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Induction of Interleukin-15 Production by HIV-1 Nef Protein: A Role in the Proliferation of Uninfected Cells
Experimental Cell Research 250, 112–121 (1999) Article ID excr.1999.4494, available online at http://www.idealibrary.com on
Induction of Interleukin-15 Production by HIV-1 Nef Protein: A Role in the Proliferation of Uninfected Cells Maria Giovanna Quaranta,* Barbara Camponeschi,* Elisabetta Straface,† Walter Malorni,† and Marina Viora* ,1 *Department of Immunology and †Department of Ultrastructures, Istituto Superiore di Sanita`, Rome, Italy
Several recent reports have provided evidence that Nef enhances human immunodeficiency virus HIV infectivity, and in vitro experiments with the nef gene have demonstrated the possible role of Nef in modulating immune responses. Exogenous Nef has been demonstrated to induce proliferation of normal human peripheral blood mononuclear cells (PBMC) and to enhance HIV-1 replication. The aim of this study was to evaluate the biological mechanisms by which Nef, used as exogenous protein, modulates cellular activation. We showed that exogenous Nef protein induces the proliferation of unstimulated and suboptimally stimulated normal human PBMC, while it has no effect on the proliferation of optimally stimulated PBMC. Moreover, the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-g, TNF-a, and IL-6 production, while, surprisingly, IL-2 production was not affected by Nef. More importantly we showed, for the first time, that Nef exerts its activating effects on PBMC proliferation through IL-15 synthesis induction by monocyte/macrophage population. In conclusion, we found that exogenous Nef protein (i) induces activation of normal PBMC, increasing their proliferative response; (ii) modulates cytokine production; (iii) exerts its activating effects through IL-15 synthesis induction; and (iv) exerts these effects entering monocyte/macrophages. Our results might suggest that Nef enhances the rate of viral replication by a novel mechanism involving the production of IL-15. © 1999 Academic Press Key Words: Nef protein; cell activation; IL-15; HIV-1.
INTRODUCTION
The HIV-1 genome contains structural (gag, pol, and env), regulatory (tat, nef, and rev), and accessory genes (vif, vpr, and vpu) [1]. Tat and Rev are known to be positive regulators essential for viral replication [2]. In 1 To whom correspondence and reprint requests should be addressed at Istituto Superiore di Sanita`, Laboratorio di Immunologia, V.le Regina Elena 299, 00161 Rome, Italy. Fax: 139-6-49902709. E-mail: [email protected].
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
contrast, the function of Nef is controversial [3]. Early studies indicated that HIV-1 Nef protein acts as a negative regulatory factor by repressing long terminal repeat-driven transcription [4, 5]. However, further studies have demonstrated that Nef acts as a positive regulator for viral replication in T cells [6]. It has been reported that Nef is required for the development of AIDS in the animal model using simian immunodeficiency virus [7, 8]. In addition, it was demonstrated that soluble Nef can be detected in sera from HIV 1 patients. In fact, Fujii et al. [9] reported that a high percentage (66%) of sera from HIV-1-infected individuals contained soluble Nef at a concentration of 5–10 ng/ml, while patients’ sera in which Nef was not detected contained high titers of anti-Nef antibodies. These data were confirmed by other groups [10, 11] and the development of cytotoxic T lymphocytes (CTL) directed against Nef protein has also been reported [12, 13]. Altogether, these data indicate that Nef plays an important role in HIV pathogenesis, and several possible functions for Nef have been suggested. For instance, Collins et al. [14] have demonstrated that HIV-1 Nef protein exerts a profound effect on MHC class I molecules in primary cells protecting infected primary cells against killing by CTL. Moreover, it has been shown that Nef modulates T-cell activation [15] and that it contributes to B cell activation occurring in association with HIV-1 infection [16]. Torres and Johnson [17] found that HIV-1 Nef protein may exert mitogenic activity similar to that of superantigens. In vitro experiments have also shown the possible role of Nef in modulating immune responses. In fact, it has been recently shown that exogenous Nef activates peripheral blood mononuclear cells (PBMC), enabling them capable to support HIV-1 replication [18]. Furthermore, Nef may act to alter normal host cell response to HIV infection by impairing signal transduction pathways [19]. Based on these findings, we decided to examine the biological mechanisms by which Nef, used as recombinant exogenous protein, modulates cellular activation. In this study, we found that exogenous Nef enters cells
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and induces activation of unstimulated and suboptimally stimulated normal human PBMC, while it did not alter optimally stimulated PBMC proliferation. In addition, we observed that Nef induces the production of cytokines relevant in AIDS pathogenesis such as IFN-g, TNF-a, and IL-6 without affecting IL-2 production. More importantly, we demonstrated that Nef protein exerts its activating effects through IL-15 synthesis induction. MATERIALS AND METHODS Reagents. Recombinant myristylated Nef (HIV-1, strain ELI) and recombinant Tat (HIV-1, strain HxB2) proteins from Escherichia coli were purchased from DBA (Intracell). Lyophilized proteins were dissolved in sterile water as recommended by the manufacturer. Aliquots were prepared and stored at 270°C. A dose–response titration curve was performed to assess the optimal concentrations of Nef and Tat. In all experiments performed Nef and Tat were used at 1 mg/ml and 1 ng/ml, respectively. Anti-Nef mAb was purchased from DBA. Cells. PBMC were isolated by Ficoll–Hypaque (Flow Laboratories) gradient separation [43] of buffy coats obtained from healthy volunteer blood donors by the Italian Red Cross Transfusion Center, Rome, Italy. Lamina propria mononuclear cells (LPMC; kindly provided by Dr. M. Boirivant) were isolated from surgical specimens obtained from bowel resection patients admitted to the VI Clinica Chirurgica, Universita` Degli Studi “La Sapienza” of Rome, for malignant and nonmalignant conditions as previously described [44]. Human monocytes were purified from PBMC by adherence to plastic dishes [45]. The population was 90% CD14 positive. Monocytes were seeded in 24-well plates at a density of 1 3 10 6 cells/ml in Iscove’s medium supplemented with 15% heat-inactivated L-glutamine and penicillin–streptomycin. Promonocytic U937 cell line was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and penicillin–streptomycin. Proliferative response. PBMC and LPMC were resuspended at 1 3 10 6/ml in complete medium (RPMI 1640 supplemented with 10% heat-inactivated human AB serum, L-glutamine, and penicillinstreptomycin) and stimulated in 96-well microtiter plates (Costar). PBMC were stimulated with 1:1,000 dilution of anti-CD2 mAb (T11 2 and T11 3; gift of Dr. Reihnertz, Dana Farber Cancer Institute, Boston, MA), 1 mg/ml of anti-CD3 mAb, 10 mg/ml of phytohemagglutinin (PHA) (Famitogen, Burroughs Wellcome), and 20 mg/ml of purified protein derivative (PPD) (from Mycobacterium tubercolosis; Statens Seruminstitut, Denmark). LPMC were optimally stimulated with 1:1000 diluition anti-CD2/1 mg/ml anti-CD28 (Becton–Dickinson) and suboptimally stimulated with 0.1 mg/ml PHA. Dose–response experiments were performed to assess the mitogen and antigen concentration giving optimal and suboptimal proliferation in vitro. In some experiments PBMC were suboptimally stimulated with the following doses of stimuli: 0.1 mg/ml PHA, 1:10,000 dilution antiCD2, 0.1 mg/ml anti-CD3, and 2.5 mg/ml PPD. In all experiments performed, exogenous Nef protein was added at the beginning of the culture period and left throughout. After 2, 3, or 5 days cultures were pulsed for 18 h with 0.5 mCi/well of [ 3H]thymidine (5 Ci/mM, Amersham International). Cells were then harvested onto glass fiber filters, and [ 3H]thymidine incorporation was measured by liquid scintillation spectroscopy. Results are expressed as mean cpm 6 SD of the mean of triplicate cultures. In some experiments Nef was preincubated with 10 mg/ml mouse anti-Nef mAb for 60 min at 37°C. Analysis of the effect of anti-IL-15 mAb (M111, Immunex Corp.) on Nef-treated cells was performed adding, at the beginning of the culture period, the anti-cytokine mAb to suboptimally PHA-stimulated PBMC in the absence or presence of exogenous Nef. After 72 h,
