Acquired T-cell sensitivity to TRAIL mediated killing during HIV infection is regulated by CXCR4-gp120 interactions

Background: Sensitivity towards apoptosis induced by ligation of the tumor necrosis factor family of death receptors is controlled in part by death receptor expression. Whereas cellular activation enhances Fas receptor expression and induces Fas sensitivity, such cellular activation neither alters TRAIL receptor expression nor induces TRAIL sensitivity. Cells infected by HIV acquire sensitivity to TRAIL induced death, although the mechanisms by which this is achieved are undefined.

Objective: To define the mechanism by which cells from HIV infected patients acquire sensitivity to TRAIL mediated killing.

Design: In vitro assessment of TRAIL receptor expression and TRAIL sensitivity.

Methods: Treatment of Jurkat T cells, peripheral blood lymphocytes from HIV negative donors, or human osteogenic seroma (HOS) cells expressing CD4, CXCR4 or CCR5 with T tropic gp120, M tropic gp120, or agonistic antibodies against CD4, CXCR4 or CCR5. TRAIL receptors were measured by flow cytometry or reverse transcription-PCR and TRAIL sensitivity was assessed by incubation with recombinant TRAIL followed by Annexin V fluorescein isothiocyanate/Propidium Iodide (PI) staining.

Results: Treatment of uninfected Jurkat T cells, as well as primary T cells with gp120 results in the upregulation of TRAIL death receptor expression and acquired sensitivity to TRAIL mediated cell death. The increase in TRAIL death receptor expression and acquisition of TRAIL sensitivity requires the chemokine coreceptor CXCR4 but not CCR5 or the CD4 receptor.

Conclusions: These results indicate that chemokine receptor interactions regulate TRAIL receptor expression and provide an explanation for the acquired T cell sensitivity to TRAIL mediated killing death during HIV infection.

Introduction

Enhanced T cell apoptosis is seen during HIV infection both in vitro and in vivo, and is induced directly or indirectly by viral proteins, host cytokines or immune effector mechanisms (reviewed in ^[1]). Amongst the biochemical signals which favor apoptosis during HIV infection are the death receptor family of apoptosis inducing proteins. Indeed, both Fas ligand/Fas (CD95) and tumor necrosis factor (TNF)/TNF receptor-type I (TNFR1) interactions have been shown to mediate apoptosis of cells from HIV infected patients in different contexts. The Fas and TNFR1 receptors transduce apoptotic signals via intracellular death domains following their ligation by Fas ligand or TNF respectively ^[2]. Another member of this family of death inducing ligands is the TNF-related apoptosis inducing ligand (TRAIL). Two death-inducing receptors for TRAIL have been defined, TRAIL-R1 and TRAIL-R2, which transmit apoptotic signals by virtue of intracytoplasmic death domains. Two other membrane receptors for TRAIL have also been defined, TRAIL-R3 and TRAIL-R4, yet they do not transduce death signals due to the absence of, or truncation of, intracytoplasmic death domains ^[3]. The regulation of TRAIL and its receptors is fundamentally different from the regulation of the Fas/Fas-ligand system and the TNF/TNFR system since normal or activated cells are insensitive to TRAIL-induced killing. Yet, a wide variety of transformed and/or virally infected cells acquire sensitivity to TRAIL-induced killing ^[3].

In the context of HIV infection, numerous groups have now established that cells from HIV infected patients are susceptible to TRAIL-induced apoptosis ^[4-7]. Previously, we demonstrated that both cells infected with HIV in vitro, as well as cells from HIV infected patients have increased expression of TRAIL-R1 and TRAIL-R2 compared to uninfected control cells, or similar cells from HIV negative patients, respectively ^[6]. The enhanced susceptibility to TRAIL mediated killing correlates with elevated levels of surface TRAIL death receptors as demonstrated in studies using melanoma cell lines ^[8]. The factor(s) which account for the observed changes in TRAIL sensitivity during HIV infection are not fully understood. The objective of the current study was to determine what factor associated with HIV infection results in enhanced TRAIL death receptor expression and enhanced sensitivity to TRAIL induced T cell apoptosis.

