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(The FASEB Journal. 2002;16:546-554.)
© 2002 FASEB

HIV-1 Tat protein induces interleukin-10 in human peripheral blood monocytes: involvement of protein kinase C-ßII and -{delta}

YAMINA BENNASSER and ELMOSTAFA BAHRAOUI1

Laboratoire d’Immuno-Virologie, EA 3038, Université Paul Sabatier, 31062 Toulouse, France

1Correspondence: Laboratoire d’Immuno-Virologie EA 3038, Université Paul Sabatier 118, route de Narbonne batiment 4R3 31062 Toulouse, France. E-mail: bahraoui{at}cict.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In HIV-infected patients, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with the disease progression toward AIDS. We have previously shown that HIV-1 Tat induces IL-10 production by human monocytes via a protein kinase C (PKC) -dependent pathway. Here we show that PKC activation by Tat is essential for IL-10 induction. Among the eight PKC isoforms present in human monocytes, we investigated which isoform(s) plays this crucial role in Tat-mediated IL-10 production and show that 1) Tat can activate PKC-{alpha}, PKC-ßII, PKC-{delta}, and PKC-{varepsilon}, 2) of these four potential candidates, only PKC-ßII, PKC-{delta}, and PKC-{varepsilon} are activated by the active domain Tat 1–45, which is responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-{alpha} and PKC-{varepsilon} by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-ßII or PKC-{delta} partially inhibits IL-10 production; and 4) the simultaneous inhibition of PKC-ßII and PKC-{delta} totally inhibits Tat-mediated IL-10. Altogether, these results suggest that the induction of IL-10 by Tat is strictly dependent on the PKC-{delta} and -ßII isoforms.—Bennasser, Y., Bahraoui, E. HIV-1 Tat protein induces interleukin 10 in human peripheral blood monocytes: involvement of protein kinase C-ßII and -{delta}.


Key Words: IL-10 • PKC isoforms • uninfected monocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV-1 INFECTION IS associated with two major disorders: immunological dysfunctions as early as the asymptomatic stage, and CD4 + T cell depletion at a late stage of infection (1 2 3 4) . Peripheral blood mononuclear cells (PBMC) of HIV-1-infected patients produce high levels of interleukin-10 (IL-10) paralleling the progressive loss of T helper function as the infection progresses toward AIDS (5 , 6) . The cellular and molecular mechanisms of this attenuation of the immune system cannot be explained solely by the direct effect of the virus on T CD4-infected cells, but would involve viral factors.

One candidate is HIV-1 Tat protein. We recently showed that by acting at the cell membrane level, HIV-1 Tat protein induces production of IL-10 by human peripheral blood monocytes (7) . HIV-1 Tat, a 14 kDa protein, is found in infected cells where it plays an essential role in viral replication (8 9 10) and as a protein secreted in the plasma of HIV-infected patients. Tat can thus act on other cells whether or not they are infected (11) . Tat contributes to immune cell disorders by inducing apoptosis of T lymphocytes and interferes with the cell-mediated immune response by inhibiting the expression of MHC class I molecule, as reported for Jurkat cells, NK cell activity, and IL-12 production by dendritic cells and monocytes. Tat protein contains a nuclear localization and a basic region, and so may exhibit an intracellular activity. We recently showed that Tat induces the production of IL-10. It acts at the level of monocyte membranes, since Tat immobilized in culture wells and the amino-terminal Tat 1–45 mutant, whose basic region is deleted, remain capable of inducing IL-10 (7) .