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cultures were pulsed for 18 h with 0.5 mCi/well of [ 3H]thymidine. Purified T lymphocytes were obtained from PBMC by immunomagnetic negative selection using immunomagnetic beads coated with mouse anti-CD19 and anti-CD14 mAb (Dynal). The resulting (unbound) T cell population contained more than 95% CD3 1 cells as assayed by flow cytometric analysis. Cytokine assays. Analysis of supernatant cytokine contents was performed on unstimulated and suboptimal anti-CD3, anti-CD2, and PHA-stimulated PBMC. After 24 h of culture, supernatants were harvested and cytokine contents were measured by ELISA (R&D Systems) according to the manufacturer’s instructions. Analysis of the intracytoplasmic IL-15 production was performed on the promonocytic U937 cell line and purified monocytes (untreated and lipopolysaccaride (LPS)-activated (1 mg/ml)) and on unstimulated and suboptimally PHA-activated PBMC. Cells (1 3 10 6) were cultured for 6 h at 37°C in the presence of 2 mM monensin (Sigma Chemicals) to avoid cytokine secretion. Cells were washed with calcium–magnesium-free Hanks’ balanced salt solution (HBSS, Hyclone) and fixed with 0.5 ml of ice-cold 4% paraformaldehyde for 10 min at 4°C. Cells were permeabilized with 1 ml HBSS/0.1% saponin (Sigma Chemicals) for 10 min and incubated with the anti-human IL-15 mAb (2.5 mg/ml) for 45 min at room temperature. A mAb direct to choleric toxin was used as control. Cells were washed, incubated with a fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin (10 mg/ml) for 30 min at 4°C, and finally resuspended in HBSS. Samples were then analyzed on a flow cytometer (Becton–Dickinson FACScan). For cytokine mRNA analysis, PBMC were cultured at 5 3 10 6/ml and suboptimally stimulated with antiCD2 mAb in the presence or absence of exogenous Nef protein. After 24 h of culture, total cellular RNA was extracted using the guanidinium isothiocyanate method [46]. RNA was reverse transcribed into cDNA as previously described [47]. The IL-2, IL-6, IFN-g, and TNF-a set of primers was purchased from Clontech. GAPDH primers were synthesized on an Applied Biosystems synthesizer (Applied Biosystems Inc.). GAPDH primer sequences were as follows: sense, 59-GTC TTC ACC ATG GAG AAG GTC-39; and antisense, 59-CAT GCC AGT GAG CTT CCC GTT CA-39. Ten microliters of cDNA was amplified by PCR in a total volume of 20 ml. The PCR mixture contained a final concentration of 13 PCR buffer (10 mM Tris–HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl 2), 50 mM dNTP, 0.1 mM of the 59 and 39 primers, and 1 unit of Taq DNA polymerase (Promega Biotec). The reaction products were amplified for 30 cycles in an automatic DNA thermal cycler (Perkin–Elmer, Cetus Corp.). Each cycle consisted of three steps: denaturation at 94°C for 45 s, annealing at 60°C for 2 min, and primer extension at 72°C for 3 min. After 30 cycles, an additional extension step was performed at 72°C for 7 min. To avoid contamination, all experiments included control PCR without cDNA. Densitometric analysis was performed as previously described [47, 48]. Analytical cytology. For intracellular Nef detection, three different experimental cell systems were used: (a) PBMC, (b) a promonocytic U937 cell line, and (c) adhering monocytes purified from PBMC. Control, treated PBMC and U937 cells were seeded on glass coverslips coated with polylysine. Monocytes were directly grown on glass coverslips. Cells were fixed with 3.7% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 (Sigma). For Nef detection cells were stained with anti-Nef mAb (final dilution of 1:20, DBA; Intracell) for 30 min at 37°C; for the negative control cells were incubated with nonrelevant IgG1 for the same time. After washing, positive and negative samples were incubated with a FITC-labeled goat anti-mouse immunoglobulin (10 mg/ml) for 30 min at 37°C. Finally, all the samples were mounted with glycerol:PBS (2:1) and observed either with a Nikon Microphot fluorescence microscope or with a Sarastro 2000 confocal laser microscope. Statistical analysis. Results were analyzed with a nonparametric test (Wilcoxon’s test). A P value (two-tailed) of less than 0.05 was considered significant.
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PBMC proliferation. Addition of exogenous Nef to unstimulated PBMC resulted in a significant proliferation (P , 0.05) (Fig. 1A). Moreover, the addition of Nef caused a significant increase (P , 0.05) in the proliferative response of PBMC stimulated with suboptimal doses of anti-CD3 (0.1 mg/ml), anti-CD2 (1:10,000 dilution), and PHA (0.1 mg/ml) (Fig. 1B). On the other hand, when exogenous Nef was added to PBMC stimulated with optimal doses of anti-CD3 (1 mg/ml), antiCD2 (1:1,000 dilution), and PHA (10 mg/ml), it did not exert any effect (Fig. 1C). The same activating effect was observed adding Nef to suboptimally PPD-stimulated PBMC, while the proliferation of optimally PPDstimulated PBMC was unchanged (data not shown). We used as control another regulatory HIV-1 protein, Tat, at the concentrations of 0.1, 1, and 10 ng/ml; the results obtained showed that recombinant Tat did not exert any effects (data not shown) on PBMC proliferative responses. The specificity of the Nef-induced proliferative response was assessed using an anti-Nef mAb (Fig. 2). Preincubation of Nef with the anti-Nef mAb abrogated the Nef-induced proliferative responses of suboptimally PHA-stimulated PBMC, confirming that the induction of PBMC proliferation was a specific Nef effect. Similar results were obtained on unstimulated PBMC proliferative responses (data not shown). In addition, the ability of the anti-Nef antibody to eliminate Nef-induced PBMC activation indicates that the Nef effect was specific and not caused by contaminants such as LPS copurifying with the Nef protein preparation. Preincubation of Nef with a mAb direct to choleric toxin (used as control) had no effect (data not shown). To determine if the observed effect of FIG. 1. Effect of Nef protein on PBMC proliferation. Normal human PBMC were stimulated, in the absence or in the presence of exogenous Nef, with (A) medium alone (unstimulated); (B) suboptimal concentration of anti-CD3 (0.1 mg/ml), anti-CD2 (1:10,000 dilution), and PHA (0.1 mg/ml); and (C) optimal concentration of antiCD3 (1 mg/ml), anti-CD2 (1:1,000 dilution), and PHA (10 mg/ml). Results are expressed as cpm 6 SD. Results shown represent means 6 SD of six experiments.
RESULTS
Nef Protein Induces Activation of Unstimulated and Suboptimally Stimulated PBMC To test the effect of Nef protein on PBMC proliferation we performed a titration curve using the following concentrations of Nef: 0.1, 1, and 10 mg/ml. Slight effects were obtained using 0.1 mg/ml, while significant effects were obtained using 1 and 10 mg/ml (data not shown). Therefore, the following experiments were performed using Nef at the concentration of 1 mg/ml. Figure 1 shows the effect of exogenous Nef protein on
FIG. 2. Effect of anti-Nef mAb on Nef-treated PBMC. Exogenous Nef was preincubated with an anti-Nef mAb (10 mg/ml) for 60 min at 37°C and then added to suboptimally PHA-stimulated PBMC. Results are expressed as cpm 6 SD. Results shown represent means 6 SD of four experiments.