Methods

Cell lines and reagents

Jurkat T cells were obtained from ATCC (Rockville, Maryland, USA) and maintained in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine and 100 U/ml of each penicillin and streptomycin (Canadian Life Technologies Products, Montreal, Canada). 293T cells stably transfected with vector control, HXB2 (T tropic) or JRFL (M tropic) gp120 as previously described ^[9], were cultured in RPMI 1640 as above. HOS cell lines (AIDS Reference and Reagent Program) were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 U/ml penicillin and streptomycin. Peripheral blood lymphocytes (PBL) from HIV negative donors were obtained following informed consent, and the protocol was reviewed and approved by the University of Ottawa and the Mayo Clinic Institutional Review Board. Peripheral blood mononuclear cells were depleted of monocytes by plastic adherence for 16-18 h (in 10% human AB serum) and further cultured in RPMI 1640 supplemented with 10% AB serum, 2 mM L-glutamine and 100 U/ml of each penicillin and streptomycin.

Cell treatments

Cells were seeded in a 96-well round bottom plate (Costar, Mississauga, Canada) and incubated for 16 h with 100 ng/ml monomeric recombinant baculovirus produced gp120 IIIb or gp120 MN as T tropic strains ^[10,11], or gp120 Ada as an M tropic strain ^[12] (Immunodiagnostics, Woburn, Massachusetts, USA), recombinant SDF-1α (R&D Systems, Minneapolis, Minnesota, USA), anti-CD4 (Leu3A), anti-CXCR4 (12G5), anti-CD3, anti-CCR5 or phytohemagglutinin (PHA; Sigma, St. Louis, Missouri, USA) at the indicated concentrations (all antibodies from BD Biosciences, San Diego, California, USA). After the indicated treatments, leucine zipper human TRAIL (Immunex Corporation, Seattle, Washington, USA) was added for an additional 8 h and apoptosis was measured by annexin V-FITC/PI staining (BD Biosciences, San Diego, California, USA). For co-culture experiments, 293T cells expressing HXB2 or JRFL gp120, or vector alone, were mixed at ratios of 1: 2 and 1: 10 with fresh PBL in 1 mL total volume and incubated at 37°C for 14 h. Cells were subsequently washed, stained as described below for flow cytometry, and fixed with 2% paraformaldehyde.

Detection of TRAIL receptors

Surface staining and RT-PCR for TRAIL receptors were performed as previously described ^[8]. For reverse transcription (RT)-PCR, β-actin was used as a loading control. In TRAIL surface detection experiments, PBL from uninfected controls were isolated and stained with anti-CD4 and anti-CD8-APC antibodies (BD Pharmingen, San Diego, California, USA) and then stained for TRAIL receptors as described below. Between 1 × 10⁵ and 1 × 10⁶ cells were resuspended in phosphate-buffered saline containing 10% human AB serum. Cells were stained with the following specific antibodies (all from Immunex Corporation): TRAIL R1 (M271), TRAIL R2 (M412), TRAIL R3 (M430), TRAIL R4 (M445) and TRAIL. Cells were incubated at 4°C, washed twice in phosphate-buffered saline and stained sequentially with anti-mouse biotinylated IgG1/2 (BD Pharmingen) antibody followed by streptavidin-phycoerythrin (BD Pharmingen). Isotype antibodies were used in all cases as controls. Fluorescent antibody cell sorting was performed on 30 000 events, and analysis performed using WinMDI software (http://pingv.salk.edu/software.html).

Statistics

Where indicated, statistical analysis was performed comparing treatment groups versus control treatment using Student's t test. Significance was achieved where P < 0.05.

