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(The FASEB Journal. 2003;17:2025-2036.)
© 2003 FASEB

HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells

MARIA GIOVANNA QUARANTA*, BENEDETTA MATTIOLI*, FRANCESCA SPADARO*, ELISABETTA STRAFACE{dagger}, LUCIANA GIORDANI*, CARLO RAMONI*, WALTER MALORNI{dagger} and MARINA VIORA*,1

* Department of Immunology,
{dagger} Department of Ultrastructures, Istituto Superiore di Sanità, 00161 Rome, Italy

1Correspondence: Department of Immunology, Istituto Superiore di Sanità Viale Regina Elena, 299 00161 Rome, Italy. E-mail: viora{at}iss.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The accessory HIV-1 Nef protein plays a key role in AIDS pathogenesis. We recently demonstrated that exogenous Nef triggers phenotypic and functional differentiation of immature dendritic cells (DCs). Here we investigated whether the Nef-induced DC differentiation occurs with morphological remodeling and have focused on the interference of Nef in the signaling pathways that regulates DC maturation. We found that exogenous Nef enters immature DCs, promoting their functional and morphological differentiation. Specifically, Nef promotes interleukin (IL) -12 release, which closely fits with nuclear factor (NF) -{kappa}B activation. Nef induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Moreover, Nef increases the capacity of DCs to form clusters with allogeneic CD4+ T cells, improving immunological synapse formation. Searching for molecules involved in Nef-triggered signaling pathways driving the DC maturation, we found that Nef targets Vav and promotes its tyrosine phosphorylation, associated with its nucleus-to-cytoplasm redistribution. This has a direct effect on Vav guanine nucleotide exchange factor activity for the small GTPase Rac1. We hypothesize that targeting Vav, Nef modulates both early signaling events (such as cytoskeletal rearrangement) and delayed responses (such as transcriptional regulation), promoting DC differentiation. Our results highlight how Nef may enhance T lymphocyte activation, thus fostering virus dissemination, manipulating the DC arm of the immune response.—Quaranta, M. G., Mattioli, B., Spadaro, F., Straface, E., Giordani, L., Ramoni, C., Malorni, W., Viora, M. HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells.


Key Words: AIDS immunopathogenesis • exogenous Nef • signal transduction • cytoskeleton rearrangement


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DENDRITIC CELLS (DCs) are the most potent antigen-presenting cells and play a crucial role in the generation and regulation of immunity (1 2 3) . Their priming ability is acquired upon maturation and is characterized by the activation of different transcriptional factors, leading to the modulation of genes involved in cytoskeleton rearrangement, antigen processing, control of migration and regulation of inflammatory responses (4 5 6 7) . Regulated migration of DCs is central to the induction of physiological immune responses and this process necessitates plasticity of the cytoskeleton. DCs, unlike other antigen-presenting cells, actively polarize their actin cytoskeleton during interaction with T cells, leading to the formation of the immunological synapse (8 , 9) . DCs are the first target of HIV and, by clustering and activating T cells, may both activate antiviral immunity and facilitate virus dissemination (10 11 12) . HIV-1 Nef protein is expressed early during infection and has been shown to be critical for viral pathogenesis in vivo (13 14 15) , enhancing viral infectivity and replication (16 , 17) . Nef exerts pleiotropic effects involving membrane-bound as well as cytoplasmic stages; depending on its intracellular localization, it interferes with cellular signal transduction pathways (18 19 20 21 22) . It is well known that Nef down-regulates the expression of MHC class I at the surface of lymphoid, monocytic and epithelial cells (23) , impairing lysis by cytotoxic T lymphocytes and thus promoting HIV’s evasion of cellular immune response (24) . Moreover, we reported that exogenous Nef inhibits the induction of a specific antibody response (25) , and this agrees the hypothesis that Nef may participate in AIDS pathogenesis by contributing to the humoral immune dysfunction. We have previously shown that exogenous Nef enters monocytes and, by up-regulating cytokine production, induces T cell bystander activation (26) . More recently, we demonstrated that Nef promotes phenotypic and functional differentiation of DCs, by up-regulating the expression of MHC class II and costimulatory/signaling molecules and the production of cytokines and chemokines. It also promotes the efficient activation of CD4+ T cells by immature DCs while down-regulating MHC class I molecules (27) . These results emphasize how Nef would favor viral spread instead of anti-viral immunity. Our hypothesis is supported by several studies indicating that endogenously expressed Nef modulates immature DCs to amplify virus replication in the DC-T cell milieu (21 , 28 29 30) . In the present study we enlarged our previous result on Nef-induced DC differentiation, analyzing the effect of exogenous Nef on DC morphological maturation. To investigate more closely how Nef could influence DC maturation, we focused on the signaling pathway involved in DC activation. A potential candidate in driving the molecular events involved in DC activation may be Vav, a protein sitting on a common pathway for cytoskeletal (31 32 33) and transcription regulation (34 , 35) that has been demonstrated to interact with Nef in human T cell lines, inducing their activation (36) . Vav activity is strictly controlled by its tyrosine phosphorylation (32 , 37) . Whereas it is clear that phosphorylated (P) -Vav localizes in the peripheral cytoplasm beneath the plasma membrane, total Vav is localized in both cytoplasm and nucleus (38) . The best-known function of Vav is its role as guanine nucleotide exchange factor (GEF) for Rho family proteins. The Rho family of GTP binding proteins, including Rho, Rac1, and Cdc42, plays a different but essential role in regulating the formation of dendritic processes in DCs (39 40 41) , and the Vav/Rac pathway is involved in nuclear factor (NF) -{kappa}B activation (34) .