IL-15 INDUCTION BY HIV-1 Nef PROTEIN
exogenous Nef was related to the cell activation state, experiments were performed by adding Nef to LPMC, which is known to have a unique state of activation and differentiation which may specifically affect or be affected by HIV infection [20]. Our results (not shown) demonstrated that Nef had no effect on LPMC proliferation. Nef Protein Increases Cytokine Production by Both Unstimulated and Suboptimally Stimulated PBMC To investigate whether the activating effect of Nef on PBMC might be mediated through modulation of cytokine network, exogenous Nef protein was added to unstimulated and suboptimally stimulated PBMC and cytokine production was measured by ELISA. Figure 3 shows the effect of Nef on cytokine supernatant contents. Addition of Nef to PBMC caused an increase of IFN–g, TNF-a, and IL-6 production in culture supernatants of both unstimulated and suboptimally stimulated PBMC. Differently, the production of IL-1b and IL-4 were not modified by exogenous Nef (data not shown). Although Nef, as previously described in the legend to Fig. 1, induced a significant increase of unstimulated and suboptimally stimulated PBMC proliferation, we unexpectedly observed that Nef did not exert any effect on IL-2 secretion, either for unstimulated or suboptimally stimulated PBMC (Fig. 3, bottom). Control cultures, performed using an optimal concentration of PHA (10 mg/ml), contained much higher IL-2 supernatant concentrations (.2,000 pg/ml) with respect to suboptimally PHA-stimulated (0.1 mg/ ml) cultures (80 pg/ml). Similar results were obtained after 48 and 72 h of culture (data not shown). ELISA results were confirmed by performing cytokine production analysis on the mRNA level at 24, 48, and 72 h (data not shown). Nef Exerts Its Activating Effects on PBMC Proliferation through IL-15 Synthesis Induction Since the observed improvement of PBMC proliferation induced by Nef was not associated with an increase of IL-2 production, we hypothesized that exogenous Nef protein acts through production of IL-15, a T cell growth factor displaying activity similar to that of IL-2 [21]. To verify this hypothesis we analyzed the effect of Nef on purified T lymphocyte proliferation; in fact, IL-15 has been shown not to be produced by lymphocytes but rather (at least among immune system cells) appears to be synthetized primarily by monocyte/ macrophages [22]. Our results (not shown) demonstrated that Nef-treated purified T lymphocytes did not proliferate, strengthening our hypothesis that Nef induces cell activation by upregulating the production of a cytokine (IL-15) produced by non-T cells. To verify whether the inductive effect of Nef on PBMC prolifer-
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ation and cytokine production was mediated by IL-15 production, experiments were performed by treating PBMC with Nef in the presence or absence of an antiIL-15 mAb. Results are illustrated in Fig. 4. Addition of anti-IL-15 mAb to control cultures induced, as expected, a reduction in the proliferative response; the addition of anti-IL-15 mAb to Nef-treated PBMC induced a statistically significant (P , 0.05) reduction in the proliferative response which is comparable to that observed in control cultures. A mAb direct to choleric toxin, used as control, did not exert any effect (data not shown). We therefore decided to measure IL-15 production by Nef-treated cells. Although activated PBMC express high levels of IL-15 mRNA, their culture supernatants did not contain meaningful quantities of IL-15, suggesting that critical posttrascriptional regulatory events affect IL-15 expression and intracellular trafficking [23]. Therefore, we measured intracellular IL-15 production by cells in the presence or absence of exogenous Nef (Fig. 5). Analysis of intracellular IL-15 production was performed using the following cells: (i) PBMC, (ii) the promonocytic U937 cell line, and (iii) enriched monocytes. Analysis of intracellular IL-15 production by unstimulated and suboptimally PHAstimulated PBMC (Fig. 5A) (performed by gating the monocyte population) demonstrated that 8.5% of Neftreated unstimulated PBMC, produced IL-15 respect to 0.06% of untreated PBMC. More importantly, we observed that even 39.2% of suboptimally PHA-stimulated PBMC treated with Nef produced IL-15, whereas the untreated cells did not produce IL-15. As illustrated in Fig. 5B, the percentage of Nef-treated U937 cells producing IL-15 rose to 9.52% compared to untreated (0.09%) and LPS-activated (1.62%) U937 cells used as positive controls. Analysis of intracellular IL-15 production by enriched monocyte population (90% CD14 1) (Fig. 5C) showed that the percentage of Nef-treated monocytes rose to 33% compared to the untreated ones (5%). The percentage of IL-15-producing cells from LPS-activated monocytes, used as a control, was 37%. A mAb direct to choleric toxin, used as control, did not exert any effect (data not shown). Nef Exerts Its Activating Effect Entering the Cell Two different sets of experiments were performed in order to assess Nef cellular effects. The first set of experiments was performed to assess the known cellular effects of Nef protein, such as downmodulation of cell surface CD4 expression [24]. The results obtained indicated that Nef was able to downmodulate expression of cell surface CD4 both on PBMC and on the U937 cell line. In fact, 60% of untreated PBMC were CD4 positive, while 40% of Nef-treated PBMC were positive (results represent means 6 SD of two experiments)
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FIG. 3. Analysis of cytokine supernatant concentration in PBMC cultures after Nef treatment. Cells were unstimulated or suboptimally stimulated with 0.1 mg/ml PHA, 0.1 mg/ml anti-CD3, and a 1:10,000 dilution of anti-CD2 in the absence or presence of Nef. After 24 h, cytokine supernatant concentration was analyzed by ELISA. Results shown represent one of three experiments performed.
and 98% of U937 cells express CD4, while just 10% of Nef-treated U937 cells were CD4 positive (results represent means 6 SD of two experiments). The second set
of experiments was performed using the immunocytochemical analysis on permeated cells to verify whether exogenous Nef exerts its activating effects entering the
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The role of the Nef protein on host immune responses is not yet well understood and conflicting data are reported in literature depending on the experimental approach used. Some authors observed that Nef inhibits cell activation pathways when it is introduced directly into PBMC [19]. Furthermore, some others reported an alteration of T cell activation and an enhanced viral production by Nef [6]. On the contrary, Nef contributes to B cell activation when it is used as
FIG. 4. Effect of anti-IL-15 mAb on Nef-treated PBMC proliferative response. Suboptimally PHA-stimulated PBMC were treated with exogenous Nef in the presence or absence of anti-human IL-15 (10 mg/ml) mAb. Results are expressed as cpm 6 SD. Results shown represent means 6 SD of four experiments.
cells. Figure 6 shows the intracellular presence of Nef on PBMC after 2 h (b) and 6 h (c) of treatment with Nef. Figure 6a represents control cells. An intracytoplasmic positivity was detectable as a cortical “ring” (Fig. 6c) or, more frequently, as dot spots (Figs. 6b and 6c). This positivity was time-dependent and evident only in some cells. Moreover, we carried out the immunocytochemical analysis using the promonocytic U937 cell line and enriched monocytes. The analysis of the intracellular labeling of Nef protein revealed a marked positivity after 2 h (Fig. 6e) and 6 h (Fig. 6f) of Nef-treated U937 cells, while untreated U937 cells were negative (Fig. 6d). It should be pointed out that this positivity was localized in the Golgi region (Fig. 6f, arrows). Finally, the analysis of enriched monocytes showed clear intracytoplasmic presence of Nef after 2 h of exposure in the entire cell population (Fig. 6h), when compared to the control (Fig. 6g). Experiments carried out by using both IgG1 as negative control and the recombinant Tat protein failed to reveal any change in the IL-15 production (data not shown). DISCUSSION
This study demonstrates that the HIV-1 recombinant Nef protein induces proliferation of both unstimulated and suboptimally stimulated normal human PBMC. In addition, we show that the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-g, TNF-a, and IL-6 production while, surprisingly, IL-2 production was unaffected by Nef. More importantly we show, for the first time, that Nef exerts its activating effects on PBMC through IL-15 synthesis induction by monocyte/macrophage population. Nef exerts these effects by entering the cells.
FIG. 5. Analysis of intracellular IL-15 production by Nef-treated cells. PBMC (A), U937 cells (B), and enriched monocytes (C) were treated with Nef for 6 h at 37°C. 2 mg/ml monensin was added to the cells at the beginning of the culture period. Suboptimally PHAstimulated PBMC, LPS-stimulated U937 cells, and LPS-stimulated monocytes were used as positive controls. Intracellular IL-15 production was analyzed by FACS. Results are expressed as % of IL-15positive cells. Results shown represent one of three experiments performed.