Analysis of signal transduction pathways strongly suggested that protein kinase C (PKC) plays a crucial role in this induction. PKC belong to the serine/threonine kinase family, a key element recruited to regulate cell responses to external stimuli (12 13 14) . When a ligand binds to a receptor on the cell surface, inositol phospholipids are hydrolyzed, producing diacylglycerol (DAG) and inositol triphosphate, both acting as ‘second messenger’ (15) . Subsequently, DAG activates the PKC, which in turn phosphorylates a range of cellular proteins (16) . Eleven PKC isoforms have been identified and classified into three groups based on their ability to be activated by Ca2+ and DAG (12) . The classical PKC-{alpha}, -ßI, -ßII, and -{gamma} isoforms are activated by Ca2+ and DAG; the novel PKC-{theta}, -{eta}, -{delta}, -{varepsilon} are Ca2+ independent but DAG dependent; finally, the atypical PKC -{zeta}, -{iota} (also named {lambda} in murine cells) are Ca2+ and DAG independent (16) . PKC are single polypeptides chains of heterogeneous size, ranging from 67.2 kDa for PKC-{iota} to 83.5 kDa for PKC-{varepsilon}; each PKC isoform is the product of a separate gene except for PKC-ßI and PKC-ßII, which are alternative spliced variants of the same gene. At the structural level, PKC contains four conserved domains (C1 to C4) and five variable regions (V1 to V5) that encode isoform-specific properties (17) . The conserved regions mediate binding to the activating cofactors: C1 for Ca2+, C2 for DAG, or to PMA when used as a pharmacological tool. At the functional level, activation of PKC is mediated by phosphatidylserine (PS) binding domains that include C2, the basic pseudosubstrate peptide, and the cysteine-rich region that contributes to the increased affinity of PKC to PS by interacting with DAG (18) . These PS binding domains have also been reported to be involved in affinity, PKC localization, membrane translocation, and binding to potential PKC receptors. In resting cells, PKC is localized in the cytoplasm in an inactive form where the pseudosubstrate sequence upstream from C1 binds to the catalytic domain, but cannot be phosphorylated since it lacks a phosphoreceptor amino acid. Upon activation, Ca2+ and/or phosphoinositides induce a conformational change, where pseudosubstrate is released and the catalytic site becomes accessible to anchoring proteins called RACK (receptor for activated kinase) or substrates STICK (substrate that interact with c-kinase). This PKC activation is accompanied by a translocation from the cytoplasm to the membrane (15) . Subsequent to membrane translocation and PKC activation, a second messenger seems to be stimulated and leads to the phosphorylation of PKC binding proteins, thus reducing their affinity for PKC and PS (19 , 20) . The translocation of PKC can be used as an index of enzyme activation (12 , 13) .

We have recently shown that PKC is required for Tat-induced IL-10 production, a highly immunosuppressive cytokine that could contribute to the immune dysregulation observed at the asymptomatic stage, as suggested by in vitro experiments showing the capacity of anti-IL-10 antibodies to restore the T helper cell response of HIV-1-infected patients to antigens (5) . Tat stimulation activates the nuclear translocation of transcription factor NF{kappa}B and induces the phosphorylation of MAP kinases ERK 1/2, two PKC substrates whose activation appears necessary for IL-10 production (7) .

Of the 11 PKC isoforms characterized, 8 are present in the human monocyte: PKC-{alpha}, -ßI, -ßII, -{delta}, -{varepsilon}, -{eta}, -{theta}, and -{zeta}. The aim of the present study was to investigate which one(s) plays a crucial role in Tat-induced IL-10 production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant HIV-1 Tat proteins
Recombinant HIV-1 Tat protein was obtained from ‘Agence Nationale de la Recherche sur le SIDA’ (Paris, France). The level of endotoxin contamination in purified HIV-1 Tat was assessed by using the Limulus amebocyte lysate assay (BioSepra, Villeneuve la Garenne, France).

HIV-Tat mutants were produced as glutathione-S-transferase (GST) fusion proteins in Escherichia coli. Wild-type GST-Tat 1–101 and Tat deleted mutants GST-Tat 1–45 were purified as described (7) . As a control, GST was purified under the same conditions and used in the same experiments. All these constructions are LPS free (<0.3 EU/µg) and biologically active, as described (21) .

Monocyte isolation
PBMC were isolated from the buffy coat of healthy HIV-negative donors in a Ficoll density gradient (Pharmacia, Piscataway, NJ). PBMC were resuspended in 60/30 complete medium (60% AIM V and 30% Iscove [Gibco-BRL, Grand Island, NY]) containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% FCS. PBMC were plated at a density of 106 cells/well in 24-well Primaria (Becton Dickinson, Rutherford, NJ) tissue culture plates. After 24 h of culture at 37°C in 5% CO2, nonadherent cells were removed, the remaining cells were washed twice, then incubated with the different compounds tested.