Results

gp120 and CXCR4 agonists upregulate TRAIL receptor expression

As T cells from HIV positive patients that are directly infected with HIV, as well as those which are not, acquire sensitivity to TRAIL mediated killing ^[6], we presumed that a soluble factor was responsible for the acquired sensitivity to TRAIL mediated killing in T cells from HIV infected patients. Of the candidate soluble factors, gp120 is a likely candidate as its crosslinking of CD4 and/or chemokine coreceptors is known to perturb host apoptosis regulatory machinery in a manner that favors apoptosis. Therefore, we examined the effect of soluble monomeric gp120 on TRAIL receptor expression. Jurkat T cells were treated with T tropic gp120 IIIb and analyzed for the expression of the four TRAIL receptors. Monomeric recombinant gp120 IIIb treatment of Jurkat T cells resulted in a 1.5-fold increase in TRAIL-R1 expression (n = 5, P < 0.03; Fig. 1a and b) and a 2.3-fold increase in TRAIL R2 expression (n = 5, P < 0.002; Fig. 1a). No significant effect on TRAIL-R3 or -R4 expression was observed (Fig. 1a and b). Since gp120 can bind to either CD4 or CXCR4, we next examined whether CD4 or CXCR4 ligation alone could alter TRAIL receptor expression. Treatment of Jurkat T cells with agonistic anti-CD4 antibodies did not alter TRAIL receptor expression (Fig. 1c). However, similar to treatments with gp120, anti-CXCR4 antibodies enhanced TRAIL-R1 (n = 5, P < 0.005; Fig. 1c) and TRAIL-R2 (n = 5, P < 0.005; data not shown) expression in a dose dependent fashion, and was confirmed by semi-quantitative RT-PCR for TRAIL-R1 mRNA (Fig. 1d).

Fig. 1

In primary T cells isolated from uninfected individuals, both gp120 IIIb and anti-CXCR4 induced a significant increase in the surface expression of all four TRAIL receptors as quantitated by flow cytometry analysis when compared to control treatments (Fig. 2a and b, n = 3, P < 0.05). The effects of gp120 or anti-CXCR4 in PBL were dose dependent in both CD4 and CD8 subsets (Fig. 2c and data not shown). Neither interleukin-2 nor PHA had an effect on TRAIL receptor expression in primary T cells or Jurkat T cells, indicating that immune activation alone was not responsible for the observed changes in TRAIL receptor levels (data not shown). In PBL treated with gp120, all four TRAIL receptors were increased, in contrast to Jurkat T cells where only TRAIL receptors 1 and 2 were increased. We therefore performed TRAIL receptor expression analysis in samples co-stained with either CD4 or CD8 markers. Within the CD4 T cell subset, increases in TRAIL receptors 1 and 2 were observed following gp120 IIIb treatment, but there were no changes in TRAIL receptors 3 and 4 (Fig. 2d). Conversely, within the CD8 subset, gp120 treatment resulted in smaller increases in TRAIL receptors 1 and 2 than within the CD4 subset. Moreover, within CD8 T cells, but not CD4 T cells, gp120 treatment resulted in increased TRAIL R3 and R4 expression (Fig. 2d).

Fig. 2

Since gp120 of different tropisms can interact with either the CXCR4 receptor (T tropic) or CCR5 receptor (M tropic), we assessed whether treatment with different forms of gp120 would impact TRAIL receptor expression. First, Jurkat T cells were incubated with recombinant monomeric baculovirus produced T tropic gp120 or M tropic gp120 and TRAIL-R2 surface expression was assessed by flow cytometry. Both T tropic and M tropic gp120 increased TRAIL-R2 expression in Jurkat T cells (Fig. 3a) (T tropic n = 3, P < 0.05; M tropic n = 3, P < 0.05). Similarly, treatment of primary T cells from healthy uninfected donors with T tropic and M tropic gp120 increased TRAIL-R2 surface expression (Fig. 3b) (T tropic n = 6, P < 0.01; M tropic n = 3, P < 0.05).