Here we demonstrate that exogenous Nef enters immature DCs and targets Vav, activating its signaling cascade via Rac1, leading to cytoskeleton rearrangement and NF-{kappa}B activation. Thus, Vav/Rac signaling pathway triggered by Nef results in activation of immature DCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Nef protein
Recombinant Nef (HIV-1, strain ELI) protein obtained from Escherichia coli was purchased from DBA (Intracell, London, UK). In all experiments performed, Nef was used at 0.1 µg/mL. To evaluate Nef uptake, FITC conjugate Nef (Bartels, Carlsbad, CA, USA) was used at 0.1 µg/mL. Nef and FITC-Nef were endotoxin-free as tested by the Limulus assay (<0.03 U/mL).

Cell culture
DCs were generated from peripheral blood mononuclear cells (PBMC) isolated by Ficoll-Hypaque (Flow Laboratories, Hornby, Ontario) gradient separation of buffy coats obtained from healthy volunteer blood donors by the Transfusion Center of Università Degli Studi La Sapienza, Rome.

Monocytes were purified from PBMC by positive selection using magnetic cell separation columns and CD14 Microbeads (Milteny Biotec, Bergisch Gladbach, Germany). Highly enriched monocytes (>95% CD14+) were cultured at 6 x 105/mL in RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum (FCS), L-glutamine, and penicillin-streptomycin, 250 ng/mL granulocyte macrophage-colony stimulating factor (GM-CSF) (PeproTech, London, England), and 100 U/mL interleukin (IL) -4 (R&D Systems, Minneapolis, MN, USA) at 37°C for 5 days. Differentiation to DC was assessed by morphologic observation and detection of specific surface markers by flow cytometry. These cells were CD14-, CD1a+, HLA-DRintermediate, HLA-ABCintermediate, CD80low, CD86low, consistent with an immature DC phenotype (data not shown). Untreated immature DCs were used as controls. After 5 days of culture, Nef or 200 ng/mL lipopolysaccharide (LPS) (Escherichia coli serotype 0111:B4, Sigma, St. Louis, MO, USA) was added to immature DCs. LPS-treated DCs were CD83+, HLA-DRhigh, HLA-ABChigh, consistent with a mature DC phenotype (data not shown).

CD4+ T cells were purified from PBMC by positive selection using magnetic cell separation columns and CD4 Microbeads (Milteny Biotec). The resulting cell preparations were at least 99% viable by trypan blue dye exclusion. The purity of the CD4+ T cells was verified as >95% by direct staining for membrane expression of CD4 (anti-CD4 monoclonal antibody (MoAb) purchased from PharMingen, San Diego, CA, USA). To assess the specificity of the Nef-induced modulation, Nef was preincubated for 60 min at 37°C with 10 µg/mL anti-Nef MoAb isotype immunoglobulin (Ig)G1 (Intracell, London, UK) or with 10 µg/mL irrelevant anti-2, 4, 6-trinitrophenyl (TNP) antibody (Intracell), displaying the same isotype of the anti-Nef MoAb.

Flow cytometry
DCs were cultured at a density of 6 x 105/mL in complete medium for 10, 30 min, 1, 2, and 6 h at 37°C in the dark in the presence of FITC-Nef or FITC-Nef preincubated with anti-Nef MoAb. Cells were treated for 10 min at 37°C with trypsin-EDTA (Life Technologies, Paisley, Scotland) to remove any externally bound protein. In some experiments, Nef uptake was performed at 4°C. Cells were washed with cold PBS and fluorescence was analyzed by a FACScan cytometer (Becton Dickinson, San José, CA, USA) equipped with a 488 argon laser. At least 10,000 events were acquired. Data were recorded and statistically analyzed by a Macintosh computer using CellQuest Software.

Cytokine assay
Analysis of IL-12p70 concentration was measured on untreated and Nef- or LPS-treated DC supernatants. Culture supernatants were collected after 24 h and IL-12p70 was measured using a sandwich ELISA (R&D Systems) according to the manufacturer’s instructions.

NF-{kappa}B activation assay
To monitor NF-{kappa}B activation, we used the NF-{kappa}B p65/NF{kappa}B p50 transcription factor assay kits (Alexis Biochemicals, San Diego, CA, USA). Briefly, we used a 96-well plate coated with oligonucleotide containing NF-{kappa}B consensus binding site. The activated NF-{kappa}B contained in cell extracts from untreated and 30 min Nef- or LPS-treated DCs specifically binds to this oligonucleotide. By using an antibody directed against the NF-{kappa}B p65 or p50 subunit, the NF-{kappa}B complex bound to the oligonucleotide was detected. A secondary antibody conjugated to horseradish peroxidase provided color development and absorbance was quantified on a spectrophotometer at 450 nm. As positive control we used a HeLa cell extract; to monitor the specificity of the assay, we used NF-{kappa}B wild-type and mutated consensus oligonucleotides, according to manufacturer’s instructions.

