Termination of an immune response requires elimination of activated T lymphocytes by activation-induced cell death (AICD). In AICD, CD95 (Apo-1/Fas) ligand (L) triggers apoptosis of CD95-positive activated T lymphocytes. In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-1 Tat) sensitizes T cells toward CD95-mediated apoptosis and up-regulates CD95L expression by affecting the cellular redox balance. In this study, we show that it is hydrogen peroxide (H2O2) that functions as an essential second messenger in TCR signaling. The H2O2 signal combined with simultaneous calcium (Ca2+) influx into the cytosol constitutes the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. We further show that HIV-1 Tat interferes with TCR signaling and induces a H2O2 signal. H2O2 generated by HIV-1 Tat combines with CD4-dependent calcium influx and causes massive T cell apoptosis. Thus, our data provide an explanation for CD4+ T lymphocyte depletion during progression of AIDS.

Apoptosis is a morphologically distinct form of cell death involved in many physiological and pathological processes (1). CD95 (Apo-1/Fas), a member of the TNFR superfamily (2, 3), and its ligand (CD95L), 3 a type II transmembrane protein of the corresponding TNF family (4), are important initiators of apoptosis (5). CD95 is widely expressed (3, 6), whereas expression of CD95L is tightly regulated (4). CD95L is highly expressed on T cells upon activation by Ag, anti-CD3 or anti-TCR Abs, or reagents that mimic T cell activation such as phorbol esters and calcium ionophores. The expression of CD95L triggers apoptosis of CD95-positive activated T lymphocytes (7, 8, 9), a phenomenon called activation-induced cell death (AICD). AICD is known to be important in maintaining peripheral lymphocyte homeostasis. Mutations in genes encoding CD95 or CD95L have been found to cause autoimmune-like symptoms and lymphadenopathy (3, 10, 11, 12, 13). Up-regulation of CD95 and CD95L expression in T lymphocytes of HIV-1-infected individuals may contribute to T cell depletion in AIDS (14, 15).

Because CD95L expression is crucial in the induction of AICD, efforts have been made to explore the connection between TCR signaling and its regulation. The first signaling events after TCR engagement are the sequential activation of tyrosine kinases, including Lck and ZAP70 (16, 17). Both Lck and ZAP70 are required for calcium (Ca2+) mobilization in T cells (18). The increase in cytosolic Ca2+ causes activation of calcineurin (19), which dephosphorylates the NF of activated T cells (NF-AT). Upon translocation into the nucleus, activated NF-AT initiates gene transcription and, therefore, it is regarded as one of the key participants in CD95L regulation (20). ZAP70 is also involved in activation of protein kinase C. The θ isoform of protein kinase C has been shown to be essential for activation-induced CD95L expression (21, 22) and for TCR-induced NF-κB activation (23). Activated NF-κB is required for high expression of CD95L in T cells (24, 25, 26, 27). Activation of T cells via TCR also leads to Ras-activated cascade of kinase activity, including Raf, Mek, ERK, and p38 MAPK. This pathway is crucial for optimal CD95L induction (28, 29) and is involved in induction of the transcription factor AP-1 (Fos/Jun) (30). Because the CD95L promoter contains an AP-1 binding site (31), AP-1 is engaged in regulation of CD95L in human T cells (32).

Reactive oxygen species (ROS) have also been shown to be important in transcriptional control of CD95L expression. It has been reported that inhibition of oxidative signals interferes with induction of CD95L expression and AICD (33, 34, 35). The transcription factors NF-κB and AP-1 can be directly activated by oxidative signals. Thus, oxidative signals may be able to influence the activity of the CD95L promoter (36, 37). However, the CD95L gene is under control of a large array of cis-acting promoter elements, which act in concert to achieve a fine degree of control over the transcriptional activity of the gene (37).

In HIV-infected patients, AIDS is characterized by a depletion of T cells that is partly due to massive apoptosis (38, 39, 40). Decreased antioxidant defense and increased lipid peroxidation have been described in serum from both HIV-positive individuals and AIDS patients, suggesting a disturbance of the redox equilibrium (41, 42). Soluble HIV-1 trans-activator of transcription (HIV-1 Tat) has been found in supernatants of HIV-infected cells as well as in the serum of HIV-infected individuals (43, 44, 45). In addition, Tat was found in the supernatant of transfected cells. It was demonstrated that Tat is internalized efficiently by surrounding cells via macropinocytosis. We (44, 46) and others (47) have shown that Tat enhances AICD of human T cells. This effect is blocked by reagents that inhibit the function of CD95 or CD95L (e.g., F(ab′)2 anti-Apo-1 Ab fragments or soluble CD95 decoys) (8). This indicates a key role of the CD95/CD95L system in TCR/Tat-enhanced apoptosis.

