisingly low in cells expressing high levels of CXCR4. It is likely that the agonist-induced downregulation of this receptor is incomplete , and the strong expression of CD4 and CXCR4 by CEM and SUP-T1 cells (Fig. 4a) allows apoptosis to occur even when a small amount of CXCR4 is available on the cell surface. Consistent with this hypothesis it has been reported that small amounts of cell surface CCR5 are sufficient to support fusion in cells expressing high levels of CD4 . Chemokines and AMD3100 block virus-cell fusion without affecting HIV-1 attachment to the cell surface [17,37]; therefore, their effect on envelope protein-induced apoptosis confirms that the induction of apoptotic signals is subsequent to the gp120-CD4 interaction and suggests that CXCR4 engagement may be a necessary event in apoptosis induction. However, several lines of evidence indicate that G-protein-mediated signalling is not involved in HIV-1 envelope protein-induced apoptosis. The fact that both an agonist and an antagonist inhibit apoptosis suggests that the blockade of CXCR4, rather than the intracellular signal associated with ligand binding, is responsible for apoptosis inhibition. In addition, in our hands envelope glycoproteins appeared to behave as neither agonists nor antagonists of CXCR4 (Fig. 3). Although several reports describe gp120 signalling through CXCR4 in neurones and CD8 T cells [26,29], others have failed to show any chemotactic/apoptotic effect of recombinant gp120 on CD4 T cells [25,26]. Furthermore, PTX, which inactivates Gi-protein signalling, did not inhibit total or single-cell apoptosis in our coculture model (Fig. 5).
As neither p56lck association with CD4 [14-16] nor Gi-protein-mediated CXCR4 signalling (Fig. 5) are essential for apoptosis induction, it may be suggested that other events associated with the fusion complex (gp120-CD4-CXCR4) may transduce apoptotic signals. The role of Gi-independent signals associated with SDF-1a, which induce CXCR4 downregulation even after PTX treatment , remains unclear, but the fact that the envelope glycoprotein complex is unable to induce CXCR4 downregulation argues against any such involvement. Membrane reorganization following gp120 binding to CD4 induces the recruitment of a variety of cell surface antigens in this complex, such as CD3, CD59, CD45, CD26 and HLA-I , leading to an aberrant T-cell receptor signalling . The involvement of some of these antigens, namely CD26 and CD45RO, in HIV-1 envelope protein-induced apoptosis has been suggested by several authors [31,40]. The fact that apoptosis can be observed at the single-cell level (Fig. 1) indicates that death induction should occur in an intermediate step between CXCR4 engagement and the irreversible membrane fusion.
CD4 T-cell apoptosis induced by envelope glycoproteins appears to be restricted to the cell surface expressed envelope glycoprotein complex, as it has been described that soluble gp120 and HIV particles are not able to induce apoptotic events . Thus, death of uninfected cells induced by envelope glycoproteins requires the expression of envelope complex on the surface of infected cells . The fact that virus-cell fusion is not able to induce apoptosis might suggest that Env-dependent or independent signals elicited by strong cell-cell contacts could also play a role in the apoptosis observed in our coculture model. According to this, adhesion molecule LFA-1 seems to be necessary for cell-cell fusion  but is not required for virus-cell fusion. Similarly, FRP-1/CD98 can modulate Env-driven cell-cell fusion  and CD7 has been implicated in both cell-cell and virus-cell fusion . It is therefore possible that one of these molecules delivers apoptotic signals in the early steps of cell-cell fusion. Further characterization of signalling associated with fusion events and gp41 functioning will be necessary to unravel the exact point of apoptosis induction by HIV-1 envelope, which seems to occur after CXCR4 engagement and before irreversible membrane fusion.
During the preparation of this manuscript, Bernt et al.  reported that stimulation of CXCR4 by the MAb 12G5 or by an immune complex formed between recombinant gp120 and anti-gp120 (IC-gp120) induces cell death in CD4 T cells. As these authors state, this is a peculiar death mechanism which is different from apoptosis induced by cell-cell contact between Env-expressing cells and uninfected CD4 T cells. Accordingly, this latter form of Env cell killing is caspase-dependent , inhibited by Bcl2 , and possesses the typical morphological and molecular hallmarks of apoptosis such as DNA fragmentation [7,8]. Although ICgp120 can mimic (gp120/gp41)n-induced oligomerization of receptors, the strong cell-cell contacts in our coculture system or alternatively, envelope complex (gp120/gp41)n-specific events could account for these differences. Whatever the case, the role of CXCR4 in (gp120/gp41)n-mediated apoptosis as described here, and in ICgp120-induced cell death  emphasizes the importance of this viral coreceptor in HIV envelope protein-mediated apoptosis. Moreover, the antagonist AMD3100 appears to be a potent inhibitor of HIV replication, cell-cell fusion, and (gp120/gp41)n-mediated apoptosis.
The mechanism of CD4 T-cell killing described here could explain, at least in part, the decline of CD4 T cells after the emergence of CXCR4 using isolates observed in vivo. However, the relevance of this mechanism is unknown. X4 strains appear to be associated with faster CD4 T-cell loss and increased cytopathogenicity [45,46] and apoptosis levels have been correlated with AIDS progression . However, the lack of increased apoptosis in PBMC after viral phenotypic switch from R5 to X4 isolates has been also reported . Apoptosis in HIV-infected individuals is probably the product of the effects of several factors, which involve viral proteins and chronic activation of immune cells. Apoptosis occurring in PBMC might be related to cell activation involving both CD4 and CD8 T-cell subsets, whereas envelope protein-induced apoptosis might be an important contribution to CD4 T-cell loss in lymphoid organs, in which strong cell-cell contacts are more likely to occur. The CD4 T-cell selectivity of the apoptotic mechanism described here correlates with the CD4 T-cell loss observed in vivo; however, it contrasts with the higher apoptosis in CD8 T cells reported in individuals infected by X4 isolates of HIV-1 . In this regard, it should be noted that the mechanisms leading to apoptosis in these subsets are different. Whereas CD4 T-cell death is the direct consequence of the interaction of HIV envelope with cell surface receptors, CD8 T cells appear to die by indirect gp120-induced upregulation of tumour necrosis factor and tumour necrosis factor receptor in macrophages and CD8 T cells, respectively . The use of functional envelope glycoproteins instead of soluble gp120 and the requirement of macrophages probably account for the restricted CD4 T-cell apoptosis observed in our coculture model. Although several mechanisms are likely to be involved in the in vivo CD4 and CD8 T-cell death during HIV infection, the involvement of a common molecule such as the chemokine receptor CXCR4 in these events may favour the therapeutic potential of targeting this chemokine receptor.
The authors thank A. M. García and A. Gutiérrez for excellent technical assistance.
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