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The Journal of Immunology, 2007, 178: 798-807.
Copyright © 2007 by The American Association of Immunologists, Inc.

IL-10 Regulation by HIV-Tat in Primary Human Monocytic Cells: Involvement of Calmodulin/Calmodulin-Dependent Protein Kinase-Activated p38 MAPK and Sp-1 and CREB-1 Transcription Factors1

Katrina Gee{ddagger},§, Jonathan B. Angel{dagger},§, Sasmita Mishra{dagger}, Maria A. Blahoianu{dagger} and Ashok Kumar2,*,{dagger},{ddagger},§

* Department of Pathology and Laboratory Medicine, {dagger} Department of Biochemistry, Microbiology, and Immunology, and {ddagger} Division of Virology and Molecular Immunology, Research Institute, Children’s Hospital of Eastern Ontario, and § Ottawa Health Research Institute and the Division of Infectious Diseases, Department of Medicine, Ottawa Hospital General Campus, University of Ottawa, Ottawa, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The anti-inflammatory cytokine, IL-10 plays an important role in HIV immunopathogenesis. The HIV accessory protein, Tat is not only critical for viral replication, but affects the host immune system by influencing cytokine production including IL-10. During HIV infection, IL-10 production by monocytic cells is up-regulated, representing a critical pathway by which HIV may induce immunodeficiency. Herein, we show that extracellular Tat-induced IL-10 expression in normal human monocytes. To understand the signaling pathways underlying HIV-Tat induced IL-10 transcription, we investigated the involvement of MAPK as well as calcium signaling and the downstream transcription factor(s). Our results suggest that Tat-induced calcium influx regulated IL-10 transcription in monocytic cells. The experiments designed to further understand the molecules involved in the calcium signaling suggested that calmodulin and calmodulin-dependent protein kinase-II (CaMK-II)-activated p38 MAPK played a role in extracellular Tat-induced IL-10 expression in primary human monocytes. Furthermore, Tat-induced IL-10 expression was regulated by p38 MAPK- and CaMK II-activated CREB-1 as well as Sp-1 transcription factors. Taken together, our results suggest that extracellular HIV-Tat induced IL-10 transcription in primary human monocytes is regulated by CREB-1 and Sp-1 transcription factors through the activation of calmodulin/CaMK-II-dependent p38 MAPK.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The anti-inflammatory cytokine IL-10 plays a key role in the pathogenesis of several autoimmune and infectious diseases (1, 2). IL-10 is produced primarily by Th0 and Th2 cells, CD8+ T cells, B cells, regulatory T cells, and monocytic cells (1, 3, 4, 5, 6). Although it facilitates the induction of Th2 cell types, it is not considered strictly a Th2 type cytokine (7). In general, IL-10 inhibits Ag-driven activity of both the Th1 and Th2 subsets (3), and down-regulates the release of reactive oxygen and nitrogen intermediates resulting in macrophage deactivation which may allow the growth of tumor cells and intracellular microbes (8). The potent inhibitory action of IL-10 on macrophages, particularly at the level of cytokine production, supports an important role for IL-10 in the regulation of T cell responses, acute inflammation and autoimmune responses (1, 2, 9, 10).

In HIV infections, IL-10 production has been shown to be altered (11, 12, 13, 14, 15, 16) indicating an important role for IL-10 in homeostasis and regulation of protective immune responses. We and others (13, 17, 18) have demonstrated that IL-10 is produced constitutively throughout the course of HIV infection, and following anti-retroviral treatment, IL-10 levels are significantly decreased. We and others have also demonstrated that HIV infection of monocytic cells in vitro results in enhanced IL-10 production (17, 19, 20, 21) which may be of significance because of the ability of IL-10 to induce immune unresponsiveness (22), to inhibit HIV replication (23, 24), and to limit viral entry as a result of its inhibitory effects on the expression of chemokine receptors on T cells (25). However, because monocytes act as viral reservoirs during HIV infection (11, 26), and because IL-10 has been shown to enhance CXCR4 expression on dendritic cells (27), increased IL-10 production may, in fact aid the virus to escape from host immune surveillance.

IL-10 production in HIV infection has been attributed at least in part to the accessory HIV Tat protein which is critical for HIV replication during active infection (28). Tat is a 14- to 16-kDa protein encoded by two exons and has a well characterized transactivating activity. It is expressed early in HIV infection where it binds a stable RNA hair pin structure (the Tat activation region) at the 5' end of HIV RNA. It recruits host transcription protein complexes to activate viral replication (29). In addition, the Tat protein modulates host cellular mechanisms thereby contributing to immune system malfunction. For example, Tat has been shown to induce T cell apoptosis (30, 31), inhibit MHC-I and MHC-II expression (32, 33), enhance epithelial VCAM-1 expression (34) and affect cytokine (IL-10, IL-12, TNF-{alpha}, and TGF-beta) production in different cell systems (35, 36, 37, 38, 39). Tat accumulates in the nucleus of infected cells and it is also secreted in nanomolar amounts into the plasma of HIV-infected patients where it can exert its effect on noninfected bystander cells (40). With regard to IL-10 induction, Badou et al. (36) have demonstrated that the critical region responsible for IL-10 stimulation was located within residues 1–45.