Ectopic expression of Tat results in strongly enhanced binding of NF-κB and human early growth response gene (Egr) to its binding sites in the CD95L promoter (36), and it was shown that Tat itself is able to bind to Egr-2 and Egr-3 and synergizes with these factors to induce expression of a CD95L promoter-driven reporter (48).

However, Tat induces a disturbance of the cellular redox equilibrium and down-regulation of the expression of mitochondrial manganese superoxide dismutase (Mn-SOD/SOD2) in human T cells (49). Therefore, Tat-enhanced AICD may operate at least in part via these mechanisms. This theory is strongly supported by studies on chimpanzees. HIV-1-infected chimpanzees show productive viral infection resembling an early infection in humans (50), but no development toward chronic T cell depletion or progression toward AIDS (51). However, in T cells of chimpanzees, HIV-1 Tat neither disturbs the redox equilibrium nor enhances AICD (46).

In the present study, we investigated involvement of ROS and Ca2+ in activation-induced expression of CD95L in T cells. Our studies show a minimal requirement for both stimuli in induction of transcription of CD95L, because neither of the two signals alone could elicit such a response. The ROS molecule responsible for activation-induced CD95L expression was identified as H2O2. The H2O2 signal was substituted by HIV-1 Tat. Thereby, we showed that Tat turns a cytosolic Ca2+ influx induced by CD4 triggering into a death-inducing signal. Thus, generation of H2O2 induced by Tat may cause depletion of CD4+ cells during progression of AIDS.

Dihydroethidium (DHE), dichlorodihydrofluorescin diacetate (DCFDA), fluo-4-acetoxymethyl ester (Fluo-4-AM), and BAPTA-AM were obtained from Molecular Probes. Ionomycin, ebselen, and glutathione-monoethyl ester were purchased from Calbiochem. N-acetylcysteine (Nac), PMA, glucose oxidase, and all other chemicals were supplied by Sigma-Aldrich. All cell culture supplies were obtained from Life Technologies. The monoclonal anti-CD3 Ab OKT3, the monoclonal anti-CD4 Ab HP 2/6, and the neutralizing anti-Tat Ab 2E12 (46) were prepared from hybridoma supernatants by Protein A affinity purification. Neutralizing anti-CD95L Ab Nok1 was obtained from BD Pharmingen.

Jurkat J16-145 is a subclone of the human T lymphoblastoid cell line Jurkat J16 that was selected for high CD3 expression. J.Bcl-2 is a Jurkat cell line overexpressing anti-apoptotic Bcl-2. J.neo is the corresponding empty vector-transfected control cell line (52). JurkatR (Rapo) is a Jurkat cell line resistant to CD95-mediated apoptosis (53). 1G5 is a cell line derived from Jurkat T cells stably transfected with a luciferase gene driven by an HIV-1 long terminal repeat (54). All Jurkat cell lines were cultured in IMDM supplemented with 10% FCS.

Human PBMCs from healthy individuals were prepared by Ficoll-Plaque density centrifugation. Adherent cells were removed by adherence to the plastic culture vessel for 1 h. T cells were isolated by rosetting with 2-amino-ethylisothyo-uronium-bromide-treated sheep RBCs as described previously (55). To isolate CD4+ or CD8+ cells, T cells were stained for CD4 or CD8 and sorted with a FACS DIVA. For activation, resting T cells (day 0) were cultured at a concentration of 2 × 106 cells/ml with 1 μg/ml PHA for 16 h (day 1). Day 1 T cells were cultured in RPMI 1640 supplemented with 10% FCS and 100 μg/ml gentamicin in the presence of 25 U/ml IL-2 for 5 (day 5) or 6 days (day 6) as described previously (55).

To induce apoptosis in Jurkat or day 6 T cells, cells were cultured in 48-well plates coated with anti-CD3 Ab (OKT3, 30 μg/ml), treated with staurosporin (1 μM), or stimulated with PMA (10 ng/ml) and ionomycin (1 μM). Cell death was assessed 24 h later by propidium iodide uptake and by a drop in the forward-to-side-scatter (FSC/SSC) profile in comparison with living cells, as described previously (56). Apoptotic nuclei were measured by determination of DNA fragmentation (8).