Fig. 3

Enhanced TRAIL Receptor expression requires CXCR4 but is independent of CD4

The ability of gp120 but not anti-CD4 to increase expression of death inducing TRAIL receptors suggests that chemokine receptors may mediate the observed effect. To formally test whether chemokine receptor stimulation causes upregulation of TRAIL and TRAIL receptors, HOS cells that do not express CD4 or chemokine receptors were stably transfected with CD4, CXCR4 or CCR5. First, we assessed the effect of either T tropic or M tropic gp120 on TRAIL-R2 expression in control HOS cells, or HOS cells expressing CD4, CCR5 or CXCR4. Only CXCR4 expressing cells upregulated TRAIL-R2 in response to gp120; and TRAIL-R2 was upregulated in response to both T tropic and M tropic gp120 (Fig. 4a). This observation suggests that CXCR4 is required for TRAIL-R2 upregulation, and is consistent with prior observations that M tropic strains of gp120 are capable of signaling through CXCR4 ^[13]. Next we assessed the expression of all four TRAIL receptors following gp120 treatment. Unstimulated parental HOS cells express all four TRAIL receptors (Fig. 4b); yet treatment of parental cells with gp120, anti-CXCR4 or anti-CCR5 did not affect TRAIL receptor expression (data not shown). HOS cells stably expressing CD4 or CXCR4 had similar levels of TRAIL receptor expression compared to parental cells. Treatment of HOS cells stably expressing CD4 alone with gp120, anti-CXCR4 or anti-CCR5 also did not alter TRAIL receptor expression (data not shown). However, HOS cells stably expressing CXCR4 and treated with T tropic gp120 IIIb, displayed a 1.4-fold increase in TRAIL-R1 (n = 3, P < 0.05) and a 1.7-fold increase in TRAIL-R2 expression (n = 3, P < 0.03; Fig. 4b), but no change in TRAIL-R3 or -R4 were observed.

Fig. 4

HIV envelope glycoprotein is present in HIV infected patients at levels estimated to be between 120 and 960 ng/ml ^[9], and may be present as a soluble monomeric form, a soluble multimeric form, multimeric virion associated forms, or expressed on the surface of infected cells. We therefore questioned whether cell associated gp120, which would cause receptor crosslinking, could still upregulate TRAIL receptor expression and, if this method of gp120 delivery would induce similar degrees of surface receptor upregulation as monomeric gp120. To recapitulate cell associated gp120, 293T cells were transfected with an empty vector (control) or vectors encoding T tropic (HXB2) or M tropic (JRFL) gp120. PBL from healthy uninfected donors were then incubated with control protein (albumin), soluble T or M tropic gp120, or with 293T cells (control) alone or 293T cells expressing T or M tropic gp120. Following incubation, PBL were identified by light scatter and analyzed for TRAIL-R2 expression. Consistently, both soluble and cell associated gp120 upregulated TRAIL-R2, however, the effect of cell associated gp120 appears to be greater than for soluble monomeric gp120 (Fig. 4c, n = 3; T tropic soluble versus T tropic cell associated, P < 0.01; M tropic soluble versus M tropic cell associated, P < 0.01).

CXCR4 Ligation sensitizes cells to TRAIL mediated killing

PBL from uninfected individuals are resistant to TRAIL killing. Since we observed a modulation of TRAIL receptor expression by gp120, and anti-CXCR4 ligation of CXCR4, we assessed whether such treatments would result in sensitization to TRAIL induced cell death. PBL were treated with gp120, or anti-CXCR4 followed by the addition of recombinant TRAIL. TRAIL induced significant death in cells pretreated with gp120 (P = 0.004, n = 3), anti-CXCR4 (P = 0.003, n = 3), but no death was observed in cells pretreated with anti CD4 (Fig. 5a, n = 3). When PBL from HIV negative donors were treated with gp120 or anti-CXCR4 followed by TRAIL treatment, and apoptosis analyzed specifically within CD4 T cells or CD8 T cells, both CD4 and CD8 T cells die, yet the magnitude of CD4 T cell death is slightly greater than that of CD8 T cells (Fig. 5b and c).

Fig. 5

Discussion

The molecular regulation of TRAIL sensitivity is impacted by several factors. Amongst these is the relative expression of death receptors (TRAIL-R1 and -R2) versus decoy receptors (TRAIL-R3, and -R4) and osteoprotegerin ^[14-19]. The determinants of sensitivity or resistance to the proapoptotic effects of TRAIL is incompletely understood. The relative concentrations of death receptors (TRAIL-R1 and -R2) to decoy receptors (TRAIL-R3 and -R4) are important in some cell types, but not sole determinant of TRAIL sensitivity. Indeed, numerous downstream apoptotic regulators including FLIP, IAPs, Akt and potentially Bcl-2 family members all impact whether a cell will display a TRAIL sensitive phenotype (reviewed in ^[20]). In the current study, we report a different and more pathophysiologically relevant explanation to promote TRAIL sensitivity via chemokine receptor ligation.