HIV-Tat mediates its biological effects by activating a multitude of signaling pathways and transcription factors. For example, Tat induces the activation of MAPK, including JNK, p38, and ERK, PI3K, and calcium signaling pathways (36, 41, 42, 43, 44). These signaling cascades are believed to be activated following interaction of Tat with various cell surface receptors including integrin receptors, members of the vascular endothelial growth factor receptor family (45, 46), the beta-chemokine receptors (CCR2 and CXCR4 (47, 48)), as well as with integrins {alpha}5beta1 and {alpha}vbeta3 (49, 50). Extracellular Tat translocates to the nucleus where it is known to activate various transcription factors including AP-1, Sp-1, CREB, and NF-{kappa}B (36, 41, 43, 51, 52, 53).

Recently, the molecular mechanism underlying murine and human IL-10 transcription following LPS-induced activation of the CD14/TLR-4 receptor complex has been investigated (4, 54). We have demonstrated that in human monocytic cells, LPS-induced IL-10 production was regulated by the Sp-1 transcription factor through the activation of p38 MAPK (4). A recent report has indicated that IL-10 production is dependent on protein tyrosine kinases and protein kinase C (PKC) activation in a murine cell line (55). In addition, factors that elevate cAMP have been suggested to be involved in the regulation of monocytic IL-10 synthesis, primarily at the mRNA level (56). However, the signaling pathways involved in IL-10 production following HIV infection and, in particular, following stimulation of monocytic cells with extracellular recombinant or intracellular Tat are not well understood. There is evidence to suggest that extracellular HIV-Tat induces IL-10 in human monocytic cells through the activation of PKC-betaII- and {delta}-dependent pathways (35, 36). Recently, we have also demonstrated that intracellular Tat induced IL-10 production in human monocytic cells by a signaling pathway involving the CREB-1 transcription factor through the activation of ERK MAPKs (57). Herein, we investigated the molecular mechanism by which recombinant endotoxin free HIV Tat regulates IL-10 production in human monocytic cells. Our results suggest for the first time, that extracellular HIV-Tat regulates IL-10 production by the calcium signaling pathway and in particular the calmodulin dependent protein kinase (CaMK)3 and the p38 MAPK through the activation of CREB-1 and Sp-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Recombinant HIV Tat obtained from the National Institutes of Health AIDS Reference and Reagent Program was treated with polymyxin B coated beads (Sigma-Aldrich) to make it endotoxin free. The endotoxin levels as tested by the LAL assay (BioWhittaker) were found to be <0.06 EU/ml. HIV-Tat was also oxidized by exposure to 3% hydrogen peroxide in PBS for 60 min before Tat addition to cell culture (36). PD98059, an inhibitor of MAP/ERK kinase-1, (Calbiochem) selectively blocks the activity of ERK MAPK and has no effect on the activity of other serine threonine protein kinases including Raf1, p38 and JNK MAPK (58, 59). The pyridinyl imidazole SB202190 (Calbiochem), a potent inhibitor of p38 MAPK, has no significant effect on the activity of ERK or JNK MAPK subgroups (58, 59). SP600125, a specific JNK inhibitor (BIOMOL), is a reversible ATP competitive inhibitor with >300-fold selectivity vs related MAPK including ERK1 and p38, and PKA and inhibitor of NF-{kappa}B kinase, IKK2 (60). The following calcium signaling inhibitors were used: EGTA (Sigma-Aldrich), a calcium chelating agent; SKF-96365 hydrochloride (Calbiochem) specifically inhibits receptor-mediated Ca2+ entry (61); aminoethoxydiphenyl borate (APB) (Calbiochem) inhibits inositol (1, 4, 5)- triphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (ER) (62); W-7 hydrochloride (W-7, Calbiochem) is a CaM antagonist; KN-93 (Calbiochem) is a specific cell-permeable inhibitor of CaMK-II; FK-506 (AG Scientific) inhibits calcineurin-binding protein and inhibits the Ca2+-dependent phosphatase; and cyclosporine A (Sigma-Aldrich) inhibits cyclophilin and calcineurin. All other chemicals used for Western blotting were obtained from Sigma.

Monocyte purification

Purified, nonactivated monocytes were obtained by a negative selection procedure involving depletion of T cells and B cells using magnetic polystyrene M-450 Dynabeads (Dynal Biotech) coated with anti-CD2 (T cells) or anti-CD19 (B cells) beads, as described earlier (4). Briefly, PBMCs (107/ml) were incubated with CD2 and CD19 Dynabeads for 30 min on ice with constant mixing followed by incubation at 37°C for 2 h. The nonadherent cells were removed and the adherent cells thus obtained contained <1% T cells and B cells as determined by flow cytometry. Cells were cultured in IMDM (Sigma) supplemented with 10% FBS (Invitrogen/Life Technologies), 100 U/ml penicillin, 100 µg/ml gentamicin, 10 mM HEPES, and 2 mM glutamine.

Cell stimulation, collection of culture supernatants, and measurement of IL-10 by ELISA

Monocytes (0.5 x 106/ml) were treated with various signal transduction inhibitors for 24 h with HIV-Tat in 24-well culture plates (Falcon, Becton Dickinson). The supernatants were frozen at –70°C and thawed at the time of analysis. IL-10 was measured by ELISA by using two different mAbs that recognize distinct epitopes, as described (4).