Protein kinase C inhibition
PKC inhibitors
Isolated monocytes were cultured in the absence or presence of HIV-1 Tat protein. HIV-1 Tat (3.6x10-5 M) was prepared as stock solutions in PBS, and further dilutions were prepared in FCS-free medium. Monocytes were preincubated for 30 min with RO-31 8220, a PKC inhibitor (22) , hispidin, a PKC-ß inhibitor (23) , or rottlerin, a PKC-{delta} inhibitor (24) ; HIV-1 Tat (10 nM) was added for an additional 24 h. A putative cytotoxic effect of the different inhibitors was tested by the trypan blue dye exclusion assay and none was found to be cytotoxic (viability was >90%) at the concentrations used.

PKC depletion
Monocytes were depleted of PKC by lengthy treatment (48 h) with PMA (phorbol-12-myristate-13-acetate) (Calbiochem), a PKC activator. PMA was prepared at 1.62 x 10-3 M in DMSO; further dilution (100 ng/ml) was prepared in FCS-free medium.

Treatment of monocytes with oligonucleotides
Phosphorothioate oligonucleotides were synthesized by Genset Oligos (Genset SA, France). The sequences of antisense oligodeoxynucleotides for PKC-{alpha}, -ßII, -{delta}, and -{varepsilon} and the control sense are listed in Table 1 . Sequences specific to each PKC isoform were selected from areas of mRNA that are relatively free of secondary structures as predicted by Mufold and screened for uniqueness using Blast (25) . In each case, the selected sequence was tested for lack of internal secondary structure and oligo pairing using Mufold (25 , 26) . Monocytes were plated at a density of 2.106 cells/ml in Iscove/AimV supplemented with 1% FCS and treated with oligonucleotide sense or antisense for 20 h. Monocytes were washed and incubated with oligonucleotide, and Tat (10 nM) was added. After 1 h, isoform-specific inhibition was assessed by Western blot; 24 h later, IL-10 production was measured by enzyme-linked immunosorbent assay (ELISA).


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  		Table 1. Antisense oligodeoxynucleotide sequences

IL-10 detection by ELISA
IL-10 production was quantified using a two-site sandwich ELISA. MAB217 (R&D Systems, Oxon, UK) monoclonal antibody (4 µg/ml) was used for capture overnight at room temperature. After three washes with PBS containing 0.05% Tween 20 (wash buffer), plates were blocked by adding 300 µl of PBS containing 1% BSA and 5% sucrose to each well for a minimum of 1 h. After three washes, culture supernatants (100 µl/well) were incubated for 2 h at room temperature. Plates were washed three times and incubated with biotinylated anti-human IL-10 polyclonal antibody (BAF217) obtained from R&D Systems for 2 h at room temperature. After washing, the bound biotinylated polyclonal antibody was visualized by an additional 20 min incubation with streptavidin peroxidase (Sigma, Saint Quentin Fallavier, France) diluted 1/16,000 in PBS-Tween-BSA. After washing, plates were incubated with the substrate O-phenylenediamine dihydrochloride plus H2O2 (Sigma). The reaction was stopped by adding 50 µl of H2SO4 (4N) to each well. Absorbance was read at 490 nm with a wavelength correction of 600 nm. Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R&D Systems). The limit of detection was 15 pg/ml.

Western analysis of PKC isoforms activation
Isolation of cytoplasmic and membrane protein extracts
After treatment with Tat or pretreatment with inhibitors PMA or oligonucleotides, monocytes were harvested and rapidly lysed at 4°C in 100 µl of hypotonic buffer A (200 mM Tris HCl, 2 mM EDTA, 1 mM DTT, 10 µg/ml leupeptin, 1 mM PMSF; pH 7.5) by repeated aspiration through a syringe fitted with a 21 gauge needle. After addition of 300 µl ice-cold sample buffer B (20 mM Tris HCl, 2 mM EDTA, 1 mM DTT, 10 µg/ml leupeptin, 1 mM PMSF, 0.33 M sucrose; pH 7.5), the lysate was centrifuged at 100,000 g at 4°C for 40 min The supernatant corresponding to the cytoplasm was collected; proteins were quantified with the Bradford assay and stored at -20°C. The membrane pellets were solubilized in 50 µl of cold sample buffer B containing 1% Triton X-100, sonicated (1 min, power 2.5), and stored at -20°C.