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FIG. 6. Immunocytochemical analysis. Detection of Nef protein inside the cell cytoplasm of PBMC (a, negative control; b, 2-h Nef-treated cells; c, 6-h Nef-treated cells); U937 cells (d, negative control; e, 2-h Nef-treated cells; f, 6-h Nef-treated cells), and purified monocytes (g, negative control; h, 2-h Nef-treated cells). Note the cortical positivity (e.g., in b and c) in some cells and the dot-spot positivity in several cells. Arrows indicate perinuclear positivity in Golgi regions.
exogenous protein [16] and it seems to exert mitogenic activity similar to that of superantigens [17]. Recently, it has been demonstrated that PBMC become activated and can support HIV-1 replication after exogenous Nef treatment [18]. It should be noted that healthy individuals develop CTL directed against Nef protein [12,
13] and that more than 70% of HIV-1 1 patients have Nef-specific antibodies [10, 11]. This suggest that Nef is a powerful immunogenic HIV-1 component. It has been demonstrated that sera from HIV-1-infected individuals contained soluble Nef at a concentration of 5–10 ng/ml [9]. Therefore, we used soluble Nef protein
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to analyze the biological mechanisms by which Nef activates uninfected cells. The previous observations concerning the effect of exogenous Nef on the activation of unstimulated normal PBMC [18] were confirmed, and this effect was shown to be exerted on suboptimally stimulated PBMC as well. On the other hand, we observed that exogenous Nef did not alter the proliferative response of optimally stimulated PBMC and of LPMC, which represent a physiologically preactivated cell population [20]. A probable explanation for these data is that Nef exerts its activating effect principally on cells that are not sufficiently stimulated, rendering them properly activated for the replication of the virus. It is important to note that the activating effect that we observed was exerted specifically by the Nef protein. In fact, (i) Nef is rapidly internalized by cells, (ii) an anti-Nef mAb completely abrogates the activating effect, and (iii) another HIV-1-regulatory protein, Tat, had no effect on our experimental conditions (not shown). Although we observed an activating effect of exogenous Nef on unstimulated and suboptimally stimulated PBMC, IL-2 production in culture supernatants was unmodified by Nef treatment. These findings contrast with previous studies reporting a role of Nef in modulating IL-2 production [25, 26]. The different results may well be explained, as above, by the different experimental approaches used in these studies (nef gene stably introduced into cells vs exogenous Nef protein). Since we observed no change in IL-2 production, we hypothesized that Nef activated cells through the production of another cell growth factor responsible for cellular activation. This cell growth factor has been demonstrated to be IL-15, a T cell growth factor with a activity similar to that of IL-2 [21], which, like IL-2, is able to stimulate T cell, B cell, and natural killer cell proliferation [27, 28]. However, IL-15, in contrast to IL-2, is not produced by lymphocytes, but instead (at least among cells of immune system) appears to be synthesized primarily by monocyte/macrophages [22]. This suggested that exogenous Nef might indirectly induce lymphocyte proliferation, increasing IL-15 production by monocyte/macrophages. We demonstrated the specific role of IL-15 in the Nef-induced proliferation by blocking Nef-induced PBMC proliferation with an anti-IL-15 mAb. Furthermore, the increased intracellular IL-15 production that we found in Nef-treated cells confirmed that Nef exerts its role by modulating this monocyte/macrophage function. A specific Nef effect on the monocyte/macrophage population was supported by our observation (not shown) that purified T lymphocytes did not proliferate when treated with Nef. It should be noted that these results are similar to those found by Chirmule et al. [16], who observed a lower stimulatory activity of exogenous Nef on B cells when monocyte/macrophages were removed from
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PBMC. The induction of IL-15 production and cell activation by Nef might explain the increase of IFN-g that we observed in Nef-treated PBMC. In fact, previous studies reported that IL-15 production by activated monocytes is important for IFN-g production [29, 30]. Furthermore, we demonstrate that there is an increase of TNF-a and IL-6 production by Nef-treated PBMC. It is noteworthy that Rouaix et al. [31] found a similar increase in TNF-a and IL-6 by peritoneal macrophages treated with a Nef peptide. Moreover, it was been demonstrated that TNF-a, IL-1b, and IL-6 can either enhance or suppress HIV-1 replication during the virus life cycle [32, 33]. Overexpression of Nef during early HIV infection promotes active virus replication and has been suggested to increase monokine/cytokine production [7, 34]. A recent report demonstrated that activation of Nef-expressing human monocytes/macrophages with PMA or LPS alterated the levels of TNF-a, IL-1b, IL-6, and IL-10 [35]. The activation of HIV-1 by cytokines is well known for latently infected cell lines or PBMC from HIV-1 carriers [36]. Thus, our findings that soluble Nef can upregulate cytokine production provides a simple and direct means to activate HIV-1. Our finding that Nef induces IL-15 production, in addition to the upregulation of IFN-g, TNF-a, and IL-6, is relevant considering that IL-15 has been recently found to be an important factor in HIV infection by stimulating the expansion of AIDS virus-specific CTL [37]. Moreover, it has been reported that both TNF-a and IL-6, which we found increased after Nef treatment, synergize in the induction of HIV expression [32]. Immunocytochemical analysis clearly demonstrated that Nef protein is able to enter the cells. In fact, we detected the presence of Nef in PBMC, in U937 promonocytic cell line and in purified monocytes. Moreover, two other points must be condidered: (i) As suggested in the literature [38], there may be an association between the Nef protein and the cytoskeleton. This appears to be confirmed by the presence of a positivity for this protein in ionic detergent-permeated cells, essentially composed of cytoskeleton remnants. (ii) This association with cytoskeleton components, primarily actin filaments [38], may at least partially explain the presence of the protein in the cell cortical regions first (2 h of Nef treatment) and in the perinuclear region later (6 h of Nef treatment). It may be reasonable to hypothesize that intracytoplasmic traffic participates in this localization and Nef-associated cytoskeleton changes are the topic of a specific study (manuscript in preparation). Taken together, our results suggest that exogenous Nef protein plays a key role in host cell activation and demonstrate, for the first time, that Nef exerts its activating function by inducing IL-15 production. Thus, this viral protein may act as a mitogen in HIV-infected individuals and may produce a stable reservoir for virus production as a result of continuous
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stimulation. It is noteworthy that a previous study by Fujinaga et al. [39] demonstrated that the extracellular soluble Nef can activate HIV-1 from latent to productive infection, indicating a key role for this protein in HIV infection. As two recent studies have demonstrated the presence of Nef protein in HIV virions [40, 41], it could be hypothesized that activation of cells may result from cell-free soluble Nef supplied by disrupted infected cells or released virions. As it has been shown that HIV requires activated cells in which to replicate [32], the identification of an HIV protein that induces cell activation is of considerable interest and may, in part, explain the role of Nef in HIV pathogenesis. A defective nef gene in long-term nonprogressor patients [42] may produce Nef protein lacking in mitogenic activity. This may explain the lack of disease progression in these patients.
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Collins, K. L., Chen, B. K., Kalams, S. A., Walker, B. D., and Baltimore, D. (1998). HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397– 401.
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Skowronski, J., Parks, D., and Mariani, R. (1993). Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J. 12, 703–713.
16.
Chirmule, N., Oyaizu, N., Saxinger, C., and Pahwa, S. (1994). Nef protein of HIV-1 has B-cell stimulatory activity. AIDS 8, 733–739.
17.
Torres, B. A., and Johnson, M. H. (1994). Identification of an HIV-1 nef peptide that binds to HLA class II antigens. Biochem. Biophys. Res. Commun. 200, 1059 –1065.
This work was supported by the Italian Ministry of Health AIDS Research Projects, Grants N°10A/S and N°10A/R.