Human T cells or Jurkat cells were stimulated either with plate-bound α-CD3 (OKT3, 30 μg/ml) or PMA (10 ng/ml) and ionomycin (1 μM) treatment. The oxidation-sensitive dyes DCFDA (5 μM) and DHE (5 μM) were added separately to samples 30 min before harvest. Incubation was terminated by washing with ice-cold PBS. ROS generation was determined as increase of DCFDA and DHE fluorescence by FACS analysis. To control for potential variations in dye uptake or de-esterification of DCFDA, the assays were also performed with oxidized forms of DCFDA (dichlorofluorescein or fluorescein diacetate; Molecular Probes). There was no difference in staining of unstimulated vs stimulated cells. The oxidized form of DHE (ethidium bromide) is relatively membrane impermeant and toxic and, therefore, it was not tested.

Jurkat and primary T cells were stained with 1 μM Fluo-4-AM, a highly specific Ca2+ indicator (Kd for Ca2+ of 345 nM) for 30 min. Thereafter, cells were treated with PMA (10 ng/ml) and/or ionomycin (1 μM), soluble anti-CD3 Abs (OKT3; 20 μg/ml), or anti-CD4 Abs (HP 2/6; 40 μg/ml). Ca2+ influx into cytosol was measured by flow cytometry.

RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. Five micrograms of total RNA was reverse-transcribed using the RT-PCR kit (Applied Biosystems). Aliquots were amplified in a DNA thermocycler with 1 U of recombinant Taq polymerase (Sigma-Aldrich) as described previously (31). Amplification products were separated by electrophoresis on 1.5% agarose gels. Primers used for detection of CD95L and β-actin transcripts were as follows: CD95L, sense 5′ (386)-ATAGGATCCATGTTTCTGCTCTTCCACCTACAGAAGGA-3′, antisense 5′ (843)-ATAGAATTCTGACCAAGAGAGGCTCAGATACGTTGAC-3′; β-actin, sense 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′, antisense 5′-CTAGAATTTGCGGTGGACGATGGAGGG-3′.

Luciferase reporter constructs containing the −0.86-kb CD95L promoter were used as described previously (57). A pRL plasmid (Promega) harboring the Renilla luc gene under control of SV40 enhancer and early promoter was used as internal control of transcription efficiency. Jurkat T cells were transfected with 1 μg of DNA of pRL plasmid and 5 μg of DNA of CD95L promoter/luciferase plasmid by electroporation according to previous work by Li-Weber et al. (36). After overnight recovering, cells were divided and treated with Nac or BAPTA-AM or were mock-treated and further cultured in the absence or presence of PMA (10 ng/ml) and/or ionomycin (1 μM) for 10 h. Luciferase activity was determined with the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer’s instructions.

Fermentation of Escherichia coli BL21 (DE3) plysS pTK1 strain expressing His-tagged HIV-1 Tat(1–86) (58) was performed at 37°C in Luria-Bertani medium supplemented with ampicillin (50 μg/ml) and chloramphenicol (30 μg/ml). Tat expression was induced with isopropyl-β-d-thiogalactopyranoside to a final concentration of 3 mM. Bacteria were lysed under denaturing conditions in 6 M GuanidineHCl, 100 mM NaH2PO4, 10 mM Tris adjusted to a pH of 8.0. The fusion of a histidine tag to Tat(1–86) sequence allows for rapid purification of the fusion protein by affinity chromatography. Tat fusion protein binds strongly to an Ni-NTA column (Qiagen). Tat was eluted with 8 M urea, 100 mM NaH2PO4, 10 mM Tris adjusted to a pH of 4.5. To obtain optimal purity, a further purification step by HPLC was applied. A 10–90% CH3CN/0.1% trifluoroacetic acid gradient was run on a C18 column (Vydac). Tat was collected and shock-frozen in liquid nitrogen and lyophilized overnight. After resuspension, purity was checked by silver-gel and Western blot.

Refolding and activity of Tat was investigated by a transactivation assay. 1G5 cells were incubated for 24 h with different amounts of Tat. Cells were harvested and luciferase activity was determined in 10 μl of cell extract using luciferase substrate (Promega) with a Duolumat LB9507, as described (59).