Western blot analysis of PKC translocation
Equal amounts of protein (10–30 µg) were subjected to 10% SDS-PAGE and separated proteins were transferred to nitrocellulose membranes. Immunoblotting was conducted with either anti-PKC antibody (1:500) or PKC isoform-specific antibodies directed against PKC-{alpha}, -ßII, -{delta}, or -{varepsilon} (1:1000) (Santa Cruz Biotechnologies, Santa Cruz, CA). The membrane was blocked with 5% milk in Tris-buffered saline with 0,05% Tween 20 (TTBS) for 1 h, washed four times with TTBS, and incubated with the primary antibody for 2 h. Immunoreactive bands were detected by incubation for 2 h with anti-rabbit or anti-mouse immunoglobulins conjugated with horseradish peroxidase (1:1000) (DAKO A/S, Roskilde, Denmark). Membranes were visualized using a chemiluminescent substrate (Pierce, Rockford, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tat-induced PKC activation is crucial for IL-10 induction
Monocytes isolated from healthy donors were cultured in the presence of Tat (10 or 100 nM) and cytoplasmic, and membrane proteins were isolated after 30 min or after 1 or 2 h. Whereas PKC is localized in the cytoplasm in unstimulated human monocytes, Tat stimulation induces (in a dose-dependent manner) PKC translocation to the membrane signifying PKC activation. This activation observed after 30 min of Tat (10 nM) stimulation reaches a peak in 1 h and decreases after 2 h of stimulation. (Fig. 1 A) Thus, HIV-1 Tat induces PKC activation in a dose- and time-dependent manner in human monocytes. To investigate the involvement of PKC activated in Tat-induced IL-10 production, monocytes were preincubated with RO-318220, a PKC inhibitor, before Tat stimulation. RO-31 8220 induces a dose-dependent inhibition of Tat-mediated IL-10 production, reaching 58% at 2.5 µM and being complete at 5 µM (Fig. 1B ). These results show that PKC activation is strictly required for Tat-induced IL-10 production. In the absence of PKC, Tat became unable to induce IL-10 in monocytes. To further characterize PKC involvement, we investigated which of the different PKC isoforms present in the monocyte play this crucial role in Tat-induced IL-10 production.



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  		Figure 1. Tat-induced PKC activation is crucial for IL-10 induction. A) PKC translocation was analyzed by Western blot. Monocytes were stimulated by Tat (10, 100 nM) or PMA (100 ng/ml) for 30 min or for 1 or 2 h. Membrane (M) and cytoplasmic (C) proteins were extracted and separated on 10% SDS page gel. PKC was visualized using an anti-PKC by chemiluminescence. B) Monocytes (106) were pretreated or not with the PKC inhibitor RO 31–8220 (2.5 and 5 µM) for 30 min. Tat was then added for 24 h. Culture supernatants were collected and the presence of IL-10 was determined by ELISA. Results are expressed in pg/ml. Representative of three independent experiments from three donors.

Tat is able to activate PKC-{alpha}, -ßII, -{delta}, and -{varepsilon}
We next investigated which isoforms of PKC are stimulated by HIV-1 Tat protein. Monocytes were incubated with Tat (10 nM) for 15, 30 min, or 1 h and PKC localization was analyzed by Western blot. Results depicted in Fig. 2 show that by as soon as 15 min, Tat induces translocation to the membrane of four PKC isoforms: PKC-{alpha} (Fig. 2A ), PKC-ßII (Fig. 2B ), PKC-{delta} (Fig. 2C ), and PKC-{varepsilon} (Fig. 2D ). As a positive control, PMA stimulation similarly activates these four isoforms. Thus, the conventional PKC-{alpha} and ßII and the nonconventional PKC-{delta} and -{varepsilon}, activated by Tat, became potential candidates for Tat-induced IL-10 production.



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  		Figure 2. HIV-1 Tat is able to activate PKC-{alpha}, -ßII, -{delta}, and -{varepsilon}. PKC translocation was analyzed by Western blot. Monocytes were stimulated by Tat (10, 100 nM) or PMA (100 ng/ml) for 15, 30 min, 1 or 2 h. Membrane (M) and cytoplasmic (C) proteins were extracted and separated on 10% SDS page gel. PKC was visualized using an anti-PKC specific to each isoform: PKC-{alpha} (A), PKC-ßII (B), PKC-{delta} (C), and PKC-{varepsilon} (D) by chemiluminescence. Results are representative of three independent experiments on the cells from three donors.