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Induction of Interleukin-15 Production by HIV-1 Nef Protein: A Role in the Proliferation of Uninfected Cells Maria Giovanna Quaranta,* Barbara Camponeschi,* Elisabetta Straface,† Walter Malorni,† and Marina Viora* ,1 *Department of Immunology and †Department of Ultrastructures, Istituto Superiore di Sanita`, Rome, Italy
Several recent reports have provided evidence that Nef enhances human immunodeficiency virus HIV infectivity, and in vitro experiments with the nef gene have demonstrated the possible role of Nef in modulating immune responses. Exogenous Nef has been demonstrated to induce proliferation of normal human peripheral blood mononuclear cells (PBMC) and to enhance HIV-1 replication. The aim of this study was to evaluate the biological mechanisms by which Nef, used as exogenous protein, modulates cellular activation. We showed that exogenous Nef protein induces the proliferation of unstimulated and suboptimally stimulated normal human PBMC, while it has no effect on the proliferation of optimally stimulated PBMC. Moreover, the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-g, TNF-a, and IL-6 production, while, surprisingly, IL-2 production was not affected by Nef. More importantly we showed, for the first time, that Nef exerts its activating effects on PBMC proliferation through IL-15 synthesis induction by monocyte/macrophage population. In conclusion, we found that exogenous Nef protein (i) induces activation of normal PBMC, increasing their proliferative response; (ii) modulates cytokine production; (iii) exerts its activating effects through IL-15 synthesis induction; and (iv) exerts these effects entering monocyte/macrophages. Our results might suggest that Nef enhances the rate of viral replication by a novel mechanism involving the production of IL-15. © 1999 Academic Press Key Words: Nef protein; cell activation; IL-15; HIV-1.
INTRODUCTION
The HIV-1 genome contains structural (gag, pol, and env), regulatory (tat, nef, and rev), and accessory genes (vif, vpr, and vpu) [1]. Tat and Rev are known to be positive regulators essential for viral replication [2]. In 1 To whom correspondence and reprint requests should be addressed at Istituto Superiore di Sanita`, Laboratorio di Immunologia, V.le Regina Elena 299, 00161 Rome, Italy. Fax: 139-6-49902709. E-mail: [email protected].
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
contrast, the function of Nef is controversial [3]. Early studies indicated that HIV-1 Nef protein acts as a negative regulatory factor by repressing long terminal repeat-driven transcription [4, 5]. However, further studies have demonstrated that Nef acts as a positive regulator for viral replication in T cells [6]. It has been reported that Nef is required for the development of AIDS in the animal model using simian immunodeficiency virus [7, 8]. In addition, it was demonstrated that soluble Nef can be detected in sera from HIV 1 patients. In fact, Fujii et al. [9] reported that a high percentage (66%) of sera from HIV-1-infected individuals contained soluble Nef at a concentration of 5–10 ng/ml, while patients’ sera in which Nef was not detected contained high titers of anti-Nef antibodies. These data were confirmed by other groups [10, 11] and the development of cytotoxic T lymphocytes (CTL) directed against Nef protein has also been reported [12, 13]. Altogether, these data indicate that Nef plays an important role in HIV pathogenesis, and several possible functions for Nef have been suggested. For instance, Collins et al. [14] have demonstrated that HIV-1 Nef protein exerts a profound effect on MHC class I molecules in primary cells protecting infected primary cells against killing by CTL. Moreover, it has been shown that Nef modulates T-cell activation [15] and that it contributes to B cell activation occurring in association with HIV-1 infection [16]. Torres and Johnson [17] found that HIV-1 Nef protein may exert mitogenic activity similar to that of superantigens. In vitro experiments have also shown the possible role of Nef in modulating immune responses. In fact, it has been recently shown that exogenous Nef activates peripheral blood mononuclear cells (PBMC), enabling them capable to support HIV-1 replication [18]. Furthermore, Nef may act to alter normal host cell response to HIV infection by impairing signal transduction pathways [19]. Based on these findings, we decided to examine the biological mechanisms by which Nef, used as recombinant exogenous protein, modulates cellular activation. In this study, we found that exogenous Nef enters cells
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and induces activation of unstimulated and suboptimally stimulated normal human PBMC, while it did not alter optimally stimulated PBMC proliferation. In addition, we observed that Nef induces the production of cytokines relevant in AIDS pathogenesis such as IFN-g, TNF-a, and IL-6 without affecting IL-2 production. More importantly, we demonstrated that Nef protein exerts its activating effects through IL-15 synthesis induction. MATERIALS AND METHODS Reagents. Recombinant myristylated Nef (HIV-1, strain ELI) and recombinant Tat (HIV-1, strain HxB2) proteins from Escherichia coli were purchased from DBA (Intracell). Lyophilized proteins were dissolved in sterile water as recommended by the manufacturer. Aliquots were prepared and stored at 270°C. A dose–response titration curve was performed to assess the optimal concentrations of Nef and Tat. In all experiments performed Nef and Tat were used at 1 mg/ml and 1 ng/ml, respectively. Anti-Nef mAb was purchased from DBA. Cells. PBMC were isolated by Ficoll–Hypaque (Flow Laboratories) gradient separation [43] of buffy coats obtained from healthy volunteer blood donors by the Italian Red Cross Transfusion Center, Rome, Italy. Lamina propria mononuclear cells (LPMC; kindly provided by Dr. M. Boirivant) were isolated from surgical specimens obtained from bowel resection patients admitted to the VI Clinica Chirurgica, Universita` Degli Studi “La Sapienza” of Rome, for malignant and nonmalignant conditions as previously described [44]. Human monocytes were purified from PBMC by adherence to plastic dishes [45]. The population was 90% CD14 positive. Monocytes were seeded in 24-well plates at a density of 1 3 10 6 cells/ml in Iscove’s medium supplemented with 15% heat-inactivated L-glutamine and penicillin–streptomycin. Promonocytic U937 cell line was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and penicillin–streptomycin. Proliferative response. PBMC and LPMC were resuspended at 1 3 10 6/ml in complete medium (RPMI 1640 supplemented with 10% heat-inactivated human AB serum, L-glutamine, and penicillinstreptomycin) and stimulated in 96-well microtiter plates (Costar). PBMC were stimulated with 1:1,000 dilution of anti-CD2 mAb (T11 2 and T11 3; gift of Dr. Reihnertz, Dana Farber Cancer Institute, Boston, MA), 1 mg/ml of anti-CD3 mAb, 10 mg/ml of phytohemagglutinin (PHA) (Famitogen, Burroughs Wellcome), and 20 mg/ml of purified protein derivative (PPD) (from Mycobacterium tubercolosis; Statens Seruminstitut, Denmark). LPMC were optimally stimulated with 1:1000 diluition anti-CD2/1 mg/ml anti-CD28 (Becton–Dickinson) and suboptimally stimulated with 0.1 mg/ml PHA. Dose–response experiments were performed to assess the mitogen and antigen concentration giving optimal and suboptimal proliferation in vitro. In some experiments PBMC were suboptimally stimulated with the following doses of stimuli: 0.1 mg/ml PHA, 1:10,000 dilution antiCD2, 0.1 mg/ml anti-CD3, and 2.5 mg/ml PPD. In all experiments performed, exogenous Nef protein was added at the beginning of the culture period and left throughout. After 2, 3, or 5 days cultures were pulsed for 18 h with 0.5 mCi/well of [ 3H]thymidine (5 Ci/mM, Amersham International). Cells were then harvested onto glass fiber filters, and [ 3H]thymidine incorporation was measured by liquid scintillation spectroscopy. Results are expressed as mean cpm 6 SD of the mean of triplicate cultures. In some experiments Nef was preincubated with 10 mg/ml mouse anti-Nef mAb for 60 min at 37°C. Analysis of the effect of anti-IL-15 mAb (M111, Immunex Corp.) on Nef-treated cells was performed adding, at the beginning of the culture period, the anti-cytokine mAb to suboptimally PHA-stimulated PBMC in the absence or presence of exogenous Nef. After 72 h,