Ca2+ influx

Ca2+ influx was performed essentially as described earlier (63). Briefly, cells were washed with Ca2+-free PBS for 5 min at room temperature and resuspended in buffer A (RPMI 1640 containing 20 mM HEPES (pH 7)). Cells were washed again and resuspended in buffer A containing 1 mM Fluo3/AM (Molecular Probes) in 1 mM Me2SO and 3.75% Pluronic F-127 solution (Sigma-Aldrich) followed by incubation in the dark for 45 min at 37°C. The reaction was stopped by adding an equal volume of buffer B (buffer A containing 5% FBS (pH 7.4)) followed by incubation for 15 min at 37°C. Cells were washed and resuspended in buffer B at a final concentration of 0.5 x 106 cells/ml followed by analysis for Ca2+ levels by the FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1fl as described earlier (63).

Western blot analysis

Phosphorylation of p38, p42/44 ERK, or JNK MAPKs was determined by Western blot analysis as previously described (4). Briefly, cells were stimulated at 37°C for 0–60 min with HIV-Tat. Cell lysates were subjected to 12% polyacrylamide SDS-PAGE followed by transfer onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were probed with either rabbit anti-phospho-p38, rabbit anti-phospho-JNK1 (both from New England Biolabs) or mouse anti-phospho-p42/44 mAb (Santa Cruz Biotechnology), followed by HRP-conjugated goat anti-rabbit or goat anti-mouse polyclonal Abs (Bio-Rad). The membranes were stripped with a buffer (62.5 mM Tris-HCl (pH 6.7), 100 mM 2-2-ME, 2% SDS, and 0.7 mM DTT) for 30 min at 50°C with gentle agitation. The membranes were washed with TBST buffer (150 mM Tris-HCl, 1 M NaCl, and 1% Tween 20) seven times followed by reprobing with rabbit polyclonal Abs specific for the unphosphorylated forms of either p38, p42 or JNK MAPKs (Santa Cruz Biotechnology). All immunoblots were visualized by ECL (Amersham Biosciences).

RNA isolation and semiquantitative reverse transcriptase-based PCR for IL-10

Total RNA was extracted using a monophase solution containing guanidine thiocyanate and phenol (Tri Reagent solution; Molecular Research Center). Total RNA (1 µg) was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (PerkinElmer Life Sciences). Equal aliquots (5 µl) of cDNA equivalent to 100 ng of RNA were subsequently amplified for IL-10 and beta-actin. The oligonucleotide primer sequences for IL-10 and beta-actin and their amplification conditions have been described previously (4). The amplified products IL-10 (204 bp) and beta-actin (610 bp) were resolved by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide staining.

EMSA

EMSAs were performed as described earlier (4, 63). Briefly, cells were treated with HIV-Tat for the time indicated in the presence or the absence of various inhibitors. The nuclear proteins (5 µg) were mixed with 32P-labeled CREB oligonucleotide probes for 20 min and the resulting complexes were separated on a 5% nondenaturing gel. The oligonucleotide probe containing sequences corresponding to the CREB binding site was as follows: 5'-CAA TTT GTC CAC GTC ACT GTG ACC-3'. The oligonucleotide probe containing sequences corresponding to the Sp-1 binding site was as follows: 5'-ATC CTG TGA CCC CGTC CTG TCC TGT-3'. To determine the specificity of the proteins binding the CREB probe, parallel EMSA reactions were incubated with 50- to 200-fold excess of unlabelled specific and nonspecific oligonucleotide probes for 20 min before the addition of labeled probe. Supershift experiments were also performed by using mouse anti-CREB-1 mAb (Santa Cruz Biotechnology).

Statistical analysis

Means were compared using the two-tailed Student’s t tests. Results are expressed as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recombinant HIV-Tat induces IL-10 production in primary human monocytes

HIV-Tat has been shown to enhance IL-10 production in human monocytic cells (35, 36). We confirmed these results by treating primary monocytes with varying concentrations of recombinant HIV Tat for 24 h. To ensure that the Tat preparation did not contain any endotoxin, it was purified by polymixin B beads. Endotoxin-free HIV Tat enhanced IL-10 production in a dose dependent manner (Fig. 1A). To further confirm the absence of endotoxins, the HIV-Tat was exposed to low concentrations of hydrogen peroxide for 20 min before addition to the cell culture, in effect neutralizing the Tat-bioactivity without inactivating the contaminating endotoxin present if any. Treatment of monocytes with oxidized Tat failed to produce IL-10 production (Fig. 1A). We have previously demonstrated that very low concentrations of LPS (1 ng/ml) induce high levels of IL-12p40 production (64). The fact that the endotoxin-free Tat did not induce IL-12p40 production in monocytic cells (Fig. 1B) further confirms that the Tat preparation used was free of endotoxin. It may be noted that washing of cells following 60–120 min of stimulation with Tat produced levels of IL-10 similar to that produced by unwashed Tat-stimulated cells (data not shown). Recombinant HIV-Tat enhanced IL-10 transcription as determined by semiquantitative RT-PCR analysis (Fig. 1C). To confirm that HIV-Tat enhanced IL-10 expression at transcriptional level, purified monocytes were treated with actinomycin D for 30 min before stimulation with Tat. Actinomycin D inhibited HIV-Tat-induced IL-10 expression as determined by RT-PCR (Fig. 1C) and ELISA (data not shown).