Fluorescence microscopy
Untreated and Nef- or LPS-treated DCs were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature (RT). For actin analyses, cells were plated on poly-L-lysine-coated coverslips for 20 min at RT. Cells were then permeabilized with 0.5% (v/v) Triton X-100 (Sigma) in PBS for 5 min at RT and stained with fluorescein-phalloidin (Sigma), a toxin capable of direct binding to F-actin. For vinculin analyses, cells were permeabilized as described above, stained with anti-vinculin antibodies (Sigma) at 37°C for 30 min and after washing in PBS cells were incubated with anti-mouse IgG fluorescein-linked whole antibody (Sigma) at 37°C for 30 min. For Nef entry detection, untreated and Nef-treated DCs were stained with anti-Nef MoAb at 37°C for 30 min and, after washing in PBS, incubated with anti-mouse IgG fluorescein-linked whole antibody (Sigma) at 37°C for 30 min. For Vav and P-Vav localization, cells were stained with anti-Vav or anti-P-Tyr-Vav antibodies (Santa Cruz Biotechnology, San Diego, CA, USA), respectively. Cells were washed in PBS and incubated with anti-rabbit IgG fluorescein-linked whole antibody (Sigma) at 37°C for 30 min. Cells were plated on coverslips by centrifugation at 2000 rpm for 15 min with Cytospin (Shandon, Germany). All samples were mounted with glycerol:PBS (2:1) analyzed by phase contrast or fluorescence with a Nikon Microphot microscope or with intensified video microscopy (IVM) by a CCD camera (Carl Zeiss, Germany).

DC/T cell conjugates analyses
DCs were left untreated or were treated for 24 h with Nef or LPS, washed three times in RPMI 1640 medium, and mixed with allogeneic CD4+ T cells (1:4). After centrifugation at low speed (600 rpm for 5 min) in a conical tube, cell mixtures were incubated at 37°C for 24 h. Fluorescence microscopy was performed as follows: DC/T cells were fixed with 3.7% formaldehyde in PBS for 10 min at RT. After washings in the same buffer, cells were plated on poly-L-lysine-coated coverslips for 20 min at RT, permeabilized with 0.5% (v/v) Triton X-100 in PBS for 5 min at RT, then incubated with a mixture 50:50 of fluorescein-phalloidin and Hoechst 33258 fluorescent dye (Molecular Probes, Leiden, The Netherlands), a DNA intercalating probe allowing detection of cell nuclei.

Scanning electron microscopy
Untreated and Nef- or LPS-treated cells were plated on poly-L-lysine-coated coverslips for 20 min at RT and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 3% (w/v) sucrose at RT for 20 min. After postfixation in 1% OsO4 for 30 min, cells were dehydrated through graded ethanols, critical point dried in CO2, and gold coated by sputtering. The samples were examined with a Cambridge 360 scanning electron microscope.

Confocal laser scanning microscopy analyses
For confocal laser scanning microscopy (CLSM) analyses, DCs untreated or treated with FITC-Nef, Nef, or LPS were seeded on poly-L-lysine hydrobromide-coated cover glass for 15 min at 37°C, fixed, and permeabilized as described above. Cells were then stained with an anti-Vav MoAb. The extensively rinsed cover glass was then mounted on the microscope slide with the ProLong reagent (Molecular Probes). Conversely FITC-Nef treated DCs were fixed and directly mounted on the microscope slide. CLSM observations were performed using a Leica TCS 4D apparatus equipped with an argon-krypton laser, 488 nm dichroic splitter, LP515 long-pass filter. Image acquisition and processing were conducted using the SCANware (Leica Lasertechnik, Heidelberg, Germany) and Adobe Photoshop software.

Western blots and affinity precipitation
For detection of total Vav and P-Vav levels, DCs were left untreated or were treated with Nef or LPS for 15 min, 1 h or 24 h, and lysed in cold lysis buffer (20 mM HEPES, 50 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.5% NP-40, containing protease and phosphatase inhibitors) for 20 min on ice. Samples were loaded on 8% SDS-PAGE and analyzed by Western blot using anti-Vav or anti-P-Vav antibodies. Immunoreactive bands were visualized by using secondary horseradish peroxidase-conjugated antibodies and Opti-4CNT Detection Kit (Bio-Rad, Hercules, CA, USA).

For affinity precipitation of active Rac1 and Cdc42, untreated and 24 h Nef- or LPS-treated DCs were lysed and immunoprecipitated using the Activation Assay (Cytoskeleton, Denver, CO, USA) according to the manufacturer’s instructions. Cells were resuspended at 107cell/mL in lysis buffer. GST-p21-activated kinase fusion protein (GST-PAK) was added to each sample, followed by incubation with glutathione beads. The complex GTP-Rac1- or GTP-Cdc42-PAK-glutathione bead was pulled down and analyzed by Western blot. Control lysates were treated with GDP or GTP{gamma}S before GST-PAK precipitation. Immunoreactive bands were visualized as described above. Densitometric analysis was performed as described previously (27) . In some experiments, DCs were pretreated for 30 min at 37°C with 10 µM PP1 [4-ammino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine] (Alexis Biochemicals) in order to inhibit Src tyrosine kinases (42) .