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cultures were pulsed for 18 h with 0.5 mCi/well of [ 3H]thymidine. Purified T lymphocytes were obtained from PBMC by immunomagnetic negative selection using immunomagnetic beads coated with mouse anti-CD19 and anti-CD14 mAb (Dynal). The resulting (unbound) T cell population contained more than 95% CD3 1 cells as assayed by flow cytometric analysis. Cytokine assays. Analysis of supernatant cytokine contents was performed on unstimulated and suboptimal anti-CD3, anti-CD2, and PHA-stimulated PBMC. After 24 h of culture, supernatants were harvested and cytokine contents were measured by ELISA (R&D Systems) according to the manufacturer’s instructions. Analysis of the intracytoplasmic IL-15 production was performed on the promonocytic U937 cell line and purified monocytes (untreated and lipopolysaccaride (LPS)-activated (1 mg/ml)) and on unstimulated and suboptimally PHA-activated PBMC. Cells (1 3 10 6) were cultured for 6 h at 37°C in the presence of 2 mM monensin (Sigma Chemicals) to avoid cytokine secretion. Cells were washed with calcium–magnesium-free Hanks’ balanced salt solution (HBSS, Hyclone) and fixed with 0.5 ml of ice-cold 4% paraformaldehyde for 10 min at 4°C. Cells were permeabilized with 1 ml HBSS/0.1% saponin (Sigma Chemicals) for 10 min and incubated with the anti-human IL-15 mAb (2.5 mg/ml) for 45 min at room temperature. A mAb direct to choleric toxin was used as control. Cells were washed, incubated with a fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin (10 mg/ml) for 30 min at 4°C, and finally resuspended in HBSS. Samples were then analyzed on a flow cytometer (Becton–Dickinson FACScan). For cytokine mRNA analysis, PBMC were cultured at 5 3 10 6/ml and suboptimally stimulated with antiCD2 mAb in the presence or absence of exogenous Nef protein. After 24 h of culture, total cellular RNA was extracted using the guanidinium isothiocyanate method [46]. RNA was reverse transcribed into cDNA as previously described [47]. The IL-2, IL-6, IFN-g, and TNF-a set of primers was purchased from Clontech. GAPDH primers were synthesized on an Applied Biosystems synthesizer (Applied Biosystems Inc.). GAPDH primer sequences were as follows: sense, 59-GTC TTC ACC ATG GAG AAG GTC-39; and antisense, 59-CAT GCC AGT GAG CTT CCC GTT CA-39. Ten microliters of cDNA was amplified by PCR in a total volume of 20 ml. The PCR mixture contained a final concentration of 13 PCR buffer (10 mM Tris–HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl 2), 50 mM dNTP, 0.1 mM of the 59 and 39 primers, and 1 unit of Taq DNA polymerase (Promega Biotec). The reaction products were amplified for 30 cycles in an automatic DNA thermal cycler (Perkin–Elmer, Cetus Corp.). Each cycle consisted of three steps: denaturation at 94°C for 45 s, annealing at 60°C for 2 min, and primer extension at 72°C for 3 min. After 30 cycles, an additional extension step was performed at 72°C for 7 min. To avoid contamination, all experiments included control PCR without cDNA. Densitometric analysis was performed as previously described [47, 48]. Analytical cytology. For intracellular Nef detection, three different experimental cell systems were used: (a) PBMC, (b) a promonocytic U937 cell line, and (c) adhering monocytes purified from PBMC. Control, treated PBMC and U937 cells were seeded on glass coverslips coated with polylysine. Monocytes were directly grown on glass coverslips. Cells were fixed with 3.7% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 (Sigma). For Nef detection cells were stained with anti-Nef mAb (final dilution of 1:20, DBA; Intracell) for 30 min at 37°C; for the negative control cells were incubated with nonrelevant IgG1 for the same time. After washing, positive and negative samples were incubated with a FITC-labeled goat anti-mouse immunoglobulin (10 mg/ml) for 30 min at 37°C. Finally, all the samples were mounted with glycerol:PBS (2:1) and observed either with a Nikon Microphot fluorescence microscope or with a Sarastro 2000 confocal laser microscope. Statistical analysis. Results were analyzed with a nonparametric test (Wilcoxon’s test). A P value (two-tailed) of less than 0.05 was considered significant.
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PBMC proliferation. Addition of exogenous Nef to unstimulated PBMC resulted in a significant proliferation (P , 0.05) (Fig. 1A). Moreover, the addition of Nef caused a significant increase (P , 0.05) in the proliferative response of PBMC stimulated with suboptimal doses of anti-CD3 (0.1 mg/ml), anti-CD2 (1:10,000 dilution), and PHA (0.1 mg/ml) (Fig. 1B). On the other hand, when exogenous Nef was added to PBMC stimulated with optimal doses of anti-CD3 (1 mg/ml), antiCD2 (1:1,000 dilution), and PHA (10 mg/ml), it did not exert any effect (Fig. 1C). The same activating effect was observed adding Nef to suboptimally PPD-stimulated PBMC, while the proliferation of optimally PPDstimulated PBMC was unchanged (data not shown). We used as control another regulatory HIV-1 protein, Tat, at the concentrations of 0.1, 1, and 10 ng/ml; the results obtained showed that recombinant Tat did not exert any effects (data not shown) on PBMC proliferative responses. The specificity of the Nef-induced proliferative response was assessed using an anti-Nef mAb (Fig. 2). Preincubation of Nef with the anti-Nef mAb abrogated the Nef-induced proliferative responses of suboptimally PHA-stimulated PBMC, confirming that the induction of PBMC proliferation was a specific Nef effect. Similar results were obtained on unstimulated PBMC proliferative responses (data not shown). In addition, the ability of the anti-Nef antibody to eliminate Nef-induced PBMC activation indicates that the Nef effect was specific and not caused by contaminants such as LPS copurifying with the Nef protein preparation. Preincubation of Nef with a mAb direct to choleric toxin (used as control) had no effect (data not shown). To determine if the observed effect of FIG. 1. Effect of Nef protein on PBMC proliferation. Normal human PBMC were stimulated, in the absence or in the presence of exogenous Nef, with (A) medium alone (unstimulated); (B) suboptimal concentration of anti-CD3 (0.1 mg/ml), anti-CD2 (1:10,000 dilution), and PHA (0.1 mg/ml); and (C) optimal concentration of antiCD3 (1 mg/ml), anti-CD2 (1:1,000 dilution), and PHA (10 mg/ml). Results are expressed as cpm 6 SD. Results shown represent means 6 SD of six experiments.
RESULTS
Nef Protein Induces Activation of Unstimulated and Suboptimally Stimulated PBMC To test the effect of Nef protein on PBMC proliferation we performed a titration curve using the following concentrations of Nef: 0.1, 1, and 10 mg/ml. Slight effects were obtained using 0.1 mg/ml, while significant effects were obtained using 1 and 10 mg/ml (data not shown). Therefore, the following experiments were performed using Nef at the concentration of 1 mg/ml. Figure 1 shows the effect of exogenous Nef protein on
FIG. 2. Effect of anti-Nef mAb on Nef-treated PBMC. Exogenous Nef was preincubated with an anti-Nef mAb (10 mg/ml) for 60 min at 37°C and then added to suboptimally PHA-stimulated PBMC. Results are expressed as cpm 6 SD. Results shown represent means 6 SD of four experiments.