Figure 1
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  		FIGURE 1. Recombinant HIV-Tat induces IL-10 expression in primary human monocytes. Monocytes (0.5 x 106 cells/ml) were isolated from healthy donors and cultured in the presence of LPS (1 µg/ml), HIV-Tat (10–500 ng/ml), or oxidized HIV Tat (500 ng/ml) for 24 h. Cell supernatants were harvested and measured for IL-10 (A) or IL-12p40 (B) by ELISA. These results represent the mean IL-10 or IL-12p40 production ± SD from a total of five separate healthy individuals. C, Monocytes (1 x 106 cells/ml) were treated with HIV-Tat (100 ng/ml) for 0.5–4 h followed by measurement of IL-10 and beta-actin mRNA by RT-PCR (left panel). The figure shown is a representative of results from three different individuals. Monocytes were also pretreated with actinomycin D for 30 min before HIV-Tat stimulation for an additional 1 h followed by measurement of IL-10 and beta-actin expression by RT-PCR. Results from two different donors are shown in the right panel.

 
HIV-Tat-induced IL-10 production is dependent on calcium influx

Previous reports have indicated a role for PKC signaling in the regulation of Tat-induced IL-10 production in human monocytes (35, 36). Because of the complex nature of intracellular signaling and because HIV-Tat is known to affect other intracellular signaling pathways, we investigated the role of calcium signaling in Tat-induced IL-10 production. We first examined whether HIV Tat was capable of inducing calcium influx. Primary human monocytes were incubated with HIV Tat for 0–12 min, and calcium influx was measured by flow cytometry. Calcium influx was observed after 4 min of stimulation (Fig. 2A). To determine whether the Tat-induced calcium flux plays a role in Tat-induced IL-10 production, cells were incubated with the calcium chelating agent EGTA at concentrations ranging from 1 to 5 µM for 2 h before treatment with HIV-Tat for 24 h and measured for IL-10 expression. EGTA partially inhibited IL-10 expression at 1 µM, and completely abolished Tat-induced IL-10 production at 2.5 µM, thus suggesting a role for calcium signaling in IL-10 production (Fig. 2B). To determine that EGTA inhibited IL-10 at the transcriptional level, IL-10 expression was analyzed by semiquantitative RT-PCR. Cells were incubated with EGTA for 2 h before stimulation with Tat for an additional 4 h. The presence of EGTA completely abolished Tat-induced IL-10 mRNA (Fig. 3B).


Figure 2
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  		FIGURE 2. HIV-Tat-induces calcium influx plays a role in Tat-induced IL-10 expression. A, HIV Tat induces calcium influx in human monocytic cells monocytes (0.5 x 106 cells/ml) were loaded with Fluo-3/AM and stimulated with HIV-Tat (50 ng/ml) for 0–12 min. The resulting calcium influx was measured by flow cytometric analysis. Top panel, Unstimulated cells. Middle panel, Stimulation with HIV-Tat (50 ng/ml) followed by the addition of EGTA. Bottom panel, Stimulation with the calcium ionophore A23187 followed by the addition of EGTA. B, Tat-induced IL-10 production is regulated by Ca2+ influx. Human monocytes (0.5 x 106 cells/ml) were pretreated with EGTA (1–5 µM) for 2 h before stimulation with HIV-Tat (50 ng/ml) for an additional 24 h. IL-10 production was measured by ELISA. The results shown are mean ± SD of three different experiments.

 

Figure 3
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  		FIGURE 3. The CaM/CaMK-II pathway is involved in HIV-Tat-induced IL-10 production. Primary human monocytes (0.5 x 106 cells/ml) (A) or (3 x 106 cells/ml) (B) were pretreated with APB (10–50 µM), W-7 (5–25 µM), or KN93 (5–20 µM) for 2 h followed by stimulation for an additional 24 h (A) or 4 h (B) with HIV Tat (50 ng/ml). A, Cell supernatants were measured for IL-10 production by ELISA. The results represent the mean IL-10 production ± SD from a total of five separate healthy individuals. B, RT-PCR for IL-10 expression was performed on cDNA isolated from the pelleted cells. beta-Actin was included as a loading control. The gels shown are a representative of three separate experiments. C, Primary human monocytes (3 x 106 cells/ml) were pretreated with W-7 (5 and 10 µM) or KN93 (5 and 10 µM) for 2 h followed by stimulation for an additional 30 min with HIV Tat (50 ng/ml). Phosphorylation of CaMK-II was measured by SDS-PAGE analysis of cell lysates. The membrane was probed using a specific anti-phospho-CaMK-II (p-CaMK-II) Ab. The membrane was stripped and reprobed using an Ab specific for CaMK-II as a loading control. The blots shown are a representative of three separate experiments.

 
To further delineate the involvement of the calcium signaling pathway in Tat-induced IL-10 production, we used commercially available specific inhibitors for signaling molecules directly involved in the calcium signaling pathway. Elevations in cytoplasmic calcium concentrations occur following stimulation by diverse stimuli that activate voltage or ligand-gated calcium channels in the surface membrane or following release of calcium present in intracellular stores, mainly in the endoplasmic reticulum (ER) (65). To determine whether calcium release from the ER regulates Tat-induced IL-10 expression, we treated cells with APB, an inhibitor of the IP3 receptor, which blocks the release of calcium from the ER by blocking IP3 receptor-gated channels (62). Prior treatment of cells with APB resulted in inhibition of Tat-induced IL-10 production (Fig. 3A), suggesting a role for the release of internal calcium stores in the induction of IL-10 expression by HIV Tat.