Correlation between PKC-ßII,-{delta}, -{varepsilon} activation and Tat-mediated IL-10 production
To evaluate the putative involvement of these four PKC isoforms in Tat-mediated IL-10 induction, we explored whether in addition to the ability of Tat to activate PKC, there was a link between PKC-{alpha}, -ßII, -{delta}, -{varepsilon} and IL-10 induction by Tat.

The use of Tat deleted mutants shows that GST-Tat 1–45 is able to induce the same amounts of IL-10 as the wild-type GST-Tat 101; so, Tat 1–45 contains the active region responsible of IL-10 induction (Fig. 3 A). Among these four potential PKC isoforms candidates, we investigated which ones were activated by Tat. Western blot analysis shows that GST-Tat 1–45 is able to induce membrane translocation of PKC-ßII,-{delta}, -{varepsilon} after 15 min of stimulation, peaking after 1 h (Fig. 3B ). In contrast, GST-Tat 1–45 remains unable to activate PKC-{alpha} even after 1 h of stimulation. These results show that the PKC-{alpha}, even though activated by Tat protein, does not seem to be involved in Tat-mediated IL-10 induction.



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  		Figure 3. NH2-terminal region Tat 1–45, responsible for IL-10 induction, activates PKC-ßII, {delta}, and -{varepsilon} but not PKC-{alpha}. A) monocytes (106) were incubated with either wild-type GST-Tat 1–101 (1, 10 nM), the recombinant mutant GST-Tat 1–45 (1, 10 nM), or (as a negative control) GST for 24 h. Culture supernatants were recovered and the presence of IL-10 was determined by ELISA. The results are expressed in pg/ml. Values are the means ± SD of three experiments from one donor. Similar results were obtained with cells from the blood of three different donors. B) Monocytes were stimulated with GST-Tat 1–45 or, as a negative control, with GST for 15 min, 30 min, or 1 h. Membrane (M) and cytoplasmic (C) proteins were extracted and separated on 10% SDS page gel. PKC-{alpha}, PKC-ßII, PKC-{delta}, and PKC-{varepsilon} were visualized by chemiluminescence using antibodies specific to each isoform.

Long-term exposure of monocytes to PMA (100 ng/ml for 48 h) renders Tat unable to induce IL-10 production (Fig. 4 A). Indeed, under these conditions, PMA treatment depletes the pool of PKC. Therefore, we investigated whether the potential candidates PKC-ßII, -{delta}, -{varepsilon} were also depleted by PMA treatment. Results depicted in Fig. 4B show that whereas in the absence of PMA, Tat activates PKC as previously shown, long-term PMA treatment depletes PKC-ßII, -{delta}, and -{varepsilon}. After long-term exposure to PMA, there is a strong down-regulation of PKC-ßII and -{varepsilon} and a complete inhibition of PKC-{delta} expression. Under these conditions, Tat was unable to activate these three isoforms even after 1 h of activation.



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  		Figure 4. PKC-ßII, PKC-{delta}, and PKC-{varepsilon} are depleted by long PMA treatment. A) To deplete the PKC pool in the cell, monocytes were pretreated with PMA (100 ng/ml), a PKC activator, for 48 h and Tat (10 nM) was added. 24 h later, culture supernatants were recovered and the presence of IL-10 was determined by ELISA. B) PKC depletion was analyzed for PKC-ßII, PKC-{delta}, and PKC-{varepsilon}. Monocytes pretreated or not with PMA were stimulated with Tat for 30 min or 1 h; membrane (M) and cytoplasmic (C) proteins were isolated and the presence and membrane translocation were visualized for each isoform using specific antibodies. Representative of two independent experiments on the cells from two donors.

Taken together, these results underscore a correlation between activation of PKC-ßII, -{delta}, and -{varepsilon} and Tat-induced IL-10 production.

