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Differential expression profiles of apoptosis-affecting genes in HIV-infected cell lines and patient T cells

Scheuring, Urban J.; Sabzevari, Helen; Corbeil, Jacques*; Theofilopoulos, Argyrios N.

Basic Science: Original Papers

Objective: To clarify the molecular mechanisms of HIV-induced apoptosis.

Design: The assessment of expression patterns for genes affecting the interrelated cell cycle and apoptosis processes in HIV-1LAI-infected T lymph oblastoid (CEM) cells, as well as CD4 and CD8 cells from HIV-infected individuals and controls.

Methods: The kinetics of HIV infection in CEM cells were defined by flow cytometry of green fluorescent protein expression from a reporter vector. Apoptosis of CEM cells was measured by propidium iodine staining and flow cytometry. Gene expression levels were determined by a multiprobe RNase protection assay.

Results: The infection and apoptosis of CEM cells were associated with enhanced expression of Bax, Bcl-2, Bcl-XL and caspase 1 (ICE). There was increased expression of Bcl-2 and caspase 1 and decreased expression of cyclin-dependent kinase inhibitor p21CIP1 in CD4 cells of HIV-infected individuals compared with uninfected controls. The CD8 cells of HIV-infected individuals exhibited increased expression of Bcl-2, Bcl-XL, Bax and caspase 1 but, in contrast to the CD4 subset, they showed elevated expression of p21CIP1 and p16INK4a compared with controls.

Conclusions: The Bax increase in CEM cells appears to be a direct effect associated with a high frequency of infection and apoptosis, because it was not found in the CD4 cells of patients. In contrast, the increase of Bax in the CD8 cells of patients seems to be an indirect effect. Increases in Bcl-2, Bcl-XL and caspase 1 in HIV-infected CEM cells may be caused by both direct and indirect mechanisms, because they also occurred in CD4 and CD8 cells of HIV-infected individuals. In addition, the low expression of p21CIP1 in the CD4 subset of HIV-infected individuals could promote apoptosis, whereas the high expression of p21CIP1 and p16INK4a in the CD8 subset may lead to a state of anergy, akin to replicative senescence.

From the The Scripps Research Institute, Department of Immunology, La Jolla, CA 92037, and *University of California at San Diego, Department of Medicine, La Jolla, CA 92093, USA. This is publication no. 11619-IMM from the Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA

Sponsorship: This work was supported by NIH grants AI34215 (A.N.T.), CA67394, 5P30A136214 UCSD Center for AIDS Research (J.C.) and a fellowship from the Deutsche Forschungsgemeinschaft, Germany (U.J.S.).

Requests for reprints to: Professor Argyrios N. Theofilopoulos, Immunology Department, The Scripps Research Institute, 10550 North Torrey Pines Road/IMM3, La Jolla, CA 92037, USA.

Date of receipt: 8 May 1998; revised: 20 October 1998; accepted: 26 October 1998.

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The hallmark of HIV-1 infection is a gradual decrease of CD4 lymphocytes, leading to AIDS. Apoptosis has been suggested to be an important mechanism of this CD4 cell depletion [1,2]. Although it has been reported that CD4 cell turnover is rapid in HIV-1-infected (HIV+) individuals and correlates with a high degree of viral replication [3,4], the majority of dying CD4 cells are not productively infected and appear to undergo bystander apoptosis [5,6]. Both CD4 and CD8 cells of HIV-infected individuals have also been reported to exhibit a propensity to undergo apoptosis in culture [7,8]. For the CD8 cells, this enhanced in-vitro cell death could be attributed to the preapoptotic signals delivered in vivo after CD8 clonal expansion by engagement with HIV and non-HIV antigens [9].

Several HIV-1 proteins modulate apoptosis, including gp120, tat and vpr. The crosslinking of CD4 receptors by gp120 appears to be one of the major apoptotic stimuli in HIV-1 infection [2], and may explain the preferential decline of helper T cells in HIV-infected individuals. Tat appears to exert dual effects, promoting or inhibiting apoptosis under certain conditions [10,11]. Vpr mediates cell cycle arrest in G2, and also affects apoptosis [12,13]. The Fas pathway has been implicated in HIV-induced lymphocyte apoptosis [14-17]. Furthermore, as in other forms of apoptosis [18,19], caspases appear to mediate the execution step of HIV-induced apoptosis [16,17,20,21]. In addition, Bcl-2 protein levels decline in lymphocytes from HIV patients [22-25] and are altered in lymphocytes exposed to certain HIV-1 proteins in vitro [26,27].