Recombinant Tat was inactivated by exposing the protein solution for 30 min to a flow of molecular oxygen (44, 49) or by boiling of the protein solution for 2 × 15 min at 95°C.

Previous studies suggested that TCR signaling may involve ROS (33, 34). To determine the kinetics of anti-CD3 Ab-induced generation of ROS, we used the cell-permeant dyes DHE and DCFDA. DHE is oxidized intracellularly by superoxide anion (O2) and DCFDA by H2O2 (60), allowing for the discrimination of the two ROS molecules O2 and H2O2.

Freshly isolated T cells from peripheral blood of healthy donors were stimulated for 6 days (then referred to as day 6 T cells) and subsequently were restimulated via TCR by plate-bound anti-CD3 Abs and analyzed for ROS production. Although the O2 signal was short-lived, the H2O2 signal persisted for at least 3 h (Fig. 1,A). However, in the T cell line Jurkat, no generation of O2 could be detected at any time point after stimulation, although robust H2O2 induction for ∼6 h was observed (Fig. 1,B). TCR stimulation of either Jurkat or day 6 T cells resulted in AICD (Fig. 2,D). To exclude the possibility that oxidative signals measured were due to mitochondrial breakdown and subsequent apoptosis, oxidative signals were analyzed in apoptosis-resistant Bcl-2-overexpressing Jurkat cells (52) and also in JurkatR cells, which show a reduced CD95 receptor expression (53). In both cell lines an oxidative signal was detectable after TCR/CD3 stimulation (Fig. 1, C and D). Intensity and persistence of the H2O2 signal in Jurkat and apoptosis-resistant cells were comparable (data not shown). From our data we conclude that generation of ROS is a direct consequence of TCR/CD3 stimulation.

To further analyze the role of ROS in TCR/CD3 signaling, reagents supplementing intracellular thiols were used. Jurkat cells were stimulated for 30 min with plate-bound anti-CD3 Abs and were cotreated with Nac. A strong reduction of the H2O2 signal was observed in presence of Nac (Fig. 2,A). A comparable inhibition of the H2O2 signal was seen also with glutathione-monoethyl ester (GSH-MEE) or ebselen (mimicking glutathione peroxidase) cotreatment (data not shown). In day 6 T cells, stimulated with anti-CD3 Abs and cotreated with Nac, we could show that Nac decreases both the H2O2 and the O2 signals (Fig. 2 B).

As previous studies suggested, oxidative signals are necessary for expression of functional CD95L (33, 34, 35). Therefore, involvement of oxidative signaling in activation-induced CD95L expression was examined by RT-PCR. Jurkat and day 6 T cells were stimulated with plate-bound anti-CD3 Abs. After 1 h, RNA was isolated, reverse transcribed, and amplified using CD95L-specific primers. CD95L transcripts were not detected in unstimulated cells, whereas TCR/CD3 triggering resulted in a strong expression of CD95L in Jurkat and day 6 T cells. Pretreatment of Jurkat cells with Nac, the less reducing scavenger GSH-MEE, and ebselen as well as pretreatment of day 6 T cells with Nac almost completely abrogated the induction of CD95L expression (Fig. 2 C).

To investigate whether AICD was affected in antioxidant-treated Jurkat and day 6 T cells, cells were stimulated with plate-bound-anti-CD3 Abs for 24 h in the absence or presence of Nac. Approximately 40% of the cells underwent apoptosis, which was markedly reduced by addition of Nac (Fig. 2,D). However, to show that CD95/CD95L-independent apoptosis was not affected by Nac treatment, staurosporine, a general kinase inhibitor, was used (61). During staurosporine treatment, ROS are generated (62, 63), but in contrast with AICD, staurosporine-induced cell death could not be inhibited by cotreatment with Nac (Fig. 2 E).