Statistical analysis
Statistical analysis was calculated using a two-tailed Student’s t test. A P value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nef is efficiently taken up by immature DCs and localizes in the cytoplasm
The uptake of Nef by DCs was evaluated by flow cytometry using FITC-conjugate Nef protein. Nef uptake was very efficient and occurred in a time-dependent fashion (Fig. 1 A). Uptake was already detectable after 10 min of treatment (21±4% of Nef-positive cells), increasing after 30 min (44±4%) and 1 h (76±6%) and reaching peak levels after 2 h (96±2%). The level of staining was reduced after 6 h (50±6%). No staining was observed in immature DCs treated with FITC-conjugated anti-IgG MoAb (data not shown) used as control for fluorescein isothiocyanate molecule. In addition, detected Nef was almost entirely intracellular because no difference was observed with trypsin-EDTA treated DCs (data not shown). Nef uptake by DCs after 2 h was greatly reduced at 4°C compared with 37°C either as percentage of stained cells or as mean fluorescence intensity. Because low temperature reduces all energy-dependent processes, including receptor-mediated endocytosis, we hypothesized that Nef uptake was likely mediated by specific receptors. Nef uptake decreased in mature DCs (LPS stimulated CD83+, HLA-DRhigh, HLA-ABChigh) and was abolished by anti-Nef MoAb treatment (data not shown).



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  		Figure 1. Nef is efficiently taken up by DCs and localizes in the cytoplasm. A) Immature DCs were left untreated (CTR) or treated with FITC-Nef at 37°C for 10, 30 min, 1, 2, and 6 h in the dark. Nef uptake after 2 h of treatment was analyzed at 4°C as well. Nef-positive cells were evaluated by flow cytometry. B) CLSM analysis of Nef distribution in immature DCs incubated for 2 h at 37°C with FITC-Nef. The insert shows a single confocal central optical cell section. C) High magnification (3200x) IVM analysis of FITC-Nef distribution in immature DCs. Nef is distributed in apical cytoplasmic regions while Golgi region appears as negative. Morphological features of this cell are visible in the corresponding phase contrast micrograph in the insert (1200x). In all panels, representative results from 1 of 5 donors giving similar results are shown.

We previously demonstrated that Nef efficiently enters monocytes (CD14+ cells) (26) . In contrast to DCs and monocytes, the uptake of Nef by T (CD4+ and CD8+), B (CD19+) and NK (CD56+) cells was much less efficient. In fact, after 2 h Nef treatment, low intracellular staining was observed in T, B and NK cells with values much lower than those obtained in DCs (1±0.4%, 4±1.1%, and 2±1.1% in T, B, and NK cells, respectively, vs. 96±2% in DCs). Thus, efficient uptake of Nef is a selective feature of monocytes and DCs, two cellular populations that have a close ontogenic relationship (43) .

We also set up experiments to investigate the subcellular localization of Nef in immature DCs using CLSM and IVM. Using CLSM analysis, after 2 h treatment Nef was detected in most cells as positive spots scattered throughout the cell cytoplasm (Fig. 1B ). Accordingly, high magnification IVM analysis clearly shows the marginalized distribution of Nef protein in DC cytoplasm (Fig. 1C ; corresponding phase contrast microscopy in the insert).

Nef enhances IL-12p70 production by the activation of NF-{kappa}B in immature DCs
We previously demonstrated that Nef induces a coordinated series of phenotypic and functional changes promoting DC differentiation (27) . According to our recent data, Nef and LPS triggered statistically significant up-regulation of IL-12p70 secretion compared with untreated DCs (Fig. 2 A). Moreover, Nef up-regulated IL-12 mRNA transcription rapidly, within 6 h of treatment (data not shown). Preincubation of Nef with a neutralizing anti-Nef MoAb, but not with an irrelevant anti-TNP antibody displaying the same isotype of the monoclonal anti-Nef, abrogated the Nef-induced effect. Thus, the up-regulation of IL-12 secretion was a Nef-specific effect.



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  		Figure 2. Nef enhances IL-12p70 release and activates NF-{kappa}B in immature DCs. A) DCs were left untreated (CTR) or were treated with Nef, LPS, Nef + {alpha}-Nef, or Nef + {alpha}-TNP for 24 h. Supernatants were assayed for the levels of IL-12p70 by ELISA. Means ± SE from 5 different donors are shown. *P < 0.05 vs. control. The effect of boiled Nef was tested and the results obtained were comparable to control values. B, C) Immature DCs were left untreated (CTR) or were treated with Nef, Nef + {alpha}-Nef or with Nef + {alpha}-TNP for 30 min. Cells were lysed and, after protein content quantification, equal amounts of lysates were used to test activated levels of both p50 (B) and p65 (C) subunits of NF-{kappa}B for each sample. HeLa cell extract was used as positive control alone or in the presence of WT or mutated (MUT) consensus oligonucleotide. Mean values ± SE from 3 different donors are shown.