In the context of HIV infection, several groups have demonstrated the involvement of TRAIL/TRAIL receptor mediated killing of lymphocytes and neurons ^[5-7,21-27]. One possible source of TRAIL appears to be HIV infected antigen presenting cells ^[28-30] stimulated by type I interferons ^[4]. However, the mechanism by which T cells from HIV infected patients acquire sensitivity to TRAIL mediated killing is unknown. In the current report, we demonstrate that gp120 interaction with CXCR4 is sufficient to render cells sensitive to TRAIL mediated killing. Moreover, such an effect is CD4 T cell specific, as these cells upregulate TRAIL receptors 1 and 2, whereas CD8 T cells upregulate the decoy receptors TRAIL-R3 and -R4 as well in response to gp120 ligation. Consistent with other research showing that M tropic (CCR5-dependent) fusion can be competitively inhibited by overexpression of CXCR4 ^[31], and that both M tropic and T tropic gp120 can initiate CXCR4 mediated signaling ^[13], our results suggest that gp120 from M tropic strains are able to bind and signal through CXCR4, causing upregulation in TRAIL-R2.

Both HIV virion particles and soluble factors (including gp120) have been suggested to induce apoptosis in both infected and uninfected T cells (reviewed in ^[32-36]. This bystander effect is thought to be a central reason for T cell depletion during HIV infection ^[32-38]. However, the precise mechanism by which this occurs is largely unknown. Antigen presenting cells such as monocytes, macrophages and dendritic cells represent the major cell types initially infected, and secrete key cytokines that are important in the regulation of primary immune responses. Activation of monocytes by HIV infection induces the release of proapoptotic factors including TNF-α, FasL and TRAIL ^[28,38,39]. One stimulus that may be responsible for initiating the production of proapoptotic signals is HIV gp120. Interaction between CXCR4 and gp120 has been shown to induce a Fas and caspase 8 independent ^[40-42] death that is associated with a rapid release of cytochrome c and loss of mitochondrial membrane potential ^[41]. The effect of gp120 on apoptotic death does not require signals from CD4, since truncated intracellular forms of CD4 and lck deficient cells do not undergo gp120 induced death ^[41,43]. Although signals transduced through CCR5 by HIV env also cause T cell death, CCR5 mediated apoptosis requires caspase 8 activation ^[44,45] indicating that chemokine coreceptor engagement has distinct downstream effects. Therefore, it is apparent that gp120 effects are receptor dependent, as well as cell type dependent. Here we define that gp120 ligation of CXCR4 has an additional effect, enhanced TRAIL death receptor expression and acquired TRAIL sensitivity.

The TRAIL/TRAIL receptor pathway has been implicated in the pathogenesis of HIV infection through its ability to induce death of both infected and uninfected T cells ^[5-7,44-46] and neurons ^[23,25-27]. Previous work by our group and others has demonstrated that TRAIL receptor expression is altered during in vitro infection with HIV and in T cells from HIV infected individuals ^[28]. The dysregulated levels of TRAIL receptors resulted in the ability of exogenous TRAIL to induce death of naive and memory T cells from the HIV infected patients. Moreover, in the HIV infected hu-PBL-NOD/SCID mouse model, TRAIL mediates death of uninfected CD4 T cells ^[24], further supporting a role for TRAIL in the pathogenesis of HIV induced immune deficiency. While it is established that a principal source of TRAIL in HIV infection is monocytoid cells, it has been undefined how such uninfected T cells become sensitive to TRAIL induced killing. Our data reconcile these observations as we demonstrate a direct effect of gp120 on the induction of TRAIL sensitivity. Such observations have implications not only for understanding mechanisms of HIV induced immune deficiency, but also for understanding the physiology of chemokine receptors as well as the regulation of TRAIL receptors during health and disease.

Acknowledgements

The authors gratefully acknowledge administrative support from Ms. Carrie Rogness and Ms. Teresa Hoff.

Sponsorship: A. Badley is supported by grants from the National Institutes of Health (R01 AI062261-01 and R01 AI40384) and the Burroughs Wellcome Award for Translational Research.