To elucidate further the molecular mechanism by which HIV induces cell death, we investigated the transcriptional induction and repression of selected genes using a multiprobe RNase protection assay (RPA). We also examined cell cycle-regulating genes, because apoptosis and the cell cycle appear to be mechanistically interrelated [28]. We studied HIV-induced apoptosis in HIV-1LAI-infected CEM cells as well as primary CD4 and CD8 T cells isolated from HIV-1 seropositive individuals and controls.

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Materials and methods

Cell culture and virus infection

CEM-green fluorescent protein (GFP) cells were infected with HIV-1LAI (titered on CEM cells at 106 TCID (tissue culture infectious dose)50/ml) at a multiplicity of infection (MOI) of 0.1, washed and resuspended at a density of 5×105 cells/ml as described [29]. Cells were harvested 24, 48 and 72h post infection, subjected to propidium iodine (PI) staining for cell cycle and apoptosis analysis and flow cytometry for determining the percentages of infected cells, or to RNA extraction (RNA Stat-60, Teltest, Friendswood, TA, USA).

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Flow cytometric analysis for productive infection and apoptosis

Aliquots of CEM-GFP cells were harvested at 24, 48 and 72h and were analysed for the expression of GFP by flow cytometry (Coulter Elite, Epics Division, Hialeah, FL, USA) as described [29]. For apoptosis and cell cycle assessment, CEM or CEM-GFP cells were washed in phosphate buffered saline (PBS), resuspended in 30% ethanol in PBS, treated with RNase A, stained with PI and subjected to flow cytometry as described [29]. Specific apoptosis was calculated as the difference between the percentages of apoptosis in HIV-infected and uninfected control cultures at the same time point.

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Patient and control materials

Peripheral blood specimens were obtained from eight patients at the Los Angeles County Hospital AIDS Clinic. The clinical stage of the patients ranged from B1 to C3, and the numbers of CD4 cells in peripheral blood from 12 to 1288/mm3. Blood samples were also obtained from four age-matched healthy control subjects. All subjects gave informed consent under the auspices of the appropriate Institutional Review Board. Mononuclear cells were separated by density gradient centrifugation, and T lymphocyte subsets were positively isolated onto magnetic beads with bound anti-CD4 or anti-CD8 monoclonal antibodies (Dynal, Lake Success, NY, USA), and subjected to RNA extraction.

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Multiprobe RNase protection assay

Riboprobes of defined length for human apoptosis- and cell cycle-affecting genes were generated by polymerase chain reaction (PCR) from thymus RNA-derived cDNA, and RPA was performed as described previously [30]. The intensity of bands representing RNA duplexes was determined on a phosphor imager (Ambis Corp., San Diego, CA, USA), calculated as cpm of band minus cpm of background and expressed as a percentage of L32 housekeeping control.

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Statistical analysis

Non-parametrically distributed values (CEM experiment) were analysed by Mann-Whitney U-test. Comparisons of values at multiple time points were conducted by one-way analysis of variance (ANOVA) with subsequent multiple comparisons, when appropriate, using the Fisher‚s protected least significant difference (PLSD). Parametrically distributed values (patient and control samples) were analysed by unpaired, two-tailed t-test. Significance was determined as P<0.05.

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Kinetics of HIV-1 infection and apoptosis in CEM cells

To characterize apoptosis and cell cycle gene alterations associated with HIV-1 infection, we utilized the CEM T cell line infected with HIV-1LAI at a high MOI. The percentage of infected cells was determined by parallel infection of a CEM reporter cell line stably transfected with the gene for GFP under the control of HIV-1-long terminal repeat (LTR) promotor [29]. This reporter cell line (CEM-GFP) permits the early detection of productive infection of single cells because the expression of GFP starts once the LTR on the reporter vector is activated by Tat, which is an early viral gene product. Forty-one, 88 and 92% of cells were GFP-positive after 24, 48 and 72h (Fig. 1A). Infection rates correlated with increasing rates of specific apoptosis defined by PI staining, which amounted to 2, 13 and 29% at 24, 48 and 72h, respectively (Fig. 1B). Infected parental CEM cells also showed increasing rates of specific apoptosis, on average 9, 12 and 44%, at the indicated time points.

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Expression of apoptosis-regulating genes in HIV-infected CEM cells

To evaluate the effect of HIV-1 infection on the expression of genes regulating apoptosis, RNA samples of uninfected and HIV-1-infected CEM cells were subjected to RPA analysis at 24, 48 and 72h after infection. Results from three experiments revealed a 0.9-, 1.8- and 2.0-fold increase in the expression of Bax in HIV-infected CEM samples compared with controls at the respective time points (Fig. 2). The increased expression of Bax was significant (P<0.05) at 72h.

The expression of Bcl-2 was also enhanced in HIV-infected CEM cultures compared with controls, i.e. 1.3-, 1.6- and 2.7-fold increases at the indicated time points (Fig. 2). The increase at 72h was statistically significant (P<0.05), as was the rise in Bcl-2 expression from 24 to 48 and 72h (P=0.01).