The rates of NF-{kappa}B activation in total lysates from untreated and Nef- or LPS-treated DCs was analyzed by measuring the levels of both p50 and p65 subunits of NF-{kappa}B, capable of binding an oligonucleotide containing the NF-{kappa}B consensus binding site. As shown in Fig. 2B, C , Nef scored a 1.8-fold increase of active p50 level and a 2.2-fold increase of p65 level compared with untreated DCs. These levels were only slightly inferior to those obtained with LPS treatment: 2.4-fold and 2.6-fold increase compared with untreated DCs for p50 and p65, respectively. These effects were lost when Nef was preincubated with anti-Nef MoAb but maintained if preincubated with anti-TNP antibody. As positive control, we showed levels of activation of NF-{kappa}B in HeLa cell extracts. The assay was specific, since incubation of HeLa cell extract in presence of a nonbound wild-type consensus oligonucleotide abolished binding of both subunits, whereas with the mutated consensus oligonucleotide the binding was equal to control HeLa cells.

Nef induces morphological changes and rearrangement of the microfilament system in immature DCs
DC maturation is accompanied by morphological changes, including acquisition of high cellular motility associated with loss of adhesive structures and cytoskeleton reorganization (4) . We investigated whether the Nef-induced DC phenotypic and functional differentiation occurs with morphological remodeling. Figure 3 shows that 24 h Nef-treated DCs developed a series of evident modifications of cell surface structures as detected by SEM analysis. In fact, while untreated immature DCs (controls) showed a roundish morphology with small microvillous structures (Fig. 3a ), Nef-treated DCs underwent a series of structural changes mainly represented by the formation of uropods and ruffles (Fig. 3b ). Similar cell surface modifications were detectable in mature DCs (Fig. 3c ). Cytoskeleton rearrangement is considered a prerequisite for the occurrence of DC morphological remodeling, associated with their maturation. Microfilament network plays a key role in the formation of both uropods and surface ruffling (4) . Thus, we analyzed the F-actin network in immature DCs. Actin filaments appeared remarkably rearranged in both Nef- and LPS-treated DCs (Fig. 3e, f ) compared with untreated DCs (Fig. 3d ). A number of cytoplasmic peripheral structures corresponding to ruffles and protrusions were detected (Fig. 3e, f ). We then evaluated the distribution of the microfilament-associated protein vinculin, a molecule important in DC adhesion, motility, and maturation (44) . We found that Nef induced a marked polarization of vinculin at one pole of DCs (Fig. 3h )—at the substrate-adhering portion of cell, i.e., at the leading edge (45) . A similar vinculin distribution was observed in LPS-treated DCs (Fig. 3i ). After 48 h, a partial depolymerization of F-actin network and loss of vinculin-containing adhesion-related structures were detected (data not shown). Preincubation of Nef with a neutralizing anti-Nef MoAb, but not with an irrelevant anti-TNP antibody, abrogated the effect of Nef. This confirms that the observed DC morphological differentiation was indeed a Nef-specific effect.



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  		Figure 3. Nef induces both morphological changes and actin cytoskeleton and vinculin rearrangement. Morphological analysis of untreated (a), Nef- (b), and LPS-treated (c) DCs was performed by SEM. Untreated DCs (CTR) displayed a round shape with small villous protrusions (a) while 24 h Nef and LPS treatments induced DC morphological remodeling characterized by uropod formation and surface ruffling (arrow) (b, c). Cytoskeleton rearrangement of untreated (d, g), Nef- (e, h), and LPS-treated (f, i) DCs was performed by IVM analysis. Actin was poorly expressed throughout the cytoplasm of untreated DCs (d); after 24 h of Nef (e) and LPS (f) treatments, actin filaments appeared remarkably rearranged at the cell periphery (arrow), strongly reminiscent of the ruffles detected by SEM analyses (b, c). Vinculin distribution in untreated DCs was characterized by isolated dot spots in the proximity of plasma membrane (g), while 24 h Nef (h) and LPS treatments (i) induced a marked polarization of vinculin in cortical DC cytoplasm. Magnification: a–c) 3700x; d–i) 3000x.

Nef improves immunological synapse formation between DCs and CD4+ T cells
As DCs actively polarize their actin cytoskeleton during interaction with T cells, we asked whether Nef affects the ability of DCs to form clusters with T cells. To this aim, allogeneic CD4+ T cells were added to untreated and Nef- or LPS-treated DCs at a fixed ratio (4:1). Cluster formation and F-actin polarization were evaluated. DCs appeared as large actin-positive cells with nuclei hidden by actin green fluorescence whereas lymphocytes were smaller than DCs with a thin subcortical actin ring and blue-labeled nuclei. When DCs were left untreated only a few DC/T cell clusters were visible, each containing few lymphocytes (Fig. 4 a). After Nef treatment, the ability of DCs to form clusters was dramatically increased and characterized by the recruitment of numerous T cells and a pronounced focal polarization of F-actin toward the DC/T cell contact sites (Fig. 4b ). Long DC protrusions forming T cell contacting area, i.e., the immunological synapse, can also be observed (Fig. 4c ). Similar results were obtained in cocultures of LPS-treated and allogeneic CD4+ T cells (Fig. 4d ).