PKC-ßII and PKC-{delta} are required for Tat-induced IL-10 production
To investigate the possible involvement of one or all PKC-ßII, -{delta}, and -{varepsilon}, these isoforms were targeted with isoform-specific antisense oligonucleotides. Monocytes were preincubated with PKC-ßII, -{delta}, and -{varepsilon} antisense oligonucleotides (5 or 15 µM) or with the corresponding sense sequence as a negative control, and stimulated by Tat (10 nM). The level of each isoform protein targeted was determined by Western blot after 1 h of stimulation. As shown in Fig. 5 A, treatment with 5 µM of PKC-{alpha} antisense oligonucleotide down-regulates PKC-{alpha} expression and the use of 15 µM oligonucleotide totally inhibits PKC-{alpha}. The use of antisense PKC-{alpha} specifically inhibits PKC-{alpha}, since PKC-ßII is still detected in the same monocytes and is activated by Tat (10 nM). (Fig. 5A , bottom). The same specificity of inhibition is observed with the other isoforms: PKC-ßII, -{delta}, and -{varepsilon} antisense oligonucleotides caused a specific down-regulation in the level of their corresponding isoform protein targeted, e.g., the antisense oligonucleotide of PKC-ßII specifically inhibited PKC-ßII but had no effect on PKC-{varepsilon}; similarly, antisense PKC-{delta} had a specific inhibitory effect on PKC-{delta} and did not affect PKC-{alpha}. The control sense sequences for PKC-{alpha}, -ßII, -{delta}, and -{varepsilon} have no effect on respective PKC isoform expression or on Tat-induced IL-10 production. The specific inhibition of PKC-{alpha} has no effect on Tat-induced IL-10 production. Similarly, PKC-{varepsilon} antisense oligonucleotide, although totally inhibiting the PKC-{varepsilon}, had no effect on IL-10 induction. In the absence of PKC-{alpha} and PKC-{varepsilon}, Tat is still able to induce IL-10 in human monocytes. These results suggest that these two isoforms are not involved in this induction. In contrast, preincubation of antisense PKC-ßII leads to a dose-dependent inhibition of Tat-induced IL-10 production, reaching 49% for 15 µM of oligonucleotide. The specific inhibition of PKC-{delta} induces a strong inhibition (69%) of Tat-mediated IL-10 production. Altogether, these results strongly suggest the involvement of PKC-ßII and PKC-{delta} in IL-10 induction.



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  		Figure 5. Effect of isoform-specific PKC inhibition with antisense oligonucleotides on Tat-induced IL-10 production. Monocytes were treated with sense or antisense oligonucleotides (5, 15 µM) specific for PKC-{alpha} (A), PKC-ßII (B), PKC-{delta} (C), PKC-{varepsilon} (D) for 20 h. Monocytes were then washed and incubated with oligonucleotide, and Tat (10 nM) was added. After 1 h, isoform-specific inhibition was assessed by Western blot (bottom); IL-10 production was measured by ELISA 24 h later (top). In each case, specific inhibition of each isoform using the specific antisense sequence (15 µM) was verified by visualizing in the same cell extracts another PKC isoform activation by Tat.

To further characterize the role of PKC-ßII and PKC-{delta} in Tat-induced IL-10 production, these isoforms were simultaneously inhibited using two approaches. Monocytes were treated with a combination of PKC-ßII and PKC-{delta} antisense oligonucleotides before Tat stimulation. The combination of control sense oligonucleotide had no effect on monocyte. When PKC-ßII and PKC-{delta} were inhibited simultaneously, Tat became unable to induce IL-10 production (Fig. 6 A). In agreement with this result, preincubation with two chemical inhibitors—rottlerin for PKC-{delta} and hispidin for PKC-ßII—inhibits Tat-induced IL-10 production by 95% (Fig. 6B ).



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  		Figure 6. Effect of PKC-ßII and PKC-{delta} coinhibition with oligonucleotides or chemical inhibitors on Tat-mediated IL-10 production. A) Monocytes were treated or not with sense or antisense oligonucleotides specific for PKC-ßII and PKC-{delta} (2.5 or 5 µM) for 20 h. Monocytes were washed and incubated with oligonucleotide; Tat (10 nM) was added for 24 h. B) Monocyte were pretreated with hispidin (a PKC-ß inhibitor) or rottlerin (a PKC-{delta} inhibitor) for 30 min, or with a combination of these two compounds, and stimulated with Tat (10 nM) for 24 h. Culture supernatants were recovered and the presence of IL-10 was determined by ELISA. Results are expressed in pg/ml. Values are the means ± SD of three experiments from one donor. Similar results were obtained with cells from the blood of three different donors.