Bcl-XL showed a 0.9-, 1.7- and 2.0-fold expression at 24, 48 and 72h. The increase was significant at 72h (P<0.05).

Similarly, the expression of caspase 1 was upregulated on average by 1.0-, 1.7- and 2.9-fold (Fig. 2), with significant increases at 72h (P<0.05) and from 24 to 72h (P=0.048).

None of the other genes tested (p53, p21, p16, cyclins A, B, C, D1, D2, D3, E, G, CDK2, CDK3 and CDK4) showed significantly modified expressions (data not shown).

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Apoptosis gene expression in CD4 and CD8 cells of HIV-infected individuals

To test whether the observed changes of gene expressions were also found in the peripheral blood mononuclear cells (PBMC) of HIV-1-infected individuals, CD4 and CD8 cells of infected and uninfected subjects were compared (Fig. 3).

The expression of Bcl-2 was significantly increased (fourfold; P=0.002) in the CD4 cells of HIV-seropositive individuals compared with controls, however, in contrast to CEM cells, there was no significant difference in the expression of Bax. The expression of Caspase 1 was increased more than threefold in the CD4 cells of HIV-positive patients, but this increase did not reach significance (P=0.093). The expression of p21CIP1 was significantly (2.7-fold; P=0.012) decreased, but without concomitant changes in p53 levels (0.33 and 0.26% of L32 in HIV-positive individuals and controls, respectively). Expression levels for the other genes examined were unchanged, except for an approximately twofold increase (P=0.032) in p16INK4a. To illustrate the results described above, autoradiographs from one control and one HIV- positive individual are shown in Fig. 4. Although alterations in the expression levels for other genes are also observed, these did not attain statistical significance in the overall analysis.

Analysis of CD8 cells from HIV-infected patients demonstrated that the expression of Bcl-2 was significantly enhanced (approximately fourfold; P=0.024) compared with controls and the expression of Bax was also significantly higher (approximately twofold; P=0.031) in this subset. Bcl-XL showed an approximately fourfold increase (P=0.0002) in HIV-positive individuals. A significant upregulation of the expression of caspase 1 (approximately sixfold; P=0.002) was also detected. In contrast to the CD4 subset, p21CIP1 RNA levels were significantly (P=0.014) elevated (approximately 15-fold) in the CD8 subset of the infected individuals, but without simultaneous changes in the expression of p53 (0.21 and 0.18% of L32 in HIV-positive individuals and controls, respectively). The expression of cyclin kinase inhibitor p16INK4a was also significantly enhanced (sixfold; P=0.010) in the CD8 cells of HIV-positive patients. The expression levels for all other tested genes were unmodified with the exception of an approximately fourfold increase in cyclin D2 (5.1 and 1.2% of L32, respectively; P=0.001). Autoradiographs are shown in Fig. 4.

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We analysed the expression levels of multiple apoptosis genes in HIV-infected CEM lymphoblastoid CD4 T cells and CD4 and CD8 T cells from HIV patients. HIV-infected CEM cells showed a rapid induction of Bax and Bcl-XL, whereas the induction of caspase 1 and Bcl-2 followed slower kinetics. The CD4 cells of HIV-positive individuals had increased Bcl-2 and caspase 1 levels and decreased p21CIP1 levels compared with uninfected individuals. The CD8 cells of HIV-positive individuals revealed enhanced expression of Bcl-2, Bcl-XL, Bax and caspase 1, but, unlike the CD4 subset, increases in p21CIP1 and p16INK4a.

The CEM culture system was characterized by a high percentage of productively infected cells that was correlated with high rates of apoptosis. The present results clearly link HIV infection-associated apoptosis with the induction of Bax. The simultaneous induction of the proapoptotic Bax [31] and the anti-apoptotic Bcl-2 and Bcl-XL genes [31] in HIV-infected CEM cells appears to be contradictory. The enhanced expression of these genes might occur either within the same cell population or in divergent cell subsets, of which one constitutes the surviving and the other the apoptotic subset. In the former possibility, it might be hypothesized that Bcl-2/Bcl-XL is highly expressed in order to protect cells transiently against death, which is ultimately induced together with the enhanced expression of Bax. This sequence of events, however, appears unlikely, because Bax increased before Bcl-2. Alternatively, under the premise of Bax and Bcl-2/Bcl-XL induction in the same cell population, it may be argued that the inhibition of apoptosis of Bcl-2/Bcl-XL may be inefficient because HIV protease may cleave and inactivate Bcl-2 [32]. The ineffectiveness of Bcl-2 in inhibiting HIV-induced apoptosis has been shown previously in Bcl-2-transfected Jurkat cells [33]. Despite this possibility, it is more probable that Bax and Bcl-2/Bcl-XL are induced in different subsets, the former in the apoptotic and the latter in the non-apoptotic subset. The increases in the expression of Bax [34] and Bcl-2 [11,26] may be triggered by Tat.