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  		Figure 4. Nef improves immunological synapse formation between DCs and allogeneic CD4+ T cells. Cluster formation and F-actin polarization in conjugates between untreated (a), Nef- (b, c), or LPS-treated (d) DCs and allogeneic CD4+ T cells were evaluated by IVM analyses. Cells were stained with an actin-labeling toxin and the nuclear staining fluorescent dye Hoechst 33258. In untreated DCs a poorly developed actin network and only a few DC/T cell clusters are visible (a). After 24 h Nef treatment, the ability of DCs to form clusters was dramatically increased and was characterized by the recruitment of numerous T cells (b). A pronounced focal polarization of F-actin toward the DC/T cell contact site was readily visible (arrow) (b). A long dendritic protrusion contacting a T cell, i.e., the immunological synapse, can be observed in (c) (arrow). Similar features can be detected in clusters obtained in cocultures of LPS-treated DCs and allogeneic CD4+ T cells (d).

Nef enhances nucleotide exchange rate of Rac1 and Cdc42
Given the key role of GTP binding Rho proteins in cytoskeleton rearrangement (39) and NF-{kappa}B activation (46) , we evaluated the activation of Rac1, a surface ruffling-associated molecule, and Cdc42, a dendrite- and pseudopod-associated molecule (47) . To directly measure activation of Rac1 and Cdc42 in total lysates, we used a fusion protein comprising a PAK CRIB domain fused to GST that specifically binds Rac-GTP and Cdc42-GTP but not the inactive GDP-bound forms. As shown in Fig. 5 , Nef treatment induced a 2.6-fold increase of active Rac1 levels and a 1.4-fold increase of active Cdc42 levels compared with controls. Notably, the Nef-induced up-regulation of GTP-Rac1 level was higher than that induced by LPS (1.6-fold increase), whereas Nef-induced up-regulation of GTP-Cdc42 level was comparable to that induced by LPS. Treatment with anti-Nef MoAb abolished the Nef-induced activation of Rac1 and Cdc42 whereas treatment with anti-TNP antibody did not. Nef-induced effects were not due to LPS contaminating the Nef preparation, since heat-inactivated Nef did not show any effects (data not shown). Furthermore, Nef and LPS treatments did not induce any variation in the quantitative expression of total Rac1 and Cdc42 proteins as assessed by Western blot and flow cytometry (data not shown).



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  		Figure 5. Nef enhances nucleotide exchange rate of Rac1 and Cdc42. DCs left untreated (CTR) or treated for 24 h with Nef or LPS were lysed and affinity precipitated with GST-PAK. Bound proteins were probed on Western blots with anti-Rac1 and anti-Cdc42 polyclonal antibodies. The lower blots show the analysis of 10% of a lysate for levels of total Rac1 or Cdc42 protein. The results are expressed as fold induction (F.I.) over the basal level of active Rac1 and Cdc42 of untreated DCs. Representative blots from 1 of 4 donors giving similar results are shown.

Nef promotes Vav nucleus-to-cytoplasm redistribution and activates Vav phosphorylation in immature DCs
To analyze whether Nef signaling influences subcellular Vav distribution, a kinetic analysis was carried out. As shown in Fig. 6 a, Vav was mainly localized in the nuclear compartment of untreated DCs. A similar distribution was observed after 15 and 30 min of Nef or LPS treatment (data not shown). After 2 h, Nef and LPS triggered translocation of Vav from the nuclear to the cytoplasmic compartment (Fig. 6b, c ). Cytospin preparation clearly showed that Vav was localized inside the nuclear matrix of untreated DCs (Fig. 6d ). After 2 h of Nef and LPS treatment, positive scattered dot spots were easily visible in the cytoplasm (Fig. 6e, f ).



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  		Figure 6. Nef promotes Vav nucleus-to-cytoplasm redistribution in immature DCs. CLSM (left panels) and IVM analyses (right panels) of Vav distribution were performed on untreated (a, d) Nef- (b, e), and LPS- (c, f) treated DCs. Vav was mainly localized in the nucleus of untreated DCs (a, d) whereas after 2 h of Nef (b, e) and LPS (c, f) treatment, a clear redistribution of Vav was detected, namely, while the DC nucleus appeared quite negative in Nef- (b, e) and LPS-treated (c, f) DCs, a well-evident cytoplasmic positivity was observed. Magnification: a–c) 1600x; d–f) 3000x.