Calmodulin (CaM), a major calcium receptor, is present in both cytoplasmic and nuclear compartments. The calcium/CaM complex regulates several downstream targets including protein kinases and phosphatases (66). To investigate the CaM arm of the calcium signaling pathway we used a specific calmodulin inhibitor, W-7 (67). The results show that W-7 inhibited Tat-induced IL-10 production in a dose-dependent manner (Fig. 3A). One major family of calcium/CaM effectors is the calmodulin-dependent protein kinases (CaMK), which includes a multifunctional kinase, CaMK-II, which phosphorylates a large number of signaling proteins. To gain further insight into the role of calcium/CaM, we examined the involvement of CaMK-II by using the CaMK-II-specific inhibitor, KN93 (67). As observed with W-7, KN93 significantly inhibited the Tat-induced IL-10 production (Fig. 3A). To determine that IL-10 production is regulated by the calcium/calmodulin signaling pathway at the transcriptional level, we pretreated primary monocytes with, KN93, W-7, or APB for 2 h before Tat treatment for a period of 4 h followed by RT-PCR analysis for IL-10 production. The results support the above observation by showing that each of APB, KN93, and W-7 inhibited Tat-induced IL-10 mRNA expression (Fig. 3B). To confirm whether HIV-Tat induced the phosphorylation of CaMK-II and whether this phosphorylation was inhibited by the CaM/CaMK-II inhibitors, monocytes were pretreated with W-7 or KN93 for 2 h before treatment with Tat for an additional 30 min. The results show that Tat induces the phosphorylation of CaMK-II (Fig. 3C) and that this phosphorylation was inhibited by W-7 and KN-93 in a dose-dependant manner.

Calcineurin is also activated by the binding of calcium to CaM, which dissociates the two proteins and allows the catalytic site of calcineurin to become accessible (63, 67). To determine whether calcineurin plays a role in Tat-induced IL-10 production, we pretreated cells with either cyclosporine A or FK506, two specific calcineurin inhibitors. Neither cyclosporine A or FK506 affected Tat-induced IL-10 production (Fig. 4A). To confirm that these inhibitors are biologically active, Jurkat T cells were pretreated with FK506 or cyclosporin A for 2 h before stimulation with PMA and ionomycin for an additional 5 min followed by analysis of expression levels of NF-AT4 in the protein lysates. PMA and ionomycin inhibit NF-AT4 phosphorylation, which can be restored by pretreatment of cells with the calcineurin inhibitors FK506 and cyclosporin A (63). Similar experiments were performed to determine the biological activity of APB. The results show that treatment of cells with either FK506, cyclosporine A or APB before stimulation with PMA/ionophore restored the expression of NFAT (Fig. 4B). These results suggest that Tat-induced IL-10 production is regulated by the CaM/CaMK-II pathway in human monocytic cells, and that calcineurin is not involved.


Figure 4
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  		FIGURE 4. FK506 and cyclosporine A do not inhibit HIV-Tat-induced IL-10 expression. A, Monocytes (0.5 x 106 cells/ml) were pretreated with FK506 (0.2–2 µg/ml) or cyclosporine A (5–20 µM) for 2 h followed by stimulation for an additional 24 h with HIV Tat (50 ng/ml). Cell supernatants were measured for IL-10 production by ELISA. The results represent the mean IL-10 production ± SD from a total of five separate healthy individuals. B, Jurkat T cells (3 x 106 cells/ml) were pretreated with APB (25 µM) FK506 (2 µg/ml) or cyclosporine A (20 µM) for 2 h followed by stimulation with PMA (50 ng/ml) and ionomycin (0.5 µg/ml) (P+I) for 5 min. Cell pellets were analyzed by western blotting for the phosphorylation levels of NF-AT4 using anti-NF-AT4 Abs. Blots were stripped and reprobed with anti-CaMK-II as a loading control. The blots shown are a representative of three separate experiments.

 
p38 MAPK is required for HIV-Tat-induced IL-10 production

We and others (4, 54) have previously demonstrated that IL-10 production by LPS-stimulated human monocytic cells is regulated through the activation of p38 MAPKs. HIV-Tat has also been shown to activate the MAPK pathway (41, 43). Therefore, it was of interest to determine whether this pathway was involved in Tat-induced IL-10 production as well. Initially, we confirmed that recombinant Tat was capable of inducing MAPK activation in primary human monocytes. Cells were treated with Tat for 0–30 min and the lysates were analyzed for phospho-p38, phospho-p42/44, and phospho-JNK activity by western blot analysis. The results show that Tat induced the phosphorylation of p42/44 ERK MAPK as early as 10 min and reached a maximum phosphorylation at 30 min poststimulation (Fig. 5A). However, Tat-induced JNK phosphorylation more rapidly with peak levels observed at 10 min poststimulation. In contrast, Tat-induced p38 phosphorylation occurred only after 30 min of stimulation (Fig. 5A). To determine whether any of the members of the MAPKs family are involved in Tat-induced IL-10 production, we used MAPK inhibitors: SB202190/SB203580, PD98059, and SP600125 for p38, p42/44, and JNK MAPK, respectively, and showed that each inhibitor blocks the phosphorylation induced by Tat of their respective kinases (Fig. 5A). To determine the involvement of MAPK in Tat-induced IL-10 production, cells were treated with each inhibitor at varying concentrations for 2 h before stimulation with HIV Tat for 24 h. The results show that both p38 inhibitors completely inhibited Tat-induced IL-10 production (Fig. 5B). Neither PD98059 nor SP600125 affected IL-10 production, therefore indicating a selective involvement of p38 MAPK in Tat-induced IL-10 production. These results were confirmed by performing RT-PCR analysis on cells that were treated with the MAPK inhibitors for 2 h before 4 h of stimulation with HIV Tat (Fig. 5C).