The present findings indicate that HIV infection is associated with the upregulation of caspase 1 at the RNA level. Although the forced expression of this protease was shown to induce apoptosis [18,19], its broader role in programmed cell death has been questioned [19]. Several studies [14,16,17,20,21], utilizing peptide inhibitors have, however, shown that ICE-like caspases participate in HIV-induced apoptosis. One possible mechanism for the induction of caspase 1 is by IFN-γ [35], which is produced at high levels by T cells of HIV-infected individuals [36-38]. The induction of IFN-γ may be caused by crosslinking of the CD4 receptor with gp120 and subsequent Fas upregulation [17,39]. Whereas the cleavage of caspase 1 participates in the execution of apoptosis, its upregulation may also sensitize cells to apoptotic stimuli as previously suggested [35].

Freshly isolated CD4 cells from HIV-positive individuals showed no alterations in the expression of Bax, in contrast to HIV-infected CEM cultures. This difference is probably the result of a high proportion of infected CEM cells undergoing apoptosis, but very few infected cells (<1%) [5] in the non-cultured CD4 cells of HIV-positive patients. On the other hand, the increase in Bax in the CD8 cells of HIV-positive individuals may mark cytotoxic T cells entering into apoptosis after their initial activation and expansion by HIV and other antigens.

The expression of Bcl-2 was increased in the CD4 and CD8 cells of HIV-infected individuals. Others have found previously [40] that freshly-isolated CD45RA+ (naive phenotype) cells displayed significantly higher Bcl-2 levels than CD45RO+ (activated/memory phenotype) cells. As some reports [41] have indicated a preferential decline of the memory phenotype cells in HIV-infected individuals, the observed increase in Bcl-2 may reflect the high proportion of CD45RA+ T cells. Alternatively, the increase in Bcl-2 may result from the preponderance of cells selected for resistance to apoptosis.

Increases in the expression of caspase 1 in the CD4 and CD8 cells of HIV-positive individuals may also occur on the basis of the induction of IFN-γ by HIV, as discussed above. Irrespective of the mechanism, however, the enhanced expression of this caspase must be the result of indirect effects, because only a minute portion of CD4 cells (<1%) and no CD8 cells are infected in these samples [5].

Proteins affecting the cell cycle process can also be components of the apoptosis circuitry [28]. The regulation of cyclin-dependent kinase inhibitor p21CIP1 may be p53-dependent or -independent [42]. We found that the latter is the case in HIV infection.

The reduced expression of p21CIP1 in the CD4 cells of HIV-positive individuals compared with controls may be mediated by indirect means and by a mechanism specific for the CD4 subset, for example by cytokines, because cytokines have been shown to affect the expression of certain cyclin-dependent kinase inhibitors [43,44].

The increased expression of p21CIP1 and p16INK4a in the CD8 cells of HIV-positive individuals may be caused by two phenomena: First, this upregulation may represent a state of anergy in CD8 cells [45], perhaps resulting from repeated stimulation by strong antigens, as observed in a variety of other systems [30,46]. Second, increased levels of cyclin-dependent kinase inhibitors may be attributed to replicative senescence [47,48]. The possibility that a considerable portion of the CD8 subset of HIV-infected individuals may be at senescence is supported by the increased frequency of activation-refractory CD8+ CD28- cells [49,50] and the decreased telomere length of CD8+ cells, especially CD8+ CD28- cells [49,51,52]. The resistance of senescent cells to cell cycle entry and apoptosis has been correlated with the overexpression of p16INK4a and p21CIP1 [47,53], and disruption of the latter gene enables cells to evade senescence [54]. Moreover, the inhibition of p21CIP1 released cellular resistance to apoptosis [55], whereas forced overexpression inhibited apoptosis [56]. On the basis of this evidence, we hypothesize that the reduced expression of p21CIP1 in CD4 versus the enhanced expression in CD8 cells of HIV-positive individuals may be a contributory factor in the selective depletion of the CD4 subset and the overall maintainance of the CD8 cells. CD8 cells at the advanced stage of the disease, however, would be expected to exhibit limited functional potential, presumably as a result of repeated engagement by ubiquitous antigens contributing to their replicative senescence.

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The authors thank Dr Robert MacPhee (USC, Los Angeles, USA) for providing patient specimens and M.K. Occhipinti for editorial assistance.

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apoptosis; cell cycle; gene transcription

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