To examine whether Nef activates Vav, we monitored the tyrosine phosphorylation of Vav. We compared levels of total and P-Vav from lysates of untreated and Nef- or LPS-treated DCs. We found that although total Vav was unchanged, the rate of phosphorylation, and therefore the level of potentially active Vav, was rapidly and transiently up-regulated by Nef. As shown in Fig. 7 A, after 15 min Nef and LPS induced 4.1-fold and 3.2-fold increase of tyrosine phosphorylation of Vav, respectively (lane 2 and 3 vs. lane 1). The increased phosphorylation of Vav induced by Nef and LPS was still evident after 1 h and decreased to control levels after 24 h. Notably, these effects were lost when Nef was preincubated with anti-Nef MoAb, but not with anti-TNP antibody, confirming that the increased Vav phosphorylation was indeed a specific effect of Nef (data not shown). IVM analyses clearly indicated that P-Vav was mainly localized in the cortical cytoplasm beneath the DC plasma membrane (Fig. 7C-E ).



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  		Figure 7. Nef induces Vav phosphorylation and its localization at the plasma membrane in immature DCs. A) DCs were left untreated (CTR) or were treated for 15 min, 1 or 24 h with Nef or LPS. Cells (107cell/mL) were lysed and proteins were probed on Western blots with anti-P-Vav or anti-Vav polyclonal antibodies. Lower blot shows the analysis of 10% of a lysate for levels of total Vav protein. Results are expressed as fold induction (F.I.) over the basal level of P-Vav of control cells. B) DCs pretreated for 30 min with 10 µM PP1 were left untreated or were treated for 15 min or 24 h with Nef. Cells (107cell/mL) were lysed and P-Vav or active Rac1 levels were determined as described above. C–E) IVM analyses of P-Vav distribution in untreated (C) or 15 min Nef- (D) and LPS-treated (E) DCs. Untreated cells were negative while both Nef- and LPS-treated DCs showed a marked polarization of P-Vav in the plasma membrane region. Representative results from 1 of 3 donors giving similar results are shown.

Since tyrosine phosphorylation of Vav is required for its GEF activity, we were interested in establishing a direct correlation between the Nef-induced Rac1 activation and Vav phosphorylation. To this aim, we used the inhibitor of Src kinase PP1 (42) in order to block the main pathway leading to Vav phosphorylation. As shown in Fig. 7B , PP1 treatment abolished the Nef-induced up-regulation of Vav phosphorylation and concomitantly abrogated the Nef-induced Rac1 activation. In both cases, activity values were similar to those of untreated DCs. Altogether, these data clearly indicate that Nef increases the level of P-Vav, which in turn activates Rac1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we outline the Nef-triggered signaling pathway leading to DC maturation. We found that Nef targets Vav and promotes its tyrosine phosphorylation associated with its nucleus-to-cytoplasm redistribution. This has a direct effect on Vav GEF activity for the small GTPase Rac1, which in turn triggers DC morphological and functional maturation. These results are summarized in the model presented in Fig. 8 .



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  		Figure 8. Signaling events in Nef-induced DC maturation. Nef enters immature DCs leading to Vav phosphorylation. This is achieved by either direct interaction with Vav or an upstream activation of Src kinases, which in turn phosphorylate Vav. P-Vav is then able to activate Rac1 and Cdc42 proteins that trigger a series of downstream signaling events. Those include rearrangement in the actin and vinculin cytoskeleton, leading to morphological changes, and NF-{kappa}B translocation to the nucleus, which yields DC phenotypical and functional differentiation. In this scenario, Nef changes the local environment of the DCs resulting in their activation.

The amount of exogenous Nef used in our experiments is close to that detected in the sera of AIDS patients (48) ; higher amounts of exogenous Nef could be present in tissue compartments such as lymph nodes, where DCs and lymphocytes closely interact (49) . Highly immunogenic soluble Nef raises both humoral (50) and cell-mediated immune responses (51 , 52) . However, exogenous Nef significance with regard to AIDS immunopathogenesis has not been completely recognized. It has been demonstrated that exogenous Nef induces normal human PBMC proliferation (53) . We have demonstrated that exogenous Nef is efficiently taken up by monocytes and activates the proliferation of PBMC through induction of IL-15 release (26) . It has been reported that Nef induces STAT-1 activation in macrophages when expressed either endogenously or after exogenous protein internalization (20) . More recently, it has been reported that exogenous Nef protein activates NF-{kappa}B, AP-1, and JNK, stimulating HIV transcription in the U937 cell line (54) . In the DC system we demonstrated that exogenous Nef induces a coordinated series of phenotypic and functional changes promoting DC differentiation (27) . Similarly, endogenously expressed Nef activates immature DCs (21 , 28 29 30) . Altogether, these data indicate that exogenous Nef induces effects comparable to those described for endogenous Nef. Nevertheless, endogenously derived Nef uncouples the secretion of inflammatory cytokines and chemokines from triggering membrane phenotypic maturation in immature DCs (28) . This contrasts with what was observed when Nef was provided exogenously (27) . Exogenous or endogenous Nef does not affect phenotype and function of mature DCs (27 , 55)

We show here that exogenous Nef is efficiently taken up by immature DCs and localizes in the cytoplasm. It has been reported that in human B and T lymphocytes, recombinant HIV-1 Nef noncovalently associates with actin (56) in a myristoylation-dependent process whereas in human astrocytes nonmyristoylated Nef colocalizes with astrocyte-specific cytoskeleton protein GFAP (57) . Therefore, we hypothesized that DCs could retain Nef in the cytoplasm, probably interfering with a cytoskeletal protein. Recombinant Nef was obtained from a bacterial host and thus was not amino-terminally myristoylated. However, the recombinant protein contains the myristoylation signal and therefore we cannot rule out an intracellular myristoylation of Nef.