Figure 5
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  		FIGURE 5. Involvement of p38 MAPK in HIV-Tat-induced IL-10 expression. A, Monocytes (3 x 106 cells/ml) were either left untreated (left panel) or were treated with either: PD98059 (25 µM) for p42/44, SP600125 (25 µM) for JNK, and SB202190 (25 µM) for p38 (right panel) followed by stimulation with HIV Tat (50 ng/ml) for 10 or 30 min as indicated in the figure. Cell pellets were lysed and analyzed for phospho-p42/44, phospho-JNK, or phospho-p38 by Western blotting by using the phospho-specific Abs to p42/44 (p-p42/44), JNK (p-JNK), or p38 (p-p38). Blots were stripped and reprobed with Abs to total p42/44, JNK, or p38. Blots shown are representative of results obtained from five individuals. B, Monocytes (0.5 x 106 cells/ml) were pretreated with either: PD98059, SP600125, or SB202190 for 2 h followed by stimulation with HIV Tat (50 ng/ml) for an additional 24 h. Cell supernatants were analyzed for IL-10 production by ELISA. The results represent the mean IL-10 production ± SD from a total of five separate healthy individuals. C, Monocytes (3 x 106 cells/ml) were pretreated with either: PD98059, SP600125, or SB202190 for 2 h followed by stimulation with HIV Tat (50 ng/ml) for an additional 4 h. Cells were harvested and analyzed for IL-10 mRNA expression by RT-PCR. beta-Actin expression was measured as a loading control. The gels shown are representative of five different individuals.

 
Activation of p38MAPK is dependent on CaMK-II activation

The MAPK pathway has been shown to be activated by the calcium pathway (68). Since the above results show that both the p38 MAPK as well as the calmodulin-CaMK-II pathways regulate HIV-Tat-induced IL-10 production, we hypothesized that Tat may phosphorylate p38 MAPK through the activation of calmodulin-CaMK-II signaling molecules. Therefore, to determine whether the calmodulin-CaMK-II activated the MAPK pathways, primary human monocytes were pretreated with the CaM and CaMK-II inhibitors, KN93 and W-7, for 2 h before 30 min stimulation with HIV-Tat followed by the analysis pf p38 phosphorylation by western blot analysis. Pretreatment of cells with W-7 as well as KN-93 inhibited Tat-induced p38 phosphorylation (Fig. 6). The p42/44 MAPK inhibitor PD98059 was used as a negative control. Overall, these results suggest that HIV-Tat induced p38 phosphorylation through the activation of intracellular calcium-calmodulin-CaMK-II signaling pathway.


Figure 6
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  		FIGURE 6. Calmodulin- and CaMK-II-dependent Tat-mediated activation of p38. Monocytes (3 x 106 cells/ml) were pretreated with W-7 (5 and 10 µM), KN93 (5 and 10 µM), and PD98059 (25 and 50 µM) for 2 h followed by stimulation for an additional 30 min with HIV Tat (50 ng/ml). Cell pellets were analyzed for phospho-p38 expression by Western blotting by using phospho-specific anti-p38 (p-p38) Abs. The membrane was stripped and reprobed with anti-p38 as a loading control. Blots shown are representative of three different experiments.

 
Involvement of CaMK-II-dependent CREB-1 and Sp-1 in HIV-Tat-induced IL-10 production

We have recently demonstrated that intracellularly expressed HIV-Tat-induced IL-10 production in human monocytic cells through the activation of the CREB-1 transcription factor (57). We have also demonstrated that LPS-induced IL-10 production in human monocytic cells is regulated by the Sp-1 transcription factor (4). Therefore, we hypothesized that Tat-induced IL-10 production in primary monocytic cells may be regulated by either CREB-1 and/or Sp-1. Since it was not possible to precisely identify the involvement of Sp-1 and CREB-1 in IL-10 production in primary monocytic cells by IL-10 promoter analysis, we investigated whether Tat was able to induce the activation of the CREB-1 and Sp-1 transcription factors in monocytic cells by gel-shift assays. Primary human monocytes were treated with Tat for 4 h, and the nuclear extracts were analyzed for binding to specific oligonucleotide probes corresponding to the CREB-1 and Sp-1 binding sequences present in the human IL-10 promoter. Following Tat stimulation, a single band was observed for CREB-1 as indicated by the arrow in Fig. 7A. Similarly, HIV Tat induced the activation of Sp-1 that was competed out by the specific cold oligonucleotides (Fig. 7B). To ensure specificity, competition with cold specific and nonspecific oligonucleotides was performed. Specificity was also confirmed by supershift analysis using anti-CREB-1 or anti-Sp-1 and isotype control Abs (Fig. 7B, left panel). The anti-Sp-1 Abs used for supershift analysis resulted in disappearance of Sp-1 band (Fig. 7B, left panel). To determine whether HIV Tat-induced IL-10 production by Sp-1 and/or CREB-1 through the activation of the p38-CaMK-II pathway, we determined if the binding of CREB-1 and Sp-1 to their binding sites on the IL-10 promoter was inhibited by the inhibitors specific for p38, CaM, and CaMK-II. Monocytes were pretreated with either EGTA, W-7, ABP, KN-93, or SB202190 for 2 h before stimulation with Tat for 4 h. The results show that all the inhibitors that inhibited Tat-induced IL-10 production in the above experiments, inhibited the binding of CREB-1 as well as Sp-1 to their specific oligonucleotide probes (Fig. 7). Taken together, these results suggest that Tat induced the expression of IL-10 in human monocytic cells by CREB-1 and Sp-1 through the activation of calmodulin-CaMK-II pathway.