One major event after TCR stimulation is the release of Ca2+ from intracellular stores. The cell-permeant dye Fluo-4-AM was used to investigate the role of Ca2+ in activation-induced CD95L expression. Jurkat cells were stained with Fluo-4-AM and treated with soluble anti-CD3 or isotype control Abs at the indicated time points. Immediately after the addition of the anti-CD3 Ab, a massive Ca2+ influx into the cytosol was detected, whereas cells treated with isotype control Abs showed no change in the amount of cytosolic Ca2+ (Fig. 3,A). We also investigated whether intracellular Ca2+ chelators could inhibit induction of CD95L mRNA expression. BAPTA-AM, a highly specific Ca2+ chelator, is activated by esterases upon uptake into the cell. Jurkat cells were stimulated with plate-bound anti-CD3 Abs in the absence or presence of BAPTA-AM. As shown in Fig. 3 B, treatment with anti-CD3 Abs resulted in CD95L expression, which is markedly reduced in cells simultaneously treated with BAPTA-AM. These data confirm that Ca2+ is crucial for activation-induced CD95L expression.

Because simultaneous treatment with PMA and ionomycin mimics T cell activation, we examined whether it was possible to induce oxidative and Ca2+ signals by these two reagents. Jurkat cells were treated with PMA and ionomycin for 30 min and stained with DCFDA. A H2O2 and a Ca2+ signal comparable to the signal observed after TCR stimulation was measured (Fig. 3, C and D). To further investigate which of the two signals was crucial for activation-induced CD95L expression, we examined the effect of Nac and BAPTA-AM on CD95L promoter activity. Luciferase reporter construct containing a −0.86-kb human CD95L promoter was used in transient transfection studies (57). Different doses of Nac or BAPTA-AM were added to the cell culture during T cell activation. Treatment with PMA/ionomycin leads to a strong increase in CD95L promoter activity, which is inhibited by addition of Nac and BAPTA-AM (Fig. 3 E). These data clearly demonstrate that PMA and ionomycin treatment induces the same pathways as does TCR/CD3 stimulation.

To separate the two signaling pathways, cells were treated with PMA or ionomycin alone. An H2O2 signal was detected after PMA treatment, but was absent in ionomycin-treated cells (Fig. 3,C). The opposite was observed for Ca2+ signaling (Fig. 3,D). Thus, PMA mimics the oxidative signal and ionomycin induces the Ca2+-dependent signaling. To investigate whether exclusive treatment with PMA or ionomycin alone is sufficient for CD95L promoter activation, cells were transiently transfected with the CD95L promoter and stimulated with PMA, ionomycin, or simultaneously with both reagents. Whereas PMA/ionomycin increased reporter activity 110-fold, PMA (5-fold increase) and ionomycin (2-fold increase) showed only a minor elevation of CD95L promoter activity (Fig. 3 E). Hence, both the oxidative signal induced by PMA and the Ca2+ signal induced by ionomycin are needed to activate the CD95L promoter.

To further analyze the synergy between Ca2+ and oxidative signals, we established a system for extracellular generation of H2O2. This was achieved by addition of glucose oxidase (GOX) into the cell culture medium. GOX converts glucose into glucoronate, during which process H2O2 is produced. The amount of H2O2 taken up by Jurkat cells was determined by DCFDA oxidation. A defined and long-lasting H2O2 pulse could be detected, which was equivalent to anti-CD3- or PMA/ionomycin-induced H2O2 signals (Fig. 4,A). We also tested whether H2O2 produced by GOX and internalized by Jurkat cells was sufficient to induce CD95L expression. Jurkat cells were treated with PMA, ionomycin, GOX, and combinations of either GOX or PMA with ionomycin. After 1 h, CD95L expression was analyzed by RT-PCR. PMA, ionomycin, and GOX alone were unable to induce CD95L mRNA expression. In contrast, combination of either PMA or GOX with ionomycin induced strong induction of CD95L mRNA (Fig. 4,B). Thus, neither the H2O2 signal induced by PMA or GOX nor the Ca2+ signal induced by ionomycin alone can induce expression of CD95L. Only the combination of both signals leads to an optimal induction of CD95L. However, treatment of Jurkat cells with higher amounts of ionomycin for extended periods of time seems to be sufficient to induce a weak CD95L expression (data not shown). In addition, it was reported that direct addition of 500 μM H2O2 can induce CD95L expression (64). This oxidative signal is not comparable with the signal induced by PMA or anti-CD3 Ab treatment. Stimulation with PMA or anti-CD3 Abs induced a moderate and long-lasting signal, whereas direct treatment with H2O2 caused an intense but short-lived oxidative signal (data not shown). Another major difference is that cells treated with 500 μM H2O2 underwent apoptosis in a CD95L-independent manner (Fig. 4 C).