These results show that PKC-ßII and PKC-{delta} play a crucial role in Tat-mediated IL-10 production.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We recently showed that HIV-1 Tat protein, by acting at the membrane level via its amino-terminal region, induces IL-10 production, a cytokine that would contribute to the development of HIV-1-associated immunological dysfunctions (7) . Analysis of the signal transduction pathways has shown that the PKC pathway is strictly required whereas calcium signaling is not involved in the induction of IL-10. Using PKA inhibitors, we showed that this pathway is not implicated (7) . Downstream from PKC, activation of NF{kappa}B and MAP kinases ERK1/2 is required.

Taking into account the role of PKC in IL-10 induction, we investigated among the eight isoforms (PKC-{alpha}, -ßI, -ßII, -{eta}, -µ, -{delta}, -{varepsilon}, and -{zeta}) present in human monocyte which one plays this crucial role. Our data show that 1) Tat is able to activate PKC-{alpha}, PKC-ßII, PKC-{delta}, and PKC-{varepsilon}, 2) among these four potential candidates, only PKC-ßII, PKC-{delta}, and PKC-{varepsilon} are activated by the active domain Tat 1–45 responsible for IL-10 production and depleted by long-term exposure to PMA, which abolishes Tat-mediated IL-10 production, 3) whereas selective inhibition of PKC-{alpha} and PKC-{varepsilon} by specific antisense oligonucleotides has no effect on Tat-mediated IL-10 induction, inhibition of either PKC-ßII or PKC-{delta} inhibits IL-10 production by 49% and 69%, respectively; and 4) the simultaneous inhibition of PKC-ßII and PKC-{delta} totally inhibits Tat-mediated IL-10 production. Altogether, these results demonstrated that the Ca2+-dependent PKC-ßII and the Ca2+-independent PKC-{delta} are the major PKC isoforms involved in Tat-induced IL-10 production. This result agrees with the ability of Tat to continue to stimulate IL-10 production in the absence of Ca2+ (unpublished observations), probably by involving the Ca2+-independent PKC {delta}.

The roles and consequences of activation of PKC-{alpha} and PKC-{varepsilon} isoforms remain to be determined. The inability of these two activated isoforms to stimulate IL-10 production may be related to their incapacity to interact with crucial substrates. In agreement with this interpretation, recent data have shown that purified PKC isoforms recognize with different affinities diverse substrates (12) . In addition, several parameters have been reported as involved in the selectivity of PKC isoforms, including differences in lipids requirements, cellular localization of the C1 domain (18) , and the availability of proteins that interact with PKCs such as RACKs, RICKs, and STICKs (12) .

In an earlier study, we showed that, downstream of the PKC, Tat could induce the phosphorylation of ERK1/2, an activation involved in Tat-induced IL-10 production (7) . According to the literature PKC-{delta}, or even PKC-ßII, is a potential candidate. Schönwasser et al. (27) reported the capacities of different PKC isoforms to activate the MAP kinase ERK1/2 pathway by transfecting Cos-7 with constitutively active PKC mutants. Whereas transfection of PKC-{delta} can activate the ERK pathway, transfection of PKC-{alpha} and PKC-{varepsilon} has no effect. Similarly, the mechanism of activation of the transcription factor NF{kappa}B, another substrate of PKC activated by Tat in human monocytes, was analyzed in the RAW264.7 murine model (28) . LPS activation of this cell line, which expresses PKC-{alpha}, -ßI, -{delta}, -{eta}, and -{zeta}, causes the nuclear translocation of NF{kappa}B involving PKC-{alpha}, -ßI, and -{delta}. Taking into account our results and the involvement of PKC-{alpha}, -ßI, and -{delta} in NF{kappa}B activation, the PKC-{delta} isoform recruited by Tat can be considered as a potential candidate in the phosphorylation cascade leading to NF{kappa}B activation.