Figure 7
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  		FIGURE 7. HIV-Tat-induced CREB-1 and Sp-1 binding to binding sites present on the human IL-10 promoter is regulated by the activation of p38 and the CaM/CaMK-II pathways. Nuclear proteins obtained from monocytes (3 x 106/ml) treated with HIV-Tat (100 ng/ml) for 4 h were incubated with 32P-labeled probes corresponding to the CREB-1 (A) and Sp-1 (B) binding sites in the human IL-10 promoter. The specificity of CREB-1 and Sp-1 binding was determined by incubating the nuclear proteins in excess of specific (CC) and nonspecific (NS CC) cold competitor CREB-1 and Sp-1 probes (left panel). Supershift analysis was performed for CREB-1 and Sp-1 by incubating the nuclear proteins with Abs specific to CREB-1 and Sp-1, respectively (left panel). The supershifted bands for CREB-1 are indicated by arrows. Monocytes were pretreated with EGTA (2.5 µM), W-7 (10 µM), 2-APB (25 µM), KN93 (10 µM), or SB202190 (25 µM) for 2 h followed by stimulation for an additional 4 h with HIV Tat (100 ng/ml) (right panel). The gels shown are representative of three different experiments from different donors.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Tat is known to accumulate in the nucleus of infected cells and it is also secreted into the plasma of HIV-infected patients where it can exert its effects on uninfected bystander cells (40). For example, Tat has been shown to induce the expression of cytokines (IL-6, TNF-{alpha}, IL-10, IL-2, IL-12), cytokine receptors (IL-4R, IL-2R), adhesion molecules, and stimulates growth as well as causes apoptosis depending on the cell type (30, 31, 32, 33, 34, 35, 36, 37, 38, 39). Hence, understanding of molecular mechanisms underlying Tat-mediated biological effects remains a central issue in AIDS pathogenesis. There is evidence that Tat activates a cascade of signaling pathways, including MAPKs, PKC, PI3K, and p56 lck tyrosine kinases, and transcription factors such as NF-{kappa}B and CREB (35, 36, 44, 51, 53). In this study, we demonstrate for the first time the involvement of the calcium signaling pathway and its cross-talk with the downstream effector, p38 MAPK, in the regulation of HIV-Tat-induced IL-10 expression. Overall, our results suggest that extracellular HIV-Tat induced IL-10 transcription involved CREB-1 and Sp-1 transcription factors through the calmodulin/CaMK-II-dependent p38 MAPK activation in primary human monocytes.

Ca2+ is an important intracellular messenger in many biological processes (69). Influx of calcium ions through ligand and voltage-gated calcium channels in the plasma membrane together with Ca2+ release from ER stores results in complex calcium signaling cascades (65, 69). Several mechanisms may control Ca2+ entry in response to external stimuli including membrane depolarization, activation of intracellular messengers, and depletion of intracellular calcium storage (65, 69). The release of Ca2+ from internal stores (ER) is controlled by Ca2+ itself or by an expanding group of messengers. For example, inositol triphosphate (IP3), produced in response to a signal from the membrane lipid phosphatidylinositol, triggers Ca2+ release from the ER after binding to the IP3 receptor (69). By using a number of inhibitors specific for the Ca2+ pathway, our results demonstrated that intracellular release of Ca2+ into the cytosol may play a critical role in the regulation of HIV-Tat induced IL-10 production.

There is little information available on the role of Ca2+ signaling pathways in Tat-mediated biological effects. HIV-Tat was shown to induce TNF-{alpha} production through the release of Ca2+ from extracellular stores in monocytic cells (70). However, Badou et al. (36) failed to link Tat-induced IL-10 production through the calcium signaling pathway. Our results clearly show that Tat induced a significant level of calcium influx and its inhibition by EGTA abrogated IL-10 production. The involvement of the calcium signaling pathway was further supported by the results using the calcium-specific inhibitors for CaM/CaMK-II. The reason for this discrepancy is not clear, but may be due to the different Tat preparation used and experimental variations. It is interesting to observe that the calcium signaling pathway involved also seems to be Ca2+ released from internal stores, as our results with APB, the inhibitor that specifically targets the release of Ca2+ from internal stores, significantly reduced Tat-induced IL-10 expression. CaM, a key signaling protein responsible for integrating the Ca2+ signal to transcription factors, is known to regulate cell cycle and related cytoskeletal functions and ion channel activity (66). Following binding to Ca2+, CaM undergoes a conformational change that renders it active and able to recognize and bind target proteins with high affinity (66). Among the possible downstream targets of CaM are calcineurin and CaMK-II (66, 69). As with other kinases, CaMK-II undergoes autophosphorylation on a threonine residue contained in a phosphopeptide common to its {alpha} and beta subunits and converts it into a Ca2+/CaM independent enzyme (66, 69). The results obtained by using specific inhibitors for CaM, calcineurin, and CaMK-II, suggested that HIV-Tat induced IL-10 production is regulated by the CaM/CaMK-II pathway, but not through calcineurin signaling.