Because HIV-1 Tat was shown to accelerate and enhance AICD (44, 46, 49) and to deplete intracellular levels of reduced glutathione (46, 49), we asked whether Tat might interfere with oxidative signaling in activation-induced CD95L expression. Jurkat cells, freshly isolated resting T cells (day 0 T cells), and day 6 T cells were incubated with recombinant Tat for 16 h. Day 0 T cells (data not shown), day 6 T cells, and Jurkat cells (Fig. 5,A) revealed an increase in intracellular H2O2, whereas no O2 generation after Tat treatment was detectable (data not shown). To clarify whether this increase of H2O2 was a direct effect of Tat, the cells were treated with neutralizing anti-Tat Abs, which clearly inhibited the formation of H2O2 induced by Tat (Fig. 5,B). Similar suppression can be achieved with the radical scavenger Nac (Fig. 5,B). To investigate whether this H2O2 shift synergizes with TCR-dependent H2O2 signals, Jurkat cells were stimulated with plate-bound anti-CD3 Abs for 30 min after a 16-h preincubation with Tat. We were able to show that this resulted in an increase of the TCR-induced H2O2 signal (Fig. 5,C). Tat did not affect the Ca2+ influx into the cytosol (Fig. 5 D). This suggested that Tat affects only the oxidative part of the TCR signal.

We further analyzed the effect of Tat on AICD by determination of specific apoptosis in Jurkat and day 6 T cells pretreated with Tat and subsequently stimulated with plate-bound anti-CD3 Abs. Specific apoptosis was enhanced by 40% in Jurkat cells and day 6 T cells. AICD in Tat-treated cells could be blocked with neutralizing anti-CD95L Abs, indicating that Tat serves to enhance the classical CD95/CD95L-dependent pathway of AICD (Fig. 5,E). In addition, Tat has to be folded correctly, because oxidation-inactivated or heat-denatured Tat failed to enhance AICD (Fig. 5 F).

Because we demonstrated that Tat induces the generation of H2O2, we investigated whether Tat was able to substitute for oxidative signals in activation-induced CD95L expression. Jurkat cells were treated with ionomycin or a combination of PMA and ionomycin after preincubation with Tat. Expression of CD95L was analyzed after 1 h by RT-PCR. Treating the cells with Tat alone did not induce CD95L expression, confirming that a singular H2O2 signal is insufficient for CD95L expression. In contrast, cotreatment of cells with Tat and ionomycin resulted in a strong induction of CD95L expression, comparable with the expression level observed in PMA and ionomycin-treated cells (Fig. 6 A).

To verify that Tat can indeed substitute for H2O2 signals in AICD, Tat-pretreated Jurkat cells were incubated with PMA or ionomycin alone or with a combination of PMA and ionomycin. Cell death was analyzed after 24 h. Induction of a single H2O2 signal by Tat or PMA failed to induce apoptosis, as did induction of Ca2+ influx by ionomycin. Significantly, only a combination of these two signals using either PMA or Tat in combination with ionomycin led to induction of apoptosis, which could partially be blocked by neutralizing anti-CD95L Abs (Fig. 6,B). Pretreatment of cells with Nac inhibited the oxidative signal induced by Tat and resulted in a markedly reduced ionomycin/Tat-induced apoptosis (Fig. 6 C).

The pathogenesis of AIDS is characterized by a massive and progressive depletion of CD4+ T cells. Previously, we have shown that CD4+ T cells pretreated with Tat and stimulated with anti-CD4 Abs undergo apoptosis (46, 49). To identify the mechanisms of Tat/CD4-induced apoptosis, we investigated whether CD4 stimulation induces a Ca2+ signal. Jurkat cells were stained with Fluo-4-AM and treated with soluble anti-CD4 Abs. After 200 s, an influx of Ca2+ into the cytosol was measured (Fig. 7,A), whereas no oxidative signal was detectable at any time point (data not shown). CD4 stimulation of Jurkat T cells results in weak CD95L expression (44) (Fig. 7,B) and up to 15% apoptosis (Fig. 7, C and D). However, CD4 stimulation did not lead to an oxidative signal and Nac was unable to block CD4-induced apoptosis (Fig. 7,D). Therefore, other mechanisms have to account for CD4-induced apoptosis. Nevertheless, CD4 engagement in combination with an oxidative signal induced by Tat led to enhanced CD95L expression (Fig. 7,B) and to a massive increase of apoptosis in Jurkat and day 6 T cells (Fig. 7, CE). The additional apoptosis caused by Tat could be inhibited by neutralizing anti-CD95L Abs (Fig. 7,C) and Nac (Fig. 7,D). To further analyze Tat/CD4-induced apoptosis, CD4+ T cells were isolated from healthy donors and prestimulated for 6 days (day 6 CD4+ T cells). CD4 stimulation without any cotreatment already induced up to 40% apoptosis in day 6 CD4+ T cells. Simultaneous treatment with Tat (H2O2 signal) and anti-CD4 Abs (Ca2+ signal), however, substantially enhanced apoptosis up to >70% (Fig. 7 F). Here, we demonstrate that CD4-induced Ca2+ influx into the cytosol in combination with an HIV-1 Tat-induced H2O2 signal leads to massive induction of apoptosis. This finding may provide an explanation for depletion of CD4+ T cells seen in AIDS.