IL-15 INDUCTION BY HIV-1 Nef PROTEIN
exogenous Nef was related to the cell activation state, experiments were performed by adding Nef to LPMC, which is known to have a unique state of activation and differentiation which may specifically affect or be affected by HIV infection [20]. Our results (not shown) demonstrated that Nef had no effect on LPMC proliferation. Nef Protein Increases Cytokine Production by Both Unstimulated and Suboptimally Stimulated PBMC To investigate whether the activating effect of Nef on PBMC might be mediated through modulation of cytokine network, exogenous Nef protein was added to unstimulated and suboptimally stimulated PBMC and cytokine production was measured by ELISA. Figure 3 shows the effect of Nef on cytokine supernatant contents. Addition of Nef to PBMC caused an increase of IFN–g, TNF-a, and IL-6 production in culture supernatants of both unstimulated and suboptimally stimulated PBMC. Differently, the production of IL-1b and IL-4 were not modified by exogenous Nef (data not shown). Although Nef, as previously described in the legend to Fig. 1, induced a significant increase of unstimulated and suboptimally stimulated PBMC proliferation, we unexpectedly observed that Nef did not exert any effect on IL-2 secretion, either for unstimulated or suboptimally stimulated PBMC (Fig. 3, bottom). Control cultures, performed using an optimal concentration of PHA (10 mg/ml), contained much higher IL-2 supernatant concentrations (.2,000 pg/ml) with respect to suboptimally PHA-stimulated (0.1 mg/ ml) cultures (80 pg/ml). Similar results were obtained after 48 and 72 h of culture (data not shown). ELISA results were confirmed by performing cytokine production analysis on the mRNA level at 24, 48, and 72 h (data not shown). Nef Exerts Its Activating Effects on PBMC Proliferation through IL-15 Synthesis Induction Since the observed improvement of PBMC proliferation induced by Nef was not associated with an increase of IL-2 production, we hypothesized that exogenous Nef protein acts through production of IL-15, a T cell growth factor displaying activity similar to that of IL-2 [21]. To verify this hypothesis we analyzed the effect of Nef on purified T lymphocyte proliferation; in fact, IL-15 has been shown not to be produced by lymphocytes but rather (at least among immune system cells) appears to be synthetized primarily by monocyte/ macrophages [22]. Our results (not shown) demonstrated that Nef-treated purified T lymphocytes did not proliferate, strengthening our hypothesis that Nef induces cell activation by upregulating the production of a cytokine (IL-15) produced by non-T cells. To verify whether the inductive effect of Nef on PBMC prolifer-
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ation and cytokine production was mediated by IL-15 production, experiments were performed by treating PBMC with Nef in the presence or absence of an antiIL-15 mAb. Results are illustrated in Fig. 4. Addition of anti-IL-15 mAb to control cultures induced, as expected, a reduction in the proliferative response; the addition of anti-IL-15 mAb to Nef-treated PBMC induced a statistically significant (P , 0.05) reduction in the proliferative response which is comparable to that observed in control cultures. A mAb direct to choleric toxin, used as control, did not exert any effect (data not shown). We therefore decided to measure IL-15 production by Nef-treated cells. Although activated PBMC express high levels of IL-15 mRNA, their culture supernatants did not contain meaningful quantities of IL-15, suggesting that critical posttrascriptional regulatory events affect IL-15 expression and intracellular trafficking [23]. Therefore, we measured intracellular IL-15 production by cells in the presence or absence of exogenous Nef (Fig. 5). Analysis of intracellular IL-15 production was performed using the following cells: (i) PBMC, (ii) the promonocytic U937 cell line, and (iii) enriched monocytes. Analysis of intracellular IL-15 production by unstimulated and suboptimally PHAstimulated PBMC (Fig. 5A) (performed by gating the monocyte population) demonstrated that 8.5% of Neftreated unstimulated PBMC, produced IL-15 respect to 0.06% of untreated PBMC. More importantly, we observed that even 39.2% of suboptimally PHA-stimulated PBMC treated with Nef produced IL-15, whereas the untreated cells did not produce IL-15. As illustrated in Fig. 5B, the percentage of Nef-treated U937 cells producing IL-15 rose to 9.52% compared to untreated (0.09%) and LPS-activated (1.62%) U937 cells used as positive controls. Analysis of intracellular IL-15 production by enriched monocyte population (90% CD14 1) (Fig. 5C) showed that the percentage of Nef-treated monocytes rose to 33% compared to the untreated ones (5%). The percentage of IL-15-producing cells from LPS-activated monocytes, used as a control, was 37%. A mAb direct to choleric toxin, used as control, did not exert any effect (data not shown). Nef Exerts Its Activating Effect Entering the Cell Two different sets of experiments were performed in order to assess Nef cellular effects. The first set of experiments was performed to assess the known cellular effects of Nef protein, such as downmodulation of cell surface CD4 expression [24]. The results obtained indicated that Nef was able to downmodulate expression of cell surface CD4 both on PBMC and on the U937 cell line. In fact, 60% of untreated PBMC were CD4 positive, while 40% of Nef-treated PBMC were positive (results represent means 6 SD of two experiments)
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FIG. 3. Analysis of cytokine supernatant concentration in PBMC cultures after Nef treatment. Cells were unstimulated or suboptimally stimulated with 0.1 mg/ml PHA, 0.1 mg/ml anti-CD3, and a 1:10,000 dilution of anti-CD2 in the absence or presence of Nef. After 24 h, cytokine supernatant concentration was analyzed by ELISA. Results shown represent one of three experiments performed.
and 98% of U937 cells express CD4, while just 10% of Nef-treated U937 cells were CD4 positive (results represent means 6 SD of two experiments). The second set
of experiments was performed using the immunocytochemical analysis on permeated cells to verify whether exogenous Nef exerts its activating effects entering the
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The role of the Nef protein on host immune responses is not yet well understood and conflicting data are reported in literature depending on the experimental approach used. Some authors observed that Nef inhibits cell activation pathways when it is introduced directly into PBMC [19]. Furthermore, some others reported an alteration of T cell activation and an enhanced viral production by Nef [6]. On the contrary, Nef contributes to B cell activation when it is used as
FIG. 4. Effect of anti-IL-15 mAb on Nef-treated PBMC proliferative response. Suboptimally PHA-stimulated PBMC were treated with exogenous Nef in the presence or absence of anti-human IL-15 (10 mg/ml) mAb. Results are expressed as cpm 6 SD. Results shown represent means 6 SD of four experiments.
cells. Figure 6 shows the intracellular presence of Nef on PBMC after 2 h (b) and 6 h (c) of treatment with Nef. Figure 6a represents control cells. An intracytoplasmic positivity was detectable as a cortical “ring” (Fig. 6c) or, more frequently, as dot spots (Figs. 6b and 6c). This positivity was time-dependent and evident only in some cells. Moreover, we carried out the immunocytochemical analysis using the promonocytic U937 cell line and enriched monocytes. The analysis of the intracellular labeling of Nef protein revealed a marked positivity after 2 h (Fig. 6e) and 6 h (Fig. 6f) of Nef-treated U937 cells, while untreated U937 cells were negative (Fig. 6d). It should be pointed out that this positivity was localized in the Golgi region (Fig. 6f, arrows). Finally, the analysis of enriched monocytes showed clear intracytoplasmic presence of Nef after 2 h of exposure in the entire cell population (Fig. 6h), when compared to the control (Fig. 6g). Experiments carried out by using both IgG1 as negative control and the recombinant Tat protein failed to reveal any change in the IL-15 production (data not shown). DISCUSSION
This study demonstrates that the HIV-1 recombinant Nef protein induces proliferation of both unstimulated and suboptimally stimulated normal human PBMC. In addition, we show that the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-g, TNF-a, and IL-6 production while, surprisingly, IL-2 production was unaffected by Nef. More importantly we show, for the first time, that Nef exerts its activating effects on PBMC through IL-15 synthesis induction by monocyte/macrophage population. Nef exerts these effects by entering the cells.
FIG. 5. Analysis of intracellular IL-15 production by Nef-treated cells. PBMC (A), U937 cells (B), and enriched monocytes (C) were treated with Nef for 6 h at 37°C. 2 mg/ml monensin was added to the cells at the beginning of the culture period. Suboptimally PHAstimulated PBMC, LPS-stimulated U937 cells, and LPS-stimulated monocytes were used as positive controls. Intracellular IL-15 production was analyzed by FACS. Results are expressed as % of IL-15positive cells. Results shown represent one of three experiments performed.