HIV-Tat has been shown to induce the phosphorylation of MAPK and regulate Tat- mediated biological effects (41, 43) such as the induction of VCAM expression in endothelial cells (34). Through the use of MAPK-specific inhibitors, our results suggest a previously unknown role for p38 MAPK in Tat-induced IL-10 expression. Since LPS is a potent mitogen capable of inducing IL-10 expression in monocytic cells, and because LPS-induced IL-10 production has been shown to be regulated by the activation of p38 MAPKs, we ensured that our Tat preparation is devoid of endotoxin contamination by purification through the use of polymyxin B beads, rendering Tat preparation biologically inactive by oxidation and by the lack of Tat-induced IL-12p40 production. The Tat concentrations used in our studies were physiologically comparable to that found in HIV infected individuals (30), particularly in lymph nodes of HIV-1 infected individuals where the Tat production would be significantly high (71). Our results also suggest a novel role for the CaMK-II-dependent p38 MAPK activation in IL-10 production. How p38 MAPK is modulated by the calcium signaling pathway is not clear. There is evidence that MAPKs can serve as substrates for the upstream calcium signaling molecules. Calcium-dependent p42/44 activation was implicated in the survival pathway induced by 7-ketocholesterol in THP-1 cells (72). Since CaMK-II has been shown to regulate the serine/theronine kinase RAF-1, upstream of p42/44 ERK MAPK activation (73), it is likely that CaMK-II may directly activate the signaling kinases involved in the activation of p38 MAPK such as MAPK kinase 3 and MAPK kinase 6.

LPS-induced IL-10 transcription in monocytic cells has previously been shown to be regulated by at least three distinct transcription factors namely, Sp-1, STAT-3, and C/EBPbeta (4, 54, 56, 74) however, the transcription factors involved in Tat-induced IL-10 production have not been well defined. Recently, we demonstrated the involvement of ERK MAPK-induced CREB-1 activation in intracellularly expressed Tat in the induction of IL-10 production in human monocytic cells (57). Additionally, we and others have demonstrated that p38 MAPK activate Sp-1 and CREB-1 transcription factors (4, 75). Herein, we describe the involvement of calcium signaling and its cross-talk with the downstream effectors, p38 MAPK, resulting in the activation of Sp-1 and CREB-1 transcription factors. Because of the difficulty in conducting promoter analysis and luciferase assays in primary cells, the involvement of these transcription factors could not be clearly defined in recombinant Tat-activated human monocytes. Moreover, promoter analysis could not be performed in monocytic cell lines such as THP-1 and U937 cells with recombinant Tat because of its inability to induce IL-10 production in these cells (data not shown). However, we analyzed the involvement of Sp-1 and CREB-1 by gel shift assays in primary monocytes, which suggests that recombinant Tat most likely regulates IL-10 production through the activation of Sp-1 and CREB-1. The Sp-1 and CREB binding activity was found to be sensitive to the same inhibitors that affected IL-10 expression, thereby indicating a role for CaMK-II and p38 MAPK-induced CREB activation in Tat-induced IL-10 production. Although promoter deletion studies did not identify any role for STAT3 or C/EBPbeta transcription factors in intracellular Tat- or LPS-induced IL-10 transcription in our previous studies (4, 57), we cannot rule out the possibility of other transactivators/coactivators such as CBP and C/EBPbeta that are known to cooperate with CREB-1 (76).

In summary, we elucidate for the first time, the signaling pathways mediated by recombinant Tat that induce IL-10 production. Our results show that recombinant HIV Tat-induced IL-10 expression in primary human monocytes via mechanisms that involve the activation of calmodulin, CaMK-II and p38 MAPK through the activation of CREB-1 and Sp-1 transcription factors. Understanding the regulation of IL-10 expression in HIV infection is important in deciphering the dysregulation of cytokine function induced by HIV and more broadly how HIV infection induces a state of immunodeficiency. The observation that Tat enhances IL-10 production in monocytic cells has several implications. First, it suggests that HIV employs Tat to enhance interactions between monocytic cells and T cells during HIV-1 infection. Since Tat is indispensable for HIV replication (28, 40), and IL-10 is known to inhibit HIV replication in monocytic cells (23, 24), Tat-induced IL-10 may prevent excessive HIV replication as a negative feedback regulator in monocytic cells. Since IL-10 is also known to prevent apoptosis in human monocytic cells (77), Tat may be a factor to prevent apoptosis of monocytic cells and thus promote the development of monocytic cells as viral reservoirs. Therefore, identification of CaMK-II and the p38 MAPK signaling pathways involved in Tat-induced IL-10 production may provide novel strategies to eliminate virus reservoirs and to restore immunological status in HIV-infected individuals.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by grants from the Ontario HIV Treatment Network, and the Canadian Institute of Health Research (to A.K.). K.G. is supported by a fellowship from the Ontario HIV Treatment Network. S.M. was supported by a fellowship from the Ontario Graduate Scholarship program, and the Ontario Graduate Scholarships in Science and Technology program. A.K. and J.B.A. are recipients of the Career Scientist Award from the Ontario HIV Treatment Network. Back

2 Address correspondence and reprint requests to Dr. Ashok Kumar, Division of Virology, Department of Pathology and Laboratory Medicine, Research Institute, Children’s Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario K1H 8L1, Canada. E-mail address: akumar{at}uottawa.ca Back

3 Abbreviations used in this paper: CaM, calmodulin; CaMK, calmodulin kinase; ER, endoplasmic reticulum; IP3, inositol (1,4,5)-triphosphate; APB, aminoethoxydiphenyl borate; PKC, protein kinase C. Back

Received for publication June 27, 2006. Accepted for publication November 7, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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