Restimulation of activated T lymphocytes by their Ag receptor induces a complex signaling network that leads to apoptosis. Early studies showed that multiple chemical antioxidants (e.g., free radical scavengers, iron chelators, and thiols) inhibited AICD when added to cultured T cells (34, 64, 65). Two recent studies extended these observations and suggested that control of AICD by ROS occurs via regulation of CD95L expression (35, 66). In the first study (35) it was suggested that O2 confers a “pro-death signal” that involved induction of CD95L expression, whereas H2O2 production regulates a proliferative “pro-life” signal that was induced by ERK activation. However, in the second study (66) it was shown that H2O2 was largely responsible for inhibition of the “pro-life” signal by ERK inactivation. Both studies tried to separate the oxidative signal by inhibitors or scavengers. However, this constitutes a problem because generation and degradation of both ROS molecules is tightly connected. Generation of H2O2 starts with a transfer of an electron to molecular oxygen (O2) and, subsequently, this one electron reduction results in production of O2. O2 in contact with protons in water is rapidly converted into H2O2 (according to the formula: 2O2 + 2H+ ↔ H2O2 + O2). The reaction can either occur spontaneously or is catalyzed by SOD1/SOD2 (67). Therefore, blocking the generation of O2 results in inhibition of H2O2 formation, and an increased degradation of H2O2 enhances the conversion of O2 into H2O2. In addition, scavengers like Nac can reduce protein disulfide bonds and may cause protein damage. This may influence CD95L expression. Therefore, we reconstituted the oxidative signaling pathway with an extracellular system for H2O2. GOX was added to the culture medium, where it converts glucose into glucoronate and thereby produces H2O2. These H2O2 molecules can substitute for oxidative signals in activation-induced CD95L expression, i.e., H2O2 and not O2 is the causative ROS and functions as a second messenger in activation-induced CD95L expression.

The finding that H2O2 alone is insufficient to induce CD95L expression called for a second signal. We had observed that blocking the intracellular Ca2+ signal by BAPTA-AM resulted in massive reduction of activation-induced CD95L expression. In addition, the Ca2+ signal alone (induced by ionomycin) was insufficient to induce optimal CD95L expression. Only both signals Ca2+ and H2O2 elicited full CD95L expression. Thus, only a combination of the Ca2+ and the H2O2 signal causes CD95L expression and AICD (Fig. 8).

Worldwide, ∼42 million individuals are currently infected with HIV. A hallmark of HIV infection is the progressive loss of CD4+ T lymphocytes. AICD is known to be involved in this process (38, 68, 69). It has been found that HIV infection increases expression of CD95L and may thereby contribute to apoptosis of T cells in AIDS (14). We and others have previously shown that CD95L mRNA expression on activated T cells was strongly increased in the presence of HIV-1 Tat (36, 44, 70). Enhancement of activation-induced CD95L expression was shown to be closely linked to a disturbance of the redox equilibrium (46, 49). This is supported by studies in HIV-1-infected chimpanzees. These animals show productive viral infection, resembling early infections in humans, but no development toward AIDS (50, 51). In addition, Tat-induced disturbance of the redox balance was not detectable in T cells of these animals (46). Some other studies have shown that Tat can directly induce apoptosis in resting T cells (47, 71). However, in our study presented here, no effect of Tat on apoptosis without any costimulus could be detected. The failure of Tat to induce CD95L expression and AICD by itself supported our theory that neither oxidative signals nor Ca2+ signals alone induce CD95L expression, because Tat is only inducing an H2O2 signal. The increase in H2O2 induced by Tat, however, interferes with TCR signaling. Thus, we showed that cells preincubated with Tat and stimulated via the TCR reveal a strong increase in the H2O2 signal in comparison with untreated cells. Such an enhanced oxidative signal combined with a Ca2+ influx into the cytosol causes a significant increase in AICD (Fig. 8). Moreover, we show for the first time that Tat completely substitutes for the oxidative signal in activation-induced CD95L expression. Neither Tat nor ionomycin alone induces CD95L expression. Only cells preincubated with Tat and subsequently treated with ionomycin reveal CD95L expression and AICD (Fig. 8). Therefore, the H2O2 signal by Tat completes the Ca2+ signal by ionomycin.