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FIG. 6. Immunocytochemical analysis. Detection of Nef protein inside the cell cytoplasm of PBMC (a, negative control; b, 2-h Nef-treated cells; c, 6-h Nef-treated cells); U937 cells (d, negative control; e, 2-h Nef-treated cells; f, 6-h Nef-treated cells), and purified monocytes (g, negative control; h, 2-h Nef-treated cells). Note the cortical positivity (e.g., in b and c) in some cells and the dot-spot positivity in several cells. Arrows indicate perinuclear positivity in Golgi regions.
exogenous protein [16] and it seems to exert mitogenic activity similar to that of superantigens [17]. Recently, it has been demonstrated that PBMC become activated and can support HIV-1 replication after exogenous Nef treatment [18]. It should be noted that healthy individuals develop CTL directed against Nef protein [12,
13] and that more than 70% of HIV-1 1 patients have Nef-specific antibodies [10, 11]. This suggest that Nef is a powerful immunogenic HIV-1 component. It has been demonstrated that sera from HIV-1-infected individuals contained soluble Nef at a concentration of 5–10 ng/ml [9]. Therefore, we used soluble Nef protein
IL-15 INDUCTION BY HIV-1 Nef PROTEIN
to analyze the biological mechanisms by which Nef activates uninfected cells. The previous observations concerning the effect of exogenous Nef on the activation of unstimulated normal PBMC [18] were confirmed, and this effect was shown to be exerted on suboptimally stimulated PBMC as well. On the other hand, we observed that exogenous Nef did not alter the proliferative response of optimally stimulated PBMC and of LPMC, which represent a physiologically preactivated cell population [20]. A probable explanation for these data is that Nef exerts its activating effect principally on cells that are not sufficiently stimulated, rendering them properly activated for the replication of the virus. It is important to note that the activating effect that we observed was exerted specifically by the Nef protein. In fact, (i) Nef is rapidly internalized by cells, (ii) an anti-Nef mAb completely abrogates the activating effect, and (iii) another HIV-1-regulatory protein, Tat, had no effect on our experimental conditions (not shown). Although we observed an activating effect of exogenous Nef on unstimulated and suboptimally stimulated PBMC, IL-2 production in culture supernatants was unmodified by Nef treatment. These findings contrast with previous studies reporting a role of Nef in modulating IL-2 production [25, 26]. The different results may well be explained, as above, by the different experimental approaches used in these studies (nef gene stably introduced into cells vs exogenous Nef protein). Since we observed no change in IL-2 production, we hypothesized that Nef activated cells through the production of another cell growth factor responsible for cellular activation. This cell growth factor has been demonstrated to be IL-15, a T cell growth factor with a activity similar to that of IL-2 [21], which, like IL-2, is able to stimulate T cell, B cell, and natural killer cell proliferation [27, 28]. However, IL-15, in contrast to IL-2, is not produced by lymphocytes, but instead (at least among cells of immune system) appears to be synthesized primarily by monocyte/macrophages [22]. This suggested that exogenous Nef might indirectly induce lymphocyte proliferation, increasing IL-15 production by monocyte/macrophages. We demonstrated the specific role of IL-15 in the Nef-induced proliferation by blocking Nef-induced PBMC proliferation with an anti-IL-15 mAb. Furthermore, the increased intracellular IL-15 production that we found in Nef-treated cells confirmed that Nef exerts its role by modulating this monocyte/macrophage function. A specific Nef effect on the monocyte/macrophage population was supported by our observation (not shown) that purified T lymphocytes did not proliferate when treated with Nef. It should be noted that these results are similar to those found by Chirmule et al. [16], who observed a lower stimulatory activity of exogenous Nef on B cells when monocyte/macrophages were removed from
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PBMC. The induction of IL-15 production and cell activation by Nef might explain the increase of IFN-g that we observed in Nef-treated PBMC. In fact, previous studies reported that IL-15 production by activated monocytes is important for IFN-g production [29, 30]. Furthermore, we demonstrate that there is an increase of TNF-a and IL-6 production by Nef-treated PBMC. It is noteworthy that Rouaix et al. [31] found a similar increase in TNF-a and IL-6 by peritoneal macrophages treated with a Nef peptide. Moreover, it was been demonstrated that TNF-a, IL-1b, and IL-6 can either enhance or suppress HIV-1 replication during the virus life cycle [32, 33]. Overexpression of Nef during early HIV infection promotes active virus replication and has been suggested to increase monokine/cytokine production [7, 34]. A recent report demonstrated that activation of Nef-expressing human monocytes/macrophages with PMA or LPS alterated the levels of TNF-a, IL-1b, IL-6, and IL-10 [35]. The activation of HIV-1 by cytokines is well known for latently infected cell lines or PBMC from HIV-1 carriers [36]. Thus, our findings that soluble Nef can upregulate cytokine production provides a simple and direct means to activate HIV-1. Our finding that Nef induces IL-15 production, in addition to the upregulation of IFN-g, TNF-a, and IL-6, is relevant considering that IL-15 has been recently found to be an important factor in HIV infection by stimulating the expansion of AIDS virus-specific CTL [37]. Moreover, it has been reported that both TNF-a and IL-6, which we found increased after Nef treatment, synergize in the induction of HIV expression [32]. Immunocytochemical analysis clearly demonstrated that Nef protein is able to enter the cells. In fact, we detected the presence of Nef in PBMC, in U937 promonocytic cell line and in purified monocytes. Moreover, two other points must be condidered: (i) As suggested in the literature [38], there may be an association between the Nef protein and the cytoskeleton. This appears to be confirmed by the presence of a positivity for this protein in ionic detergent-permeated cells, essentially composed of cytoskeleton remnants. (ii) This association with cytoskeleton components, primarily actin filaments [38], may at least partially explain the presence of the protein in the cell cortical regions first (2 h of Nef treatment) and in the perinuclear region later (6 h of Nef treatment). It may be reasonable to hypothesize that intracytoplasmic traffic participates in this localization and Nef-associated cytoskeleton changes are the topic of a specific study (manuscript in preparation). Taken together, our results suggest that exogenous Nef protein plays a key role in host cell activation and demonstrate, for the first time, that Nef exerts its activating function by inducing IL-15 production. Thus, this viral protein may act as a mitogen in HIV-infected individuals and may produce a stable reservoir for virus production as a result of continuous
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stimulation. It is noteworthy that a previous study by Fujinaga et al. [39] demonstrated that the extracellular soluble Nef can activate HIV-1 from latent to productive infection, indicating a key role for this protein in HIV infection. As two recent studies have demonstrated the presence of Nef protein in HIV virions [40, 41], it could be hypothesized that activation of cells may result from cell-free soluble Nef supplied by disrupted infected cells or released virions. As it has been shown that HIV requires activated cells in which to replicate [32], the identification of an HIV protein that induces cell activation is of considerable interest and may, in part, explain the role of Nef in HIV pathogenesis. A defective nef gene in long-term nonprogressor patients [42] may produce Nef protein lacking in mitogenic activity. This may explain the lack of disease progression in these patients.
11.
Sabatier, J. M., Clerget-Raslain, B., Fontan, G., Fenuillet, E., Rochat, H., Granier, C., Gluckman, J., Van Rietschoten, J., Montagnier, L., and Bahraoui, E. (1989). Use of synthetic peptides for detection of antibodies against the nef regulating protein in sera of HIV-infected patients. AIDS 3, 215–220.
12.
Lucchiari, M., Niedermann, G., Leipner, C., Meyerhans, A., Eichmann, K., and Maier, B. (1994). Human immune response to HIV-1 Nef. I. CD45RO 2 T lymphocytes of non-infected donors contain cytotoxic T lymphocyte precursor at high frequency. Int. Immunol. 6, 1739 –1749.
13.
Lucchiari-Hartz, M., Bauer M., Niedermann, G., Maier, B., Meyerhans, A., and Eichmann, K. (1996). Human immune response to HIV-1 Nef. II. Induction of HIV-1/HIV-2 Nef crossreactive cytotoxic T lymphocytes of non-infected healthy individuals. Int. Immunol. 8, 577–584.
14.
Collins, K. L., Chen, B. K., Kalams, S. A., Walker, B. D., and Baltimore, D. (1998). HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature 391, 397– 401.
15.
Skowronski, J., Parks, D., and Mariani, R. (1993). Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J. 12, 703–713.
16.
Chirmule, N., Oyaizu, N., Saxinger, C., and Pahwa, S. (1994). Nef protein of HIV-1 has B-cell stimulatory activity. AIDS 8, 733–739.
17.
Torres, B. A., and Johnson, M. H. (1994). Identification of an HIV-1 nef peptide that binds to HLA class II antigens. Biochem. Biophys. Res. Commun. 200, 1059 –1065.
This work was supported by the Italian Ministry of Health AIDS Research Projects, Grants N°10A/S and N°10A/R.
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