The observation that Nef is efficiently internalized by DCs led us to evaluate its effects on these cells. DCs pick up extracellular Nef possibly released by virions (58 , 59) or by apoptotic HIV-infected lymphocytes in those tissues (i.e., lymph nodes) where high levels of HIV replication is associated with close interaction between DCs and T lymphocytes. The effects of Nef on immature DCs deserve attention considering that mucosal DCs and blood DC-SIGN+ DCs represent the first HIV-1 targets upon sexual transmission (60) and transmission via blood (61) , respectively. Once DCs are mature, they activate CD4+ T cells, promote virus amplification, and spread. Hence, we performed an extensive study using immature DCs as the closest model for in vivo primary HIV infection to investigate the effect of Nef on DC maturation. We found that Nef enhances IL-12p70 release by immature DCs and this result is in line with our initial study (27) . Here we found that Nef cytoskeletal rearrangement in immature DCs render them more like their mature counterpart. Thus, Nef activates immature DCs manipulating their phenotypical, morphological, and functional developmental program.

It has been demonstrated that unlike other antigen-presenting cells, DCs actively polarize their actin cytoskeleton during interaction with T cells (8) . DC cytoskeletal rearrangement was critical for both the clustering and activation of resting T cells, playing a key role in the establishment of the immunological synapse. We found that the Nef-induced actin rearrangement increases the capacity of DCs to form clusters with T cells, improving the immunological synapse formation. Moreover, DC-T cell interaction could be influenced by cytokine and chemokine secretion profiles (here and our previous study, ref 27 ) required for recruitment and successful activation of T cells. The increase of DC-T cells clustering may also involve up-regulation of DC-SIGN expression induced by Nef as reported by N. Sol-Foulon et al. (29) . Experiments are under way to assess this phenomenon in our system.

Differences in gene expression are indicative of morphological, phenotypical, and functional changes induced in a cell by environmental factors and perturbations (62 63 64) . In particular, NF-{kappa}B activation has been shown to be required for DC maturation. We found that Nef induces NF-{kappa}B activation, and our result concurs with previous reports indicating that both endogenously expressed and exogenous Nef activate NF-{kappa}B in immature DCs and the U937 promonocyte cell line, respectively (21 , 54) . Therefore, the Nef-triggered activation of immature DCs as monitored by up-regulation of surface molecules, secretion of soluble factors and immunostimulatory capacity closely fits with the activation of the NF-{kappa}B transcriptional factor (Fig. 2 and ref 27 ).

Moreover, we show that Nef drives DC maturation by targeting Vav, a molecule that sits on a common pathway for cytoskeletal and transcriptional regulation. The interaction of Nef with Vav has recently been described in T cells (36) . Here we found that in immature DCs, Vav is mainly localized in the nuclear compartment. A nuclear localization of Vav in T cell lymphomas, granulocytes, and megakaryoblastic cells even in the absence of any stimulus has been reported (65 , 66) . Nef signaling induces nucleus to cytoplasm redistribution of Vav and its phosphorylation. Via its DH domain, Vav has been shown to catalyze the GDP-GTP exchange of Rho family GTPases, including Rho, Rac1, and Cdc42. It is well known that Rho family GTPases play a different but essential role in both cytoskeleton rearrangement and NF-{kappa}B activation (37 , 34) . We demonstrate that Nef induces Rac1 and, to a lesser extent, Cdc42 activation in immature DCs, likely ascribable to the increased Vav GEF-activity.

It is well established that Nef can interact through its SH3 homology domain with cellular Src kinases such as Lck and Hck in monocytes/macrophages facilitating infection in the T cell compartment (67) . To verify whether the Nef-induced nucleotide-exchange rate of Rac1 is enhanced by Vav tyrosine phosphorylation, we treated immature DCs with PP1, which inhibits the Src kinases (42) and therefore the major Vav-activating pathway. We found that upon inhibition of Src kinase activity, nucleotide exchange on Rac1 was correlated with Vav phosphorylation. These results strongly suggest that Nef triggers a central Src family tyrosine kinase cascade with a network of downstream signaling that may be ascribed to Vav phosphorylation.

This work outlines a novel Nef-induced, Vav-dependent signaling pathway by which HIV-1 may exploit the DC arm of the immune system favoring nonspecific T cell activation, increasing the pool of lymphocytes permissive to infection. Therefore, Nef plays a crucial role in the immunopathogenesis of AIDS ensuring viral replication and dissemination.

In conclusion, the Nef-induced signaling pathway regulating DC functional and morphological differentiation could provide opportunities for therapeutic manipulation of immune responses in vivo.


   ACKNOWLEDGMENTS
 
This work was supported by a grant from the National Research Project on AIDS to M.V. and W.M.

Received for publication March 18, 2003. Accepted for publication June 26, 2003.


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 RESULTS
 DISCUSSION
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