This capacity of extracellular Tat to recruit PKCs seems to depend on the cell type. Borgatti et al. (29) showed that stimulating the neuron line PC12 with Tat selectively activated PKC-{alpha}, -{varepsilon}, and -{zeta}, among the -{alpha}, -ßI, -ßII, -{delta}, -{varepsilon}, -{eta}, -{theta}, and -{zeta} isoforms present. Even though PKC isoforms -ß II and -{delta} are present in the PC12 line, they are not activated by Tat. This activation selectivity between PC12 cells and human monocytes could be explained by the involvement of partners upstream from PKCs, such as phosphoinositide-dependent kinase, which plays an essential role in cell signaling by regulating the activation state of the different PKC isoforms, or downstream partners such as RACKs and STICKs or direct substrates (12) whose expression and localization may depend not only on the cell type, but also on the differentiation state of the cell. Monick et al. showed that differentiation of monocytes to alveolar macrophages is accompanied by a considerable decrease in the level of PKC-ßII, whose expression is totally inhibited in alveolar macrophages (30) . This differential expression of PKC is accompanied by the loss or acquisition of new functions, as shown by activation of ERK2 kinase and AP-1 in monocytes stimulated with PMA and not in alveolar macrophages treated under the same conditions (30) . The correlation between PKC and differentiation has been observed in several cell systems such as a variant of the HL-60 line, which again can differentiate into mature myeloid cells after PMA stimulation when it is stimulated by dihydroxy-vitamin D3, which activates PKC-ß (31) . In agreement, Tonetti et al. showed that prior transfection of the PKC-ßI and -ßII isoforms transforms this PMA-resistant HL-60 line into cells that are sensitive to differentiation by PMA (32) . Nevertheless, this correlation is disputed by other workers who invoke the resistance of the HL-60 variant at a step downstream of PKC (33) . In disagreement with the direct role of PKC-ß in the differentiation of the HL-60 line, Mischak et al. showed that the expression profile of PKC-ß isoforms is identical in resistant and sensitive HL-60 cells after PMA treatment (34) .

This difference in tissue-specific expression and activation of PKC should imply a different role for each isoform in signal transduction. Even though peptide motifs corresponding to the optimal substrate of the different isoforms have been described, no substrate specificity could be attributed to each isoform (35) . This selectivity could instead be related to the subcellular localization of PKC isoforms and their partners (12) .

Activation of several PKC isoforms by the same ligand can, in some cases, cause antagonist functions. For example, in NIH 3T3 cells, PKC-{delta} inhibits cell proliferation whereas PKC-{varepsilon} stimulates it (18) . In our experiments, stimulation of PKC-{alpha} and -{varepsilon} by Tat did not inhibit the activator effect of PKC-ßII and -{delta}, since in the presence of specific antisense inhibitors of PKC-{alpha} and -{varepsilon}, the level of IL-10 production does not increase. However, since HIV-1 Tat is involved in the activation and modulation of several genes (IL-1, IL-6, MnSOD) (36 , 37) and functions (angiogenesis, immunosuppression, transformation, and oncogenicity) (38) , Tat-activated PKC-{alpha} and PKC-{varepsilon} may be involved in such signaling pathways. In fact, different PKC isoforms have distinct roles in signal transduction pathways: for example, overexpression of PKC-{varepsilon} is associated with oncogenic transformation (39) . Tat protein is involved in HIV-1-associated Kaposi sarcoma by acting on endothelial cell spreading (40) .

In summary, we have shown that HIV-1 Tat activates four PKC isoforms in human monocytes: PKC-{alpha}, PKC-ßII, PKC-{delta}, and PKC-{varepsilon}. This study introduces a way to explore the role of PKCs in signaling pathways in Tat-activated monocytes and to the search for the partners of PKC-ßII and -{delta} involved in production of IL-10, an immunosuppressive cytokine that participates in the deregulation of the immune system as early as the asymptomatic stage of HIV infection.


   ACKNOWLEDGMENTS
 
This work was supported by Agence Nationale de Recherche sur le SIDA, SIDACTION, and Ministère de la Recherche et de la Technologie. We thank Dr. Moncef Benkirane for the gift of HIV-Tat mutants.

Received for publication September 25, 2001. Revision received January 2, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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