One of the principal cellular targets of HIV infection is CD4+ T lymphocytes. During progression toward AIDS, CD4+ T cells gradually decrease. To explain why in AIDS CD4+ T cells decrease, specifically it was reasonable to invoke the CD4 surface receptor. Therefore, the role of CD4 stimulation and Ca2+ signaling was analyzed. CD4 triggering induces a Ca2+ influx into the cytosol. Consequently, Jurkat or day 6 T cells undergo rapid apoptosis when cotreated with Tat. Hence, the oxidative signal induced by HIV-1 Tat turns a Ca2+ influx induced by CD4 triggering into a death signal. In vivo CD4 might effectively be triggered by HIV gp120.

Taken together, HIV-1 Tat interferes with TCR signaling. It enhances the oxidative signal and thereby significantly increases activation-induced CD95L expression and AICD. Tat substitutes for the H2O2 signal and combines with the Ca2+ influx into the cytosol induced by CD4 triggering. These two stimuli together are sufficient for induction of CD95L expression and apoptosis of CD4+ T cells in vitro. High concentrations of Tat might be found particularly in the lymph nodes of HIV-infected individuals, where productively infected cells are most frequent (72, 73). Thus, Tat may enhance the exaggerated AICD of CD4+ T cells seen during progression of AIDS.

However, it is evident that apart from Tat, other HIV proteins have also been shown to exhibit proapoptotic functions. The HIV viral protein R is able to induce apoptosis via a direct mechanism on the mitochondrial permeability transition pore (74). Viral protein U and negative factor increase susceptibility of HIV-1-infected cells to CD95-induced apoptosis (75, 76), and the envelope gene products gp160/gp120 induce caspase-3 activation (77), down-regulate Bcl-2 (78), and enhance CD4-induced apoptosis (79, 80, 81). In addition, it was shown that HIV-dependent up-regulation of CD95L in macrophages mediates apoptosis of uninfected T cells (82). Thus, in the context of HIV infection, up-regulation of CD95L and activation of caspases is one part of the equation; the other part is the increased sensitivity to CD95L-induced apoptosis (70, 83).

Nevertheless, because immune reconstitution is a widely accepted aim of HIV therapy, cysteine/antioxidant supplementation at a sufficient quantity and treatment with neutralizing anti-Tat Abs may be considered as an additional therapy for HIV-infected individuals and AIDS patients in the future.

We gratefully acknowledge Drs. R. Frank, P. Rosch, and G. Moldenhauer for their generous gift of plasmids and Abs. We thank Dr. D. Wegman for helping to establish the Tat purification protocol and Dr. B. Fritzsching for isolating CD4+ T cells. We also thank M. Diwo, S. Aschenbrenner, and E. Fromm for technical assistance and Drs. D. Macasev, R. Arnold, and D. Klemke as well as S. Fas, C. Fritsch, A. Golks, and A. Hong for critical reading of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Wilhelm Sander Stiftung, the Deutsche Forschungsgemeinschaft, and the European Community.

3

Abbreviations used in this paper: L, ligand; AICD, activation-induced cell death; ROS, reactive oxygen species; Tat, trans-activator of transcription; Egr, human early growth response gene identical with Z-225; SOD, superoxide dismutase; DHE, dihydroethidium; DCFDA, dichlorodihydrofluorescin diacetate; Fluo-4-AM, fluo-4-acetoxymethyl ester; Nac, N-acetylcysteine; FSC/SSC, forward-to-side-scatter; GSH-MEE, glutathione-monoethyl ester; GOX, glucose oxidase.

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